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Posts Tagged ‘lymphomas’


Treatment of Lymphomas [2.4.4C]

Larry H. Bernstein, MD, FCAP, Author, Curator, Editor

http://pharmaceuticalinnovation.com/2015/8/11/larryhbern/Treatment-of-Lymphomas-[2.4.4C]

 

Lymphoma treatment

Overview

http://www.emedicinehealth.com/lymphoma/page8_em.htm#lymphoma_treatment

The most widely used therapies are combinations of chemotherapyand radiation therapy.

  • Biological therapy, which targets key features of the lymphoma cells, is used in many cases nowadays.

The goal of medical therapy in lymphoma is complete remission. This means that all signs of the disease have disappeared after treatment. Remission is not the same as cure. In remission, one may still have lymphoma cells in the body, but they are undetectable and cause no symptoms.

  • When in remission, the lymphoma may come back. This is called recurrence.
  • The duration of remission depends on the type, stage, and grade of the lymphoma. A remission may last a few months, a few years, or may continue throughout one’s life.
  • Remission that lasts a long time is called durable remission, and this is the goal of therapy.
  • The duration of remission is a good indicator of the aggressiveness of the lymphoma and of the prognosis. A longer remission generally indicates a better prognosis.

Remission can also be partial. This means that the tumor shrinks after treatment to less than half its size before treatment.

The following terms are used to describe the lymphoma’s response to treatment:

  • Improvement: The lymphoma shrinks but is still greater than half its original size.
  • Stable disease: The lymphoma stays the same.
  • Progression: The lymphoma worsens during treatment.
  • Refractory disease: The lymphoma is resistant to treatment.

The following terms to refer to therapy:

  • Induction therapy is designed to induce a remission.
  • If this treatment does not induce a complete remission, new or different therapy will be initiated. This is usually referred to as salvage therapy.
  • Once in remission, one may be given yet another treatment to prevent recurrence. This is called maintenance therapy.

Chemotherapy

Many different types of chemotherapy may be used for Hodgkin lymphoma. The most commonly used combination of drugs in the United States is called ABVD. Another combination of drugs, known as BEACOPP, is now widely used in Europe and is being used more often in the United States. There are other combinations that are less commonly used and not listed here. The drugs that make up these two more common combinations of chemotherapy are listed below.

ABVD: Doxorubicin (Adriamycin), bleomycin (Blenoxane), vinblastine (Velban, Velsar), and dacarbazine (DTIC-Dome). ABVD chemotherapy is usually given every two weeks for two to eight months.

BEACOPP: Bleomycin, etoposide (Toposar, VePesid), doxorubicin, cyclophosphamide (Cytoxan, Neosar), vincristine (Vincasar PFS, Oncovin), procarbazine (Matulane), and prednisone (multiple brand names). There are several different treatment schedules, but different drugs are usually given every two weeks.

The type of chemotherapy, number of cycles of chemotherapy, and the additional use of radiation therapy are based on the stage of the Hodgkin lymphoma and the type and number of prognostic factors.

Adult Non-Hodgkin Lymphoma Treatment (PDQ®)

http://www.cancer.gov/cancertopics/pdq/treatment/adult-non-hodgkins/Patient/page1

Key Points for This Section

Adult non-Hodgkin lymphoma is a disease in which malignant (cancer) cells form in the lymph system.

Because lymph tissue is found throughout the body, adult non-Hodgkin lymphoma can begin in almost any part of the body. Cancer can spread to the liver and many other organs and tissues.

Non-Hodgkin lymphoma in pregnant women is the same as the disease in nonpregnant women of childbearing age. However, treatment is different for pregnant women. This summary includes information on the treatment of non-Hodgkin lymphoma during pregnancy

Non-Hodgkin lymphoma can occur in both adults and children. Treatment for children, however, is different than treatment for adults. (See the PDQ summary on Childhood Non-Hodgkin Lymphoma Treatment for more information.)

There are many different types of lymphoma.

Lymphomas are divided into two general types: Hodgkin lymphoma and non-Hodgkin lymphoma. This summary is about the treatment of adult non-Hodgkin lymphoma. For information about other types of lymphoma, see the following PDQ summaries:

Age, gender, and a weakened immune system can affect the risk of adult non-Hodgkin lymphoma.

If cancer is found, the following tests may be done to study the cancer cells:

  • Immunohistochemistry : A test that uses antibodies to check for certain antigens in a sample of tissue. The antibody is usually linked to a radioactive substance or a dye that causes the tissue to light up under a microscope. This type of test may be used to tell the difference between different types of cancer.
  • Cytogenetic analysis : A laboratory test in which cells in a sample of tissue are viewed under a microscope to look for certain changes in the chromosomes.
  • Immunophenotyping : A process used to identify cells, based on the types of antigens ormarkers on the surface of the cell. This process is used to diagnose specific types of leukemia and lymphoma by comparing the cancer cells to normal cells of the immune system.

Certain factors affect prognosis (chance of recovery) and treatment options.

The prognosis (chance of recovery) and treatment options depend on the following:

  • The stage of the cancer.
  • The type of non-Hodgkin lymphoma.
  • The amount of lactate dehydrogenase (LDH) in the blood.
  • The amount of beta-2-microglobulin in the blood (for Waldenström macroglobulinemia).
  • The patient’s age and general health.
  • Whether the lymphoma has just been diagnosed or has recurred (come back).

Stages of adult non-Hodgkin lymphoma may include E and S.

Adult non-Hodgkin lymphoma may be described as follows:

E: “E” stands for extranodal and means the cancer is found in an area or organ other than the lymph nodes or has spread to tissues beyond, but near, the major lymphatic areas.

S: “S” stands for spleen and means the cancer is found in the spleen.

Stage I adult non-Hodgkin lymphoma is divided into stage I and stage IE.

  • Stage I: Cancer is found in one lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen).
  • Stage IE: Cancer is found in one organ or area outside the lymph nodes.

Stage II adult non-Hodgkin lymphoma is divided into stage II and stage IIE.

  • Stage II: Cancer is found in two or more lymph node groups either above or below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
  • Stage IIE: Cancer is found in one or more lymph node groups either above or below the diaphragm. Cancer is also found outside the lymph nodes in one organ or area on the same side of the diaphragm as the affected lymph nodes.

Stage III adult non-Hodgkin lymphoma is divided into stage III, stage IIIE, stage IIIS, and stage IIIE+S.

  • Stage III: Cancer is found in lymph node groups above and below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
  • Stage IIIE: Cancer is found in lymph node groups above and below the diaphragm and outside the lymph nodes in a nearby organ or area.
  • Stage IIIS: Cancer is found in lymph node groups above and below the diaphragm, and in the spleen.
  • Stage IIIE+S: Cancer is found in lymph node groups above and below the diaphragm, outside the lymph nodes in a nearby organ or area, and in the spleen.

In stage IV adult non-Hodgkin lymphoma, the cancer:

  • is found throughout one or more organs that are not part of a lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen), and may be in lymph nodes near those organs; or
  • is found in one organ that is not part of a lymphatic area and has spread to organs or lymph nodes far away from that organ; or
  • is found in the liver, bone marrow, cerebrospinal fluid (CSF), or lungs (other than cancer that has spread to the lungs from nearby areas).

Adult non-Hodgkin lymphomas are also described based on how fast they grow and where the affected lymph nodes are in the body.  Indolent & aggressive.

The treatment plan depends mainly on the following:

  • The type of non-Hodgkin’s lymphoma
  • Its stage (where the lymphoma is found)
  • How quickly the cancer is growing
  • The patient’s age
  • Whether the patient has other health problems
  • If there are symptoms present such as fever and night sweats (see above)
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Hematologic Malignancies , Table of Contents

Writer and Curator:  Larry H. Bernstein, MD, FCAP

Hematologic Malignancies 

Not excluding lymphomas [solid tumors]

The following series of articles are discussions of current identifications, classification, and treatments of leukemias, myelodysplastic syndromes and myelomas.

2.4 Hematological Malignancies

2.4.1 Ontogenesis of blood elements

Erythropoiesis

White blood cell series: myelopoiesis

Thrombocytogenesis

2.4.2 Classification of hematopoietic cancers

Primary Classification

Acute leukemias

Myelodysplastic syndromes

Acute myeloid leukemia

Acute lymphoblastic leukemia

Myeloproliferative Disorders

Chronic myeloproliferative disorders

Chronic myelogenous leukemia and related disorders

Myelofibrosis, including chronic idiopathic

Polycythemia, including polycythemia rubra vera

Thrombocytosis, including essential thrombocythemia

Chronic lymphoid leukemia and other lymphoid leukemias

Lymphomas

Non-Hodgkin Lymphoma

Hodgkin lymphoma

Lymphoproliferative disorders associated with immunodeficiency

Plasma Cell dyscrasias

Mast cell disease and Histiocytic neoplasms

Secondary Classification

Nuance – PathologyOutlines

2.4.3 Diagnostics

Computer-aided diagnostics

Back-to-Front Design

Realtime Clinical Expert Support

Regression: A richly textured method for comparison and classification of predictor variables

Converting Hematology Based Data into an Inferential Interpretation

A model for Thalassemia Screening using Hematology Measurements

Measurement of granulocyte maturation may improve the early diagnosis of the septic state.

The automated malnutrition assessment.

Molecular Diagnostics

Genomic Analysis of Hematological Malignancies

Next-generation sequencing in hematologic malignancies: what will be the dividends?

Leveraging cancer genome information in hematologic malignancies.

p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies

Genomic approaches to hematologic malignancies

2.4.4 Treatment of hematopoietic cancers

2.4.4.1 Treatments for leukemia by type

2.4.4..2 Acute lymphocytic leukemias

2.4..4.3 Treatment of Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia

Gene-Expression Patterns in Drug-Resistant Acute Lymphoblastic Leukemia Cells and Response to Treatment

Leukemias Treatment & Management

Treatments and drugs

2.4.5 Acute Myeloid Leukemia

New treatment approaches in acute myeloid leukemia: review of recent clinical studies

Novel approaches to the treatment of acute myeloid leukemia.

Current treatment of acute myeloid leukemia

Adult Acute Myeloid Leukemia Treatment (PDQ®)

2.4.6 Treatment for CML

Chronic Myelogenous Leukemia Treatment (PDQ®)

What`s new in chronic myeloid leukemia research and treatment?

4.2.7 Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia Treatment (PDQ®)

Results from the Phase 3 Resonate™ Trial

Typical treatment of chronic lymphocytic leukemia

4.2.8 Lymphoma treatment

4.2.8.1 Overview

4.2.8.2 Chemotherapy

………………………………..

Chapter 6

Total body irradiation (TBI)

Bone marrow (BM) transplantation

Autologous stem cell transplantation

Hematopoietic stem cell transplantation

Supportive Therapies

Blood transfusions

Erythropoietin

G-CSF (granulocyte-colony stimulating factor)

Plasma exchange (plasmapheresis)

Platelet transfusions

Steroids

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Hematological Cancer Classification

Author and Curator: Larry H. Bernstein, MD, FCAP

 

 

Introduction to leukemias and lymphomas

 

2.4.1 Ontogenesis of the blood elements: hematopoiesis

http://www.britannica.com/EBchecked/topic/69747/blood-cell-formation

Blood cells are divided into three groups: the red blood cells (erythrocytes), the white blood cells (leukocytes), and the blood platelets (thrombocytes). The white blood cells are subdivided into three broad groups: granulocytes, lymphocytes, and monocytes.

Blood cells do not originate in the bloodstream itself but in specific blood-forming organs, notably the marrow of certain bones. In the human adult, the bone marrow produces all of the red blood cells, 60–70 percent of the white cells (i.e., the granulocytes), and all of the platelets. The lymphatic tissues, particularly the thymus, the spleen, and the lymph nodes, produce the lymphocytes (comprising 20–30 percent of the white cells). The reticuloendothelial tissues of the spleen, liver, lymph nodes, and other organs produce the monocytes (4–8 percent of the white cells). The platelets, which are small cellular fragments rather than complete cells, are formed from bits of the cytoplasm of the giant cells (megakaryocytes) of the bone marrow.

In the human embryo, the first site of blood formation is the yolk sac. Later in embryonic life, the liver becomes the most important red blood cell-forming organ, but it is soon succeeded by the bone marrow, which in adult life is the only source of both red blood cells and the granulocytes. Both the red and white blood cells arise through a series of complex, gradual, and successive transformations from primitive stem cells, which have the ability to form any of the precursors of a blood cell. Precursor cells are stem cells that have developed to the stage where they are committed to forming a particular kind of new blood cell.

In a normal adult the red cells of about half a liter (almost one pint) of blood are produced by the bone marrow every week. Almost 1 percent of the body’s red cells are generated each day, and the balance between red cell production and the removal of aging red cells from the circulation is precisely maintained.

Cells-in-the-Bone-Marrow-1024x747

http://interactive-biology.com/wp-content/uploads/2012/07/Cells-in-the-Bone-Marrow-1024×747.png

Erythropoiesis

http://www.interactive-biology.com/3969/erythropoiesis-formation-of-red-blood-cells/

Erythropoiesis – Formation of Red Blood Cells

Because of the inability of erythrocytes (red blood cells) to divide to replenish their own numbers, the old ruptured cells must be replaced by totally new cells. They meet their demise because they don’t have the usual specialized intracellular machinery, which controls cell growth and repair, leading to a short life span of 120 days.

This short life span necessitates the process erythropoiesis, which is the formation of red blood cells. All blood cells are formed in the bone marrow. This is the erythrocyte factory, which is soft, highly cellar tissue that fills the internal cavities of bones.

Erythrocyte differentiation takes place in 8 stages. It is the pathway through which an erythrocyte matures from a hemocytoblast into a full-blown erythrocyte. The first seven all take place within the bone marrow. After stage 7 the cell is then released into the bloodstream as a reticulocyte, where it then matures 1-2 days later into an erythrocyte. The stages are as follows:

  1. Hemocytoblast, which is a pluripotent hematopoietic stem cell
  2. Common myeloid progenitor, a multipotent stem cell
  3. Unipotent stem cell
  4. Pronormoblast
  5. Basophilic normoblast also called an erythroblast.
  6. Polychromatophilic normoblast
  7. Orthochromatic normoblast
  8. Reticulocyte

These characteristics can be seen during the course of erythrocyte maturation:

  • The size of the cell decreases
  • The cytoplasm volume increases
  • Initially there is a nucleus and as the cell matures the size of the nucleus decreases until it vanishes with the condensation of the chromatin material.

Low oxygen tension stimulates the kidneys to secrete the hormone erythropoietin into the blood, and this hormone stimulates the bone marrow to produce erythrocytes.

Rarely, a malignancy or cancer of erythropoiesis occurs. It is referred to as erythroleukemia. This most likely arises from a common myeloid precursor, and it may occur associated with a myelodysplastic syndrome.

Summary of erythrocyte maturation

White blood cell series: myelopoiesis

http://www.nlm.nih.gov/medlineplus/ency/presentations/100151_3.htm

http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/15220.jpg

There are various types of white blood cells (WBCs) that normally appear in the blood: neutrophils (polymorphonuclear leukocytes; PMNs), band cells (slightly immature neutrophils), T-type lymphocytes (T cells), B-type lymphocytes (B cells), monocytes, eosinophils, and basophils. T and B-type lymphocytes are indistinguishable from each other in a normal slide preparation. Any infection or acute stress will result in an increased production of WBCs. This usually entails increased numbers of cells and an increase in the percentage of immature cells (mainly band cells) in the blood. This change is referred to as a “shift to the left” People who have had a splenectomy have a persistent mild elevation of WBCs. Drugs that may increase WBC counts include epinephrine, allopurinol, aspirin, chloroform, heparin, quinine, corticosteroids, and triamterene. Drugs that may decrease WBC counts include antibiotics, anticonvulsants, antihistamine, antithyroid drugs, arsenicals, barbiturates, chemotherapeutic agents, diuretics and sulfonamides.   (Updated by: David C. Dugdale, III, MD)

https://www.med-ed.virginia.edu/courses/path/innes/nh/wcbmaturation.cfm

Note that the mature forms of the myeloid series (neutrophils, eosinophils, basophils), all have lobed (segmented) nuclei. The degree of lobation increases as the cells mature.

The earliest recognizable myeloid cell is the myeloblast (10-20m dia) with a large round to oval nucleus. There is fine diffuse immature chromatin (without clumping) and a prominant nucleolus.

The cytoplasm is basophilic without granules. Although one may see a small golgi area adjacent to the nucleus, granules are not usually visible by light microscopy. One should not see blast cells in the peripheral blood.

myeloblast x100b

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20myeloblast%20x100b.jpeg

The promyelocyte (10-20m) is slightly larger than a blast. Its nucleus, although similar to a myeloblast shows slight chromatin condensation and less prominent nucleoli. The cytoplasm contains striking azurophilic granules or primary granules. These granules contain myeloperoxidase, acid phosphatase, and esterase enzymes. Normally no promyelocytes are seen in the peripheral blood.

At the point in development when secondary granules can be recognized, the cell becomes a myelocyte.

promyelocyte x100

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20promyelocyte%20×100%20a.jpeg

Myelocytes (10-18m) are not normally found in the peripheral blood. Nucleoli may not be seen in the late myelocyte. Primary azurophilic granules are still present, but secondary granules predominate. Secondary granules (neut, eos, or baso) first appear adjacent to the nucleus. In neutrophils this is the “dawn” of neutrophilia.

Metamyelocytes (10-18m) have kidney shaped indented nuclei and dense chromatin along the nuclear membrane. The cytoplasm is faintly pink, and they have secondary granules (neutro, eos, or baso). Zero to one percent of the peripheral blood white cells may be metamyelocytes (juveniles).

metamyelocyte x100

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20metamyelocyte%20×100.jpeg

Bands, slightly smaller than juveniles, are marked by a U-shaped or deeply indented nucleus.

band neutrophilx100a

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20band%20x100a.jpeg

Segmented (segs) or polymorphonuclear (PMN) leukocytes (average 14 m dia) are distinguished by definite lobation with thin thread-like filaments of chromatin joining the 2-5 lobes. 45-75% of the peripheral blood white cells are segmented neutrophils.

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20neutrophil%20×100%20d.jpeg

Thrombocytogenesis

The incredible journey: From megakaryocyte development to platelet formation

Kellie R. Machlus1,2 and Joseph E. Italiano Jr
JCB 2013; 201(6): 785-796
http://dx.doi.org:/10.1083/jcb.201304054

Large progenitor cells in the bone marrow called megakaryocytes (MKs) are the source of platelets. MKs release platelets through a series of fascinating cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Then, in a cytoskeletal-driven process, they extend long branching processes, designated proplatelets, into sinusoidal blood vessels where they undergo fission to release platelets.

megakaryocyte production of platelets

http://dm5migu4zj3pb.cloudfront.net/manuscripts/26000/26891/medium/JCI0526891.f4.jpg

platelets and the immune continuum nri2956-f3

http://www.nature.com/nri/journal/v11/n4/images/nri2956-f3.jpg

2.4.2 Classification of hematological malignancies
Practical Diagnosis of Hematologic Disoreders. 4th edition. Vol 2.
Kjeldsberg CR, Ed.  ASCP Press.  2006. Chicago, IL.

2.4.2.1 Primary Classification

Acute leukemias

Myelodysplastic syndromes

Acute myeloid leukemia

Acute lymphoblastic leukemia

Myeloproliferative Disorders

Chronic myeloproliferative disorders

Chronic myelogenous leukemia and related disorders

Myelofibrosis, including chronic idiopathic

Polycythemia, including polycythemia rubra vera

Thrombocytosis, including essential thrombocythemia

Chronic lymphoid leukemia and other lymphoid leukemias

Lymphomas

Non-Hodgkin Lymphoma

Hodgkin lymphoma

Lymphoproliferative disorders associated with immunodeficiency

Plasma Cell dyscrasias

Mast cell disease and Histiocytic neoplasms

2.4.2.2 Secondary Classification

2.4.2.3 Nuance – PathologyOutlines
Nat Pernick, Ed.

Leukemia – Acute

Primary referencesacute leukemia-generalAML generalAML classificationtransient abnormal myelopoiesis

Recurrent genetic abnormalities: AML with t(6;9)AML with t(8;21)AML with 11q23 abnormalitiesAML with inv(16) or t(16;16)AML with Down syndromeAML with FLT3 mutationsAML with myelodysplastic related changesAML therapy relatedAPL microgranular variantAPL with t(15;17)APL with t(V;17)APL therapy related

AML not otherwise categorized: minimally differentiated (M0)without maturation (M1)with maturation (M2)M3myelomonocyticmonoblastic and monocyticerythroidmegakaryoblasticCD13/CD33 negativebasophilicmyeloid sarcomaacute panmyelosis with myelofibrosiswith Philadelphia chromosomewith pseudo Chediak-Higashi anomalyhypocellular

ALL: generalWHO classificationwith eosinophilia

PreB ALL: generalt(9;22)t(v;11q23)t(1;19)t(5;14)t(12;21)hyperdiploidyhypodiploidymature B ALL/Burkitt

Other ALL: T ALLambiguous lineagemixed phenotype

AML and related malignancies

Acute myeloid leukemias with recurrent genetic abnormalities:

  • AML with t(8;21)(q22;q22); RUNX1-RUNX1T1
  • AML with inv(16)(p13.1;q22) or t(16;16)(p13.1;q22); CBF&beta-MYH11
  • Acute promyelocytic leukemia with t(15;17)(q22;q12); PML/RAR&alpha and variants
  • AML with t(9;11)(p22;q23); MLLT3-MLL
  • AML with t(6;9)(p23;q34); DEK-NUP214
  • AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1
  • AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1
  • AML with mutated NPM1*
  • AML with mutated CEBPA*

* provisional

Acute myeloid leukemia with myelodysplasia related changes

Therapy related acute myeloid leukemia

  • Alkylating agent related
  • Topoisomerase II inhibitor related (some maybe lymphoid)

Acute myeloid leukemia not otherwise categorized:

  • AML minimally differentiated (M0)
  • AML without maturation (M1)
  • AML with maturation (M2)
  • Acute myelomonocytic leukemia (M4)
  • Acute monoblastic and monocytic leukemia (M5a, M5b)
  • Acute erythroid leukemia (M6)
  • Acute megakaryoblastic leukemia (M7)
  • Acute basophilic leukemia
  • Acute panmyelosis with myelofibrosis

Myeloid Sarcoma

Myeloid proliferations related to Down syndrome:

  • Transient abnormal myelopoeisis
  • Myeloid leukemia associated with Down syndrome

Blastic plasmacytoid dentritic cell neoplasm:

Acute leukemia of ambiguous lineage:

  • Acute undifferentiated leukemia
  • Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); BCR-ABL1
  • Mixed phenotype acute leukemia with t(v;11q23); MLL rearranged
  • Mixed phenotype acute leukemia, B/myeloid, NOS
  • Mixed phenotype acute leukemia, T/myeloid, NOS
  • Mixed phenotype acute leukemia, NOS, rare types
  • Other acute leukemia of ambiguous lineage
  • References: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue (IARC, 2008), Discovery Medicine 2010, eMedicine

Acute lymphocytic leukemia

General
=================================================================

  • WHO classification system includes former FAB classifications ALL-L1 and L2
    ● FAB L3 is now considered Burkitt lymphoma

WHO classification of acute lymphoblastic leukemia
=================================================================

Precursor B lymphoblastic leukemia / lymphoblastic lymphoma:
● ALL with t(9;22)(q34;q11.2); BCR-ABL (Philadelphia chromosome)
● ALL with t(v;11q23) (MLL rearranged)
● ALL with t(1;19)(q23;p13.3); TCF3-PBX1 (E2A-PBX1)
● ALL with t(12;21)(p13;q22); ETV6-RUNX1 (TEL-AML1)
● Hyperdiploid > 50
● Hypodiploid
● t(5;14)(q31;q32); IL3-IGH

Precursor T lymphoblastic leukemia / lymphoma

Additional references
=================================================================

Mixed phenotype acute leukemia (MPAL)

General
=================================================================

  • De novo acute leukemia containing separate populations of blasts of more than one lineage (bilineal or bilineage), or a single population of blasts co-expressing antigens of more than one lineage (biphenotypic)Excludes:
    ● Acute myeloid leukemia (AML) with recurrent translocations t(8;21), t(15;17) or inv(16)
    ● Leukemias with FGFR1 mutations
    ● Chronic myelogenous leukemia (CML) in blast crisis
    ● Myelodysplastic syndrome (MDS)-related AML and therapy-related AML, even if they have MPAL immunophenotypeCriteria for biphenotypic leukemia:
    ● Score of 2 or more for each of two separate lineages:The European Group for the Immunological Classification of Leukemias (EGIL) scoring system2008 WHO classification of acute leukemias of ambiguous lineage 

Prognosis
=================================================================

  • Poor, overall survival of 18 months
    ● Young age, normal karyotype and ALL induction therapy are associated with favorable survival
    ● Ph+ is a predictor for poor prognosis
    ● Bone marrow transplantation should be considered in first remission

Major Categories

MPAL with t(9;22)(q34;q11.2); BCR-ABL1
=================================================================

  • 20% of all MPAL
    ● Blasts with t(9;22)(q34;q11.2) translocation or BCR-ABL1 rearrangement (Ph+) without history of CML
    ● Majority in adults
    ● High WBC counts● Most of the cases B/myeloid phenotype
    ● Rare T/myeloid, B and T lineage, or trilineage leukemiasMorphology:
    ● Many cases show a dimorphic blast population, one resembling myeloblasts and the other lymphoblastsCytogenetic abnormalities:
    ● Conventional karyotyping for t(9;22), FISH or PCR for BCR-ABL1 translocation
    ● Additional complex karyotypes
    ● Ph+ is a poor prognostic factor for MPAL, with a reported median survival of 8 months
    ● Worse than patients of all other types of MPAL

MPAL with t(v;11q23); MLL rearranged
=================================================================

  • Meeting the diagnostic criteria for MPAL with blasts bearing a translocation involving the 11q23 breakpoint (MLL gene)
    ● MPAL with MLL rearranged rare
    ● More often seen in children and relatively common in infancy
    ● High WBC counts
    ● Poor prognosis
    ● Dimorphic blast population, with one resembling monoblasts and the other resembling lymphoblasts
    ● Lymphoblast population often shows a CD19+, CD10- B precursor immunophenotype, frequently CD15+
    ● Expression of other B markers usually weak
    ● Translocations involving MLL gene include t(4;11)(q21;q23), t(11;19)(q23;p13), and t(9;11)(p22;q23)
    ● Cases with chromosome 11q23 deletion should not be classified in this category

B cell acute lymphoblastic leukemia (ALL) / lymphoblastic lymphoma (LBL)

General

=================================================================

  • Current 2008 WHO classification: B lymphoblastic leukemia / lymphoma, NOS or B lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities
  • See also lymphomas: B cell chapter
  • Also called B cell acute lymphoblastic leukemia / lymphoblastic lymphoma, pre B ALL / LBL
  • Usually children
  • B acute lymphoblastic leukemia presents with pancytopenia due to extensive marrow involvement, stormy onset of symptoms, bone pain due to marrow expansion, hepatosplenomegaly due to neoplastic infiltration, CNS symptoms due to meningeal spread and testicular involvement
  • B acute lymphoblastic lymphoma often presents with cutaneous nodules, bone or nodal involvement, < 25% lymphoblasts in bone marrow and peripheral blood; aleukemic cases are usually asymptomatic
  • Depending on specific leukemia, arises in either hematopoietic stem cell or B-cell progenitor
  • Tumors are derived from pre-germinal center naive B cells with unmutated VH region genes
  • Have multiple immunophenotyping aberrancies relative to normal B cell precursors (hematogones); at relapse, 73% show loss of 1+ aberrance and 60% show new aberrancies (Am J Clin Pathol 2007;127:39)

Prognostic features

=================================================================

  • Favorable prognosis: age 1-10 years, female, white; preB phenotype, hyperdiploidy>50, t(12,21), WBC count at presentation <50×108/L, non-traumatic tap with no blasts in CNS, rapid response to chemotherapy < 5% blasts on morphology on day 15, remission status after induction <5% blasts on morphology and <0.01% blast on flow or PCR, CD10+
  • Intermediate prognosis: hyperdiploidy 47-50, diploid, 6q- and rearrangements of 8q24
  • Unfavorable prognosis: under age 1 (usually have 11q23 translocations) or over age 10; t(9;22) (but not if age 59+ years, Am J Clin Pathol 2002;117:716); male, > 50×108/L WBC at presentation, hypodiploidy, near tetraploidy, 17p- and MLL rearrangements t(v;11q23); CD10-; non-traumatic tap with > 5% blasts or traumatic tap (7%); also increased microvessel staining using CD105 in children (Leuk Res 2007;31:1741), MDR1 expression in children (Oncol Rep 2004;12:1201) and adults (Blood 2002;100:974), 25%+ blasts on morphology on day 15, remission status after induction ≥ 5% blasts on morphology and ≥ 0.1% blasts on flow or PCR

Case reports

=================================================================

  • 12 month old girl and 13 month old boy with mature phenotype but no translocations (Arch Pathol Lab Med 2003;127:1340)
  • 56 year old man with ALL arising from follicular lymphoma (Arch Pathol Lab Med 2002;126:997)
  • 76 year old man with basal cell carcinoma (Diagn Pathol 2007;2:32)
  • With hemophagocytic lymphohistiocytosis (Pediatr Blood Cancer 2008;50:381)

Treatment

================================================================

  • Chemotherapy cures more children than adults; adolescents benefit from intensive regimens (Hematology Am Soc Hematol Educ Program 2005:123)

Micro description

=================================================================

  • Bone marrow smears: small to intermediate blast-like cells with scant, variably basophilic cytoplasm, round / oval or convoluted nuclei, fine chromatin and indistinct nucleoli; frequent mitotic figures; may have “starry sky” appearance similar to Burkitt lymphoma; may have large lymphoblasts with 1-4 prominent nucleoli resembling myeloblasts; usually no sclerosis
  • Bone marrow biopsy: usually markedly hypercellular with reduction of trilinear maturation; cells have minimal cytoplasm, medium sized nuclei that are often convoluted, moderately dense chromatin and indistinct nucleoli, brisk mitotic activity
  • Other tissues: may have “starry sky” appearance similar to Burkitt lymphoma; collagen dissection, periadipocyte growth pattern and single cell linear filing

Chronic Leukemia

Chronic Myeloid Neoplasms

Myelodysplastic syndromes (MDS): general, WHO classification, childhood, refractory anemia, refractory anemia with ringed sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts, 5q-syndrome, therapy related, unclassified, arsenic toxicity

Myeloproliferative neoplasms (MPN): general, WHO classification, chronic eosinophilic leukemia, chronic myelogenous leukemia, chronic neutrophilic leukemia, essential thrombocythemia, hypereosinophilic syndrome, mast cell disease, polycythemia vera, primary myelofibrosis, unclassifiable

MDS/MPN: general, WHO classification, atypical CML, chronic myelomonocytic leukemia (CMML), chronic myelomonocytic leukemia with eosinophilia, juvenile myelomonocytic leukemia, unclassifiable

Myeloid neoplasms associated with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1: PDGFRA, PDGFRB, FGFR1

Miscellaneous: transient myeloproliferative disorder of Downís syndrome

Lymphoma and plasma cell neoplasms

Lymph nodes: normal development-generalB cellsT cellsNK cellsnormal histologygrossing lymph nodesfeatures to report

Molecular testing: theoryFISHnorthern blotPCRsouthern blot

Non-Hodgkin lymphoma: generalcytogeneticsstagingstaging-pediatricmorphologic clueshemophagocytic syndromechemotherapeutic atypia

B cell disorders: generalpost-rituximabbone marrow biopsyclassification-historicalWHO classification

B cell lymphoma subtypes: age-related EBV-associatedALK positive large cellBurkittunclassifiable-intermediate between Burkitt and diffuse large B cell lymphomaCLL
diffuse large B cell: 
diffuse-NOSCD5+T cell / histiocyte richprimary cutaneous-generalprimary cutaneous-legprimary sites-other
follicular: 
generalchildhoodcutaneousGI
hairy cell leukemiaHCL variantintravascular large B celllymphomatoid granulomatosislymphoplasmacyticmantle cell-classicmantle cell-blastoidmarginal zone-generalmarginal zone-MALTMALT-primary sitesmarginal zone-nodalmediastinal (thymic)plasmablasticpre B lymphoblastic leukemia/lymphomaprimary effusionprolymphocytic leukemiapyothorax associatedSLLsplenic marginal zonesplenic lymphoma with villous lymphocytes

Plasma cell neoplasms: generalmyelomaplasmacytomaheavy chain diseaseprimary amyloidosisMGUSOsteosclerotic myeloma (POEMS)cryoglobulinemia

T/NK cell disorders: generalWHO classificationadult T cellaggressive NK cell leukemiaanaplastic large cell ALK+ALK-angioimmunoblastic T cellblastic plasmacytoidchronic lymphoproliferative disorders of NK cellscutaneous CD4+ small/medium sized T cell lymphomacutaneous CD30 positive T cell lymphoproliferative disorderscutaneous gamma delta T cell lymphomaenteropathyepidermotropic CD8+ T cell lymphomahepatosplenicindolent T cell proliferationsmycosis fungoidesNK/T cell lymphoma-nasal typenodal CD8+ cytotoxic T cellnonB nonT lymphoblasticperipheral T cell lymphoma, NOSprimary effusion lymphomaSezary syndromestagingsubcutaneous panniculitis-likeT cell large granular lymphocytic leukemiaT cell lymphoblastic leukemia/lymphomaT cell prolymphocytic leukemia

Hodgkin lymphoma: general/stagingclassiclymphocyte depletedlymphocyte rich classicalmixed cellularitynodular lymphocyte predominantnodular sclerosis

Post-transplantation: generalWHO classificationplasmacytic hyperplasia/IM-like lesionspolymorphic B cell lymphoproliferative disordersmonomorphic B cell lymphoproliferative disordersothergraft versus host disease

Other: AIDS associated-generalAIDS associated-examplesEBV+ T cell lymphoproliferative disorders of childhoodprimary immune disorders related

Myeloproliferative neoplasms (MPN)

WHO 2008 – Myeloproliferative neoplasms (MPN) 

General
=================================================================

  • Chronic myelogenous leukemia
    ● Polycythemia vera
    ● Essential thrombocythemia
    ● Primary myelofibrosis
    ● Chronic neutrophilic leukemia
    ● Chronic eosinophilic leukemia, not otherwise categorized
    ● Mast cell disease
    ● MPNs, unclassifiable

WHO 2001 – Chronic myeloproliferative diseases 

Definition
=================================================================

  • Chronic myelogenous leukemia (Philadelphia chromosome, t(9;22)(q34;q11), BCR-ABL positive)
    ● Chronic neutrophilic leukemia
    ● Chronic eosinophilic leukemia (and the hypereosinophilic syndrome)
    ● Polycythemia vera
    ● Chronic idiopathic myelofibrosis (with extramedullary hematopoiesis)
    ● Essential thrombocythemia
    ● Chronic myeloproliferative disease, unclassifiable

Additional references
=================================================================

The World Health Organization (WHO) classification of the myeloid neoplasms  James W. Vardiman, Nancy Lee Harris, and Richard D. Brunning
Blood 2002; 100(7)  http://dx.doi.org/10.1182/blood-2002-04-1199

Lymphoma – Non B cell neoplasms

T/NK cell disorders/WHO classification (2008)

Principles of classification
=================================================================

  • Based on all available information (morphology, immunophenotype, genetics, clinical)
    ● No one antigenic marker is specific for any neoplasm (except ALK1)
    ● Immune profiling less helpful in subclassification of T cell lymphomas then B cell lymphomas
    ● Certain antigens commonly associated with specific disease entities but not entirely disease specific
    ● CD30: common in anaplastic large cell lymphoma but also classic Hodgkin lymphoma and other B and T cell lymphomas
    ● CD56: characteristic for nasal NK/T cell lymphoma, but also other T cell neoplasms and plasma cell disorders
    ● Variation of immunophenotype within a given disease (hepatosplenic T cell lymphoma: usually γδ but some are αβ)
    ● Recurrent genetic alterations have been identified for many B cell lymphomas but not for most T cell lymphomas
    ● No attempt to stratify lymphoid malignancies by grade
    ● Recognize the existence of grey zone lymphomas
    ● This multiparameter approach has been validated in international studies as highly reproducible

WHO 2008 classification of tumors of hematopoietic and lymphoid tissues (T/NK)
=================================================================

Precursor T-lymphoid neoplasms
● T lymphoblastic leukemia/lymphoma, 9837/3

Mature T cell and NK cell neoplasms
● T cell prolymphocytic leukemia, 9834/3
● T cell large granular lymphocytic leukemia, 9831/3
● Chronic lymphoproliferative disorder of NK cells, 9831/3
● Aggressive NK cell leukemia, 9948/3
● Systemic EBV-positive T cell lymphoproliferative disease of childhood, 9724/3
● Hydroa vacciniforme-like lymphoma, 9725/3
● Adult T cell leukemia/lymphoma, 9827/3
● Extranodal NK/T cell lymphoma, nasal type, 9719/3
● Enteropathy-associated T cell lymphoma, 9717/3
● Hepatosplenic T cell lymphoma, 9716/3
● Subcutaneous panniculitis-like T cell lymphoma, 9708/3
● Mycosis fungoides, 9700/3
● Sézary syndrome, 9701/3
● Primary cutaneous CD30-positive T cell lymphoproliferative disorders
● Lymphomatoid papulosis, 9718/1
● Primary cutaneous anaplastic large cell lymphoma, 9718/3
● Primary cutaneous gamma-delta T cell lymphoma, 9726/3
● Primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T cell lymphoma, 9709/3
● Primary cutaneous CD4-positive small/medium T cell lymphoma, 9709/3
● Peripheral T cell lymphoma, NOS, 9702/3
● Angioimmunoblastic T cell lymphoma, 9705/3
● Anaplastic large cell lymphoma, ALK-positive, 9714/3
● Anaplastic large cell lymphoma, ALK-negative, 9702/3

Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia Staging
Author: Sandy D Kotiah, MD; Chief Editor: Jules E Harris, MD
Medscape Sep 6, 2013
http://emedicine.medscape.com/article/2006578-overview

General considerations in the staging of chronic lymphocytic leukemia (CLL) and the revised Rai (United States) and Binet (Europe) staging systems for CLL are provided below.[1, 2, 3]

See Chronic Leukemias: 4 Cancers to Differentiate, a Critical Images slideshow, to help detect chronic leukemias and determine the specific type present.

General considerations

  • CLL and small lymphocytic lymphoma (SLL) are different manifestations of the same disease; SLL is diagnosed when the disease is mainly nodal, and CLL is diagnosed when the disease is seen in the blood and bone marrow
  • CLL is diagnosed by > 5000 monoclonal lymphocytes/mm3 for longer than 3mo; the bone marrow usually has more than 30% monoclonal lymphocytes and is either normocellular or hypercellular
  • Monoclonal B lymphocytosis is a precursor form of CLL that is defined by a monoclonal B cell lymphocytosis < 5000 monoclonal lymphocytes/mm3; all lymph nodes smaller than 1.5 cm; no anemia; and no thrombocytopenia

Revised Rai staging system (United States)

Low risk (formerly stage 0)[1] :

  • Lymphocytosis, lymphocytes in blood > 15000/mcL, and > 40% lymphocytes in the bone marrow

Intermediate risk (formerly stages I and II):

  • Lymphocytosis as in low risk with enlarged node(s) in any site, or splenomegaly or hepatomegaly or both

High risk (formerly stages III and IV):

  • Lymphocytosis as in low risk and intermediate risk with disease-related anemia (hemoglobin level < 11.0 g/dL or hematocrit < 33%) or platelets < 100,000/mcL

Binet staging system (Europe)

Stage A:

  • Hemoglobin ≥ 10 g/dL, platelets ≥ 100,000/mm3, and < 3 enlarged areas

Stage B:

  • Hemoglobin ≥ 10 g/dL, platelets ≥ 100,000/mm3, and ≥ 3 enlarged areas

Stage C:

  • Hemoglobin < 10 g/dL, platelets < 100,000/mm3, and any number of enlarged areas

Read Full Post »


Hematologic Malignancies [2.4.3]

Writer and Curator:  Larry H. Bernstein, MD, FCAP

Updated on 4/14/2016

Hematologic Malignancies 

Not excluding lymphomas [solid tumors]

The following series of articles are discussions of current identifications, classification, and treatments of leukemias, myelodysplastic syndromes and myelomas.

6.2 Hematological Malignancies

6.2.1 Ontogenesis of blood elements

6.2.1.1 Erythropoiesis

6.2.1.2 White blood cell series: myelopoiesis

6.2.1.3 Thrombocytogenesis

6.2.2 Classification of hematopoietic cancers

6.2.2.1 Primary Classification

6.2.2.1.1 Acute leukemias

6.2.2.1.1 Myelodysplastic syndromes

6.2.2.1.2 Acute myeloid leukemia

6.2.2.1.3 Acute lymphoblastic leukemia

6.2.2.2 Myeloproliferative Disorders

6.2.2.2.1 Chronic myeloproliferative disorders

6.2.2.2.2 Chronic myelogenous leukemia and related disorders

6.2.2.2.3 Myelofibrosis, including chronic idiopathic

6.2.2.2.4 Polycythemia, including polycythemia rubra vera

6.2.2.2.5 Thrombocytosis, including essential thrombocythemia

6.2.2.3 Chronic lymphoid leukemia and other lymphoid leukemias

6.2.2.4 Lymphomas

6.2.2.4.1 Non-Hodgkin Lymphoma

6.2.2.4.2 Hodgkin lymphoma

6.2.2.5 Lymphoproliferative disorders associated with immunodeficiency

6.2.2.6 Plasma Cell dyscrasias

6.2.2.7 Mast cell disease and Histiocytic neoplasms

6.2.3 Secondary Classification

6.2.3.1 Nuance – PathologyOutlines

6.2.3.1..1-8

6.2.4 Diagnostics

6.2.4.1 Computer-aided diagnostics

6.2.4.1.1 Back-to-Front Design

6.2.4.1.2 Realtime Clinical Expert Support

6.2.4.1.3 Regression: A richly textured method for comparison and classification of predictor variables

6.2.4.1.4 Converting Hematology Based Data into an Inferential Interpretation

6.2.4.1.5 A model for Thalassemia Screening using Hematology Measurements

6.2.4.1.6 Measurement of granulocyte maturation may improve the early diagnosis of the septic state.

6.2.4.1.7 The automated malnutrition assessment.

6.2.4.2 Molecular Diagnostics

6.2.4.2.1 Genomic Analysis of Hematological Malignancies

6.2.4.2.2 Next-generation sequencing in hematologic malignancies: what will be the dividends?

6.2.4.2.3 Leveraging cancer genome information in hematologic malignancies.

6.2.4.2.4 p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies

6.2.4.2.5 Genomic approaches to hematologic malignancies

6.2.5  Treatment of hematopoietic cancers

6.2.5.1 Treatments for leukemia by type

6.2.5.1.1 Acute lymphocytic leukemias

6.2.5.1.2 Treatment of Acute Lymphoblastic Leukemia

6.2.5.1.3 Acute Lymphoblastic Leukemia

6.2.5.1.4 Gene-Expression Patterns in Drug-Resistant Acute Lymphoblastic Leukemia Cells and Response to Treatment

6.2.5.1.5 Leukemias Treatment & Management

6.2.5.1.6 Treatments and drugs

6.2.5.2 Acute Myeloid Leukemia

6.2.5.2.1 New treatment approaches in acute myeloid leukemia: review of recent clinical studies

6.2.5.2.2 Novel approaches to the treatment of acute myeloid leukemia.

6.2.5.2.3 Current treatment of acute myeloid leukemia

6.2.5.2.4 Adult Acute Myeloid Leukemia Treatment (PDQ®)

6.2.5.3 Treatment for CML

6.2.5.3.1 Chronic Myelogenous Leukemia Treatment (PDQ®)

6.2.5.3.2 What`s new in chronic myeloid leukemia research and treatment?

6.2.5.4 Chronic Lymphocytic Leukemia

6.2.5.4.1 Chronic Lymphocytic Leukemia Treatment (PDQ®)

6.2.5.4.2 Results from the Phase 3 Resonate™ Trial

6.2.5.4.3 Typical treatment of chronic lymphocytic leukemia

6.2.5.5 Lymphoma treatment

6.2.5.5.1 Overview

6.2.5.5.2 Chemotherapy

6.2.6 Primary treatments

6.2.6.1 Total body irradiation (TBI)

6.2.6.2 Bone marrow (BM) transplantation

6.2.6.2.1 Autologous stem cell transplantation

6.2.6.2.2  Hematopoietic stem cell transplantation

6.2.7 Supportive Therapies

6.2.7.1  Blood transfusions

6.2.7.2  Erythropoietin

6.2.7.3  G-CSF (granulocyte-colony stimulating factor)

6.2.7.4  Plasma exchange (plasmapheresis)

6.2.7.5  Platelet transfusions

6.2.7.6  Steroids

6.2.1 Ontogenesis of the blood elements: hematopoiesis

http://www.britannica.com/EBchecked/topic/69747/blood-cell-formation

Blood cells are divided into three groups: the red blood cells (erythrocytes), the white blood cells (leukocytes), and the blood platelets (thrombocytes). The white blood cells are subdivided into three broad groups: granulocytes, lymphocytes, and monocytes.

Blood cells do not originate in the bloodstream itself but in specific blood-forming organs, notably the marrow of certain bones. In the human adult, the bone marrow produces all of the red blood cells, 60–70 percent of the white cells (i.e., the granulocytes), and all of the platelets. The lymphatic tissues, particularly the thymus, the spleen, and the lymph nodes, produce the lymphocytes (comprising 20–30 percent of the white cells). The reticuloendothelial tissues of the spleen, liver, lymph nodes, and other organs produce the monocytes (4–8 percent of the white cells). The platelets, which are small cellular fragments rather than complete cells, are formed from bits of the cytoplasm of the giant cells (megakaryocytes) of the bone marrow.

In the human embryo, the first site of blood formation is the yolk sac. Later in embryonic life, the liver becomes the most important red blood cell-forming organ, but it is soon succeeded by the bone marrow, which in adult life is the only source of both red blood cells and the granulocytes. Both the red and white blood cells arise through a series of complex, gradual, and successive transformations from primitive stem cells, which have the ability to form any of the precursors of a blood cell. Precursor cells are stem cells that have developed to the stage where they are committed to forming a particular kind of new blood cell.

In a normal adult the red cells of about half a liter (almost one pint) of blood are produced by the bone marrow every week. Almost 1 percent of the body’s red cells are generated each day, and the balance between red cell production and the removal of aging red cells from the circulation is precisely maintained.

Cells-in-the-Bone-Marrow-1024x747

Cells-in-the-Bone-Marrow-1024×747

http://interactive-biology.com/wp-content/uploads/2012/07/Cells-in-the-Bone-Marrow-1024×747.png

6.2.1.1 Erythropoiesis

http://www.interactive-biology.com/3969/erythropoiesis-formation-of-red-blood-cells/

Erythropoiesis – Formation of Red Blood Cells

Because of the inability of erythrocytes (red blood cells) to divide to replenish their own numbers, the old ruptured cells must be replaced by totally new cells. They meet their demise because they don’t have the usual specialized intracellular machinery, which controls cell growth and repair, leading to a short life span of 120 days.

This short life span necessitates the process erythropoiesis, which is the formation of red blood cells. All blood cells are formed in the bone marrow. This is the erythrocyte factory, which is soft, highly cellar tissue that fills the internal cavities of bones.

Erythrocyte differentiation takes place in 8 stages. It is the pathway through which an erythrocyte matures from a hemocytoblast into a full-blown erythrocyte. The first seven all take place within the bone marrow. After stage 7 the cell is then released into the bloodstream as a reticulocyte, where it then matures 1-2 days later into an erythrocyte. The stages are as follows:

  1. Hemocytoblast, which is a pluripotent hematopoietic stem cell
  2. Common myeloid progenitor, a multipotent stem cell
  3. Unipotent stem cell
  4. Pronormoblast
  5. Basophilic normoblast also called an erythroblast.
  6. Polychromatophilic normoblast
  7. Orthochromatic normoblast
  8. Reticulocyte

These characteristics can be seen during the course of erythrocyte maturation:

  • The size of the cell decreases
  • The cytoplasm volume increases
  • Initially there is a nucleus and as the cell matures the size of the nucleus decreases until it vanishes with the condensation of the chromatin material.

Low oxygen tension stimulates the kidneys to secrete the hormone erythropoietin into the blood, and this hormone stimulates the bone marrow to produce erythrocytes.

Rarely, a malignancy or cancer of erythropoiesis occurs. It is referred to as erythroleukemia. This most likely arises from a common myeloid precursor, and it may occur associated with a myelodysplastic syndrome.

Summary of erythrocyte maturation

6.2.1.2 White blood cell series: myelopoiesis

http://www.nlm.nih.gov/medlineplus/ency/presentations/100151_3.htm

http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/15220.jpg

There are various types of white blood cells (WBCs) that normally appear in the blood: neutrophils (polymorphonuclear leukocytes; PMNs), band cells (slightly immature neutrophils), T-type lymphocytes (T cells), B-type lymphocytes (B cells), monocytes, eosinophils, and basophils. T and B-type lymphocytes are indistinguishable from each other in a normal slide preparation. Any infection or acute stress will result in an increased production of WBCs. This usually entails increased numbers of cells and an increase in the percentage of immature cells (mainly band cells) in the blood. This change is referred to as a “shift to the left” People who have had a splenectomy have a persistent mild elevation of WBCs. Drugs that may increase WBC counts include epinephrine, allopurinol, aspirin, chloroform, heparin, quinine, corticosteroids, and triamterene. Drugs that may decrease WBC counts include antibiotics, anticonvulsants, antihistamine, antithyroid drugs, arsenicals, barbiturates, chemotherapeutic agents, diuretics and sulfonamides.   (Updated by: David C. Dugdale, III, MD)

https://www.med-ed.virginia.edu/courses/path/innes/nh/wcbmaturation.cfm

Note that the mature forms of the myeloid series (neutrophils, eosinophils, basophils), all have lobed (segmented) nuclei. The degree of lobation increases as the cells mature.

The earliest recognizable myeloid cell is the myeloblast (10-20m dia) with a large round to oval nucleus. There is fine diffuse immature chromatin (without clumping) and a prominant nucleolus.

The cytoplasm is basophilic without granules. Although one may see a small golgi area adjacent to the nucleus, granules are not usually visible by light microscopy. One should not see blast cells in the peripheral blood.

myeloblast x100b

myeloblast x100b

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20myeloblast%20x100b.jpeg

The promyelocyte (10-20m) is slightly larger than a blast. Its nucleus, although similar to a myeloblast shows slight chromatin condensation and less prominent nucleoli. The cytoplasm contains striking azurophilic granules or primary granules. These granules contain myeloperoxidase, acid phosphatase, and esterase enzymes. Normally no promyelocytes are seen in the peripheral blood.

At the point in development when secondary granules can be recognized, the cell becomes a myelocyte.

promyelocyte x100

promyelocyte x100

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20promyelocyte%20×100%20a.jpeg

Myelocytes (10-18m) are not normally found in the peripheral blood. Nucleoli may not be seen in the late myelocyte. Primary azurophilic granules are still present, but secondary granules predominate. Secondary granules (neut, eos, or baso) first appear adjacent to the nucleus. In neutrophils this is the “dawn” of neutrophilia.

Metamyelocytes (10-18m) have kidney shaped indented nuclei and dense chromatin along the nuclear membrane. The cytoplasm is faintly pink, and they have secondary granules (neutro, eos, or baso). Zero to one percent of the peripheral blood white cells may be metamyelocytes (juveniles).

metamyelocyte x100

metamyelocyte x100

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20metamyelocyte%20×100.jpeg

Bands, slightly smaller than juveniles, are marked by a U-shaped or deeply indented nucleus.

band neutrophilx100a

band neutrophilx100a

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20band%20x100a.jpeg

Segmented (segs) or polymorphonuclear (PMN) leukocytes (average 14 m dia) are distinguished by definite lobation with thin thread-like filaments of chromatin joining the 2-5 lobes. 45-75% of the peripheral blood white cells are segmented neutrophils.

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20neutrophil%20×100%20d.jpeg

6.2.1.3 Thrombocytogenesis

The incredible journey: From megakaryocyte development to platelet formation

Kellie R. Machlus1,2 and Joseph E. Italiano Jr
JCB 2013; 201(6): 785-796
http://dx.doi.org:/10.1083/jcb.201304054

Large progenitor cells in the bone marrow called megakaryocytes (MKs) are the source of platelets. MKs release platelets through a series of fascinating cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Then, in a cytoskeletal-driven process, they extend long branching processes, designated proplatelets, into sinusoidal blood vessels where they undergo fission to release platelets.

megakaryocyte production of platelets

megakaryocyte production of platelets

http://dm5migu4zj3pb.cloudfront.net/manuscripts/26000/26891/medium/JCI0526891.f4.jpg

platelets and the immune continuum nri2956-f3

platelets and the immune continuum nri2956-f3

http://www.nature.com/nri/journal/v11/n4/images/nri2956-f3.jpg

6.2.2 Classification of hematological malignancies
Practical Diagnosis of Hematologic Disoreders. 4th edition. Vol 2.
Kjeldsberg CR, Ed.  ASCP Press.  2006. Chicago, IL.

6.2.2.1 Primary Classification

6.2.2.1.1 Acute leukemias

6.2.2.1.1 Myelodysplastic syndromes

6.2.2.1.2 Acute myeloid leukemia

6.2.2.1.3 Acute lymphoblastic leukemia

6.2.2.2 Myeloproliferative Disorders

6.2.2.2.1 Chronic myeloproliferative disorders

6.2.2.2.2 Chronic myelogenous leukemia and related disorders

6.2.2.2.3 Myelofibrosis, including chronic idiopathic

6.2.2.2.4 Polycythemia, including polycythemia rubra vera

6.2.2.2.5 Thrombocytosis, including essential thrombocythemia

6.2.2.3 Chronic lymphoid leukemia and other lymphoid leukemias

6.2.2.4 Lymphomas

6.2.2.4.1 Non-Hodgkin Lymphoma

6.2.2.4.2 Hodgkin lymphoma

6.2.2.5 Lymphoproliferative disorders associated with immunodeficiency

6.2.2.6 Plasma Cell dyscrasias

6.2.2.7 Mast cell disease and Histiocytic neoplasms

6.2.3 Secondary Classification

6.2.3.1 Nuance – PathologyOutlines
Nat Pernick, Ed.

http://www.pathologyoutlines.com/leukemia.html

This site is up-to-date and revised periodically. It is the best site for pathology information.

6.2.4 Diagnostics

6.2.4.1 Computer-aided diagnostics

6.2.4.1.1 Back-to-Front Design

Robert Didner
Bell Laboratories

Decision-making in the clinical setting
Didner, R  Mar 1999  Amer Clin Lab

Mr. Didner is an Independent Consultant in Systems Analysis, Information Architecture (Informatics) Operations Research, and Human Factors Engineering (Cognitive Psychology),  Decision Information Designs, 29 Skyline Dr., Morristown, NJ07960, U.S.A.; tel.: 973-455-0489; fax/e-mail: bdidner@hotmail.com

A common problem in the medical profession is the level of effort dedicated to administration and paperwork necessitated by various agencies, which contributes to the high cost of medical care. Costs would be reduced and accuracy improved if the clinical data could be captured directly at the point they are generated in a form suitable for transmission to insurers or machine transformable into other formats. Such a capability could also be used to improve the form and the structure of information presented to physicians and support a more comprehensive database linking clinical protocols to outcomes, with the prospect of improving clinical outcomes. Although the problem centers on the physician’s process of determining the diagnosis and treatment of patients and the timely and accurate recording of that process in the medical system, it substantially involves the pathologist and laboratorian, who interact significantly throughout the in-formation-gathering process. Each of the currently predominant ways of collecting information from diagnostic protocols has drawbacks. Using blank paper to collect free-form notes from the physician is not amenable to computerization; such free-form data are also poorly formulated, formatted, and organized for the clinical decision-making they support. The alternative of preprinted forms listing the possible tests, results, and other in-formation gathered during the diagnostic process facilitates the desired computerization, but the fixed sequence of tests and questions they present impede the physician from using an optimal decision-making sequence. This follows because:

  • People tend to make decisions and consider information in a step-by-step manner in which intermediate decisions are intermixed with data acquisition steps.
  • The sequence in which components of decisions are made may alter the decision outcome.
  • People tend to consider information in the sequence it is requested or displayed.
  • Since there is a separate optimum sequence of tests and questions for each cluster of history and presenting symptoms, there is no one sequence of tests and questions that can be optimal for all presenting clusters.
  • As additional data and test results are acquired, the optimal sequence of further testing and data acquisition changes, depending on the already acquired information.

Therefore, promoting an arbitrary sequence of information requests with preprinted forms may detract from outcomes by contributing to a non-optimal decision-making sequence. Unlike the decisions resulting from theoretical or normative processes, decisions made by humans are path dependent; that is, the out-come of a decision process may be different if the same components are considered in a different sequence.

Proposed solution

This paper proposes a general approach to gathering data at their source in computer-based form so as to improve the expected outcomes. Such a means must be interactive and dynamic, so that at any point in the clinical process the patient’s presenting symptoms, history, and the data already collected are used to determine the next data or tests requested. That de-termination must derive from a decision-making strategy designed to produce outcomes with the greatest value and supported by appropriate data collection and display techniques. The strategy must be based on the knowledge of the possible outcomes at any given stage of testing and information gathering, coupled with a metric, or hierarchy of values for assessing the relative desirability of the possible outcomes.

A value hierarchy

  • The numbered list below illustrates a value hierarchy. In any particular instance, the higher-numbered values should only be considered once the lower- numbered values have been satisfied. Thus, a diagnostic sequence that is very time or cost efficient should only be considered if it does not increase the likelihood (relative to some other diagnostic sequence) that a life-threatening disorder may be missed, or that one of the diagnostic procedures may cause discomfort.
  • Minimize the likelihood that a treatable, life-threatening disorder is not treated.
  • Minimize the likelihood that a treatable, discomfort-causing disorder is not treated.
  • Minimize the likelihood that a risky procedure(treatment or diagnostic procedure) is inappropriately administered.
  • Minimize the likelihood that a discomfort-causing procedure is inappropriately administered.
  • Minimize the likelihood that a costly procedure is inappropriately administered.
  • Minimize the time of diagnosing and treating thepatient.8.Minimize the cost of diagnosing and treating the patient.

The above hierarchy is relative, not absolute; for many patients, a little bit of testing discomfort may be worth a lot of time. There are also some factors and graduations intentionally left out for expository simplicity (e.g., acute versus chronic disorders).This value hierarchy is based on a hypothetical patient. Clearly, the hierarchy of a health insurance carrier might be different, as might that of another patient (e.g., a geriatric patient). If the approach outlined herein were to be followed, a value hierarchy agreed to by a majority of stakeholders should be adopted.

Efficiency

Once the higher values are satisfied, the time and cost of diagnosis and treatment should be minimized. One way to do so would be to optimize the sequence in which tests are performed, so as to minimize the number, cost, and time of tests that need to be per-formed to reach a definitive decision regarding treatment. Such an optimum sequence could be constructed using Claude Shannon’s information theory.

According to this theory, the best next question to ask under any given situation (assuming the question has two possible outcomes) is that question that divides the possible outcomes into two equally likely sets. In the real world, all tests or questions are not equally valuable, costly, or time consuming; therefore, value(risk factors), cost, and time should be used as weighting factors to optimize the test sequence, but this is a complicating detail at this point.

A value scale

For dynamic computation of outcome values, the hierarchy could be converted into a weighted value scale so differing outcomes at more than one level of the hierarchy could be readily compared. An example of such a weighted value scale is Quality Adjusted Life Years (QALY).

Although QALY does not incorporate all of the factors in this example, it is a good conceptual starting place.

The display, request, decision-making relationship

For each clinical determination, the pertinent information should be gathered, organized, formatted, and formulated in a way that facilitates the accuracy, reliability, and efficiency with which that determination is made. A physician treating a patient with high cholesterol and blood pressure (BP), for example, may need to know whether or not the patient’s cholesterol and BP respond to weight changes to determine an appropriate treatment (e.g., weight control versus medication). This requires searching records for BP, certain blood chemicals (e.g., HDLs, LDLs, triglycerides, etc.), and weight from several

sources, then attempting to track them against each other over time. Manually reorganizing this clinical information each time it is used is extremely inefficient. More important, the current organization and formatting defies principles of human factors for optimally displaying information to enhance human information-processing characteristics, particularly for decision support.

While a discussion of human factors and cognitive psychology principles is beyond the scope of this paper, following are a few of the system design principles of concern:

  • Minimize the load on short-term memory.
  • Provide information pertinent to a given decision or component of a decision in a compact, contiguous space.
  • Take advantage of basic human perceptual and pat-tern recognition facilities.
  • Design the form of an information display to com-plement the decision-making task it supports.

F i g u re 1 shows fictitious, quasi-random data from a hypothetical patient with moderately elevated cholesterol. This one-page display pulls together all the pertinent data from six years of blood tests and related clinical measurements. At a glance, the physician’s innate pattern recognition, color, and shape perception facilities recognize the patient’s steadily increasing weight, cholesterol, BP, and triglycerides as well as the declining high-density lipoproteins. It would have taken considerably more time and effort to grasp this information from the raw data collection and blood test reports as they are currently presented in independent, tabular time slices.

Design the formulation of an information display to complement the decision-making task.

The physician may wish to know only the relationship between weight and cardiac risk factors rather than whether these measures are increasing or decreasing, or are within acceptable or marginal ranges. If so, Table 1 shows the correlations between weight and the other factors in a much more direct and simple way using the same data as in Figure 1. One can readily see the same conclusions about relations that were drawn from Figure 1.This type of abstract, symbolic display of derived information also makes it easier to spot relationships when the individual variables are bouncing up and down, unlike the more or less steady rise of most values in Figure 1. This increase in precision of relationship information is gained at the expense of other types of information (e.g., trends). To display information in an optimum form then, the system designer must know what the information demands of the task are at the point in the task when the display is to be used.

Present the sequence of information display clusters to complement an optimum decision-making strategy.

Just as a fixed sequence of gathering clinical, diagnostic information may lead to a far from optimum outcome, there exists an optimum sequence of testing, considering information, and gathering data that will lead to an optimum outcome (as defined by the value hierarchy) with a minimum of time and expense. The task of the information system designer, then, is to provide or request the right information, in the best form, at each stage of the procedure. For ex-ample, Figure 1 is suitable for the diagnostic phase since it shows the current state of the risk factors and their trends. Table 1, on the other hand, might be more appropriate in determining treatment, where there may be a choice of first trying a strict dietary treatment, or going straight to a combination of diet plus medication. The fact that Figure 1 and Table 1 have somewhat redundant information is not a problem, since they are intended to optimally provide information for different decision-making tasks. The critical need, at this point, is for a model of how to determine what information should be requested, what tests to order, what information to request and display, and in what form at each step of the decision-making process. Commitment to a collaborative relationship between physicians and laboratorians and other information providers would be an essential requirement for such an undertaking. The ideal diagnostic data-collection instrument is a flexible, computer-based device, such as a notebook computer or Personal Digital Assistant (PDA) sized device.

Barriers to interactive, computer-driven data collection at the source

As with any major change, it may be difficult to induce many physicians to change their behavior by interacting directly with a computer instead of with paper and pen. Unlike office workers, who have had to make this transition over the past three decades, most physicians’ livelihoods will not depend on converting to computer interaction. Therefore, the transition must be made attractive and the changes less onerous. Some suggestions follow:

  1. Make the data collection a natural part of the clinical process.
  2. Ensure that the user interface is extremely friendly, easy to learn, and easy to use.
  3. Use a small, portable device.
  4. Use the same device for collection and display of existing information (e.g., test results and his-tory).
  5. Minimize the need for free-form written data entry (use check boxes, forms, etc.).
  6. Allow the entry of notes in pen-based free-form (with the option of automated conversion of numeric data to machine-manipulable form).
  7. Give the physicians a more direct benefit for collecting data, not just a means of helping a clerk at an HMO second-guess the physician’s judgment.
  8. Improve administrative efficiency in the office.
  9. Make the data collection complement the clinical decision-making process.
  10. Improve information displays, leading to better outcomes.
  11. Make better use of the physician’s time and mental effort.

Conclusion

The medical profession is facing a crisis of information. Gathering information is costing a typical practice more and more while fees are being restricted by third parties, and the process of gathering this in-formation may be detrimental to current outcomes. Gathered properly, in machine-manipulable form, these data could be reformatted so as to greatly improve their value immediately in the clinical setting by leading to decisions with better outcomes and, in the long run, by contributing to a clinical data warehouse that could greatly improve medical knowledge. The challenge is to create a mechanism for data collection that facilitates, hastens, and improves the outcomes of clinical activity while minimizing the inconvenience and resistance to change on the part of clinical practitioners. This paper is intended to provide a high-level overview of how this may be accomplished, and start a dialogue along these lines.

References

  1. Tversky A. Elimination by aspects: a theory of choice. Psych Rev 1972; 79:281–99.
  2. Didner RS. Back-to-front design: a guns and butter approach. Ergonomics 1982; 25(6):2564–5.
  3. Shannon CE. A mathematical theory of communication. Bell System Technical J 1948; 27:379–423 (July), 623–56 (Oct).
  4. Feeny DH, Torrance GW. Incorporating utility-based quality-of-life assessment measures in clinical trials: two examples. Med Care 1989; 27:S190–204.
  5. Smith S, Mosier J. Guidelines for designing user interface soft-ware. ESD-TR-86-278, Aug 1986.
  6. Miller GA. The magical number seven plus or minus two. Psych Rev 1956; 65(2):81–97.
  7. Sternberg S. High-speed scanning in human memory. Science 1966; 153: 652–4.

Table 1

Correlation of weight with other cardiac risk factors

Cholesterol 0.759384
HDL 0.53908
LDL 0.177297
BP-syst. 0.424728
BP-dia. 0.516167
Triglycerides 0.637817

Figure 1  Hypothetical patient data.

(not shown)

6.2.4.1.2 Realtime Clinical Expert Support

https://pharmaceuticalintelligence.com/2015/05/10/realtime-clinical-expert-support/

6.2.4.1.3 Regression: A richly textured method for comparison and classification of predictor variables

https://pharmaceuticalintelligence.com/2012/08/14/regression-a-richly-textured-method-for-comparison-and-classification-of-predictor-variables/

6.2.4.1.4 Converting Hematology Based Data into an Inferential Interpretation

Larry H. Bernstein, Gil David, James Rucinski and Ronald R. Coifman
In Hematology – Science and Practice
Lawrie CH, Ch 22. Pp541-552.
InTech Feb 2012, ISBN 978-953-51-0174-1
https://www.researchgate.net/profile/Larry_Bernstein/publication/221927033_Converting_Hematology_Based_Data_into_an_Inferential_Interpretation/links/0fcfd507f28c14c8a2000000.pdf

6.2.4.1.5 A model for Thalassemia Screening using Hematology Measurements

https://www.researchgate.net/profile/Larry_Bernstein/publication/258848064_A_model_for_Thalassemia_Screening_using_Hematology_Measurements/links/0c9605293c3048060b000000.pdf

A model for automated screening of thalassemia in hematology (math study).

Kneifati-Hayek J, Fleischman W, Bernstein LH, Riccioli A, Bellevue R.
Lab Hematol. 2007; 13(4):119-23. http://dx.doi.org:/10.1532/LH96.07003.

The results of 398 patient screens were collected. Data from the set were divided into training and validation subsets. The Mentzer ratio was determined through a receiver operating characteristic (ROC) curve on the first subset, and screened for thalassemia using the second subset. HgbA2 levels were used to confirm beta-thalassemia.

RESULTS: We determined the correct decision point of the Mentzer index to be a ratio of 20. Physicians can screen patients using this index before further evaluation for beta-thalassemia (P < .05).

CONCLUSION: The proposed method can be implemented by hospitals and laboratories to flag positive matches for further definitive evaluation, and will enable beta-thalassemia screening of a much larger population at little to no additional cost.

6.2.4.1.6 Measurement of granulocyte maturation may improve the early diagnosis of the septic state.

Bernstein LH, Rucinski J. Clin Chem Lab Med. 2011 Sep 21;49(12):2089-95.
http://dx.doi.org:/10.1515/CCLM.2011.688.

6.2.4.1.7 The automated malnutrition assessment.

David G, Bernstein LH, Coifman RR. Nutrition. 2013 Jan; 29(1):113-21.
http://dx.doi.org:/10.1016/j.nut.2012.04.017

6.2.4.2 Molecular Diagnostics

6.2.4.2.1 Genomic Analysis of Hematological Malignancies

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy that occurs in children. Although more than 90% of children with ALL now survive to adulthood, those with the rarest and high-risk forms of the disease continue to have poor prognoses. Through the Pediatric Cancer Genome Project (PCGP), investigators in the Hematological Malignancies Program are identifying the genetic aberrations that cause these aggressive forms of leukemias. Here we present two studies on the genetic bases of early T-cell precursor ALL and acute megakaryoblastic leukemia.

  • Early T-Cell Precursor ALL Is Characterized by Activating Mutations
  • The CBFA2T3-GLIS2Fusion Gene Defines an Aggressive Subtype of Acute Megakaryoblastic Leukemia in Children

Early T-cell precursor ALL (ETP-ALL), which comprises 15% of all pediatric T-cell leukemias, is an aggressive disease that is typically resistant to contemporary therapies. Children with ETP-ALL have a high rate of relapse and an extremely poor prognosis (i.e., 5-year survival is approximately 20%). The genetic basis of ETP-ALL has remained elusive. Although ETP-ALL is associated with a high burden of DNA copy number aberrations, none are consistently found or suggest a unifying genetic alteration that drives this disease.

Through the efforts of the PCGP, Jinghui Zhang, PhD (Computational Biology), James R. Downing, MD (Pathology), Charles G. Mullighan, MBBS(Hons), MSc, MD (Pathology), and colleagues analyzed the whole-genome sequences of leukemic cells and matched normal DNA from 12 pediatric patients with ETP-ALL. The identified genetic mutations were confirmed in a validation cohort of 52 ETP-ALL specimens and 42 non-ETP T-lineage ALLs (T-ALL).

In the journal Nature, the investigators reported that each ETP-ALL sample carried an average of 1140 sequence mutations and 12 structural variations. Of the structural variations, 51% were breakpoints in genes with well-established roles in hematopoiesis or leukemogenesis (e.g., MLH2,SUZ12, and RUNX1). Eighty-four percent of the structural variations either caused loss of function of the gene in question or resulted in the formation of a fusion gene such as ETV6-INO80D. The ETV6 gene, which encodes a protein that is essential for hematopoiesis, is frequently mutated in leukemia. Among the DNA samples sequenced in this study, ETV6 was altered in 33% of ETP-ALL but only 10% of T-ALL cases.

6.2.4.2.2 Next-generation sequencing in hematologic malignancies: what will be the dividends?

Jason D. MerkerAnton Valouev, and Jason Gotlib
Ther Adv Hematol. 2012 Dec; 3(6): 333–339.
http://dx.doi.org:/10.1177/2040620712458948

The application of high-throughput, massively parallel sequencing technologies to hematologic malignancies over the past several years has provided novel insights into disease initiation, progression, and response to therapy. Here, we describe how these new DNA sequencing technologies have been applied to hematolymphoid malignancies. With further improvements in the sequencing and analysis methods as well as integration of the resulting data with clinical information, we expect these technologies will facilitate more precise and tailored treatment for patients with hematologic neoplasms.

6.2.4.2.3 Leveraging cancer genome information in hematologic malignancies.

Rampal R1Levine RL.
J Clin Oncol. 2013 May 20; 31(15):1885-92.
http://dx.doi.org:/10.1200/JCO.2013.48.7447

The use of candidate gene and genome-wide discovery studies in the last several years has led to an expansion of our knowledge of the spectrum of recurrent, somatic disease alleles, which contribute to the pathogenesis of hematologic malignancies. Notably, these studies have also begun to fundamentally change our ability to develop informative prognostic schema that inform outcome and therapeutic response, yielding substantive insights into mechanisms of hematopoietic transformation in different tissue compartments. Although these studies have already had important biologic and translational impact, significant challenges remain in systematically applying these findings to clinical decision making and in implementing new technologies for genetic analysis into clinical practice to inform real-time decision making. Here, we review recent major genetic advances in myeloid and lymphoid malignancies, the impact of these findings on prognostic models, our understanding of disease initiation and evolution, and the implication of genomic discoveries on clinical decision making. Finally, we discuss general concepts in genetic modeling and the current state-of-the-art technology used in genetic investigation.

6.2.4.2.4 p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies

E Wattel, C Preudhomme, B Hecquet, M Vanrumbeke, et AL.
Blood, (Nov 1), 1994; 84(9): pp 3148-3157
http://www.bloodjournal.org/content/bloodjournal/84/9/3148.full.pdf

We analyzed the prognostic value of p53 mutations for response to chemotherapy and survival in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic lymphocytic leukemia (CLL). Mutations were detected by single-stranded conformation polymorphism (SSCP) analysis of exons 4 to 10 of the P53 gene, and confirmed by direct sequencing. A p53 mutation was found in 16 of 107 (15%) AML, 20 of 182 (11%) MDS, and 9 of 81 (11%) CLL tested. In AML, three of nine (33%) mutated cases and 66 of 81 (81%) nonmutated cases treated with intensive chemotherapy achieved complete remission (CR) (P = .005) and none of five mutated cases and three of six nonmutated cases treated by low-dose Ara C achieved CR or partial remission (PR) (P = .06). Median actuarial survival was 2.5 months in mutated cases, and 15 months in nonmutated cases (P < lo-‘). In the MDS patients who received chemotherapy (intensive chemotherapy or low-dose Ara C), 1 of 13 (8%) mutated cases and 23 of 38 (60%) nonmutated cases achieved CR or PR (P = .004), and median actuarial survival was 2.5 and 13.5 months, respectively (P C lo-’). In all MDS cases (treated and untreated), the survival difference between mutated cases and nonmutated cases was also highly significant. In CLL, 1 of 8 (12.5%) mutated cases treated by chemotherapy (chlorambucil andlor CHOP andlor fludarabine) responded, as compared with 29 of 36 (80%) nonmutated cases (P = .02). In all CLL cases, survival from p53 analysis was significantly shorter in mutated cases (median 7 months) than in nonmutated cases (median not reached) (P < IO-’). In 35 of the 45 mutated cases of AML, MDS, and CLL, cytogenetic analysis or SSCP and sequence findings showed loss of the nonmutated P53 allele. Our findings show that p53 mutations are a strong prognostic indicator of response to chemotherapy and survival in AML, MDS, and CLL. The usual association of p53 mutations to loss of the nonmutated P53 allele, in those disorders, ie, to absence of normal p53 in tumor cells, suggests that p53 mutations could induce drug resistance, at least in part, by interfering with normal apoptotic pathways in tumor cells.

6.2.4.2.5 Genomic approaches to hematologic malignancies

Benjamin L. Ebert and Todd R. Golub
Blood. 2004; 104:923-932
https://www.broadinstitute.org/mpr/publications/projects/genomics/Review%20Genomics%20of%20Heme%20Malig,%20Blood%202004.pdf

In the past several years, experiments using DNA microarrays have contributed to an increasingly refined molecular taxonomy of hematologic malignancies. In addition to the characterization of molecular profiles for known diagnostic classifications, studies have defined patterns of gene expression corresponding to specific molecular abnormalities, oncologic phenotypes, and clinical outcomes. Furthermore, novel subclasses with distinct molecular profiles and clinical behaviors have been identified. In some cases, specific cellular pathways have been highlighted that can be therapeutically targeted. The findings of microarray studies are beginning to enter clinical practice as novel diagnostic tests, and clinical trials are ongoing in which therapeutic agents are being used to target pathways that were identified by gene expression profiling. While the technology of DNA microarrays is becoming well established, genome-wide surveys of gene expression generate large data sets that can easily lead to spurious conclusions. Many challenges remain in the statistical interpretation of gene expression data and the biologic validation of findings. As data accumulate and analyses become more sophisticated, genomic technologies offer the potential to generate increasingly sophisticated insights into the complex molecular circuitry of hematologic malignancies. This review summarizes the current state of discovery and addresses key areas for future research.

6.2.4.3 Flow cytometry

Introduction to Flow Cytometry: Blood Cell Identification

Dana L. Van Laeys
https://www.labce.com/flow_cytometry.aspx

No other laboratory method provides as rapid and detailed analysis of cellular populations as flow cytometry, making it a valuable tool for diagnosis and management of several hematologic and immunologic diseases. Understanding this relevant methodology is important for any medical laboratory scientist.

Whether you have no previous experience with flow cytometry or just need a refresher, this course will help you to understand the basic principles, with the help of video tutorials and interactive case studies.

Basic principles include:

  1. Immunophenotypic features of various types of hematologic cells
  2. Labeling cellular elements with fluorochromes
  3. Blood cell identification, specifically B and T lymphocyte identification and analysis
  4. Cell sorting to isolate select cell population for further analysis
  5. Analyzing and interpreting result reports and printouts

6.2.5 Treatments

6.2.5.1 Treatments for leukemia by type

6.2.5.1.1 Acute lymphocytic leukemias

6.2.5.1.1.1 Treatment of Acute Lymphoblastic Leukemia

Ching-Hon Pu, and William E. Evans
N Engl J Med Jan 12, 2006; 354:166-178
http://dx.doi.org:/10.1056/NEJMra052603

Although the overall cure rate of acute lymphoblastic leukemia (ALL) in children is about 80 percent, affected adults fare less well. This review considers recent advances in the treatment of ALL, emphasizing issues that need to be addressed if treatment outcome is to improve further.

6.2.5.1.1.2 Acute Lymphoblastic Leukemia

Ching-Hon Pui, Mary V. Relling, and James R. Downing
N Engl J Med Apr 8, 2004; 350:1535-1548
http://dx.doi.org:/10.1056/NEJMra023001

This comprehensive survey emphasizes how recent advances in the knowledge of molecular mechanisms involved in acute lymphoblastic leukemia have influenced diagnosis, prognosis, and treatment.

6.2.5.1.1.3 Gene-Expression Patterns in Drug-Resistant Acute Lymphoblastic Leukemia Cells and Response to Treatment

Amy Holleman, Meyling H. Cheok, Monique L. den Boer, et al.
N Engl J Med 2004; 351:533-42

Childhood acute lymphoblastic leukemia (ALL) is curable with chemotherapy in approximately 80 percent of patients. However, the cause of treatment failure in the remaining 20 percent of patients is largely unknown.

Methods We tested leukemia cells from 173 children for sensitivity in vitro to prednisolone, vincristine, asparaginase, and daunorubicin. The cells were then subjected to an assessment of gene expression with the use of 14,500 probe sets to identify differentially expressed genes in drug-sensitive and drug-resistant ALL. Gene-expression patterns that differed according to sensitivity or resistance to the four drugs were compared with treatment outcome in the original 173 patients and an independent cohort of 98 children treated with the same drugs at another institution.

Results We identified sets of differentially expressed genes in B-lineage ALL that were sensitive or resistant to prednisolone (33 genes), vincristine (40 genes), asparaginase (35 genes), or daunorubicin (20 genes). A combined gene-expression score of resistance to the four drugs, as compared with sensitivity to the four, was significantly and independently related to treatment outcome in a multivariate analysis (hazard ratio for relapse, 3.0; P=0.027). Results were confirmed in an independent population of patients treated with the same medications (hazard ratio for relapse, 11.85; P=0.019). Of the 124 genes identified, 121 have not previously been associated with resistance to the four drugs we tested.

Conclusions  Differential expression of a relatively small number of genes is associated with drug resistance and treatment outcome in childhood ALL.

6.2.5.1.1.4 Leukemias Treatment & Management

Author: Lihteh Wu, MD; Chief Editor: Hampton Roy Sr
http://emedicine.medscape.com/article/1201870-treatment

The treatment of leukemia is in constant flux, evolving and changing rapidly over the past few years. Most treatment protocols use systemic chemotherapy with or without radiotherapy. The basic strategy is to eliminate all detectable disease by using cytotoxic agents. To attain this goal, 3 phases are typically used, as follows: remission induction phase, consolidation phase, and maintenance therapy phase.

Chemotherapeutic agents are chosen that interfere with cell division. Tumor cells usually divide more rapidly than host cells, making them more vulnerable to the effects of chemotherapy. Primary treatment will be under the direction of a medical oncologist, radiation oncologist, and primary care physician. Although a general treatment plan will be outlined, the ophthalmologist does not prescribe or manage such treatment.

  • The initial treatment of ALL uses various combinations of vincristine, prednisone, and L-asparaginase until a complete remission is obtained.
  • Maintenance therapy with mercaptopurine is continued for 2-3 years following remission.
  • Use of intrathecal methotrexate with or without cranial irradiation to cover the CNS varies from facility to facility.
  • Daunorubicin, cytarabine, and thioguanine currently are used to obtain induction and remission of AML.
  • Maintenance therapy for 8 months may lengthen remission. Once relapse has occurred, AML generally is curable only by bone marrow transplantation.
  • Presently, treatment of CLL is palliative.
  • CML is characterized by a leukocytosis greater than 100,000 cells. Emergent treatment with leukopheresis sometimes is necessary when leukostastic complications are present. Otherwise, busulfan or hydroxyurea may control WBC counts. During the chronic phase, treatment is palliative.
  • When CML converts to the blastic phase, approximately one third of cases behave as ALL and respond to treatment with vincristine and prednisone. The remaining two thirds resemble AML but respond poorly to AML therapy.
  • Allogeneic bone marrow transplant is the only curative therapy for CML. However, it carries a high early mortality rate.
  • Leukemic retinopathy usually is not treated directly. As the hematological parameters normalize with systemic treatment, many of the ophthalmic signs resolve. There are reports that leukopheresis for hyperviscosity also may alleviate intraocular manifestations.
  • When definite intraocular leukemic infiltrates fail to respond to systemic chemotherapy, direct radiation therapy is recommended.
  • Relapse, manifested by anterior segment involvement, should be treated by radiation. In certain cases, subconjunctival chemotherapeutic agents have been injected.
  • Optic nerve head infiltration in patients with ALL is an emergency and requires prompt radiation therapy to try to salvage some vision.

6.2.5.1.1.5 Treatments and drugs

http://www.mayoclinic.org/diseases-conditions/leukemia/basics/
treatment/con-20024914

Common treatments used to fight leukemia include:

  • Chemotherapy. Chemotherapy is the major form of treatment for leukemia. This drug treatment uses chemicals to kill leukemia cells.

Depending on the type of leukemia you have, you may receive a single drug or a combination of drugs. These drugs may come in a pill form, or they may be injected directly into a vein.

  • Biological therapy. Biological therapy works by using treatments that help your immune system recognize and attack leukemia cells.
  • Targeted therapy. Targeted therapy uses drugs that attack specific vulnerabilities within your cancer cells.

For example, the drug imatinib (Gleevec) stops the action of a protein within the leukemia cells of people with chronic myelogenous leukemia. This can help control the disease.

  • Radiation therapy. Radiation therapy uses X-rays or other high-energy beams to damage leukemia cells and stop their growth. During radiation therapy, you lie on a table while a large machine moves around you, directing the radiation to precise points on your body.

You may receive radiation in one specific area of your body where there is a collection of leukemia cells, or you may receive radiation over your whole body. Radiation therapy may be used to prepare for a stem cell transplant.

  • Stem cell transplant. A stem cell transplant is a procedure to replace your diseased bone marrow with healthy bone marrow.

Before a stem cell transplant, you receive high doses of chemotherapy or radiation therapy to destroy your diseased bone marrow. Then you receive an infusion of blood-forming stem cells that help to rebuild your bone marrow.

You may receive stem cells from a donor, or in some cases you may be able to use your own stem cells. A stem cell transplant is very similar to a bone marrow transplant.

6.2.5.1.2 Acute Myeloid Leukemia

6.2.5.1.2.1 New treatment approaches in acute myeloid leukemia: review of recent clinical studies.

Norsworthy K1Luznik LGojo I.
Rev Recent Clin Trials. 2012 Aug; 7(3):224-37.
http://www.ncbi.nlm.nih.gov/pubmed/22540908

Standard chemotherapy can cure only a fraction (30-40%) of younger and very few older patients with acute myeloid leukemia (AML). While conventional allografting can extend the cure rates, its application remains limited mostly to younger patients and those in remission. Limited efficacy of current therapies and improved understanding of the disease biology provided a spur for clinical trials examining novel agents and therapeutic strategies in AML. Clinical studies with novel chemotherapeutics, antibodies, different signal transduction inhibitors, and epigenetic modulators demonstrated their clinical activity; however, it remains unclear how to successfully integrate novel agents either alone or in combination with chemotherapy into the overall therapeutic schema for AML. Further studies are needed to examine their role in relation to standard chemotherapy and their applicability to select patient populations based on recognition of unique disease and patient characteristics, including the development of predictive biomarkers of response. With increasing use of nonmyeloablative or reduced intensity conditioning and alternative graft sources such as haploidentical donors and cord blood transplants, the benefits of allografting may extend to a broader patient population, including older AML patients and those lacking a HLA-matched donor. We will review here recent clinical studies that examined novel pharmacologic and immunologic approaches to AML therapy.

6.2.5.1.2.2 Novel approaches to the treatment of acute myeloid leukemia.

Roboz GJ1
Hematology Am Soc Hematol Educ Program. 2011:43-50.
http://dx.doi.org:/10.1182/asheducation-2011.1.43.

Approximately 12 000 adults are diagnosed with acute myeloid leukemia (AML) in the United States annually, the majority of whom die from their disease. The mainstay of initial treatment, cytosine arabinoside (ara-C) combined with an anthracycline, was developed nearly 40 years ago and remains the worldwide standard of care. Advances in genomics technologies have identified AML as a genetically heterogeneous disease, and many patients can now be categorized into clinicopathologic subgroups on the basis of their underlying molecular genetic defects. It is hoped that enhanced specificity of diagnostic classification will result in more effective application of targeted agents and the ability to create individualized treatment strategies. This review describes the current treatment standards for induction, consolidation, and stem cell transplantation; special considerations in the management of older AML patients; novel agents; emerging data on the detection and management of minimal residual disease (MRD); and strategies to improve the design and implementation of AML clinical trials.

Age ≥ 60 years has consistently been identified as an independent adverse prognostic factor in AML, and there are very few long-term survivors in this age group.5 Poor outcomes in elderly AML patients have been attributed to both host- and disease-related factors, including medical comorbidities, physical frailty, increased incidence of antecedent myelodysplastic syndrome and myeloproliferative disorders, and higher frequency of adverse cytogenetics.28 Older patients with multiple poor-risk factors have a high probability of early death and little chance of long-term disease-free survival with standard chemotherapy. In a retrospective analysis of 998 older patients treated with intensive induction at the M.D. Anderson Cancer Center, multivariate analysis identified age ≥ 75 years, unfavorable karyotype, poor performance status, creatinine > 1.3 mg/dL, duration of antecedent hematologic disorder > 6 months, and treatment outside a laminar airflow room as adverse prognostic indicators.29 Patients with 3 or more of these factors had expected complete remission rates of < 20%, 8-week mortality > 50%, and 1-year survival < 10%. The Medical Research Council (MRC) identified cytogenetics, WBC count at diagnosis, age, and de novo versus secondary disease as critical factors influencing survival in > 2000 older patients with AML, but cautioned in their conclusions that less objective factors, such as clinical assessment of “fitness” for chemotherapy, may be equally important in making treatment decisions in this patient population.30 It is hoped that data from comprehensive geriatric assessments of functional status, cognition, mood, quality of life, and other measures obtained during ongoing cooperative group trials will improve our ability to predict how older patients will tolerate treatment.

6.5.1.2.3 Current treatment of acute myeloid leukemia.

Roboz GJ1.
Curr Opin Oncol. 2012 Nov; 24(6):711-9.
http://dx.doi.org:/10.1097/CCO.0b013e328358f62d.

The objectives of this review are to discuss standard and investigational nontransplant treatment strategies for acute myeloid leukemia (AML), excluding acute promyelocytic leukemia.

RECENT FINDINGS: Most adults with AML die from their disease. The standard treatment paradigm for AML is remission induction chemotherapy with an anthracycline/cytarabine combination, followed by either consolidation chemotherapy or allogeneic stem cell transplantation, depending on the patient’s ability to tolerate intensive treatment and the likelihood of cure with chemotherapy alone. Although this approach has changed little in the last three decades, increased understanding of the pathogenesis of AML and improvements in molecular genomic technologies are leading to novel drug targets and the development of personalized, risk-adapted treatment strategies. Recent findings related to prognostically relevant and potentially ‘druggable’ molecular targets are reviewed.

SUMMARY: At the present time, AML remains a devastating and mostly incurable disease, but the combination of optimized chemotherapeutics and molecularly targeted agents holds significant promise for the future.

6.5.1.2.4  Adult Acute Myeloid Leukemia Treatment (PDQ®)
http://www.cancer.gov/cancertopics/pdq/treatment/adultAML/healthprofessional/page9

About This PDQ Summary

This summary is reviewed regularly and updated as necessary by the PDQ Adult Treatment Editorial Board, which is editorially independent of the National Cancer Institute (NCI). The summary reflects an independent review of the literature and does not represent a policy statement of NCI or the National Institutes of Health (NIH).

Board members review recently published articles each month to determine whether an article should:

  • be discussed at a meeting,
  • be cited with text, or
  • replace or update an existing article that is already cited.

Treatment Option Overview for AML

Successful treatment of acute myeloid leukemia (AML) requires the control of bone marrow and systemic disease and specific treatment of central nervous system (CNS) disease, if present. The cornerstone of this strategy includes systemically administered combination chemotherapy. Because only 5% of patients with AML develop CNS disease, prophylactic treatment is not indicated.[13]

Treatment is divided into two phases: remission induction (to attain remission) and postremission (to maintain remission). Maintenance therapy for AML was previously administered for several years but is not included in most current treatment clinical trials in the United States, other than for acute promyelocytic leukemia. (Refer to the Adult Acute Myeloid Leukemia in Remission section of this summary for more information.) Other studies have used more intensive postremission therapy administered for a shorter duration of time after which treatment is discontinued.[4] Postremission therapy appears to be effective when given immediately after remission is achieved.[4]

Since myelosuppression is an anticipated consequence of both the leukemia and its treatment with chemotherapy, patients must be closely monitored during therapy. Facilities must be available for hematologic support with multiple blood fractions including platelet transfusions and for the treatment of related infectious complications.[5] Randomized trials have shown similar outcomes for patients who received prophylactic platelet transfusions at a level of 10,000/mm3 rather than 20,000/mm3.[6] The incidence of platelet alloimmunization was similar among groups randomly assigned to receive pooled platelet concentrates from random donors; filtered, pooled platelet concentrates from random donors; ultraviolet B-irradiated, pooled platelet concentrates from random donors; or filtered platelets obtained by apheresis from single random donors.[7] Colony-stimulating factors, for example, granulocyte colony–stimulating factor (G-CSF) and granulocyte-macrophage colony–stimulating factor (GM-CSF), have been studied in an effort to shorten the period of granulocytopenia associated with leukemia treatment.[8] If used, these agents are administered after completion of induction therapy. GM-CSF was shown to improve survival in a randomized trial of AML in patients aged 55 to 70 years (median survival was 10.6 months vs. 4.8 months). In this Eastern Cooperative Oncology Group (ECOG) (EST-1490) trial, patients were randomly assigned to receive GM-CSF or placebo following demonstration of leukemic clearance of the bone marrow;[9] however, GM-CSF did not show benefit in a separate similar randomized trial in patients older than 60 years.[10] In the latter study, clearance of the marrow was not required before initiating cytokine therapy. In a Southwest Oncology Group (NCT00023777) randomized trial of G-CSF given following induction therapy to patients older than 65 years, complete response was higher in patients who received G-CSF because of a decreased incidence of primary leukemic resistance. Growth factor administration did not impact on mortality or on survival.[11,12] Because the majority of randomized clinical trials have not shown an impact of growth factors on survival, their use is not routinely recommended in the remission induction setting.

The administration of GM-CSF or other myeloid growth factors before and during induction therapy, to augment the effects of cytotoxic therapy through the recruitment of leukemic blasts into cell cycle (growth factor priming), has been an area of active clinical research. Evidence from randomized studies of GM-CSF priming have come to opposite conclusions. A randomized study of GM-CSF priming during conventional induction and postremission therapy showed no difference in outcomes between patients who received GM-CSF and those who did not receive growth factor priming.[13,14][Level of evidence: 1iiA] In contrast, a similar randomized placebo-controlled study of GM-CSF priming in patients with AML aged 55 to 75 years showed improved disease-free survival (DFS) in the group receiving GM-CSF (median DFS for patients who achieved complete remission was 23 months vs. 11 months; 2-year DFS was 48% vs. 21%), with a trend towards improvement in overall survival (2-year survival was 39% vs. 27%, = .082) for patients aged 55 to 64 years.[15][Level of evidence: 1iiDii]

References

  1. Kebriaei P, Champlin R, deLima M, et al.: Management of acute leukemias. In: DeVita VT Jr, Lawrence TS, Rosenberg SA: Cancer: Principles and Practice of Oncology. 9th ed. Philadelphia, Pa: Lippincott Williams & Wilkins, 2011, pp 1928-54.
  2. Wiernik PH: Diagnosis and treatment of acute nonlymphocytic leukemia. In: Wiernik PH, Canellos GP, Dutcher JP, et al., eds.: Neoplastic Diseases of the Blood. 3rd ed. New York, NY: Churchill Livingstone, 1996, pp 283-302.
  3. Morrison FS, Kopecky KJ, Head DR, et al.: Late intensification with POMP chemotherapy prolongs survival in acute myelogenous leukemia–results of a Southwest Oncology Group study of rubidazone versus adriamycin for remission induction, prophylactic intrathecal therapy, late intensification, and levamisole maintenance. Leukemia 6 (7): 708-14, 1992. [PUBMED Abstract]
  4. Cassileth PA, Lynch E, Hines JD, et al.: Varying intensity of postremission therapy in acute myeloid leukemia. Blood 79 (8): 1924-30, 1992. [PUBMED Abstract]
  5. Supportive Care. In: Wiernik PH, Canellos GP, Dutcher JP, et al., eds.: Neoplastic Diseases of the Blood. 3rd ed. New York, NY: Churchill Livingstone, 1996, pp 779-967.
  6. Rebulla P, Finazzi G, Marangoni F, et al.: The threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia. Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto. N Engl J Med 337 (26): 1870-5, 1997. [PUBMED Abstract]
  7. Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med 337 (26): 1861-9, 1997. [PUBMED Abstract]
  8. Geller RB: Use of cytokines in the treatment of acute myelocytic leukemia: a critical review. J Clin Oncol 14 (4): 1371-82, 1996. [PUBMED Abstract]
  9. Rowe JM, Andersen JW, Mazza JJ, et al.: A randomized placebo-controlled phase III study of granulocyte-macrophage colony-stimulating factor in adult patients (> 55 to 70 years of age) with acute myelogenous leukemia: a study of the Eastern Cooperative Oncology Group (E1490). Blood 86 (2): 457-62, 1995. [PUBMED Abstract]
  10. Stone RM, Berg DT, George SL, et al.: Granulocyte-macrophage colony-stimulating factor after initial chemotherapy for elderly patients with primary acute myelogenous leukemia. Cancer and Leukemia Group B. N Engl J Med 332 (25): 1671-7, 1995. [PUBMED Abstract]
  11. Dombret H, Chastang C, Fenaux P, et al.: A controlled study of recombinant human granulocyte colony-stimulating factor in elderly patients after treatment for acute myelogenous leukemia. AML Cooperative Study Group. N Engl J Med 332 (25): 1678-83, 1995. [PUBMED Abstract]
  12. Godwin JE, Kopecky KJ, Head DR, et al.: A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest oncology group study (9031). Blood 91 (10): 3607-15, 1998. [PUBMED Abstract]
  13. Buchner T, Hiddemann W, Wormann B, et al.: GM-CSF multiple course priming and long-term administration in newly diagnosed AML: hematologic and therapeutic effects. [Abstract] Blood 84 (10 Suppl 1): A-95, 27a, 1994.
  14. Löwenberg B, Boogaerts MA, Daenen SM, et al.: Value of different modalities of granulocyte-macrophage colony-stimulating factor applied during or after induction therapy of acute myeloid leukemia. J Clin Oncol 15 (12): 3496-506, 1997. [PUBMED Abstract]
  15. Witz F, Sadoun A, Perrin MC, et al.: A placebo-controlled study of recombinant human granulocyte-macrophage colony-stimulating factor administered during and after induction treatment for de novo acute myelogenous leukemia in elderly patients. Groupe Ouest Est Leucémies Aiguës Myéloblastiques (GOELAM). Blood 91 (8): 2722-30, 1998. [PUBMED Abstract]

6.2.5.1.3 Treatment for CML

6.2.5.1.3.1 Chronic Myelogenous Leukemia Treatment (PDQ®)

http://www.cancer.gov/cancertopics/pdq/treatment/CML/Patient/page4

Treatment Option Overview

Key Points for This Section

There are different types of treatment for patients with chronic myelogenous leukemia.

Six types of standard treatment are used:

  1. Targeted therapy
  2. Chemotherapy
  3. Biologic therapy
  4. High-dose chemotherapy with stem cell transplant
  5. Donor lymphocyte infusion (DLI)
  6. Surgery

New types of treatment are being tested in clinical trials.

Patients may want to think about taking part in a clinical trial.

Patients can enter clinical trials before, during, or after starting their cancer treatment.

Follow-up tests may be needed.

There are different types of treatment for patients with chronic myelogenous leukemia.

Different types of treatment are available for patients with chronic myelogenous leukemia (CML). Some treatments are standard (the currently used treatment), and some are being tested in clinical trials. A treatment clinical trial is a research study meant to help improve current treatments or obtain information about new treatments for patients with cancer. When clinical trials show that a new treatment is better than the standard treatment, the new treatment may become the standard treatment. Patients may want to think about taking part in a clinical trial. Some clinical trials are open only to patients who have not started treatment.

Six types of standard treatment are used:

Targeted therapy

Targeted therapy is a type of treatment that uses drugs or other substances to identify and attack specific cancer cells without harming normal cells. Tyrosine kinase inhibitors are targeted therapy drugs used to treat chronic myelogenous leukemia.

Imatinib mesylate, nilotinib, dasatinib, and ponatinib are tyrosine kinase inhibitors that are used to treat CML.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Chemotherapy

Chemotherapy is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. When chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy). When chemotherapy is placed directly into the cerebrospinal fluid, an organ, or a body cavity such as the abdomen, the drugs mainly affect cancer cells in those areas (regional chemotherapy). The way the chemotherapy is given depends on the type and stage of the cancer being treated.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Biologic therapy

Biologic therapy is a treatment that uses the patient’s immune system to fight cancer. Substances made by the body or made in a laboratory are used to boost, direct, or restore the body’s natural defenses against cancer. This type of cancer treatment is also called biotherapy or immunotherapy.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

High-dose chemotherapy with stem cell transplant

High-dose chemotherapy with stem cell transplant is a method of giving high doses of chemotherapy and replacing blood-forming cells destroyed by the cancer treatment. Stem cells (immature blood cells) are removed from the blood or bone marrow of the patient or a donor and are frozen and stored. After the chemotherapy is completed, the stored stem cells are thawed and given back to the patient through an infusion. These reinfused stem cells grow into (and restore) the body’s blood cells.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Donor lymphocyte infusion (DLI)

Donor lymphocyte infusion (DLI) is a cancer treatment that may be used after stem cell transplant.Lymphocytes (a type of white blood cell) from the stem cell transplant donor are removed from the donor’s blood and may be frozen for storage. The donor’s lymphocytes are thawed if they were frozen and then given to the patient through one or more infusions. The lymphocytes see the patient’s cancer cells as not belonging to the body and attack them.

Surgery

Splenectomy

6.2.5.1.3.2 What`s new in chronic myeloid leukemia research and treatment?

http://www.cancer.org/cancer/leukemia-chronicmyeloidcml/detailedguide/leukemia-chronic-myeloid-myelogenous-new-research

Combining the targeted drugs with other treatments

Imatinib and other drugs that target the BCR-ABL protein have proven to be very effective, but by themselves these drugs don’t help everyone. Studies are now in progress to see if combining these drugs with other treatments, such as chemotherapy, interferon, or cancer vaccines (see below) might be better than either one alone. One study showed that giving interferon with imatinib worked better than giving imatinib alone. The 2 drugs together had more side effects, though. It is also not clear if this combination is better than treatment with other tyrosine kinase inhibitors (TKIs), such as dasatinib and nilotinib. A study going on now is looking at combing interferon with nilotinib.

Other studies are looking at combining other drugs, such as cyclosporine or hydroxychloroquine, with a TKI.

New drugs for CML

Because researchers now know the main cause of CML (the BCR-ABL gene and its protein), they have been able to develop many new drugs that might work against it.

In some cases, CML cells develop a change in the BCR-ABL oncogene known as a T315I mutation, which makes them resistant to many of the current targeted therapies (imatinib, dasatinib, and nilotinib). Ponatinib is the only TKI that can work against T315I mutant cells. More drugs aimed at this mutation are now being tested.

Other drugs called farnesyl transferase inhibitors, such as lonafarnib and tipifarnib, seem to have some activity against CML and patients may respond when these drugs are combined with imatinib. These drugs are being studied further.

Other drugs being studied in CML include the histone deacetylase inhibitor panobinostat and the proteasome inhibitor bortezomib (Velcade).

Several vaccines are now being studied for use against CML.

6.2.5.1.4. Chronic Lymphocytic Leukemia

6.2.5.1.4.1 Chronic Lymphocytic Leukemia Treatment (PDQ®)

General Information About Chronic Lymphocytic Leukemia

Key Points for This Section

  1. Chronic lymphocytic leukemia is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell).
  2. Leukemia may affect red blood cells, white blood cells, and platelets.
  3. Older age can affect the risk of developing chronic lymphocytic leukemia.
  4. Signs and symptoms of chronic lymphocytic leukemia include swollen lymph nodes and tiredness.
  5. Tests that examine the blood, bone marrow, and lymph nodes are used to detect (find) and diagnose chronic lymphocytic leukemia.
  6. Certain factors affect treatment options and prognosis (chance of recovery).
  7. Chronic lymphocytic leukemia is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell).

Chronic lymphocytic leukemia (also called CLL) is a blood and bone marrow disease that usually gets worse slowly. CLL is one of the most common types of leukemia in adults. It often occurs during or after middle age; it rarely occurs in children.

http://www.cancer.gov/images/cdr/live/CDR755927-750.jpg

Anatomy of the bone; drawing shows spongy bone, red marrow, and yellow marrow. A cross section of the bone shows compact bone and blood vessels in the bone marrow. Also shown are red blood cells, white blood cells, platelets, and a blood stem cell.

Anatomy of the bone. The bone is made up of compact bone, spongy bone, and bone marrow. Compact bone makes up the outer layer of the bone. Spongy bone is found mostly at the ends of bones and contains red marrow. Bone marrow is found in the center of most bones and has many blood vessels. There are two types of bone marrow: red and yellow. Red marrow contains blood stem cells that can become red blood cells, white blood cells, or platelets. Yellow marrow is made mostly of fat.

Leukemia may affect red blood cells, white blood cells, and platelets.

Normally, the body makes blood stem cells (immature cells) that become mature blood cells over time. A blood stem cell may become a myeloid stem cell or a lymphoid stem cell.

A myeloid stem cell becomes one of three types of mature blood cells:

  1. Red blood cells that carry oxygen and other substances to all tissues of the body.
  2. White blood cells that fight infection and disease.
  3. Platelets that form blood clots to stop bleeding.

A lymphoid stem cell becomes a lymphoblast cell and then one of three types of lymphocytes (white blood cells):

  1. B lymphocytes that make antibodies to help fight infection.
  2. T lymphocytes that help B lymphocytes make antibodies to fight infection.
  3. Natural killer cells that attack cancer cells and viruses.
Blood cell development. CDR526538-750

Blood cell development. CDR526538-750

http://www.cancer.gov/images/cdr/live/CDR526538-750.jpg

Blood cell development; drawing shows the steps a blood stem cell goes through to become a red blood cell, platelet, or white blood cell. A myeloid stem cell becomes a red blood cell, a platelet, or a myeloblast, which then becomes a granulocyte (the types of granulocytes are eosinophils, basophils, and neutrophils). A lymphoid stem cell becomes a lymphoblast and then becomes a B-lymphocyte, T-lymphocyte, or natural killer cell.

Blood cell development. A blood stem cell goes through several steps to become a red blood cell, platelet, or white blood cell.

In CLL, too many blood stem cells become abnormal lymphocytes and do not become healthy white blood cells. The abnormal lymphocytes may also be called leukemia cells. The lymphocytes are not able to fight infection very well. Also, as the number of lymphocytes increases in the blood and bone marrow, there is less room for healthy white blood cells, red blood cells, and platelets. This may cause infection, anemia, and easy bleeding.

This summary is about chronic lymphocytic leukemia. See the following PDQ summaries for more information about leukemia:

  • Adult Acute Lymphoblastic Leukemia Treatment.
  • Childhood Acute Lymphoblastic Leukemia Treatment.
  • Adult Acute Myeloid Leukemia Treatment.
  • Childhood Acute Myeloid Leukemia/Other Myeloid Malignancies Treatment.
  • Chronic Myelogenous Leukemia Treatment.
  • Hairy Cell Leukemia Treatment

Older age can affect the risk of developing chronic lymphocytic leukemia.

Anything that increases your risk of getting a disease is called a risk factor. Having a risk factor does not mean that you will get cancer; not having risk factors doesn’t mean that you will not get cancer. Talk with your doctor if you think you may be at risk. Risk factors for CLL include the following:

  • Being middle-aged or older, male, or white.
  • A family history of CLL or cancer of the lymph system.
  • Having relatives who are Russian Jews or Eastern European Jews.

Signs and symptoms of chronic lymphocytic leukemia include swollen lymph nodes and tiredness.

Usually CLL does not cause any signs or symptoms and is found during a routine blood test. Signs and symptoms may be caused by CLL or by other conditions. Check with your doctor if you have any of the following:

  • Painless swelling of the lymph nodes in the neck, underarm, stomach, or groin.
  • Feeling very tired.
  • Pain or fullness below the ribs.
  • Fever and infection.
  • Weight loss for no known reason.

Tests that examine the blood, bone marrow, and lymph nodes are used to detect (find) and diagnose chronic lymphocytic leukemia.

The following tests and procedures may be used:

Physical exam and history : An exam of the body to check general signs of health, including checking for signs of disease, such as lumps or anything else that seems unusual. A history of the patient’s health habits and past illnesses and treatments will also be taken.

Complete blood count (CBC) with differential : A procedure in which a sample of blood is drawn and checked for the following:

The number of red blood cells and platelets.

The number and type of white blood cells.

The amount of hemoglobin (the protein that carries oxygen) in the red blood cells.

The portion of the blood sample made up of red blood cells.

6.2.5.1.4.2 Results from the Phase 3 Resonate™ Trial

Significantly improved progression free survival (PFS) vs ofatumumab in patients with previously treated CLL

  • Patients taking IMBRUVICA® had a 78% statistically significant reduction in the risk of disease progression or death compared with patients who received ofatumumab1
  • In patients with previously treated del 17p CLL, median PFS was not yet reached with IMBRUVICA® vs 5.8 months with ofatumumab (HR 0.25; 95% CI: 0.14, 0.45)1

Significantly prolonged overall survival (OS) with IMBRUVICA® vs ofatumumab in patients with previously treated CLL

  • In patients with previously treated CLL, those taking IMBRUVICA® had a 57% statistically significant reduction in the risk of death compared with those who received ofatumumab (HR 0.43; 95% CI: 0.24, 0.79; P<0.05)1

6.2.5.1.4.3 Typical treatment of chronic lymphocytic leukemia

http://www.cancer.org/cancer/leukemia-chroniclymphocyticcll/detailedguide/leukemia-chronic-lymphocytic-treating-treatment-by-risk-group

Treatment options for chronic lymphocytic leukemia (CLL) vary greatly, depending on the person’s age, the disease risk group, and the reason for treating (for example, which symptoms it is causing). Many people live a long time with CLL, but in general it is very difficult to cure, and early treatment hasn’t been shown to help people live longer. Because of this and because treatment can cause side effects, doctors often advise waiting until the disease is progressing or bothersome symptoms appear, before starting treatment.

If treatment is needed, factors that should be taken into account include the patient’s age, general health, and prognostic factors such as the presence of chromosome 17 or chromosome 11 deletions or high levels of ZAP-70 and CD38.

Initial treatment

Patients who might not be able to tolerate the side effects of strong chemotherapy (chemo), are often treated with chlorambucil alone or with a monoclonal antibody targeting CD20 like rituximab (Rituxan) or obinutuzumab (Gazyva). Other options include rituximab alone or a corticosteroid like prednisione.

In stronger and healthier patients, there are many options for treatment. Commonly used treatments include:

  • FCR: fludarabine (Fludara), cyclophosphamide (Cytoxan), and rituximab
  • Bendamustine (sometimes with rituximab)
  • FR: fludarabine and rituximab
  • CVP: cyclophosphamide, vincristine, and prednisone (sometimes with rituximab)
  • CHOP: cyclophosphamide, doxorubicin, vincristine (Oncovin), and prednisone
  • Chlorambucil combined with prednisone, rituximab, obinutuzumab, or ofatumumab
  • PCR: pentostatin (Nipent), cyclophosphamide, and rituximab
  • Alemtuzumab (Campath)
  • Fludarabine (alone)

Other drugs or combinations of drugs may also be also used.

If the only problem is an enlarged spleen or swollen lymph nodes in one region of the body, localized treatment with low-dose radiation therapy may be used. Splenectomy (surgery to remove the spleen) is another option if the enlarged spleen is causing symptoms.

Sometimes very high numbers of leukemia cells in the blood cause problems with normal circulation. This is calledleukostasis. Chemo may not lower the number of cells until a few days after the first dose, so before the chemo is given, some of the cells may be removed from the blood with a procedure called leukapheresis. This treatment lowers blood counts right away. The effect lasts only for a short time, but it may help until the chemo has a chance to work. Leukapheresis is also sometimes used before chemo if there are very high numbers of leukemia cells (even when they aren’t causing problems) to prevent tumor lysis syndrome (this was discussed in the chemotherapy section).

Some people who have very high-risk disease (based on prognostic factors) may be referred for possible stem cell transplant (SCT) early in treatment.

Second-line treatment of CLL

If the initial treatment is no longer working or the disease comes back, another type of treatment may help. If the initial response to the treatment lasted a long time (usually at least a few years), the same treatment can often be used again. If the initial response wasn’t long-lasting, using the same treatment again isn’t as likely to be helpful. The options will depend on what the first-line treatment was and how well it worked, as well as the person’s health.

Many of the drugs and combinations listed above may be options as second-line treatments. For many people who have already had fludarabine, alemtuzumab seems to be helpful as second-line treatment, but it carries an increased risk of infections. Other purine analog drugs, such as pentostatin or cladribine (2-CdA), may also be tried. Newer drugs such as ofatumumab, ibrutinib (Imbruvica), and idelalisib (Zydelig) may be other options.

If the leukemia responds, stem cell transplant may be an option for some patients.

Some people may have a good response to first-line treatment (such as fludarabine) but may still have some evidence of a small number of leukemia cells in the blood, bone marrow, or lymph nodes. This is known as minimal residual disease. CLL can’t be cured, so doctors aren’t sure if further treatment right away will be helpful. Some small studies have shown that alemtuzumab can sometimes help get rid of these remaining cells, but it’s not yet clear if this improves survival.

Treating complications of CLL

One of the most serious complications of CLL is a change (transformation) of the leukemia to a high-grade or aggressive type of non-Hodgkin lymphoma called diffuse large cell lymphoma. This happens in about 5% of CLL cases, and is known as Richter syndrome. Treatment is often the same as it would be for lymphoma (see our document called Non-Hodgkin Lymphoma for more information), and may include stem cell transplant, as these cases are often hard to treat.

Less often, CLL may transform to prolymphocytic leukemia. As with Richter syndrome, these cases can be hard to treat. Some studies have suggested that certain drugs such as cladribine (2-CdA) and alemtuzumab may be helpful.

In rare cases, patients with CLL may have their leukemia transform into acute lymphocytic leukemia (ALL). If this happens, treatment is likely to be similar to that used for patients with ALL (see our document called Leukemia: Acute Lymphocytic).

Acute myeloid leukemia (AML) is another rare complication in patients who have been treated for CLL. Drugs such as chlorambucil and cyclophosphamide can damage the DNA of blood-forming cells. These damaged cells may go on to become cancerous, leading to AML, which is very aggressive and often hard to treat (see our document calledLeukemia: Acute Myeloid).

CLL can cause problems with low blood counts and infections. Treatment of these problems were discussed in the section “Supportive care in chronic lymphocytic leukemia.”

6.2.5.1.5 Lymphoma treatment

 6.2.5.1.5.1 Overview

http://www.emedicinehealth.com/lymphoma/page8_em.htm#lymphoma_treatment

The most widely used therapies are combinations of chemotherapyand radiation therapy.

  • Biological therapy, which targets key features of the lymphoma cells, is used in many cases nowadays.

The goal of medical therapy in lymphoma is complete remission. This means that all signs of the disease have disappeared after treatment. Remission is not the same as cure. In remission, one may still have lymphoma cells in the body, but they are undetectable and cause no symptoms.

  • When in remission, the lymphoma may come back. This is called recurrence.
  • The duration of remission depends on the type, stage, and grade of the lymphoma. A remission may last a few months, a few years, or may continue throughout one’s life.
  • Remission that lasts a long time is called durable remission, and this is the goal of therapy.
  • The duration of remission is a good indicator of the aggressiveness of the lymphoma and of the prognosis. A longer remission generally indicates a better prognosis.

Remission can also be partial. This means that the tumor shrinks after treatment to less than half its size before treatment.

The following terms are used to describe the lymphoma’s response to treatment:

  • Improvement: The lymphoma shrinks but is still greater than half its original size.
  • Stable disease: The lymphoma stays the same.
  • Progression: The lymphoma worsens during treatment.
  • Refractory disease: The lymphoma is resistant to treatment.

The following terms to refer to therapy:

  • Induction therapy is designed to induce a remission.
  • If this treatment does not induce a complete remission, new or different therapy will be initiated. This is usually referred to as salvage therapy.
  • Once in remission, one may be given yet another treatment to prevent recurrence. This is called maintenance therapy.

6.2.5.1.5.2 Chemotherapy

Many different types of chemotherapy may be used for Hodgkin lymphoma. The most commonly used combination of drugs in the United States is called ABVD. Another combination of drugs, known as BEACOPP, is now widely used in Europe and is being used more often in the United States. There are other combinations that are less commonly used and not listed here. The drugs that make up these two more common combinations of chemotherapy are listed below.

ABVD: Doxorubicin (Adriamycin), bleomycin (Blenoxane), vinblastine (Velban, Velsar), and dacarbazine (DTIC-Dome). ABVD chemotherapy is usually given every two weeks for two to eight months.

BEACOPP: Bleomycin, etoposide (Toposar, VePesid), doxorubicin, cyclophosphamide (Cytoxan, Neosar), vincristine (Vincasar PFS, Oncovin), procarbazine (Matulane), and prednisone (multiple brand names). There are several different treatment schedules, but different drugs are usually given every two weeks.

The type of chemotherapy, number of cycles of chemotherapy, and the additional use of radiation therapy are based on the stage of the Hodgkin lymphoma and the type and number of prognostic factors.

6.2.5.1.5.3 Adult Non-Hodgkin Lymphoma Treatment (PDQ®)

http://www.cancer.gov/cancertopics/pdq/treatment/adult-non-hodgkins/Patient/page1

Key Points for This Section

Adult non-Hodgkin lymphoma is a disease in which malignant (cancer) cells form in the lymph system.

Because lymph tissue is found throughout the body, adult non-Hodgkin lymphoma can begin in almost any part of the body. Cancer can spread to the liver and many other organs and tissues.

Non-Hodgkin lymphoma in pregnant women is the same as the disease in nonpregnant women of childbearing age. However, treatment is different for pregnant women. This summary includes information on the treatment of non-Hodgkin lymphoma during pregnancy

Non-Hodgkin lymphoma can occur in both adults and children. Treatment for children, however, is different than treatment for adults. (See the PDQ summary on Childhood Non-Hodgkin Lymphoma Treatment for more information.)

There are many different types of lymphoma.

Lymphomas are divided into two general types: Hodgkin lymphoma and non-Hodgkin lymphoma. This summary is about the treatment of adult non-Hodgkin lymphoma. For information about other types of lymphoma, see the following PDQ summaries:

Age, gender, and a weakened immune system can affect the risk of adult non-Hodgkin lymphoma.

If cancer is found, the following tests may be done to study the cancer cells:

  • Immunohistochemistry : A test that uses antibodies to check for certain antigens in a sample of tissue. The antibody is usually linked to a radioactive substance or a dye that causes the tissue to light up under a microscope. This type of test may be used to tell the difference between different types of cancer.
  • Cytogenetic analysis : A laboratory test in which cells in a sample of tissue are viewed under a microscope to look for certain changes in the chromosomes.
  • Immunophenotyping : A process used to identify cells, based on the types of antigens ormarkers on the surface of the cell. This process is used to diagnose specific types of leukemia and lymphoma by comparing the cancer cells to normal cells of the immune system.

Certain factors affect prognosis (chance of recovery) and treatment options.

The prognosis (chance of recovery) and treatment options depend on the following:

  • The stage of the cancer.
  • The type of non-Hodgkin lymphoma.
  • The amount of lactate dehydrogenase (LDH) in the blood.
  • The amount of beta-2-microglobulin in the blood (for Waldenström macroglobulinemia).
  • The patient’s age and general health.
  • Whether the lymphoma has just been diagnosed or has recurred (come back).

Stages of adult non-Hodgkin lymphoma may include E and S.

Adult non-Hodgkin lymphoma may be described as follows:

E: “E” stands for extranodal and means the cancer is found in an area or organ other than the lymph nodes or has spread to tissues beyond, but near, the major lymphatic areas.

S: “S” stands for spleen and means the cancer is found in the spleen.

Stage I adult non-Hodgkin lymphoma is divided into stage I and stage IE.

  • Stage I: Cancer is found in one lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen).
  • Stage IE: Cancer is found in one organ or area outside the lymph nodes.

Stage II adult non-Hodgkin lymphoma is divided into stage II and stage IIE.

  • Stage II: Cancer is found in two or more lymph node groups either above or below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
  • Stage IIE: Cancer is found in one or more lymph node groups either above or below the diaphragm. Cancer is also found outside the lymph nodes in one organ or area on the same side of the diaphragm as the affected lymph nodes.

Stage III adult non-Hodgkin lymphoma is divided into stage III, stage IIIE, stage IIIS, and stage IIIE+S.

  • Stage III: Cancer is found in lymph node groups above and below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
  • Stage IIIE: Cancer is found in lymph node groups above and below the diaphragm and outside the lymph nodes in a nearby organ or area.
  • Stage IIIS: Cancer is found in lymph node groups above and below the diaphragm, and in the spleen.
  • Stage IIIE+S: Cancer is found in lymph node groups above and below the diaphragm, outside the lymph nodes in a nearby organ or area, and in the spleen.

In stage IV adult non-Hodgkin lymphoma, the cancer:

  • is found throughout one or more organs that are not part of a lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen), and may be in lymph nodes near those organs; or
  • is found in one organ that is not part of a lymphatic area and has spread to organs or lymph nodes far away from that organ; or
  • is found in the liver, bone marrow, cerebrospinal fluid (CSF), or lungs (other than cancer that has spread to the lungs from nearby areas).

Adult non-Hodgkin lymphomas are also described based on how fast they grow and where the affected lymph nodes are in the body.  Indolent & aggressive.

The treatment plan depends mainly on the following:

  • The type of non-Hodgkin’s lymphoma
  • Its stage (where the lymphoma is found)
  • How quickly the cancer is growing
  • The patient’s age
  • Whether the patient has other health problems
  • If there are symptoms present such as fever and night sweats (see above)

6.2.5.1.6 Primary treatments

6.2.5.1.6.1 Radiation Therapy for leukemias and lymphomas

http://www.lls.org/treatment/types-of-treatment/radiation-therapy

Radiation therapy, also called radiotherapy or irradiation, can be used to treat leukemia, lymphoma, myeloma and myelodysplastic syndromes. The type of radiation used for radiotherapy (ionizing radiation) is the same that’s used for diagnostic x-rays. Radiotherapy, however, is given in higher doses.

Radiotherapy works by damaging the genetic material (DNA) within cells, which prevents them from growing and reproducing. Although the radiotherapy is directed at cancer cells, it can also damage nearby healthy cells. However, current methods of radiotherapy have been improved upon, minimizing “scatter” to nearby tissues. Therefore its benefit (destroying the cancer cells) outweighs its risk (harming healthy cells).

When radiotherapy is used for blood cancer treatment, it’s usually part of a treatment plan that includes drug therapy. Radiotherapy can also be used to relieve pain or discomfort caused by an enlarged liver, lymph node(s) or spleen.

Radiotherapy, either alone or with chemotherapy, is sometimes given as conditioning treatment to prepare a patient for a blood or marrow stem cell transplant. The most common types used to treat blood cancer are external beam radiation (see below) and radioimmunotherapy.
External Beam Radiation

External beam radiation is the type of radiotherapy used most often for people with blood cancers. A focused radiation beam is delivered outside the body by a machine called a linear accelerator, or linac for short. The linear accelerator moves around the body to deliver radiation from various angles. Linear accelerators make it possible to decrease or avoid skin reactions and deliver targeted radiation to lessen “scatter” of radiation to nearby tissues.

The dose (total amount) of radiation used during treatment depends on various factors regarding the patient, disease and reason for treatment, and is established by a radiation oncologist. You may receive radiotherapy during a series of visits, spread over several weeks (from two to 10 weeks, on average). This approach, called dose fractionation, lessens side effects. External beam radiation does not make you radioactive.

6.2.5.1.6.2 bone marrow (BM) transplantation

http://www.nlm.nih.gov/medlineplus/ency/article/003009.htm

There are three kinds of bone marrow transplants:

Autologous bone marrow transplant: The term auto means self. Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment. The stem cells are stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments, your stems cells are put back in your body to make (regenerate) normal blood cells. This is called a rescue transplant.

Allogeneic bone marrow transplant: The term allo means other. Stem cells are removed from another person, called a donor. Most times, the donor’s genes must at least partly match your genes. Special blood tests are done to see if a donor is a good match for you. A brother or sister is most likely to be a good match. Sometimes parents, children, and other relatives are good matches. Donors who are not related to you may be found through national bone marrow registries.

Umbilical cord blood transplant: This is a type of allogeneic transplant. Stem cells are removed from a newborn baby’s umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Umbilical cord blood cells are very immature so there is less of a need for matching. But blood counts take much longer to recover.

Before the transplant, chemotherapy, radiation, or both may be given. This may be done in two ways:

Ablative (myeloablative) treatment: High-dose chemotherapy, radiation, or both are given to kill any cancer cells. This also kills all healthy bone marrow that remains, and allows new stem cells to grow in the bone marrow.

Reduced intensity treatment, also called a mini transplant: Patients receive lower doses of chemotherapy and radiation before a transplant. This allows older patients, and those with other health problems to have a transplant.

A stem cell transplant is usually done after chemotherapy and radiation is complete. The stem cells are delivered into your bloodstream usually through a tube called a central venous catheter. The process is similar to getting a blood transfusion. The stem cells travel through the blood into the bone marrow. Most times, no surgery is needed.

Donor stem cells can be collected in two ways:

Bone marrow harvest. This minor surgery is done under general anesthesia. This means the donor will be asleep and pain-free during the procedure. The bone marrow is removed from the back of both hip bones. The amount of marrow removed depends on the weight of the person who is receiving it.

Leukapheresis. First, the donor is given 5 days of shots to help stem cells move from the bone marrow into the blood. During leukapheresis, blood is removed from the donor through an IV line in a vein. The part of white blood cells that contains stem cells is then separated in a machine and removed to be later given to the recipient. The red blood cells are returned to the donor.

Why the Procedure is Performed

A bone marrow transplant replaces bone marrow that either is not working properly or has been destroyed (ablated) by chemotherapy or radiation. Doctors believe that for many cancers, the donor’s white blood cells can attach to any remaining cancer cells, similar to when white cells attach to bacteria or viruses when fighting an infection.

Your doctor may recommend a bone marrow transplant if you have:

Certain cancers, such as leukemia, lymphoma, and multiple myeloma

A disease that affects the production of bone marrow cells, such as aplastic anemia, congenital neutropenia, severe immunodeficiency syndromes, sickle cell anemia, thalassemia

Had chemotherapy that destroyed your bone

6.2.5.1.6.2.1 Autologous stem cell transplantation

6.2.5.1.6.2.1.1 Phase II trial of 131I-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas

O.W Press,  F Appelbaum,  P.J Martin, et al.
http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(95)92225-3/abstract

25 patients with relapsed B-cell lymphomas were evaluated with trace-labelled doses (2·5 mg/kg, 185-370 MBq [5-10 mCi]) of 131I-labelled anti-CD20 (B1) antibody in a phase II trial. 22 patients achieved 131I-B1 biodistributions delivering higher doses of radiation to tumor sites than to normal organs and 21 of these were treated with therapeutic infusions of 131I-B1 (12·765-29·045 GBq) followed by autologous hemopoietic stem cell reinfusion. 18 of the 21 treated patients had objective responses, including 16 complete remissions. One patient died of progressive lymphoma and one died of sepsis. Analysis of our phase I and II trials with 131I-labelled B1 reveal a progression-free survival of 62% and an overall survival of 93% with a median follow-up of 2 years. 131I-anti-CD20 (B1) antibody therapy produces complete responses of long duration in most patients with relapsed B-cell lymphomas when given at maximally tolerated doses with autologous stem cell rescue.

6.2.5.2.6.2.1.2 Autologous (Self) Transplants

http://www.leukaemia.org.au/treatments/stem-cell-transplants/autologous-self-transplants

An autologous transplant (or rescue) is a type of transplant that uses the person’s own stem cells. These cells are collected in advance and returned at a later stage. They are used to replace stem cells that have been damaged by high doses of chemotherapy, used to treat the person’s underlying disease.

In most cases, stem cells are collected directly from the bloodstream. While stem cells normally live in your marrow, a combination of chemotherapy and a growth factor (a drug that stimulates stem cells) called Granulocyte Colony Stimulating Factor (G-CSF) is used to expand the number of stem cells in the marrow and cause them to spill out into the circulating blood. From here they can be collected from a vein by passing the blood through a special machine called a cell separator, in a process similar to dialysis.

Most of the side effects of an autologous transplant are caused by the conditioning therapy used. Although they can be very unpleasant at times it is important to remember that most of them are temporary and reversible.

6.2.5.2.6.2.1.3  Hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient’s own stem cells are used) or allogeneic (the stem cells come from a donor).

Hematopoietic Stem Cell Transplantation

Author: Ajay Perumbeti, MD, FAAP; Chief Editor: Emmanuel C Besa, MD
http://emedicine.medscape.com/article/208954-overview

Hematopoietic stem cell transplantation (HSCT) involves the intravenous (IV) infusion of autologous or allogeneic stem cells to reestablish hematopoietic function in patients whose bone marrow or immune system is damaged or defective.

The image below illustrates an algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy.

An algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy: If a matched sibling donor is not available, then a MUD is selected; if a MUD is not available, then choices include a mismatched unrelated donor, umbilical cord donor(s), and a haploidentical donor.

6.2.5.3 Supportive Therapies

6.2.5.3.1  Blood transfusions – risks and complications of a blood transfusion

  • Allogeneic transfusion reaction (acute or delayed hemolytic reaction)
  • Allergic reaction
  • Viruses Infectious Diseases

The risk of catching a virus from a blood transfusion is very low.

HIV. Your risk of getting HIV from a blood transfusion is lower than your risk of getting killed by lightning. Only about 1 in 2 million donations might carry HIV and transmit HIV if given to a patient.

Hepatitis B and C. The risk of having a donation that carries hepatitis B is about 1 in 205,000. The risk for hepatitis C is 1 in 2 million. If you receive blood during a transfusion that contains hepatitis, you’ll likely develop the virus.

Variant Creutzfeldt-Jakob disease (vCJD). This disease is the human version of Mad Cow Disease. It’s a very rare, yet fatal brain disorder. There is a possible risk of getting vCJD from a blood transfusion, although the risk is very low. Because of this, people who may have been exposed to vCJD aren’t eligible blood donors.

  • Fever
  • Iron Overload
  • Lung Injury
  • Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a condition in which white blood cells in the new blood attack your tissues.

6.2.5.3.2  Erythropoietin

Erythropoietin, (/ɨˌrɪθrɵˈpɔɪ.ɨtɨn/UK /ɛˌrɪθr.pˈtɪn/) also known as EPO, is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. It is a cytokine (protein signaling molecule) for erythrocyte (red blood cell) precursors in the bone marrow. Human EPO has a molecular weight of 34 kDa.

Also called hematopoietin or hemopoietin, it is produced by interstitial fibroblasts in the kidney in close association with peritubular capillary and proximal convoluted tubule. It is also produced in perisinusoidal cells in the liver. While liver production predominates in the fetal and perinatal period, renal production is predominant during adulthood. In addition to erythropoiesis, erythropoietin also has other known biological functions. For example, it plays an important role in the brain’s response to neuronal injury.[1] EPO is also involved in the wound healing process.[2]

Exogenous erythropoietin is produced by recombinant DNA technology in cell culture. Several different pharmaceutical agents are available with a variety ofglycosylation patterns, and are collectively called erythropoiesis-stimulating agents (ESA). The specific details for labelled use vary between the package inserts, but ESAs have been used in the treatment of anemia in chronic kidney disease, anemia in myelodysplasia, and in anemia from cancer chemotherapy. Boxed warnings include a risk of death, myocardial infarction, stroke, venous thromboembolism, and tumor recurrence.[3]

6.2.5.3.4  G-CSF (granulocyte-colony stimulating factor)

Granulocyte-colony stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream.

There are different types, including

  • Lenograstim (Granocyte)
  • Filgrastim (Neupogen, Zarzio, Nivestim, Ratiograstim)
  • Long acting (pegylated) filgrastim (pegfilgrastim, Neulasta) and lipegfilgrastim (Longquex)

Pegylated G-CSF stays in the body for longer so you have treatment less often than with the other types of G-CSF.

6.2.5.3.5  Plasma exchange (plasmapheresis)

http://emedicine.medscape.com/article/1895577-overview

Plasmapheresis is a term used to refer to a broad range of procedures in which extracorporeal separation of blood components results in a filtered plasma product.[1, 2] The filtering of plasma from whole blood can be accomplished via centrifugation or semipermeable membranes.[3] Centrifugation takes advantage of the different specific gravities inherent to various blood products such as red cells, white cells, platelets, and plasma.[4] Membrane plasma separation uses differences in particle size to filter plasma from the cellular components of blood.[3]

Traditionally, in the United States, most plasmapheresis takes place using automated centrifuge-based technology.[5] In certain instances, in particular in patients already undergoing hemodialysis, plasmapheresis can be carried out using semipermeable membranes to filter plasma.[4]

In therapeutic plasma exchange, using an automated centrifuge, filtered plasma is discarded and red blood cells along with replacement colloid such as donor plasma or albumin is returned to the patient. In membrane plasma filtration, secondary membrane plasma fractionation can selectively remove undesired macromolecules, which then allows for return of the processed plasma to the patient instead of donor plasma or albumin. Examples of secondary membrane plasma fractionation include cascade filtration,[6] thermofiltration, cryofiltration,[7] and low-density lipoprotein pheresis.

The Apheresis Applications Committee of the American Society for Apheresis periodically evaluates potential indications for apheresis and categorizes them from I to IV based on the available medical literature. The following are some of the indications, and their categorization, from the society’s 2010 guidelines.[2]

  • The only Category I indication for hemopoietic malignancy is Hyperviscosity in monoclonal gammopathies

6.2.5.3.6  Platelet transfusions

6.2.5.3.6.1 Indications for platelet transfusion in children with acute leukemia

Scott Murphy, Samuel Litwin, Leonard M. Herring, Penelope Koch, et al.
Am J Hematol Jun 1982; 12(4): 347–356
http://onlinelibrary.wiley.com/doi/10.1002/ajh.2830120406/abstract;jsessionid=A6001D9D865EA1EBC667EF98382EF20C.f03t01
http://dx.doi.org:/10.1002/ajh.2830120406

In an attempt to determine the indications for platelet transfusion in thrombocytopenic patients, we randomized 56 children with acute leukemia to one of two regimens of platelet transfusion. The prophylactic group received platelets when the platelet count fell below 20,000 per mm3 irrespective of clinical events. The therapeutic group was transfused only when significant bleeding occurred and not for thrombocytopenia alone. The time to first bleeding episode was significantly longer and the number of bleeding episodes were significantly reduced in the prophylactic group. The survival curves of the two groups could not be distinguished from each other. Prior to the last month of life, the total number of days on which bleeding was present was significantly reduced by prophylactic therapy. However, in the terminal phase (last month of life), the duration of bleeding episodes was significantly longer in the prophylactic group. This may have been due to a higher incidence of immunologic refractoriness to platelet transfusion. Because of this terminal bleeding, comparison of the two groups for total number of days on which bleeding was present did not show a significant difference over the entire study period.

6.2.5.3.6.2 Clinical and laboratory aspects of platelet transfusion therapy
Yuan S, Goldfinger D
http://www.uptodate.com/contents/clinical-and-laboratory-aspects-of-platelet-transfusion-therapy

INTRODUCTION — Hemostasis depends on an adequate number of functional platelets, together with an intact coagulation (clotting factor) system. This topic covers the logistics of platelet use and the indications for platelet transfusion in adults. The approach to the bleeding patient, refractoriness to platelet transfusion, and platelet transfusion in neonates are discussed elsewhere.

Pooled platelets – A single unit of platelets can be isolated from every unit of donated blood, by centrifuging the blood within the closed collection system to separate the platelets from the red blood cells (RBC). The number of platelets per unit varies according to the platelet count of the donor; a yield of 7 x 1010 platelets is typical [1]. Since this number is inadequate to raise the platelet count in an adult recipient, four to six units are pooled to allow transfusion of 3 to 4 x 1011 platelets per transfusion [2]. These are called whole blood-derived or random donor pooled platelets.

Advantages of pooled platelets include lower cost and ease of collection and processing (a separate donation procedure and pheresis equipment are not required). The major disadvantage is recipient exposure to multiple donors in a single transfusion and logistic issues related to bacterial testing.

Apheresis (single donor) platelets – Platelets can also be collected from volunteer donors in the blood bank, in a one- to two-hour pheresis procedure. Platelets and some white blood cells are removed, and red blood cells and plasma are returned to the donor. A typical apheresis platelet unit provides the equivalent of six or more units of platelets from whole blood (ie, 3 to 6 x 1011 platelets) [2]. In larger donors with high platelet counts, up to three units can be collected in one session. These are called apheresis or single donor platelets.

Advantages of single donor platelets are exposure of the recipient to a single donor rather than multiple donors, and the ability to match donor and recipient characteristics such as HLA type, cytomegalovirus (CMV) status, and blood type for certain recipients.

Both pooled and apheresis platelets contain some white blood cells (WBC) that were collected along with the platelets. These WBC can cause febrile non-hemolytic transfusion reactions (FNHTR), alloimmunization, and transfusion-associated graft-versus-host disease (ta-GVHD) in some patients.

Platelet products also contain plasma, which can be implicated in adverse reactions including transfusion-related acute lung injury (TRALI) and anaphylaxis. (See ‘Complications of platelet transfusion’ .)

6.2. +  Steroids

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The History of Hematology and Related Sciences

Curator: Larry H. Bernstein, MD, FCAP

 

The History of Hematology and Related Sciences: A Historical Review of Hematological Diagnosis from 1880 -1980

 

Blood Description: The Analysis of Blood Elements a Window into Diseases

Diagnosing bacterial infection (BI) remains a challenge for the attending physician. An ex vivo infection model based on human fixed polymorphonuclear neutrophils (PMNs) gives an autofluorescence signal that differs significantly between stimulated and unstimulated cells. We took advantage of this property for use in an in vivo pneumonia mouse model and in patients hospitalized with bacterial pneumonia. A 2-fold decrease was observed in autofluorescence intensity for cytospined PMNs from broncho-alveolar lavage (BAL) in the pneumonia mouse model and a 2.7-fold decrease was observed in patients with pneumonia when compared with control mice or patients without pneumonia, respectively. This optical method provided an autofluorescence mean intensity cut-off, allowing for easy diagnosis of BI. Originally set up on a confocal microscope, the assay was also effective using a standard epifluorescence microscope. Assessing the autofluorescence of PMNs provides a fast, simple, cheap and reliable method optimizing the efficiency and the time needed for early diagnosis of severe infections. Rationalized therapeutic decisions supported by the results from this method can improve the outcome of patients suspected of having an infection.

Monsel A, Le´cart S, Roquilly A, Broquet A, Jacqueline C, et al. (2014) Analysis of Autofluorescence in Polymorphonuclear Neutrophils: A New Tool for Early Infection Diagnosis. PLoS ONE 9(3): e92564.
http://dx.doi.org:/10.1371/journal.pone.0092564

This study was designed to validate or refute the reliability of total lymphocyte count (TLC) and other hematological parameters as a substitute for CD4 cell counts. Participants consisted of two groups, including 416 antiretroviral naive (G1) and 328 antiretroviral experienced (G2) patients. CD4+ T cell counts were performed using a Cyflow machine. Hematological parameters were analyzed using a hematology analyzer. The median ± SEM CD4 count (range) of participants in G1 was 199 ± 10.9 (5–1840 cells/μL) and the median ± SEM TLC (range) was 1. 61 ± 0.05 (0.07–6.63 × 103/μL). The corresponding values among G2 were 421 ± 15.8 (13–1801) and 2.13 ± 0.04 (0.06–5.58), respectively. Using a threshold value of 1.2 × 103/μL for TLC alone, the sensitivity of G1 was 88.4% (specificity (SP) 67.4%, the positive predictive value (PPV) 53.5% and negative predictive value (NPV) of 93.2% for CD4 , 200 cells/μL, the sensitivity for G2 was 83.3%, SP 85.3%, PPV 23.8%, and NPV of 93.2%. Using multiple parameters, including TLC , 1.2 × 103/μL, hemoglobin , 10 g/dL, and platelets , 150 × 103/L, the sensitivity increased to 96.0% (SP, 82.7%; PPV, 80%; NPV, 96.7%) among G1, while no change was observed in the G2 cohort. TLC , 1.2 × 103/μL alone is an insensitive predictor of CD4 count of , 200 cells/μL. Incorporating hemoglobin , 10 g/dL, and platelets , 150 × 103/L enhances the ability of TLC , 1.2 × 103/μL to predict CD4 count , 200 cells/μL among the antiretroviral-naïve cohort. We recommend the use of multiple, inexpensively measured hematological parameters in the form of an algorithm for predicting CD4 count level.

Evaluating Total Lymphocyte Counts and Other Hematological Parameters as a Substitute for CD4 Counts in the Management of HIV Patients in Northeastern Nigeria. BA Denue, AU Abja, IM Kida, AH Gabdo, AA Bukar and CB Akawu.
Retrovirology: Research and Treatment 2013:5 9–16 http://dx.doi.org:/10.4137/RRT.S11562

Sepsis is a syndrome that results in high morbidity and mortality. We investigated the delta neutrophil index (DN) as a predictive marker of early mortality in patients with gram-negative bacteremia. Retrospective study. The DN was measured at onset of bacteremia and 24 hours and 72 hours later. The DN was calculated using an automatic hematology analyzer. Factors associated with 10-day mortality were assessed using logistic regression. A total of 172 patients with gram-negative bacteremia were included in the analysis; of these, 17 patients died within 10 days of bacteremia onset. In multivariate analysis, Sequental organ failure assessment scores (odds ratio [OR]: 2.24, 95% confidence interval [CI]: 1.31 to 3.84; P = 0.003), DN-day 1 ≥ 7.6% (OR: 305.18, 95% CI: 1.73 to 53983.52; P = 0.030) and DN-day 3 ≥ DN day 1 (OR: 77.77, 95% CI: 1.90 to 3188.05; P = 0.022) were independent factors associated with early mortality in gram-negative bacteremia. Of four multivariate models developed and tested using various factors, the model using both DN-day 1 ≥ 7.6% and DN-day 3 ≥ DN-day 1 was most predictive early mortality. DN may be a useful marker of early mortality in patients with gram-negative bacteremia. We found both DN-day 1 and DN trend to be significantly associated with early mortality.

Delta Neutrophil Index as a Prognostic Marker of Early Mortality in Gram Negative Bacteremia. HW Kim, JH Yoon, SJ Jin, SB Kim, NS Ku, SJ Jeong,
et al. Infect Chemother 2014;46(2):94-102. pISSN 2093-2340·eISSN 2092-6448
http://dx.doi.org/10.3947/ic.2014.46.2.94
Various indices derived from red blood cell (RBC) parameters have been described for distinguishing thalassemia and iron deficiency. We studied the microcytic to hypochromic RBC ratio as a discriminant index in microcytic anemia and compared it to traditional indices in a learning set and confirmed our findings in a validation set. The learning set comprised samples from 371 patients with microcytic anemia mean cell volume (MCV < 80 fL), which were measured on a CELL-DYN Sapphire analyzer and various discriminant functions calculated. Optimal cutoff values were established using ROC analysis. These values were used in the validation set of 338 patients. In the learning set, a microcytic to hypochromic RBC ratio >6.4 was strongly indicative of thalassemia (area under the curve 0.948). Green-King and England-Fraser indices showed comparable area under the ROC curve. However, the microcytic to hypochromic ratio had the highest sensitivity (0.964). In the validation set, 91.1% of microcytic patients were correctly classified using the M/H ratio. Overall, the microcytic to hypochromic ratio as measured in CELL-DYN Sapphire performed equally well as the Green-King index in identifying thalassemia carriers, but with higher sensitivity, making it a quick and inexpensive screening tool.
Differential diagnosis of microcytic anemia: the role of microcytic and hypochromic erythrocytes. E. Urrechaga, J.J.M.L. Hoffmann, S. Izquierdo, J.F. Escanero. Intl Jf Lab Hematology Aug 2014. http://dx.doi.org:/10.1111/ijlh.12290

Achievement of complete response (CR) to therapy in chronic lymphocytic leukemia (CLL) has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry [and polymerase chain reaction methods] can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10–4), using the above-mentioned sophisticated techniques, is the current cutoff for minimal residual disease (MRD). Tumor burdens lower than 10–4 are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.

Minimal Residual Disease Surveillance in Chronic Lymphocytic Leukemia by Fluorescence-Activated Cell Sorting. S Ringelstein-Harlev, R Fineman.
Rambam Maimonides Med J. Oct 2014   5 (4)  e0027. http://dx.doi.org:/10.5041/RMMJ.10161

Natural Killer cells (CD3-CD16+CD56+) are a major players in innate immunity, both as direct cytotoxic effectors as well as regulators for other innate immunity cell types. We have shown that, using the FlowCellect™ human NK cell characterization kit, one can achieve accurate phenotyping on a variety of sample types, including whole blood samples. Using the same kit to perform an NK cell cytotoxicity test, we demonstrate that unbound K562 target cells can be clearly distinguished from those that have been engaged by CD56+ NK cells, and each of these populations can be further investigated for viability using the eFluor 660® dye.

Analysis of NK cell subpopulations in whole blood

Analysis of NK cell subpopulations in whole blood

Analysis of NK cell subpopulations in whole blood

A

Proportion of K562 target cells bound to NK cells

Proportion of K562 target cells bound to NK cells

In a 5:1 effector cell:target cell population, 8% of the K562 cells were bound to NK cells (Figure 3B). 84% of the bound K562 cells were viable (Figure 3C) stained with fixable viability dye), while 96% of the unbound K562 cells were viable (Figure 3D). (B,C,D not shown)

Characterization of Natural Killer Cells Using Flow Cytometry.
EMD Millipore is a division of Merck KGaA, Darmstadt, Germany.

Red blood cell distribution width (RDW) is increased in liver disease. Its clinical significance, however, remains largely unknown. The aim of this study was to identify whether RDW was a prognostic index for liver disease. Retrospective: 33 patients with non-cirrhotic HBV chronic hepatitis, 125 patients with liver cirrhosis after HBV infection, 81 newly diagnosed primary epatocellular carcinoma (pHCC) patients, 17 alcoholic liver cirrhosis patients and 42 patients with primary biliary cirrhosis (PBC). Sixty-six healthy individuals represented the control cohort. The relationship between RDW on admission and clinical features: The association between RDW and hospitalization outcome was estimated by receiver operating curve (ROC) analysis and a multivariable logistic regression model. Increased RDW was observed in liver disease patients. RDW was positively correlated with serum bilirubin and creatinine levels, prothrombin time, and negatively correlated with platelet counts and serum albumin concentration. A subgroup analysis, considering the different etiologies, revealed similar findings. Among the patients with liver cirrhosis, RDW increased with worsening of Child-Pugh grade. In patients with PBC, RDW positively correlated with Mayo risk score. Increased RDW was associated with worse hospital outcome, as shown by the AUC [95% confidence interval (CI)] of 0.76 (0.67 – 0.84). RDW above 15.15% was independently associated with poor hospital outcome after adjustment for serum bilirubin, platelet count, prothrombin time, albumin and age, with the odds ratio (95% CI) of 13.29 (1.67 – 105.68). RDW is a potential prognostic index for liver disease.

Red blood cell distribution width is a potential prognostic index for liver disease
Z Hua , Y Suna , Q Wanga , Z Han , Y Huang , X Liu , C Ding, et al.
Clin Chem Lab Med 2013; 51(7):1403–1408.
http://dx.doi.org:/10.1515/cclm-2012-0704

Blood Plasma and Red Blood Cells

Whole blood consists of red and white blood cells, as well as platelets suspended in a liquid referred to as blood plasma. According to the American Red Cross, plasma is 92% water and makes up 55% of blood volume. The permeability of blood plasma is equal to 1.

Red blood cells make up slightly lower blood volume than blood plasma — about 45% of whole blood. As you probably already know, these types of blood cells contain hemoglobin, which in turn consists of iron that helps transport oxygen throughout the body. The permeability of red blood cells is slightly less than 1,
(1 – 3.9e-6). Or to put it in words, red blood cell particles are diamagnetic.

Due to their magnetic properties, red blood cells may be separated from the plasma via a magnetophoretic approach. If the blood were to be in a channel subject to a magnetophoretic force, we could control where the red blood cells and the plasma go within the channels. In other words, because the red blood cells have different permeability, they can be separated from the flow channel. However, such methodology is beyond the year 1980.

Timeline of Major Hematology Landmarks

1877 Paul Ehrlich develops techniques to stain blood cells to improve microscopic visualization.

1897 The Diseases of Infancy and Childhood contains a 20-page chapter on diseases of the blood and is the first American pediatric medical textbook to provide significant hematologic information.

1821–1902 Rudolph Virchow, during a long and illustrious career, demonstrates the importance of fibrin in the blood coagulation process, coins the terms embolism and thrombosis, identifies the disease leukemia, and theorizes that leukocytes are made in response to inflammation.

1901 Karl Landsteiner and colleagues identify blood groups of A, B, AB, and O.

1907 Ludvig Hektoen suggests that the safety of transfusion might be improved by crossmatching blood between donors and patients to exclude incompatible mixtures. Reuben Ottenberg performs the first blood transfusion using blood typing and crossmatching in New York. Ottenberg also observes the Mendelian inheritance of blood groups and recognizes the “universal” utility of group O donors.

1910 The first clinical description of sickle cell published in medical literature.

1914 Sodium citrate is found to prevent blood from clotting, allowing blood to be stored between collection and transfusion.

1924 Pediatrics is the first comprehensive American publication on pediatric hematology.

1925 Alfred P. Hart performs the first exchange transfusion.

1925 Thomas Cooley describes a Mediterranean hematologic syndrome of anemia, erythroblastosis, skeletal disorders, and splenomegaly that is later called Cooley’s anemia and now thalassemia.

1936 Chicago’s Cook County Hospital establishes the first true “blood bank” in the United States.

1938 Dr. Louis Diamond (known as the “father of American pediatric hematology”) along with Dr. Kenneth Blackfan describes the anemia still known as Diamond-Blackfan anemia.

1941 The Atlas of the Blood of Children is published by Blackfan, Diamond, and Leister.

1945 Coombs, Mourant, and Race describe the use of antihuman globulin (later known as the “Coombs Test”) to identify “incomplete” antibodies.

1954 The blood product cryoprecipitate is developed to treat bleeds in people with hemophilia.

1950s The “butterfly” needle and intercath are developed, making IV access easier and safer.

1961 The role of platelet concentrates in reducing mortality from hemorrhage in cancer patients is recognized.

1962 The first antihemophilic factor concentrate to treat coagulation disorders in hemophilia patients is developed through fractionation.

1969 S. Murphy and F. Gardner demonstrate the feasibility of storing platelets at room temperature, revolutionizing platelet transfusion therapy.

1971 Hepatitis B surface antigen testing of blood begins in the United States.

1972 Apheresis is used to extract one cellular component, returning the rest of the blood to the donor.

1974 Hematology of Infancy and Childhood is published by Nathan and Oski.

As I write today my hospital celebrates its 150th anniversary. Great Ormond Street Children’s Hospital was founded on 14 February 1852 by the visionary Dr Charles West followed his belief that hospital care allied to research in children’s diseases would reduce child mortality from above 50% by the age of 15 years. It is foolish to believe that we can progress in medicine without a knowledge of the past and that much of life is based upon experience. When putting together a series of articles on the history of haematology, initially published in BJH, this was the main raison d’être, along with the belief that the practice of medicine has become increasingly serious but should also be fun and interesting and even occasionally uplifting to the spirit.

The central problem of any survey of the history of haematology is usually the question of balance. Achieving a degree of balance among themes and topics that will be satisfactory to practicing haematologists/physicians with an interest in blood diseases is essentially impossible. Our preference has been for themes of general interest rather than those of a purely scientific view into a field that has led the way in understanding the molecular basis of human disease.

  1. M. Hann, London, 2002; O. P. Smith, Dublin, 2002.

Origins of the Discipline `Neonatal Haematology’, 1925-75

In every modern neonatal intensive care unit (NICU), haematological problems are encountered daily. Many of these problems involve varieties of anaemia, neutropenia or thrombocytopenia that are unique to NICU patients. A characteristic aspect of these unique problems is that, if the neonate survives, the haematological problem will remit and will not recur later in life, nor will it evolve into a chronic illness (although the problem might occur in a future newborn sibling). This characteristic comes about because the common haematological problems of NICU patients are not genetic defects but are environmental stresses (such as infection, alloimmunization or a variety of maternal illnesses) that are imposed on a developmentally immature haematopoietic system.

In the USA, and in some parts of Europe, the unique haematological problems that occur among NICU patients are diagnosed and treated by neonatologists, not by paediatric haematologists. Although these haematological conditions were generally first described by haematologists, the conditions occur, obviously, in neonates. Thus, the neonatologist, who is familiar with intensive care management of neonates, has also become familiar with the diagnosis and management of the neonate’s common haematological disorders. A growing number of neonatologists have sought specific additional training in haematology, with the goals of discovering the mechanisms underlying the unique haematological problems of NICU patients and improving the management and outcome of the patients who have these conditions. These physicians have remained as neonatologists and they do not practice paediatric haematology, although their research contributions certainly come under the purview of haematology, or more precisely under the discipline of `neonatal haematology’. In many places in Europe, it is the haematologists rather than the neonatologists who have an academic and clinical interest in neonatal haematology.

The roots of the discipline of neonatal haematology can be traced to the early application of haematological methods to animal and human embryos and fetuses, such as found in the reports of Maximow (1924) and Wintrobe & Schumacker (1936). The clinical underpinnings of this discipline include reports of anaemia (Fikelstein, 1911) and jaundice (Blomfeld, 1901; YlppoÈ, 1913) among neonates.

Before the 1930s, very few studies and very few published clinical case reports originated from premature nurseries. Such nurseries had dubious beginnings, which were criticized by some physicians as more resembling circus exhibitions than medical care wards (Bonar, 1932). These units generally had mortality rates greatly exceeding 50% on the day of admission, with the majority of the first-day survivors having late deaths or serious long-term morbidity.

It was not until publication of the review of premature nursery care at the Children’s Hospital of Michigan, in 1932, that it was clear that some units had instituted systematic attempts to monitor and improve outcomes. A special care nursery had been established at the Children’s Hospital in 1926 and, in 1932, Drs Marsh Poole and Thomas Cooley reported their experience in that unit (Poole & Cooley, 1932). The report included  incubator design with temperature and humidity control, growth curves of patients on various feeding practices, mortality statistics and attempts to determine causes of death.

At the time premature nursery care was beginning to merit academic credentials, reports were published of haematological problems that were unique to the neonate. These papers included the seminal publication on erythroblastosis fetalis by Drs Diamond (Fig 1), Blackfan and Baty (Diamond et al, 1932), and the report of sepsis neonatorum at the Yale New Haven Hospital by Ethyl C. Dunham (Fig 2) (Dunham,

1933).

The first major textbook devoted to clinical haematology, as well as the first textbook of neonatology, contained very little information about what are today’s common NICU haematological problems. For instance, in the first edition of Clinical Hematology by Dr Maxwell M. Wintrobe (Fig 3), of the Johns Hopkins University Hospital (Wintrobe, 1942), several topics related to paediatric haematology were reviewed, but discussions of the haematological problems of neonates were limited to three – erythroblastosis fetalis, haemorrhagic disease of the newborn and the `anaemia of prematurity’. Similarly, Premature Infants: A Manual for

Physicians, the original neonatology textbook, published in 1948 by Dr Ethyl C. Dunham (Fig 2; Dunham, 1948), had only a few pages devoted to haematological problems – the same three discussed by Dr Wintrobe. Also, the classic neonatology text book, `The Physiology of the Newborn Infant’, published in 1945 by Dr Clement A. Smith, contained almost no discussion of haematological problems (Smith, 1945). hrombocytopenia, which is now diagnosed among 25-30% of NICU patients, and neutropenia, now diagnosed in 8-10% of NICU patients, were not mentioned.

The first article published in Paediatrics (1948) dealing with a neonatal haematological problem was in volume two, in which Dr Diamond detailed his technique for performing a replacement transfusion (which later became known as an `exchange’ transfusion) as a treatment for erythroblastosis fetalis (Diamond, 1949). The second paper published by Paediatrics containing aspects of neonatal haematology was 1 year later, when Sliverman & Homan (1949) described leucopenia among neonates with sepsis. Most of the 25 infants they described, who were treated at Babies Hospital in New York over an 11-year period, had `late-onset’ sepsis, beginning after 3 days of life. They reported 14 neonates with Escherichia coli sepsis and four with streptococcal or staphylococcal sepsis, and observed that leucopenia occurred occasionally among these patients but was uncommon. (Indeed, today neutropenia remains uncommon in `late-onset’ sepsis, but common in congenital or `early onset’ sepsis.)

Louis K. Diamond, MD, at Children's Hospital, Boston,

Louis K. Diamond, MD, at Children’s Hospital, Boston,

Louis K. Diamond, MD, at Children’s Hospital, Boston, MA. , date unknown (obtained with the kind assistance of Charles F. Simmons, MD, Harvard University).

Diagnosing neutropenia, anaemia or thrombocytopenia in a neonate obviously requires knowledge of the expected normal range for neutrophil concentration, haematocrit and platelet concentration in the appropriate reference population. Early contributions to neonatal haematology included the publications of these reference ranges. The landmark studies included the range of blood leucocyte and neutrophil concentrations in neonates published in 1935 by Dr Katsuji Kato from the Department of Paediatrics at the University of Chicago (Kato, 1935). He tabulated the leucocyte concentrations and differential counts of 1081 children, ranging from birth to 15 years of age. A striking finding of his report (Fig 4) was the very high neutrophil counts during the first hours and days of life. Blood neutrophil concentrations among neonates with infections were published during the early and mid-1970s by Dr Marietta Xanthou (Fig 5) at the Hammersmith Hospital in London (Xanthou, 1970, 1972), and by Drs Barbara Manroe and Charles Rosenfeld (Fig 6) at the University of Texas Southwestern Medical Center in Dallas, Texas (Manroe et al, 1977).

Normal values for haemoglobin, haematocrit, erythrocyte indices and leucocyte concentrations were refined by DeMarsh et al (1942, 1948), and in a series of publications in the early 1950s in Archives of Diseases of Children by Gairdner et al (1952a, b). These were followed by observations on human fetal haematopoiesis by Thomas and Yoffey in the British Journal of Haematology (Thomas & Yoffey, 1962, 1964), and by the work on blood volume during the 1960s (Usher et al, 1963, Usher & Lind, 1965; Yao et al, 1967, 1968). Normal ranges for blood platelet counts in ill and well preterm and term infants were published in the early 1970s (Sell et al, 1973; Corrigan, 1974).

The first publication addressing the problem of neutropenia accompanying fatal early onset bacterial sepsis was that of Tygstrup et al (1968). This was a report of a near-term male with congenital Listeria sepsis who lived for only 4 h. The platelet count was 80*109/l and the leucocyte count was 13´7*109/l, but no granulocytes were observed on the differential count, which consisted of 84% lymphocytes, 8% monocytes and 8% leucocyte precursors. A sternal marrow aspirate was taken of the infant shortly before death that revealed myeloblasts, promyelocytes and myelocytes, but no band or segmented neutrophils.

An important advance in understanding the blood neutrophil count during neonatal sepsis occurred with the back-to-back papers in Archives of Diseases of Childhood in 1972 by Dr Marietta Xanthou of Hammersmith Hospital, London (Xanthou, 1972), and Drs Gregory and Hey of Babies’ Hospital, Newcastle upon Tyne (Gregory & Hey, 1972). Both papers reported that neonates who had life threatening (or indeed fatal) infections became neutropenic prior to death. Dr Xanthou reported 35 ill preterm and term babies within their first 28 d of life. Twenty-four were ill but not infected, and these had normal blood neutrophil concentrations and morphology. However, among the 11 who were ill with a bacterial infection, neutrophilia was observed in the survivors, but neutropenia, a `left shift’, and toxic granulation were observed in the non-survivors. Consistent with this observation, Gregory and Hey reported three neonates who died with overwhelming bacterial sepsis and noted that all had profound neutropenia. Neutrophilia was common among the survivors and neutropenia, a “left shift’, and specific neutrophil morphological changes were seen among those who subsequently died.

A pivotal publication that launched the search for mechanistic information and successful treatments was that of Dr Barbara Manroe, a fellow in Neonatal Medicine, and her mentor Dr Charles Rosenfeld (Fig 6) from the University of Texas, South-western, Parkland Hospital in Dallas, Texas (Manroe et al, 1977). They evaluated 45 neonates who had culture-proven group B streptococcal infection and found that 39 had abnormal leucocyte counts: 25 neutrophilia and 14 neutropenia, and that 41 had a `left shift’. This paper was the first to quantify the `left shift’ using a method that has since become popular in neonatology – the ratio of immature neutrophils to total neutrophils on the differential cell count.

From these beginning, hundreds of studies using experimental models and clinical observations and trials were published, detailing the kinetic and molecular mechanisms accounting for this common variety of neutropenia. Marked improvements in the survival of neonates with this condition have come about through combined efforts, including early maternal screening for GBS carriage, early anti-microbial administration to ill neonates, non-specific antibody administration and a variety of measures to improve supportive care of neonates with early onset sepsis.

In the early 1930s, Dr Helen Mackay worked as a paediatrician in Mother’s Hospital, a maternity hospital located in the north-east section of London. Acting on the observation of Lichtenstein (1921) that infants of subnormal birth weight regularly became anaemic in the first months of life, she measured and reported serial heel-stick haemoglobin levels on 150 infants during their first 6 months. Thirty-nine of these infants weighed under five pounds at birth (six were under four pounds), 52 weighed five to six pounds, and 59 weighed six pounds and upwards. She showed that babies of the lightest birth weights had the most rapid fall in haemoglobin and that these fell to lower levels than those of babies of heavier birth weight (MacKay et al, 1935). Figure 7 contrasts this fall in babies weighing `3-4 lbs odd at birth’ with those weighing `5 lbs odd at birth’.

Her attempts to prevent the anaemia of prematurity failed,  but her work constituted the first clear definition of the `anaemia of prematurity’ and showed that iron administration did not prevent this condition. In the early 1950s, Douglas Gairdner, John Marks and Janet D. Roscoe, of the Department of Pathology of Cambridge Maternity Hospital, published pioneering studies in blood formation in infancy (Gairdner et al, 1952a, b). Studying 105 blood samples and 102 bone marrow samples, they concluded that `erythropoiesis ceases when the oxygen saturation just after birth increases from about 65% in the umbilical vein to .95% just after birth’. Publications by Dr Irving Schulman, in the mid- to late 1950s, defined three phases of the anaemia of prematurity and provided a mechanistic explanation for the anaemia (Schulman & Smith, 1954; Schulman, 1959). His work illustrated that the early and intermediate phases of this anaemia occur in the face of relative iron excess and are unaffected by prophylactic iron administration.

Haemoglobin levels during the first 25 weeks of life among

Haemoglobin levels during the first 25 weeks of life among

Haemoglobin levels during the first 25 weeks of life among neonates in London [by permission; Archives Diseases of Children, (MacKay, 1935)].

In 1963, Dr Sverre Halvorsen of the Department of Paediatrics at Rikshospatalet in Oslo, Norway (Fig 9), provided an underlying explanation for the observations made by MacKay, Gairdner and Schulman (Halvorson, 1963). He observed that, compared with the blood of healthy adults, umbilical cord blood of healthy neonates had a high erythropoietin concentration, but the concentration was considerably higher in the plasma of severely erythroblastotic, anaemic infants. Among the healthy infants, erythropoietin levels fell to unmeasurably low concentrations after delivery, but levels remained elevated in hypoxic and cyanotic infants. Dr Per Haavardsholm Finne, also of the Children’s Department, Paediatric Research Institute and Department of Obstetrics and Gynaecology at Rikshospitalet in Oslo, observed high oncentrations of erythropoietin in the amniotic fluid and the umbilical cord blood after fetal hypoxia (Finne, 1964, 1967).

In subsequent studies, Dr Halvorsen observed lower plasma erythropoietin concentrations in the cord blood of preterm infants at delivery than in term neonates at delivery (Halvorsen & Finne, 1968). These observations supported the concept of Gairdner et al (1952a, b) that the postnatal fall in erythropoiesis (the `physiologic anaemia’ of neonates) is as a result of an increase in oxygen delivery to tissues following birth and is mediated by a fall in circulating erythropoietin concentration. The observations gave rise to the postulate that the `anaemia of prematurity’ was an exaggeration of this physiological anaemia and involved a limitation of preterm infants to appropriately increase erythropoietin production.

Many landmark reports of haematological findings of neonates that were published between 1925 and 1975 were not detailed in this review because they were outside the restricted topics selected.

Robert D. Christensen, MD, Gainesville, FL
Brit J Haem 2001; 113: 853-860

Towards Molecular Medicine; Reminiscences of the Haemoglobin Field

When historians of medicine in the twentieth century start to piece together the complex web of events that led from a change of emphasis of medical research from studies of patients and their organs to disease at the levels of cells and molecules they will undoubtedly have their attention drawn to the haemoglobin field, particularly the years that followed Linus Pauling’s seminal paper in 1949 which described sickle-cell anaemia as a `molecular disease’. These are personal reminiscences of some of the highlights of those exciting times, and of those who made them happen.

One of my first patients serving the RAMC was a Nepalese Ghurka child who was kept alive from the first few months of life with regular blood transfusion without a diagnosis. Henry Kunkel published a paper which described how, using electrophoresis in slabs of starch, he had found a minor component of human haemoglobin (Hb), Hb A2, the proportion of which was elevated in some carriers of thalassaemia. After several weeks spent knee deep in potato starch, we found that the Ghurka child’s parents had increased Hb A2 levels and, hence, that she was likely to be homozygous for thalassaemia. I was hauled up before the Director General of Medical Services for the Far East Land Forces and told that I could be court marshalled for not getting permission from the War House (Office) to publish information about military personnel. `And, in any case’, he added, `it is bad form to tell the world that one of our pukka regiments has bad genes; don’t do it again’.

Just before the end of my National Service I arranged to go to Johns Hopkins Hospital in Baltimore to train in genetics and haematology. I was told that I was wasting my time working on haemoglobin because there was `nothing left to do’. `Start exploring red cell enzymes’, he suggested. On arriving in Baltimore in 1960 it turned out that human genetics, and the haemoglobin field in particular, were bubbling with excitement and potential. The only lessons for those contemplating careers in medical research from this chapter of academic and military gaffs are that, regardless of the working conditions, when there are sick people there are always interesting research questions to be asked.

The excitement of the haemoglobin field in 1960 reflected the chance amalgamation of several disciplines in the 1950s, particularly X-ray crystallography, protein chemistry, human genetics and haematology.

From the early 1930s the structure of proteins became one of the central problems of biochemistry. At that time, the only way of tackling this problem was by X-ray crystallography. In 1937 Felix Haurowitz suggested to Max Perutz (Fig 1) that an X-ray study of haemoglobin might be a good subject for his doctoral thesis. He was given some large crystals of horse methaemoglobin which gave excellent Xray diffraction patterns.

Max Perutz

Max Perutz

However, there was a major snag; an X-ray diffraction pattern provided only half the information required to solve the structure of a protein, that is the amplitudes of diffracted rays, while the other half, their phases, could not be determined. But in 1953, they discovered that it could be solved in two dimensions by comparison of the diffraction patterns of a crystal of native haemoglobin with that of haemoglobin reacted with mecuribenzoate, which combines with its two reactive sulphydryl groups. In short, to solve the structure in three dimensions required the comparison of the diffraction patterns of at least three crystals, one native and two with heavy atoms combined with different sites on the haemoglobin molecule. In 1959 this approach yielded the first three-dimensional model of haemoglobin, at 5´5 AÊ resolution.

Protein chemistry evolved side-by-side with X-ray crystallography during the 1950s. In 1951 Fred Sanger solved the structure of insulin, a remarkable tour de force which showed that proteins have unique chemical structures and amino acid sequences. Sanger had perfected methods for fractionation and characterization of small peptides by paper chromatography or electrophoresis. In 1956 Vernon Ingram (Fig 2), who, like Max Perutz, was a refugee from Germany, was set the task of studying the structure of haemoglobin from patients with sickle-cell anaemia. Ingram separated the peptides produced after globin had been hydrolysed with the enzyme trypsin, which cuts only at lysine and arginine residues. Although these amino acids accounted for 60 residues per mol of haemoglobin, only 30 tryptic peptides were obtained, indicating that haemoglobin consists of two identical half molecules. Re-examination of the amino-terminal sequences of haemoglobin by groups in the United States and Germany showed 2 mols of valine ± leucine and 2 mols of valine ± histidine ± leucine per mol of globin. These findings, which were in perfect agreement with the X-ray crystallographic results, suggested that haemoglobin is a tetramer composed of two pairs of unlike peptide chains, which were called α and β.

A seminal advance, and one which was to mark the beginning of molecular medicine, was the chance result of an overnight conversation on a train journey between Denver and Chicago. Linus Pauling, the protein chemist, and William Castle (Fig 3), one of the founding fathers of experimental haematology, were returning from a meeting in Denver and Castle mentioned to Pauling that he and his colleagues had noticed that when red cells from patients with sickle-cell anaemia are deoxygenated and sickle they show birefringence in polarized light.

Five generations of Boston haematology. Seated is William Castle. Standing (left to right) are Stuart Orkin, David Nathan and Alan Michelson. The picture on the left is of Dean David Edsall of Harvard Medical School who established the Thorndyke Laboratory at the Boston City Hospital. He was succeeded by Dean Peabody, who recruited both George Minot, who won the Nobel Prize for his work on pernicious anaemia, and William Castle, who should have also received it.

Pauling guessed that this might reflect a structural difference between normal and sickle-cell haemoglobin which could be detected by a change in charge. He gave this problem to one of his postdoctoral students, a young medical graduate called Harvey Itano. At that time they knew that a Swede, Arne Tiselius, had invented a machine for separating proteins according to their charge by electrophoresis. As there was no machine of this kind in Pauling’s laboratory, Itano and his colleagues set to and built one. Eventually they found that the haemoglobin of patients with sickle-cell anaemia behaves differently to that of normal people in an electric field, indicating that it must have a different amino acid composition. Even better, the haemoglobin of sickle-cell carriers was a mixture of both types of haemoglobin. This work was published in Science in 1949, under the title `Sickle-cell anaemia: a molecular disease’.

Perutz and Crick suggested to Ingram that he should apply Sanger’s techniques of peptide analysis to see if he could find any difference between normal and sickle cell haemoglobin. After digesting haemoglobin with trypsin, Ingram separated the peptides by electrophoresis and chromatography in two dimensions to produce what he later called `fingerprints’. He recalls that his first efforts looked like a watercolour that had been left out in the rain. But gradually things improved and he was able to show that the fingerprints of Hbs A and S were identical except for the position of one peptide. Using a method that had been developed a few years earlier by Pehr Edman, which allowed a peptide to be degraded one amino acid at a time in a stepwise fashion, Ingram found that this difference was due to the substitution of valine for glutamic acid at position 6 in the β chain of Hb S.

As well as demonstrating how a crippling disease can result from only a single amino acid difference in the haemoglobin molecule, this beautiful work had broader implications for molecular genetics. Although nothing was known about the nature of the genetic code at the time, the findings were compatible with the notion that the primary product of the β-globin gene is a peptide chain, a further development of the one-gene-one-enzyme concept, suggested earlier by Beadle and Tatum from their studies of Neurospora, and a prelude to the later studies of Yanofsky on Escherichia coli, which were to confirm this principle.

With the advent of simple filter paper electrophoresis, haemoglobin analysis became the province of clinical research laboratories during the 1950s and `new’ abnormal haemoglobins appeared almost by the week. Although many scientists were involved it was Hermann Lehmann (Fig 4) who became the father figure. Like Handel, Hermann was born in Halle and, also like the composer, made his home in Great Britain. He came to England as a refugee and at the beginning of the Second World War had a short period of internment as a `friendly alien’ at Huyton, close to Liverpool, an experience shared with many others, including Max Perutz. He travelled widely during his later war service in the RAMC and developed a wide international network which enabled him to discover 81 haemoglobin variants during his career.

Harvey Itano and Elizabeth Robinson showed that Hb Hopkins 2 is an a chain variant. Hence, it was now clear that there must be at least two unlinked loci involved in regulating haemoglobin production, a and b. The discovery of the λ and δ chains of Hbs F and A2, respectively, meant that there must be at least four loci involved. Subsequent family studies and analyses of unusual variants resulting from the production of δβ or λβ fusion chains led to the ordering of the non-α globin genes.

It had been known for some years that children with severe forms of thalassaemia might have persistent production of HbF and it was found later that some carriers might have elevated levels of Hb A2. The seminal observation in favour of this notion came from the study of patients who had inherited the sickle-cell gene from one parent and thalassaemia from the other. Sickle-cell thalassaemia was first described by Ezio Silvestroni and his wife Ida Bianco in 1946, although at the time they could not have known the full significance of their finding.  Phillip Sturgeon and his colleagues in the USA found that the pattern of haemoglobin production in patients with sickle-cell thalassaemia is quite different to that of heterozygotes for the sickle-cell gene; the effect of the thalassaemia gene is to reduce the amount of Hb A to below that of Hb S, i.e. exactly the  opposite to the ratio observed in sickle-cell carriers. As it was known that the sickle-cell mutation occurs in the β globin gene, it could be inferred that the action of the thalassaemia gene was to reduce the amount of β globin production from the normal allele. Indeed, from the few family studies available in 1960 there was a hint that this form of thalassaemia might be an allele of the β globin gene. Another major observation that was made in the mid-50 s was the association of unusual tetramer haemoglobins, β4 (Hb H) and λ4 (Hb Bart’s), with a thalassaemia phenotype. In 1959 Vernon Ingram and Tony Stretton proposed in a seminal article that there are two major classes, α and β, just as there are two major types of structural haemoglobin variants. They extended the ideas of Linus Pauling and Harvey Itano, who had suggested that defective globin synthesis in thalassaemia might be due to `silent’ mutations of the β globin genes, and postulated that the defects might lie outside the structural gene in the area of DNA in the connecting unit. work on the interactions of thalassaemia and haemoglobin variants in the late 1950s had moved the field to a considerably higher level of understanding than is apparent in the earlier papers of Pauling and Itano. In any case, in their paper Ingram and Stretton generously acknowledged the ideas of other workers, including Lehmann, Gerald, Neel and Ceppellini, that had allowed them to develop their conceptual framework of the general nature of thalassaemia. This interpretation of events, and the input of scientists from many different disciplines into these concepts, is supported by the published discussions of several conferences on haemoglobin held in the late 1950s.

Historical Review. Towards Molecular Medicine; Reminiscences of the Haemoglobin Field. D. J. Weatherall, Weatherall Institute of Molecular Medicine, University of Oxford. Brit J  Haem 115:729-738.

The Emerging Understanding of Sickle Cell Disease

The first indisputable case of sickle cell disease in the literature was described in a dental student studying in Chicago between 1904 and 1907 (Herrick, 1910). Coming from the north of the island of Grenada in the eastern Caribbean, he was first admitted to the Presbyterian Hospital, Chicago, in late December 1904 and a blood test showed the features characteristic of homozygous sickle cell (SS) disease. It was a happy coincidence that he was under the care of Dr James Herrick (Fig 1) and his intern Dr Ernest Irons because both had an interest in laboratory investigation and Herrick had previously presented a paper on the value of blood examination in reaching a diagnosis (Herrick, 1904-05). The resulting blood test report by Dr Irons described and contained drawings of the abnormal red cells (Fig 2) and the photomicrographs, showing irreversibly sickled cells.

People with positive sickle tests were divided into asymptomatic cases, `latent sicklers’, and those with features of the disease, `active sicklers’, and it was Dr Lemuel Diggs of Memphis who first clearly distinguished symptomatic cases called sickle cell anaemia from the latent asymptomatic cases which were termed the sickle cell trait (Diggs et al, 1933).

Prospective data collection in 29 cases of the disease showed sickling in all 42 parents tested (Neel, 1949), providing strong support for the theory of homozygous inheritance. A Colonial Medical Officer working in Northern Rhodesia (Beet, 1949) reached similar conclusions at the same time with a study of one large family (the Kapokoso-Chuni pedigree). The implication that sickle cell anaemia should occur in all communities in which the sickle cell trait was common and that its frequency would be determined by the prevalence of the trait did not appear to fit the observations from Africa. Despite a sickle cell trait prevalence of 27% in Angola, Texeira (1944) noted the active form of the disease to be `extremely rare’ and similar observations were made from East Africa. Lehmann and Raper (1949, 1956) found a positive sickling test in 45% of one community, from which homozygous inheritance would have predicted that nearly 10% of children had SS disease, yet not a single case was found. The discrepancy led to a hypothesis that some factor inherited from non-black ancestors in America might be necessary for expression of the disease (Raper, 1950).

The explanation for this apparent discrepancy gradually emerged. Working with the Jaluo tribe in Kenya, Foy et al (1951) found five cases of sickle cell anaemia among very young children and suggested that cases might be dying at an age before those sampled in surveys. A similar hypothesis was advanced by Jelliffe (1952) and was supported by data from the then Belgian Congo (Lambotte-Legrand Lambotte-Legrand, 1951, Lambotte-Legrand, 1952, Vandepitte, 1952). Although most cases were consistent with the concept of homozygous inheritance, exceptions continued to occur. Patients with a non-sickling parent of Mediterranean ancestry were later recognized to have sickle cell-β thalassaemia (Powell et al, 1950; Silvestroni & Bianco, 1952; Sturgeon et al, 1952; Neel et al, 1953a), a condition also widespread in African and Indian subjects that presents a variable syndrome depending on the molecular basis of the β thalassaemia mutation and the amount of HbA produced.

Phenotypically, there are two major groups in subjects of African origin, sickle cell-β+ thalassaemia manifesting 20-30% HbA and mutations at 229(A,G) or 288(C,T), and sickle cell-β0 thalassaemia with no HbA and mutations at IVS2-849(A,G) or IVS2-1(G,A). In Indian subjects, a more severe β thalassaemia mutation IVS1-5(G,C) results in a sickle cell-β+ thalassaemia condition with 3-5% HbA and a relatively severe clinical course.

Other double heterozygote conditions causing sickle cell disease include sickle cell-haemoglobin C (SC) disease, (Kaplan et al, 1951; Neel et al, 1953b), sickle cellhaemoglobin O Arab (Ramot et al, 1960), sickle cellhaemoglobin Lepore Boston (Stammatoyannopoulos & Fessas, 1963) and sickle cell-haemoglobin D Punjab (Cooke & Mack, 1934). The latter condition was first described in siblings in 1934, who were reinvestigated for confirmation of HbD (Itano, 1951), the clinical features reported (Sturgeon et al, 1955) and who were finally identified as HbD Punjab (Babin et al, 1964), representing a remarkable example of longitudinal observation and investigation in the same family over 30 years.

The maintenance of high frequencies of the sickle cell trait in the presence of almost obligatory losses of homozygotes in Equatorial Africa implied that there was either a very high frequency of HbS arizing by fresh mutations or that the sickle cell trait conveyed a survival advantage in the African environment. There followed a remarkable period in the 1950s when three prominent scientists were each addressing this problem in East Africa, Dr Alan Raper and Dr Hermann Lehmann in Uganda and Dr Anthony Allison in Kenya. It was quickly calculated that mutation rates were far too low to balance the loss of HbS genes from deaths of homozygotes (Allison, 1954a). An increased fertility of heterozygotes was proposed (Foy et al, 1954; Allison, 1956a) but never convincingly demonstrated. Raper (1949) was the first to suggest that the sickle cell trait might have a survival advantage against some adverse condition in the tropics and Mackey & Vivarelli (1952) suggested that this factor might be malaria. The close geographical association between the distribution of malaria and the sickle cell gene supported this concept (Allison, 1954b) and led to an exciting period in the history of research in sickle cell disease.

The first observations on malaria and the sickle cell trait were from Northern Rhodesia where Beet (1946, 1947) noted that malarial parasites were less frequent in blood films from subjects with the sickle cell trait. Allison (1954c) drew attention to this association, concluding that persons with the sickle cell trait developed malaria less frequently and less severely than those without the trait. This communication marked the beginning of a considerable controversy.Two studies failed to document differences in parasite densities between `sicklers’ and `non-sicklers’ (Moore et al, 1954; Archibald & Bruce-Chwatt, 1955) and Beutler et al (1955) were unable to reproduce the inoculation experiments of Allison (1954c). Raper (1955) speculated that some feature of Allison’s observations had accentuated a difference of lesser magnitude and postulated that the sickle cell trait might inhibit the establishment of malaria in non-immune subjects. The conflicting results in these and other studies appear to have occurred because the protective effect of the sickle cell trait was overshadowed by the role of acquired immunity. Examination of young children before the development of acquired immunity confirmed both lower parasite rates and densities in children with the sickle cell trait (Colbourne & Edington, 1956; Edington & Laing, 1957; Gilles et al, 1967) and it is now generally accepted that the sickle cell trait confers some protection against falciparum malaria during a critical period of early childhood between the loss of passively acquired immunity and the development of active immunity (Allison, 1957; Rucknagel & Neel, 1961; Motulsky, 1964). The mechanism of such an effect is still debated, although possible factors include selective sickling of parasitized red cells (Miller et al, 1956; Luzzatto et al, 1970) resulting in their more effective removal by the reticulo-endothelial system, inhibition of parasite growth by the greater potassium loss and low pH of sickled red cells (Friedman et al, 1979), and greater endothelial adherence of parasitized red cells (Kaul et al, 1994).

The occurrence of the sickle cell mutation and the survival advantage conferred by malaria together determine the primary distribution of the sickle cell gene. Equatorial Africa is highly malarial and the sickle cell mutation appears to have arisen independently on at least three and probably four separate occasions in the African continent, and the mutations were subsequently named after the areas where they were first described and designated the Senegal, Benin, Bantu and Cameroon haplotypes of the disease (Kulozik et al, 1986; Chebloune et al, 1988; Lapoumeroulie et al, 1992). The disease seen in North and South America, the Caribbean and the UK is predominantly of African origin and mostly of the Benin haplotype, although the Bantu is proportionately more frequent in Brazil (Zago et al, 1992). It is therefore easy to understand the common misconception held in these areas that the disease is of African origin.

However, the sickle cell gene is widespread around the Mediterranean, occurring in Sicily, southern Italy, northern Greece and the south coast of Turkey, although these are all of the Benin haplotype and so, ultimately, of African origin. In the Eastern province of Saudi Arabia and in central India, there is a separate independent occurrence of the HbS gene, the Asian haplotype. The Shiite population of the Eastern Province traditionally marry first cousins, tending to increase the prevalence of SS disease above that expected from the gene frequency (Al-Awamy et al, 1984). Furthermore, extensive surveys performed by the Anthropological Survey of India estimate an average sickle cell trait frequency of 15% across the states of Orissa, Madhya Pradesh and Masharastra which, with the estimated population of 300 million people, implies that there may be more cases of sickle cell disease born in India than in Africa. The Asian haplotype of sickle cell disease is generally associated with very high frequencies of alpha thalassaemia and high levels of fetal haemoglobin, both factors believed to ameliorate the severity of the disease.

The promotion of sickling by low oxygen tension and acid conditions was first recognized by Hahn & Gillespie (1927) and further investigated by others (Lange et al, 1951; Allison, 1956b; Harris et al, 1956). The morphological and some functional characteristics of irreversibly sickled cells were described (Diggs & Bibb, 1939; Shen et al, 1949), but the essential features of the polymerization of reduced HbS molecules had to await the developments of electron microscopy (Murayama, 1966; Dobler & Bertles, 1968; Bertles & Dobler, 1969; White & Heagan, 1970) and Xray diffraction (Perutz & Mitchison, 1950; Perutz et al, 1951). The early observations on the inducement of sickling by hypoxia led to the first diagnostic tests utilizing sealed chambers in which oxygen was removed by white cells (Emmel, 1917), reducing agents such as sodium metabisulphite (Daland & Castle, 1948) or bacteria such as Escherichia coli (Raper, 1969). These slide sickling tests are very reliable with careful sealing and the use of positive controls, but require a microscope and some expertise in its use. An alternative method of detecting HbS utilizes its relative insolubility in hypermolar phosphate buffers (Huntsman et al, 1970), known as the solubility test. Both the slide sickle test and the solubility test detect the presence of HbS, but fail to make the vital distinction between the sickle cell trait and forms of sickle cell disease. This requires the process of haemoglobin electrophoresis, which detects the abnormal mobility of HbS, HbC and many other abnormal haemoglobins within an electric field.

The contributions of several workers on the determinants of sickling (Daland & Castle, 1948), birefringence of deoxygenated sickled cells (Sherman, 1940) the lesser degree of sickling in very young children which implied that it was a feature of adult haemoglobin (Watson, 1948) led Pauling to perform Tiselius moving boundary electrophoresis on haemoglobin solutions from subjects with sickle cell anaemia and the sickle cell trait. The demonstration of electrophoretic and, hence, implied chemical differences between normal, sickle cell trait and sickle cell disease led to the proposal that it was a molecular disease (Pauling et al, 1949). The chance encounter between Castle and Pauling who shared a train compartment returning from a meeting in Denver in 1945, its background and implications, has passed into the folklore of medical research (Conley, 1980; Feldman & Tauber, 1997).

The nature of this difference was soon elucidated. The haem groups appeared identical, suggesting that the difference resided in the globin, but early chemical analyses revealed no distinctive differences (Schroeder et al, 1950; Huisman et al, 1955). Analyses of terminal amino acids also failed to reveal differences, although an excess of valine in HbS was noted but considered an experimental error (Havinga, 1953). The development of more sensitive methods of fingerprinting combining high voltage electrophoresis and chromatography allowed the identification of the essential difference between HbA and HbS. This method enabled the separation of constituent peptides and demonstrated that a peptide in HbS was more positively charged than in HbA (Ingram, 1956). This peptide was found to contain less glutamic acid and more valine, suggesting that valine had replaced glutamic acid (Ingram, 1957). The sequence of this peptide was shown to be Val-His-Leu-Thr-Pro-Val-Glu-Lys in HbS instead of the Val-His-Leu-Thr-Pro-Glu-Glu-Lys in HbA (Hunt & Ingram, 1958), a sequence which was subsequently identified as the amino-terminus of the b chain (Hunt & Ingram, 1959). This amino acid substitution was consistent with the genetic code and was subsequently found to be attributable to the nucleotide change from GAG to GTG (Marotta et al, 1977).

Haemolysis and anaemia. The presence of anaemia and jaundice in the first four cases suggested accelerated haemolysis, which was supported by elevated reticulocyte counts (Sydenstricker et al, 1923) and expansion of the bone marrow (Sydenstricker et al, 1923; Graham, 1924). The bone changes of medullary expansion and cortical thinning were noted in early radiological reports (Vogt & Diamond, 1930; LeWald, 1932; Grinnan, 1935). Drawing on a comparison of sickle cell disease and hereditary spherocytosis, Sydenstricker (1924) introduced the term `haemolytic crisis’ that has persisted in the literature to this day, despite the lack of evidence for such an entity in sickle cell disease. The increased requirements of folic acid and the consequence of a deficiency leading to megaloblastic change was not noted until much later (Zuelzer & Rutzky, 1953; Jonsson et al, 1959; MacIver & Went, 1960).

The haemoglobin level in SS disease of African origin is typically between 6 and 9 g/dl and is well tolerated, partly because of a marked shift in the oxygen dissociation curve (Scriver & Waugh, 1930; Seakins et al, 1973) so that HbS within the red cell behaves with a low oxygen affinity. This explains why patients at their steady state haemoglobin levels rarely show classic symptoms of anaemia and fail to benefit clinically from blood transfusions intended to improve oxygen delivery.

Graham R. Serjeant
Sickle Cell Trust, Kingston, Jamaica
Brit J Haem 2001; 112: 3-18

The Immune Haemolytic Anaemias

The growth in knowledge of the scientific basis of haemolytic anaemias, which have been a main interest of the author, has been remarkable, as have consequent advances in the practice of medicine since the mid-1930s. At that time, the cause and mechanism of important disorders such as the acquired antibody determined (immune) haemolytic anaemias, haemolytic disease of the newborn, hereditary spherocytosis and paroxysmal nocturnal haemoglobinuria were unknown or but partially understood.

According to Crosby (1952), William Hunter of London, in an article on pernicious anaemia published in 1888, was the first to use the term `haemolytic’ to denote an anaemia caused by excessive blood destruction. By the turn of the century, the term was being widely used in clinical literature. Peyton Rous, in his comprehensive review `Destruction of the red blood corpuscles in health and disease’ (Rous, 1923), concluded that the generally held view in the early 1930s was that about one-fifteenth of the erythrocyte mass was destroyed daily. Rous was aware of the pioneer work of Winifred Ashby (1919), who, by following the survival of serologically distinct but compatible transfused erythrocytes, had found that normal erythrocytes might live for up to 100 d in the recipients’ circulation. Subsequent work using radioactive chromium (51Cr) as an erythrocyte label, showed that Ashby’s data and conclusions were in fact correct, i.e. that normal erythrocytes in health circulate in the peripheral blood for approximately 110 d. Erythrocyte labelling with 51Cr also had a further advantage over the Ashby method in addition to enabling the life-span of the patients’ erythrocytes to be assessed in the circulation by surface counting, to detect and measure the accumulation of radioactivity in the spleen and liver, and thereby assess the organs’ role in haemolysis

In the first decade of the twentieth century Widal et al (1908a) and Le Gendre & Brulea (1909) reported that autohaemoagglutination was a striking finding in some cases of icteare heamolytique acquis, and also Chauffard & Trosier (1908) and Chauffard & Vincent (1909) had described the presence of haemolysins in the serum of patients suffering from intense haemolysis. The conclusion was that abnormal immune processes, i.e. the development of auto-antibodies damaging the patients’ own erythrocytes, might play a part in the genesis of some cases of acquired haemolytic anaemia. This was indeed antedated by the classic observations of Donath & Landsteiner (1904) and Eason (1906) on the mechanism of haemolysis in paroxysmal cold haemoglobinuria.

That blood might auto-agglutinate when chilled had been described by Landsteiner (1903) and that an unusual degree of the phenomenon might complicate some types of respiratory disease was reported by Clough & Richter (1918) and later by Wheeler et al (1939). A few years later Peterson et al (1943) and Horstmann & Tatlock (1943) reported that cold auto-agglutinins at high titres were frequently found in the serum of patients who had suffered from the then so called primary atypical pneumonia.

Stats & Wasserman’s (1943) review on cold haemagglutination was a valuable contribution to contemporary knowledge. They listed in a table as many as 94 references to papers published between 1890 and 1943 in which cold haemagglutination had been described. In 32 of the papers the patients referred to had suffered from increased haemolysis

Recognition that cold auto-antibodies played an important role in the pathogenesis of some cases of haemolytic anaemia led to the concept that auto-immune haemolytic anaemia (AIMA) might usefully be classified into warm antibody or cold-antibody types, according to whether the patient is forming (warm) antibodies which react (perhaps optimally) at body temperature or (cold) antibodies which react strongly at low temperatures (e.g. 48C) but progressively less well as the temperature is raised and are perhaps inactive at 37oC. The clinical syndrome suffered by the patient would depend not only on the amount of antibody produced but also on its temperature requirement. Another important advance in understanding has been the realization that both types of AIHA could develop in association with a wide range of underlying disorders (secondary AIHA) as well as `idiopathically’, i.e. for no obvious cause (primary AIHA). The author’s own experience was summarized in a review (Dacie & Worlledge, 1969): 99 out of 210 cases of warm AIHA were judged to be secondary as were 39 out of 85 cases of cold AIHA. Petz & Garratty (1980), summarized the data from six centres: 55% out of a total of 656 cases had been reported as secondary. They listed the disorders with which warm antibody AIHA had been associated as chronic lymphocytic leukaemia, Hodgkin’s disease, non-Hodgkin’s lymphomas, thymomas, multiple myeloma, Waldenstrom’s macroglobulinaemia, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, infectious disease/ childhood viral disorders, hypogammaglobulinaemia, dysglobulinaemias, other immune deficiency syndromes, and ulcerative colitis.

Conley (1981), in an interesting review of warm-antibody AIHA patients seen at the Johns Hopkins Hospital, emphasized how important it was to carry out a careful enquiry into the patient’s past history and also to undertake a prolonged follow-up. He stated that a retrospective review of 33 patients whose illnesses in the past have been designated `idiopathic” had revealed an associated immunologically related disorder in 19 of them. An additional three patients had developed a lymphoma 2±10 years after they had developed AIHA. As already referred to, warm-antibody AIHA is now known to complicate a wide range of underlying diseases, particularly malignant lymphoproliferative disorders, other auto-immune disorders and immune deficiency syndromes. What proportion of patients suffering from a lymphoproliferative disorder develop AIHA is an interesting question. Duehrsen et al (1987) stated that this had occurred in 12 out of 637 patients. Early data on the incidence of a positive DAT in SLE were provided by Harvey et al (1954) – in six out of 34 patients tested the DAT had been positive. Later, Mongan et al (1967), who had studied a large number of patients suffering from a variety of connective tissue disorders, reported that the DAT had been positive in 15 out of 23 patients with SLE, none of whom, however, had suffered from overt haemolytic anaemia. It has also been realized since the 1960s that warm-antibody AIHA may develop in patients suffering from a variety of immune deficiency syndromes, both congenital and acquired.

It was in the mid-1960s that it was realized that, in a significant proportion of patients thought to have `idiopathic’ warm-antibody AIHA, the development of the causal auto-antibodies had been triggered in some way by a drug the patient was taking. The first drug implicated was the antihypertensive drug a-methyldopa (Aldomet) (Carstairs et al, 1966a,b). Following the finding that treating hypertensive patients with a-methyldopa led to the formation of anti-erythrocyte auto-antibodies in a significant percentage of patients, renewed interest was taken in the possibility that other drugs might have the same effect. Two main hypotheses have been advanced in relation to how certain drugs in some patients appear to have caused the development of anti-erythrocyte auto-antibodies. One hypothesis was that the drug or its metabolites act on the immune system so as to impair immune tolerance; the other was that the drug affects antigens at the erythrocyte surface in such a way that a normally active immune system responds by developing anti-erythrocyte antibodies. Clearly, too, the patient’s individuality must be an important factor, for only a proportion of patients receiving the same dosage of the offending drug for the same period of time develop a positive DAT and only a small percentage develop overt AIHA.

An interesting development in the history of the immune haemolytic anaemias was the realization in the mid-1950s that, rather rarely, haemolysis was brought about by the patient developing antibodies that were directed against a drug the patient had been taking and that the erythrocytes were in some way secondarily involved. The first drug to be implicated was Fuadin (stibophen), which had been used to treat a patient with schistosomiasis (Harris, 1954, 1956). The patient’s serum contained an antibody that agglutinated his own or normal erythrocytes and/or sensitized them to agglutination by antiglobulin sera; however, this occurred only in the presence of the drug.

In the late 1940s, several accounts of patients with AIHA who had persistently low platelet counts were published, e.g. Fisher (1947) and Evans & Duane (1949); and it was suggested that the patients might have been forming autoantibodies directed against platelets. This concept was further developed by Evans et al (1951). Eight out of their 18 patients with AIHA were thrombocytopenic; four had clinically obvious purpura. Evans et al (1951) suggested that there exists `a spectrum-like relationship between acquired haemolytic anaemia and thrombocytopenic purpura’; also that `on the one hand, acquired haemolytic anaemia with sensitization of the red cells is often accompanied with thrombocytopenia, while, on the other hand, primary thrombocytopenic purpura is frequently accompanied with red cell sensitization with or without haemolytic anaemia’. Many further case reports of AIHA accompanied by severe thrombocytopenia have since been published

There are two features in the blood film of a patient with an acquired haemolytic anaemia which indicate that he or she is suffering from AIHA; one is auto-agglutination, the other is erythrophagocytosis. Spherocytosis, although often present to a marked degree, is of course found in other types of haemolytic anaemia.

The pioneer French observations on auto-agglutination already referred to were generally overlooked until the late 1930s, and serological studies seem seldom to have been undertaken until the publication of Dameshek & Schwartz’s (1938b) report in which they described the presence of `haemolysins’ in cases of acute apparently acquired haemolytic anaemia. Dameshek & Schwartz (1940) summarized contemporary knowledge in an extensive review. They concluded that it was not improbable that haemolysins of various types and `dosages’ were in fact responsible for many cases of human haemolytic anaemias, including congenital haemolytic anaemia, which they suggested might be caused by the `more or less continued action of an haemolysin’.

Six years were to pass before the concept that an abnormal immune mechanism played a decisive role in some cases of acquired haemolytic anaemia was clearly demonstrated by Boorman et al (1946), who reported that the erythrocytes of five patients with acquired acholuric jaundice had been agglutinated by an antiglobulin serum, i.e. that the newly described antiglobulin reaction or Coombs test (Coombs et al, 1945) was positive, while the test had been negative in 28 patients suffering from congenital acholuric jaundice. This work aroused great interest and was soon confirmed.

Until the 1950s, the auto-antibodies responsible for AIHA were generally concluded to be `non-specific’. According to Wiener et al (1953), `Red cell auto-antibodies react not only with the individual’s own red cells but also with the erythrocytes of all other human beings. The substances on the red blood cell envelope with which the auto-antibodies combine are agglutinogens like the ABO, MN and RhHr systems, except that, in the former case, the blood factors with which the auto-antibodies react are not type specific but are shared by all human beings.’ They suggested that the auto-antibodies might be directed to the `nucleus of the RhHr substance’. Earlier work had, however, indicated that the sensitivity of normal group-compatible erythrocytes to a patient’s auto-antibody might vary considerably (Denys & van den Broucke, 1947; Kuhns & Wagley, 1949). That auto-antibodies might have a clearly defined Rh specificity, e.g. anti-e, was described by Race & Sanger (1954) in the second edition of their book. Referring to Wiener et al (1953), they wrote: `This beautifully clear investigation made the present authors realize that a curious result obtained by one of them (Ruth Sanger) in 1953 in Australia had after all been true; the serum of a man who had died of a haemolytic anaemia 3000 miles away contained anti-e; his cells were clearly CDe-cde’. A similar finding, i.e. an auto-anti-e, was described by Weiner et al (1953).

A further development in the unravelling of a complicated story was the realization that some of the antibodies which appeared to be specific were reacting with more basic antigens, although showing a preference for specific antigens, i.e. some specific auto-antibodies appeared to be less specific than their allo-antibody counterparts. Moreover, some antibodies, reacting with specific antigens, have been shown to be partially or completely absorbable by antigen negative cells.

Many apparently `non-specific’ antidl antibodies have been shown to be not strictly `nonspecific’ but to react with antigens of very high frequency, e.g. to be anti-Wrb, anti-Ena, anti-LW or anti-U. Issitt et al (1980)) listed six additional very common antigens that had been identified as targets for anti-dl auto-antibodies, i.e. Hr, Hro, Rh34, Rh29, Kpb and K13.

In relation to human acquired haemolytic anaemia, the discovery in the late 1940s and 1950s that many cases were apparently brought about by the development of damaging anti-erythrocyte antibodies led to intense interest and speculation into the why and how of auto-antibody formation. Of seminal importance at the time were the experiments and theoretical arguments of Burnet (Burnet & Fenner, 1949; Burnet, 1957, 1959, 1972) and the studies on transplantation immunity of Medawar (Billingham et al, 1953; Medawar, 1961). Of particular interest, too, was the report by Bielschowsky et al (1959) of the occurrence of AIHA in an inbred strain of mice – the NZB/BL strain. Remarkably, by the time the mice were 9-months-old the DAT was positive in almost every mouse. Burnet (1963) referred to the gift of the mice to the Walter and Eliza Hall Institute of Medical Research, Melbourne as `the finest gift the Institute has ever received’.

Exactly how is it that auto-antibodies reacting with an erythrocyte surface antigen result in the cell’s premature destruction? The possible role of auto-agglutination in bringing about haemolysis was emphasized by Castle and colleagues as the result of a series of studies carried out in the 1940s and 1950s. As summarized by Castle et al (1950), an antibody which appears to be incapable of causing `lysis in vitro might bring about the following sequence of events in vivo. (1) Red cell agglutination in the peripheral blood; (2) red cell sequestration and separation from plasma in tissue capillaries; (3) ischaemic injury of tissue cells with release of substances that increase the osmotic and mechanical fragilities of red cells locally; (4) local osmotic lysis of red cells or subsequent escape of mechanically fragile red cells into the blood stream where the traumatic motion of the circulation causes their destruction’.

We can expect, as the years pass, that more and more will be known as to the intricate mechanisms that bring about self-tolerance and the mechanisms underlying the occurrence of auto-immune disorders in general, including the role of infectious agents, drugs and genetic factors. Patients with immune haemolytic anaemias can be expected to benefit from the new knowledge; for in parallel with a better understanding as to how immune self-tolerance breaks down will hopefully be the development of more effective drugs and therapies aimed at controlling the breakdown.

The Immune Haemolytic Anaemias: A Century of Exciting Progress in Understanding.  Sir John Dacie, Emeritus Professor of Haematology.
Brit J Haem 2001; 114: 770-785.

A History of Pernicious Anaemia

This is a review of the ideas and observations that have led to our current understanding of pernicious anaemia (PA). PA is a megaloblastic anaemia (MA) due to atrophy of the mucosa of the body of the stomach which, in turn, is brought about by autoimmune factors.

A case report by Osler & Gardner (1877) in Montreal could be that of PA. This anaemic patient had numbness of the fingers, hands and forearms; the red blood cells were large; at autopsy the gastric mucosa appeared atrophic and the marrow had large numbers of erythroblasts with finely granular nuclei. The increased marrow cellularity had also been noted by Cohnheim (1876).

Ehrlich (1880) (Fig 1) distinguished between cells he termed megaloblasts present in the blood in PA from normoblasts present in anaemia as a result of blood loss. Not only were large red blood cells noted in PA, but irregular red cells, ? poikilocytes, were reported in wet blood preparations by Quincke (1877). Megaloblasts in the marrow during life were first noted by Zadek (1921). Hypersegmented neutrophils in peripheral blood in PA were described by Naegeli (1923) and came to be widely recognized after Cooke’s study (Cooke, 1927). The giant metamyelocytes in the marrow were described by Tempka & Braun (1932).

Paul Ehrlich

Paul Ehrlich

Fig 1. Paul Ehrlich (Wellcome Institute Library, London).

The association between PA and spinal cord lesions was described by Lichtheim (1887) and a full account was published by Russell et al (1900), who coined the term `subacute combined degeneration of the spinal cord’ (SCDC) although they were not convinced of its relation to PA. Arthur Hurst at Guy’s Hospital, London, confirmed the association of the neuropathy with PA and added, too, the association of loss of hydrochloric acid in the gastric juice (Hurst & Bell, 1922). Cabot (1908) found that numbness and tingling of the extremities were present in almost all of his 1200 patients and 10% had ataxia. William Hunter (1901) noted the prevalence of a sore tongue in PA, which was present in 40% of Cabot’s series.

In 1934, the Nobel Prize in medicine and physiology was awarded to Whipple, Minot and Murphy. Was there ever an award more deserved? They saved the lives of their patients and pointed the way forward for further research. What was there in liver that was lacking in patients with PA? The effect of liver in restoring the anaemia in Whipple’s iron-deficient dogs was by supplying iron which is  abundant in liver.

Liver given by mouth also provides Cbl and folic acid. But patients with PA cannot absorb Cbl, although some 1% of an oral dose can cross the intestinal mucosa by passive diffusion; this, presumably, is what happened when large amounts of liver were eaten. Beef liver contains about 110 mg of Cbl per 100 g and about 140 mg of folate per 100 g. Cbl is stable and generally resistant to heat; folate is labile unless preserved with reducing agents. The daily requirement of Cbl by man is l-2 mg. The liver diet, if consumed, had enough of these haematinics to provide a response in most MAs.

George Richard Minot

George Richard Minot

George Richard Minot (Wellcome Institute Library, London).

The availability of liver extracts brought about interest in the nature of the haematological response. An optimal response required a peak rise of reticulocytes 5±7 d after the injection of liver extract and the height of the peak was greatest in those with severe anaemia; the flood of reticulocytes was as a result of a synchronous maturation of a vast number of megaloblasts into red cells. There is a steady rise in the red cell count to reach 3 x 1012/l in the 3rd week (Minot & Castle, 1935). Many liver extracts did not have enough antianaemic factor to achieve this and some assayed by the author had only 1-2 mg of Cbl.  It took another 22 years for a pure antianaemic factor to be isolated, although, admittedly, the Second World War intervened; in 1948, an American group led by Karl Folkers and an English group led by E. Lester-Smith published, within weeks of each other, the isolation of a red crystalline substance termed vitamin B12 and subsequently renamed cobalamin.

The structure of this red crystalline compound was studied by the nature of its degradation products and by X-ray crystallography. It soon became apparent that there was a cobalt atom at the heart of the structure and this heavy atom was of great aid to the crystallographers, so much so that, with additional information from the chemists, they were the first to come up with the complete structure. To quote Dorothy Hodgkin: `To be able to write down a chemical structure very largely from purely crystallographic evidence on the arrangement of atoms in space – and the chemical structure of a quite formidably large molecule at that – is for any crystallographer, something of a dream-like situation’. As Lester-Smith (1965) pointed out, it also required some 10 million calculations. In 1964, Dorothy Hodgkin was awarded the Nobel Prize for chemistry.

Barker et al (1958) published an account of the metabolism of glutamate by a Clostridium. The glutamate underwent an isomerization and an orange-coloured co-enzyme was involved that turned out to be Cbl with a deoxyadenosyl group attached to the cobalt.

This Cbl co-enzyme, deoxyadenosylCbl, is the major form of Cbl in tissues; it is also extremely sensitive to light, being changed rapidly to hydroxoCbl. DeoxyadenosylCbl is concerned with the metabolism of methylmalonic acid in man (Flavin & Ochoa, 1957). The other functional form of Cbl is methylCbl involved in conversion of homocysteine to methionine (Sakami & Welch, 1950). Both these pathways are impaired in PA in relapse.

Cbl consists of a ring of four pyrrole units very similar to that present in haem. These, however, have the cobalt atom in the centre instead of iron and the ring is called the corrin nucleus. The cobalamins have a further structure, a base, termed benzimidazole, set at right angles to the corrin nucleus and this may have a link to the cobalt atom (base on position).

By the time Cbl had been isolated from liver it was already known that it was also present in fermentation flasks growing bacteria such as streptomyces species. Other organisms gave higher yields so that kilogram quantities of pure Cbl were obtained; these sources have replaced liver in the production of Cbl. By adding radioactive form of cobalt to the fermentation flasks instead of ordinary cobalt, labelled Cbl became available (Chaiet et al, 1950). The importance of labelled Cbl is that it made it possible to carry out Cbl absorption tests in patients, to design isotope dilution assays for serum Cbl, to design ways of assaying intrinsic factor (IF), to detect antibodies to IF and even to measure glomerular filtratration rate, as free Cbl is excreted by the glomerulus without any reabsorption by the renal tubules.

William Castle at the Thorndike Memorial Laboratory, Boston City Hospital, devised experiments to explore the relationship between gastric juice, the anti-anaemic factor that Castle assumed, correctly, was also present in beef, and the response in PA. The question Castle asked was `Was it possible that the stomach of the normal person could derive something from ordinary food that for him was equivalent to eating liver?’.

The experiment in untreated patients with PA consisted of two consecutive periods of 10 d or more during which daily reticulocyte counts were made. During the first period of 10 d, the PA patient received 200 g of lean beef muscle (steak) each day. There was no reticulocyte response. During the second period, the contents of the stomach of a healthy man were recovered 1 h after the ingestion of 300 g of steak; about 100 g could not be recovered. The gastric contents were incubated for a few hours until liquefied and then given to the PA patient through a tube. This was done daily. On day 6 there was a rise in reticulocytes reaching a peak on day 10, followed by a rise in the red cell count. The response was similar to that obtained with large amounts of oral liver.

Thus, Castle concluded that a reaction was taking place between an unknown intrinsic factor (IF) in the gastric juice and an unknown extrinsic factor in beef muscle. Whereas Minot & Murphy (1926) found that 200-300 g of liver daily was needed to get a response in PA, 10 g liver was adequate when incubated with 10-20 ml normal gastric juice (Reiman & Fritsch, 1934). Castle’s extrinsic factor is the same as the anti-anaemic factor that is Cbl, and IF is needed for its absorption. Presumably the gastric juice in PA lacks IF.

The elegant studies of Hoedemaeker et al (1964) in Holland using autoradiography of frozen sections of human stomach incubated with [57Co]-Cbl showed that IF was produced in the gastric parietal cell. The binding of Cbl to

the parietal cell was abolished by first incubating the section with a serum containing antibodies to IF. The parietal cell in man is thus the source of both hydrochloric acid and IF. The parietal cell is the only source of IF in man as a total gastrectomy is invariably followed by a MA due to Cbl deficiency. IF is a glycoprotein with a molecular weight of 45 000.

Assay of protein fractions of serum after electrophoresis showed that endogenous Cbl is in the position of α-1 globulin. Chromatography of serum after addition of [57Co]-Cbl on Sephadex G-200 showed that Cbl was attached to two proteins, one eluting before the albumin termed transcobalamin I (TCI) and the other after the albumin termed transcobalamin II (TCII). Charles Hall showed that, when labelled Cbl given by mouth is absorbed, it first appears in the position of TCII and later in the position of TCI as well (Hall and Finkler, l965). They concluded that TCII is the prime Cbl transport protein carrying Cbl from the gut into the blood and then to the liver from where it is redistributed by both new TCII as well as TCI. Congenital absence of a functional TCII causes a severe MA in the first few months of life owing to an inability to transport Cbl. Most of the Cbl in serum is on TCI because it has a relatively long half-life of 9±10 d, whereas the half-life of TCII is about 1.5 h. Thus, in assaying the serum Cbl level, it is mainly TCI-Cbl that is being assayed.

With the availability of labelled Cbl, Cbl absorption tests began to be widely used in the 1950s. The commonest method was the urinary excretion test described by Schilling (1953). Here, an oral dose of radioactive Cbl is followed by an injection of 1000 mg of cyano-Cbl. The free cyano-Cbl is largely excreted into the urine over the next 24 h and carries with it about one third of the absorbed labelled Cbl.

Parietal cell antibodies (Taylor et al, 1962) are present in serum in 76-93% of different series of PAs and in the serum of 36% of the relatives of PA patients. The antibody is present in sera from 32% of patients with myxoedema, 28% of patients with Graves’ disease, 20% of relatives of thyroid patients and 23% of patients with Addison’s disease. Parietal cell antibodies are found in between 2-16% of controls, the high 16% figure being in elderly women. There is a higher frequency of PA in women, the female to male ratio being 1.7 to 1.0. The parietal cell antibody is probably important in the production of gastric atrophy. Thyroid antibodies are present in sera from 55% of PAs, in sera from 50% of PA relatives, in 87% of sera from myxoedema patients, in 53% of sera in Graves’ disease and in 46% of relatives of patients with thyroid disease.

There is a high frequency of PA among those disorders that have antibodies against the target organ. Thus, among 286 patients with myxoedema, 9.0% also had PA (Chanarin, 1979), as compared with a frequency of PA of about 1 per 1000 (0.01%) in the general population. Of 102 consecutive patients with vitiligo,
eight also had PA.

Patients with acquired hypogammaglobulinaemia are unable to make humoral antibodies; nevertheless, one third have PA as well. This cannot be as a result of action of IF antibodies and must be because of specific cell-mediated immunity. Tai & McGuigan (1969) demonstrated lymphocyte transformation in the presence of IF in six out of 16 PA patients and Chanarin & James (1974) found 10 out of 51 tests were positive.

Twenty-five patients with PA were tested for the presence of humoral IF antibody in serum and gastric juice and for cell-mediated immunity against IF. All but one gave positive results in one or more tests. It was concluded that these findings establish the autoimmune nature of PA and that the immunity is not merely an interesting byproduct.

Patients with PA treated with steroids show a reversal of the abnormal findings characterizing the disease. If they are still megaloblastic, the anaemia will respond in the first instance (Doig et al, 1957), but in the longer term Cbl neuropathy may be precipitated. The absorption of Cbl improves and may become `normal’ (Frost & Goldwein, 1958). There is a return of IF in the gastric juice (Kristensen and Friis, 1960) and a decline in the amount of IF antibody in serum (Taylor, 1959). In some patients there is return of acid in the gastric juice. Gastric biopsy shows a return of parietal and chief cells (Ardeman & Chanarin, 1965b; Jeffries, 1965). All this is as a result of suppression of cell-mediated immunity against the parietal cell and against IF. Withdrawal of steroids leads to a slow return to the status quo.

The author has dipped freely into the two volumes by the late M. M. Wintrobe. These are: Wintrobe, M.M. (1985) Hematology, the Blossoming of a Science. Lea & Febinge

A History of Pernicious Anaemia
I. Chanarin, Richmond, Surrey
Brit J Haem 111: 407-415
History of Folic Acid

1928 Lucy Wills studied macrocytic anaemia in pregnancy in Bombay, India

1932 Janet Vaughn studied macrocytic anemia associated with coeliac disease and idiopathic steatorrhea (1932) showed a response to marmite

1941 Folic acid extracted from spinach and is a growth factor for S. Faecalis

1941 pteroylglutamic acid synthesized at Amer Cyanamide – Pteridine ring, paraminobenzoic acid, glutamine –  PGA differed from natural compound in some respects

1945 PGA resolved the macrocytic anemia, but not the neuropathy

1979 Stokstad and associates at Berkeley obtained the first purified mammalian enzymes involved in synthesis

Folate antagonists inhibit tumor growth (Hitchings and Elion)(Nobel)

  • Misincorporation of uracil instead of thymine into DNA

Sidney Farber introduced Aminopterine and also Methotrexate for treatment of childhood lymphoblastic leukemia

  • MTX inhibits DHFR enzyme (dihydrofolate reductase) necessary for THF

Wellcome introduces trimethoprim (antibacterial), and also pyramethoprime (antimalarial)

Homocysteine isolated by Du Vineaud, but it was not noticed

Finkelstein and Mudd demonstrated the importance of remethylation for tHy and worked out the transsulfuration pathway

  1. Function of methyl THF is remethylation of homocysteine
  2. Synthesized by MTHFR
Metabolism of folate

Metabolism of folate

Metabolism of folate

Allosterically regulated by S-adenosyl methionine (Stokstad)

MTHF also inhibits glycine methyl transferase controlling excess SAM – transmethylation

JD Finkelstein

JD Finkelstein

James D Finkelstein

  • Homocysteinuria – mental retardation, skeletal malformation, thromboembolic disease; deficiency of cystathionine synthase (controls trans-sulfuration)
  • NTDs – pregnancy
  • Hyperhomocysteinemia and VD

AD Hoffbrand and DG Weir
Brit J Haem 2001; 113: 579-589

The History of Haemophilia in the Royal Families of Europe Queen Victoria.

On 17 July 1998 a historic ceremony of mourning and commemoration took place in the ancestral church of the Peter and Paul Fortress in St Petersburg. President Boris Yeltsin, in a dramatic eleventh-hour change of heart, decided to represent his country when the bones of the last emperor, Tsar Nicholas II, and his family were laid to rest 80 years to the day after their assassination in Yekaterinberg (Binyon, 1998). He described it as ‘ironic that the Orthodox Church, for so long the bedrock of the people’s faith, should find it difficult to give this blessing the country had expected’. ‘I have studied the results of DNA testing carried out in England and abroad and am convinced that the remains are those of the Tsar and his family’ (The Times, 1998a). Unfortunately, politicians and the hierarchy of the Russian Orthodox Church had argued about what to do with the bones previously stored in plastic bags in a provincial city mortuary. Politics, ecclesiastical intrigue, secular ambition, and emotions had fuelled the debate. Yeltsin and the Church wanted to honour a man many consider to be a saint, but many of the older generation are opposed to the rehabilitation of a family which symbolizes the old autocracy.

Our story starts, almost inevitably, with Queen Victoria of England who had nine children by Albert, Prince of Saxe-Coburg-Gotha. Victoria was certainly an obligate carrier for haemophilia as over 20 individuals subsequently inherited the condition (Figs 1 and 2). Princess Alice (1843–78) was Victoria’s third child and second daughter. Having married the Duke of Hesse at an early age, Alice went on to have seven children, one of whom, Frederick (‘Frittie’) was a haemophiliac who died at the age of 3 following a fall from a window.

Prince Leopold with Sir William Jenner at Balmoral in 1877

Prince Leopold with Sir William Jenner at Balmoral in 1877

Prince Leopold with Sir William Jenner at Balmoral in 1877. (Hulton Deutsch Collection Ltd.)

Alexandra was the sixth child and was only 6 years old when her mother and youngest sister died. ‘Sunny’, as she became known, was a favourite of Queen Victoria, who as far as possible directed her upbringing from across the channel: Alexandra (Alix) was forced to eat her baked apples and rice pudding with the same regularity as her English cousins. Alix visited her older sister Elizabeth (Ella) on her marriage to Grand Duke Serge and met Tsarevich Nicholas for the first time: she was 12 and not impressed. Five years later they met again and Alix fell in love, but by now she had been confirmed in the Lutheran Church and religion became the solemn core of her life.

Victoria had other aspirations for Alix. She hoped that she would marry her grandson Albert Victor (The Duke of Clarence) and the eldest son of the Prince of Wales (later Edward VII). The Duke was an unimpressive young man who was somewhat deaf and had limited intellectual abilities. If this arrangement had proceeded then Alix’s haemophilia carrier status would have been introduced into the British Royal Family and the possibility of a British monarch with haemophilia might have become a reality; however, the Duke died in 1892.

Nicholas and Alexandra. Alix and Nicholas were married in 1894 one week after the death of Nicholas’s father (Alexander III). In the same way that Victoria, with her personal aspirations of a marriage between Alix and the Duke of Clarence, had not considered the possibility of haemophilia, neither did the St Petersburg hierarchy consider a marriage to Nicholas undesirable. Haemophilia was already well recognized in Victoria’s descendants. Her youngest son, Leopold, had already died, as had Frittie her grandson. The inheritance of haemophilia had been known for some time since its description by John Conrad Otto (Otto, 1803). However, it was as late as 1913 before the first royal marriage was declined because of the risk of haemophilia, when the Queen of Rumania decided against an association between her son, Crown Prince Ferdinand, and Olga, the eldest daughter of Nicholas and Alexandra. The Queen of Rumania was herself a granddaughter of Queen Victoria and therefore a potential haemophilia carrier!

Alix was received into the Russian Orthodox Church, taking the name of Alexandra Fedorova. The first duty of a Tsarina was to maintain the dynasty and produce a male heir, but between 1895 and 1901 Alix produced four princesses, Olga, Tatiana, Maria and Anastasia. Failure to produce a son made Alix increasingly neurotic and she had at least one false pregnancy. However, in early 1904 she was definitely pregnant.

For a month or so all seemed well with little Alexis, but it was then noticed that the Tsarevitch was bleeding excessively from the umbilicus (a relatively uncommon feature of haemophilia). At first the diagnosis was not admitted by the parents, but eventually the truth had to be faced although even then only by the doctors and immediate family. Alix was grief stricken: ‘she hardly knew a day’s happiness after she realized her boy’s fate’. As a newly diagnosed haemophilia carrier she dwelt morbidly on the fact that she had transmitted the disease. These feelings are well known to some haemophiliac mothers but the situation was different in Russia in the early twentieth century. The people regarded any defect as divine intervention. The Tsar, as head of the Church and leader of the people, must be free of any physical defect, so the Tsarevich’s haemophilia was concealed. The family retreated into greater isolation and were increasingly dominated by the young heir’s affliction (Fig 3).

Up to a third of haemophiliac males do not have a family history of the condition. This is usually thought to be the result of a relatively high mutation rate occurring in either affected males or female carriers. None of Queen Victoria’s ancestors, for many generations, showed any evidence of haemophilia. Victoria was therefore either a victim of a mutation, or the Duke of Kent was not her father.The mutation is unlikely to have been in her mother, Victoire, who had a son and daughter by her first marriage, and there is no sign of haemophilia in their numerous descendants.

Victoire was under considerable pressure to produce an heir. The year before Victoria was born, Princess Charlotte, the only close heir to the throne, had died and the Duke of Kent had somewhat reluctantly agreed to marry Victoire with the aim of producing an heir. The postulate that the Queen’s gardener had a limp has not been substantiated!

The Duke of Kent had no evidence of haemophilia (he was 51 when Victoria was born) but did inherit another condition from his father (George III): porphyria. While a young man in Gibralter he suffered bilious attacks which were recognized as being similar to his father’s complaint.

Had Queen Victoria carried the gene for porphyria we might expect that she would have at least as many descendants with this condition as had haemophilia. Until recently only two possible cases of porphyria have been suggested amongst Victoria’s descendants: Kaiser Wilhelm’s sister and niece (MacAlpine & Hunter, 1969), but they could have inherited it from their Hohenzollern ancestor, Frederick the Great. A recent television programme (Secret History, 1998) claims to have identified two more cases in Victoria’s descendants, Princess Victoria, the Queen’s eldest daughter, and Prince William of Gloucester, nephew of George V. If these two cases are correct then they would tend to confirm that Victoria was indeed the daughter of the Duke of Kent, but the apparent lack of more cases in Victoria’s extended family is difficult to understand. The gene for acute intermittent porphyria has been isolated on chromosome 11. There is still plenty of scope for further genetic analysis on the European Royal Families!

We can only speculate as to the impact on European events over the last 150 years if the marriages within the Royal houses had been different. What is evident is the dramatic effect of haemophilia on the Royal Princes and their families.

Empress Alexandra at the Tsarevich’s bedside during a haemophiliac crisis

Empress Alexandra at the Tsarevich’s bedside during a haemophiliac crisis

Empress Alexandra at the Tsarevich’s bedside during a haemophiliac crisis in 1912. (Radio Times Hulton Picture Library.)

Richard F. Stevens
Royal Manchester Children’s Hospital
Brit J Haem 1999, 105, 25–32

`The longer you can look back ± the further you can look forward’: Winston Churchill in an address to The Royal College of Physicians, London 1944. At the time that Churchill was speaking in 1944, leukaemia was a fatal disease that had been identified 100 years before. The disease was described as the dreaded leukaemias, sinister and poorly understood.

Thomas Hodgkin chose a career in medicine and enrolled as a pupil at Guy’s Hospital in London. Being a Quaker, however, he could not enter the English universities of Oxford and Cambridge and decided to follow the medical courses at Edinburgh. At that times, Aristotelian and Hippocratic medicine were greatly influencing British physicians. Hodgkin, still a medical student, wrote a paper `On the Uses of the Spleen’ where he reported his beliefs on the purposes of the spleen: to regulate fluid volume, clean impurities from the body, supply expandability to the portal system. The subject was a presage of the disease that bears his name.

Hodgkin interrupted his studies at Edinburgh to spend a year in Paris where he met many people who had a great influence in his life and future activities. Among them, were Laennec (Hodgkin played an important role in bringing the stethoscope to Great Britain); Baron von Humboldt who introduced Hodgkin to the field of anthropology; Baron Cuvier, a distinguished anatomist and palaeontologist; and Thomas A. Bowditch, whose expeditions to Africa had a great impact on Hodgkin’s future activities.

In 1825, Thomas Hodgkin returned to London to join the staff at Guy’s Hospital, and in 1826 he was made `Inspector of the Dead’ and `Curator of the Museum of Morbid Anatomy’. In developing the museum he had accumulated, by 1829, over 1600 specimens demonstrating the effects of disease. The correlation of clinical disease to pathological material was quite new: from analyses of pathological specimens Hodgkin was able to describe appendicitis with perforation and peritonitis, the local spread of cancer to draining lymph nodes, noting that the tumour had similar characteristics at both sides, and features of other diseases.

In his historic paper `On Some Morbid Appearances of the Absorbent Glands and Spleen’ (Hodgkin, 1832), he briefly described the clinical histories and gross postmortem findings on six patients from the experience at Guy’s Hospital and included another case sent to him in a detailed drawing by his friend Carswell (Fig 2). In the very first paragraph he wrote: `The morbid alterations of structure which I am about to describe are probably familiar to many practical morbid anatomists, since they can scarcely have failed to have fallen under their observation in the course of cadaveric inspection’. Hodgkin’s studies had convinced him that he was dealing with a primary disease of the absorbent (lymphatic) glands. `This enlargement of the glands appeared to be a primitive affection of those bodies, rather than the result of an irritation propagated to them from some ulcerated surface or other inflamed texture – Unless the word inflammation be allowed to have a more indefinite and loose eaning, this affection – can hardly be attributed to that cause’ was stated on pages 85 and 86 of his 1832 paper. Hodgkin also mentioned that the first reference that he could find to this or similar disease was in fact by Malpighi in 1666.

Wilks (1865) described the disease in detail and, made aware by Bright that the first observations were done by Hodgkin, linked his name permanently to this new entity in a paper entitled `Cases of Enlargement of the Lymphatic Glands and Spleen (or Hodgkin’s Disease) with Remarks’ (Fig 3).

In 1837 Thomas Hodgkin was the outstanding candidate for the position of Assistant Physician at Guy’s Hospital in succession to Thomas Addison who had been promoted to Physician. After 10 years spent as Inspector of the Dead, he had published a great deal, including a two-volume work entitled The Morbid Anatomy of Serous and Mucous Membrane.

Hodgkin, acting in his other capacity, had sent Benjamin Harrison a report on the terrible consequences to native Indians of monopoly trading and on the inhuman treatment they received from officials of the Hudson Bay Company, of which Harrison was the financier. when the opportunity to appoint an Assistant Physician occurred, Harrison exercised an autocratic rule over the hospital and presided at the appointment made by the General Court. Thomas Hodgkin did not get the job and the next day he resigned all his appointments at Guy’s Hospital. Social medicine, medical problems associated with poverty, antislavery, concern for underpriviledged groups such as American Indians and Africans, as well as a strong sense of responsibility defined his life after this separation.

Sternberg (1898) and Reed (1902) are generally credited with the first definitive and thorough descriptions of the histopathology of Hodgkin’s disease. Based on the findings observed in her case series, Dorothy Reed concluded `We believe then, from the descriptions in the literature and the findings in 8 cases examined, that Hodgkin’s disease has a peculiar and typical histological picture and could thus rightly be considered a histopathological disease entity’.

During the successive decades, pathologists began to describe a broader spectrum of histological features. However, it was Jackson and Parker who, in scientific papers and in their well-known book Hodgkin’s Disease and Allied Disorders (Jackson & Parker, 1947), presented the first serious effort at a histopathological classification. They assigned the name `Hodgkin’s granuloma’ to the main body of typical cases. A much more malignant variant, usually characterized by a great abundance of pleomorphic and anaplastic Reed-Sternberg cells and seen in a relativelysmall number of cases was named `Hodgkin’s sarcoma’. A third, similarly infrequent, variant characterized by an extremely slow clinical evolution, a relative paucity of Reed-Sternberg cells and a great abundance of lymphocytes was termed `Hodgkin’s paragranuloma’. It was only approximately 20 years later that Lukes & Butler (1966) reported a characteristic subtype of the heterogeneous `granuloma’ category, to which they assigned the name `nodular sclerosis’. They also proposed a new histopathological classification, still in use to date, with an appreciably greater prognostic relevance and usefulness than the

previous Jackson-Parker classification.

The first human bone marrow transfusion was given to a patient with aplastic anemia in 1939.9 This patient received daily blood transfusions, and an attempt to raise her leukocyte and platelet counts was made using intravenous injection of bone marrow. After World War II and the use of the atomic bomb, researchers tried to find ways to restore the bone marrow function in aplasia caused by radiation exposure. In the 1950s, it was proven in a mouse model that marrow aplasia secondary to radiation can be overcome by syngeneic marrow graft.10 In 1956, Barnes and colleagues published their experiment on two groups of mice with acute leukemia: both groups were irradiated as anti-leukemic therapy and both were salvaged from marrow aplasia by bone marrow transplantation.

The topics of leukemias and lymphomas will not be discussed further in  this discussion.

The related references are:

Leukaemia – A Brief Historical Review from Ancient Times to 1950
British Journal of Haematology, 2001, 112, 282-292

The Story of Chronic Myeloid Leukaemia
British Journal of Haematology, 2000, 110, 2-11

Historical Review of Lymphomas
British Journal of Haematology 2000, 109, 466-476

Historical Review of Hodgkin’s Disease
British Journal of Haematology, 2000, 110, 504-511

Multiple Myeloma: an Odyssey of Discovery
British Journal of Haematology, 2000, 111, 1035-1044

The History of Blood Transfusion
British Journal of Haematology, 2000, 110, 758-767

Hematopoietic Stem Cell Transplantation—50 Years of Evolution and Future Perspectives. Henig I, Zuckerman T.
Rambam Maimonides Med J 2014;5 (4):e0028.
http://dx.doi.org/10.5041/RMMJ.10162

Landmarks in the history of blood transfusion.

1666 Richard Lower (Oxford) conducts experiments involving transfusion of blood from one animal to another

1667 Jean Denis (Paris) transfuses blood from animals to humans

1818 James Blundell (London) is credited with being the first person to transfuse blood from one human to another

1901 Karl Landsteiner (Vienna) discovers ABO blood groups. Awarded Nobel Prize for Medicine in 1930

1908 Alexis Carrel (New York) develops a surgical technique for transfusion, involving anastomosis of vein in the recipient with artery in the donor. Awarded Nobel Prize for Medicine in 1912

1915 Richard Lewinsohn (New York) develops 0.2% sodium citrate as anticoagulant

1921 The first blood donor service in the world was established in London by Percy Oliver

1937 Blood bank established in a Chicago hospital by Bernard Fantus

1940 Landsteiner and Wiener (New York) identify Rhesus antigens in man

1940 Edwin Cohn (Boston) develops a method for fractionation of plasma proteins. The following year, albumin produced by this method was used for the first time to treat victims of the Japanese attack on Pearl Harbour

1945 Antiglobulin test devised by Coombs (Cambridge), which also facilitated identification of several other antigenic systems such as Kell (Coombs et al, 1946), Duffy (Cutbush et al, 1950) and Kidd (Cutbush et al, 1950)

1948 National Blood Transfusion Service (NBTS) established in the UK

1951 Edwin Cohn (Boston) and colleagues develop the first blood cell separator

1964 Judith Pool (Palo Alto, California) develops cryoprecipitate for the treatment of haemophilia

1966 Cyril Clarke (Liverpool) reports the use of anti-Rh antibody to prevent haemolytic disease of the newborn

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