CLINICAL LABORATORY DIAGNOSTICS OF HEMOBLASTOSIS

Leukemia appears to be a clonal disease resulting from the abnormal uncontrolled proliferation of a single stem cell from which a new clone of cells develops. For some unknown reason, these abnormal cells have a selective growth advantage over normal cells. Present data indicate that these cells are functionally different and biochemically abnormal.

Etiology is Unknown: Possible Causes:

A. Viruses are the proven etiological agent in some animal leukemias, and may well be the causative agent in some human leukemias as well.

B. Marrow damage due to irradiation increases the frequency of some leukemias, but not others.

C. A variety of chemicals and drugs have been implicated as possible etiological agents of leukemia, especially benzene.

D. Possible genetic factors have been implicated, especially in Chronic Lymphocytic Leukemia.

Leukemia Classification

Leukemias are classified into 2 major groups. Chronic in which the onset is insidious, the disease is usually less aggressive, and the cells involved are usually more mature cells. Acute in which the onset is usually rapid, the disease is very aggressive, and the cells involved are usually poorly differentiated with many blasts.

Both acute and chronic leukemias are further classified according to the prominent cell line involved in the expansion:

1. If the prominent cell line is of the myeloid series it is a myelocytic leukemia (sometimes also called granulocytic).

2. If the prominent cell line is of the lymphoid series it is a lymphocytic leukemia

Therefore, there are four basic types of leukemia.

I. Acute myelocytic leukemia AML - (includes myeloblastic, promyelocytic, monocytic, myelomonocytic, erythrocytic, and megakaryocytic)

II. Acute lymphocytic leukemia ALL - (includes T cell, B cell, and Null cell)

III. Chronic myelocytic leukemia CML - (includes myelocytic and myelomonocytic)

IV. Chronic lymphocytic leukemia CLL - (includes plasmocytic multiple myeloma, Hairy cell, prolymphocytic, large granular cell lymphocytic, Sezarys syndrome, and circulating lymphoma)

Acute

Chronic

Lymphoid

ALL

CLL

Myeloid

AML

CML

 

The acute leukemias are divided into 2 categories, depending upon their cell of origin. Leukemia evolving from the myeloid/granulocyte cell line is called acute

myelogenous leukemia (AML). Lymphocytic precursors give rise to acute lymphocytic leukemia (ALL). Each year in the United States, approximately 10,000 persons develop AML and 3000 develop ALL.

AML is the most common type of acute leukemia in adults, accounting for 80% of new cases. AML is un-common in children. The incidence increases steadily with age, with a sharp increase after 45 years. ALL is the most common malignant disease affecting chil-dren, accounting for approximately 30% of all child-hood cancers.

ALL has a bimodal age distribution, peaking in children between 3 and 5 years of age and again in persons older than 65 years.

Acute leukemias can occur in all age groups

n     ALL is more common in children

n     AML is more common in adults

Chronic leukemias are usually a disease of adults

n     CLL is extremely rare in children and unusual before the age of 40

n     CML has a peak age of 30-50

Age Distribution: optimum ages for development:

1. ALL: 3‑4 years old

2. AML: 15‑20 years old

3. CLL: 50 years old

4. CML: 20‑50 years old

5. Monocytic: middle age (rarely before 30)

Comparison of acute and chronic leukemias:

Acute Chronic

Age all ages usually adults

Clinical onset sudden insidious

Course (untreated) 6 mo. or less 2-6 years

Leukemic cells immature >30% blasts more mature cells

Anemia prominent mild

Thrombocytopenia prominent mild

WBC count variable increased

Lymphadenopathy mild present; often prominent

Splenomegaly mild present; often prominent

Acute Leukemia

Is a result of:

Malignant transformation of a stem cell leading to unregulated proliferation and

arrest in maturation at the primitive blast stage. Remember that a blast is the most immature cell that can be recognized as committed to a particular cell line.

Clinical features

Leukemic proliferation, accumulation, and invasion of normal tissues, including the liver, spleen, lymph nodes, central nervous system, and skin, cause lesions ranging from rashes to tumors. Failure of the bone marrow and normal hematopoiesis may result in pancytopenia with death from hemorrhaging and infections.

Pathophysiology of the clinical manifestations of acute leukemias

1. Marrow failure due to infiltration

-anemia -fatigue, pallor,

-thrombocytopenia -bleeding, spontaneous bruising

-neutropenia-infections, sepsis

2. Infiltration of other organs

-liver, spleen, lymph nodes (particularly in ALL)

-lymphadenopathy

-hepatosplenomegaly

-mediastinal masses(T-ALL)

-gums gum hypertrophy (monocytic subtype of AML)

-bone pain, esp. in children with ALL

-any organ or tissue

-skin-leukemia cutis

-soft tissue -chloromas

-testis

-CNS

-solid organs

3. Leukostasis (only seen with WBC >>50x109 /L)

-CNS -strokes

-lungs -pulmonary infiltrates, hypoxemia

4. Constitutional symptoms

-fevers, sweats are common

-weight loss uncommon

other

-exposure of substances that can initiate coagulation can cause DIC

Lab evaluation

At the time of presentation, the leukocyte count in a patient with acute leukemia is generally elevated, but it may be normal or reduced. Fewer than 20% of patients have a leukocyte count greater than 100 ×103/mm3.

Peripheral blood smears show blasts in most cases.

Other notable findings include anemia and thrombocy-topenia; these result from the increased percentage of blasts in the bone marrow, which leaves little room for erythroid and megakaryocytic precursors. Severe throm-bocytopenia (fewer than 50 ×103platelets/mm3) is pres-ent in more than half of patients presenting with acute leukemia. Abnormal results of coagulation tests (ie, hypofibrinogenemia, elevated fibrin split products, defi-ciency of coagulation factors) are seen in patients pre-senting with signs and symptoms of DIC.

Serum electrolyte levels are typically normal in patients with acute leukemia. Lactate dehydrogenase and uric acid levels may be elevated due to rapid cell

turnover; this can lead to urate nephropathy and acute renal failure. Rapid lysis of tumor cells, especially when chemotherapy is instituted, can result in tumor lysis syndrome, manifested by hypocalcemia, hyperkalemia, hyperphosphatemia, and hyperuricemia.

DIAGNOSIS OF LEUKEMIA

The diagnosis of acute leukemia requires that blasts comprise 30 % or more of bone marrow cells or circu-lating white cells.

(For AML, the new WHO classifica-tion proposes to change this to 20 % blasts.) For differ-entiating ALL and AML, a bone marrow aspirate and

biopsy are necessary.

A peripheral blood smear may provide clues to the type of acute leukemia. Myeloblasts exhibit great vari-ability in size, abundant pale blue cytoplasm with

azurophilic (bluish) granules, and distinct nucleoli. The presence of Auer rods, appearing as pink strands within the cytoplasm of the myeloblast, is characteristic of AML. Lymphoblasts tend to be small, with scant cytoplasm and indistinct nucleoli.

Because it is extremely difficult to characterize leu-kemia as lymphoblastic or myeloid based on morpho-logic appearance of blasts, additional analyses of the

blasts are necessary, including cytochemical staining, phenotypic analyses via flow cytometry, and molecular

evaluation for chromosomal abnormalities (ie, cytogenetic studies).

The most important cytochemical stains for deter-mining lineage are myeloperoxidase, Sudan black B, and the esterases. Positive myeloperoxidase reaction or staining with Sudan black B in 3% or more of blast cells indicates myeloid origin. Acid phosphatase is present in early T cells, and demonstration of its activity can dif-ferentiate T-cell ALL from nonT-cell ALL. Generally, lymphoblasts stain with terminal deoxynucleotidyltrans-ferase (Tdt), although a small percentage of myelo-blasts may be positive as well.

Immunophenotyping by flow cytometry will con-firm the diagnosis of leukemia or establish the diagno-sis in cases in which morphology and cytochemical

stains are equivocal. Cytogenetic studies are important because two thirds of patients diagnosed with AML or ALL and 90 % of patients with secondary leukemia will have leukemic blasts showing clonal chromosomal abnormalities.

The chromosomal abnormalities differ between AML and ALL and among the various sub-types. Immunophenotyping and cytogenetic analyses

assist in risk stratification and provide information that has important clinical, prognostic, and treatment implications.

The lab diagnosis is based on two things:

1. Finding a significant increase in the number of immature cells in the bone marrow including blasts, promyelocytes, promonocytes (> 30 % blasts is diagnostic)

2. Identification of the cell lineage of the leukemic cells

Peripheral blood:

Anemia (normochromic, normocytic)

Decreased platlets

Variable WBC count:

The degree of peripheral blood involvement determines classification:

n     Leukemic increased WBCs due to blasts

n     Subleukemic blasts without increased WBCs

n     Aleukemic decreased WBCs with no blasts

Classification of the immature cells involved may be done by:

Morphology an experienced morphologist can look at the size of the blast, the amount of cytoplasm, the nuclear chromatin pattern, the presence of nucleoli and the presence of auer rods (are a pink staining, splinter shaped inclusion due to a rod shaped alignment of primary granules found only in myeloproliferative processes) to identify the blast type:

n     AML the myeloblast is a large blast with a moderate amount of cytoplasm, fine lacey chromatin, and prominent nucleoli. 10-40% of myeloblasts contain auer rods.

n     ALL in contrast to the myeloblast, the lymphoblast is a small blast with scant cytoplasm, dense chromatin, indistinct nucleoli, and no auer rods.

Cytochemistry

Cytochemistry help to classify the lineage of a leukemic cell (myeloid versus lymphoid). Myeloperoxidase is found in the primary granules of granulocytic cells starting at the late blast stage. Monocytes may be weakly positive.

Sudan black stains phospholipids, neutral fats and sterols found in primary and secondary granules of granulocytic cells and to a lesser extent in monocytic lysosomes.

Rare positives occur in lymphoid cells. Nonspecific esterase is used to identify monocytic cells which are diffusely positive. T lymphocytes may have focal staining.

Acid phosphatase may be found in myeloblasts and lymphoblasts. T lymphocytes have a high level of acid phosphatase and this can be used to help make a diagnosis of acute T-lymphocytic leukemia.

Leukocyte alkaline phosphatase is located in the tertiary granules of segmented neutrophils, bands and metamyelocytes. The LAP score is determined by counting 100 mature neutrophils and bands. Each cell is graded from 0 to 5. The total LAP score is calculated by adding up the scores for each cell.

The differential diagnosis of acute leukemia

The differential diagnosis of acute leukemia in-cludes other conditions in which patients present with an elevated leukocyte count, anemia, and thrombocy-topenia. These include leukemoid reactions and deep-seated infections, which may be associated with an ele-vated leukocyte count and a left shift. Infection with Epstein-Barr virus may cause severe lymphocytosis with atypical lymphocytes present on peripheral smear. The diagnosis can be made easily by the absence of blasts in these conditions.

Patients with acute leukemia may also present with low leukocyte counts together with anemia and throm-bocytopenia. The differential diagnosis of this presen-tation includes the primary bone marrow diseases of myelodysplastic syndrome and aplastic anemia. Infil-tration of the bone marrow by other neoplasms, in-cluding solid tumors and hematologic malignancies, may also present with anemia and/or thrombocytope-nia. Immature forms of leukocytes resembling blasts may be seen in severe megaloblastic anemia due to folate and vitamin B 12 deficiency.

Chronic Lymphocytic Leukemia

Neoplastic proliferation of small mature-appearing lymphocytes

>99 % are B-cell derived

Older patients (> 40)

Disease may be discovered incidentally

Fatigue, weakness, weight loss, anorexia, and/or recurrent infections may occur

Variable splenomegaly and non tender lymphadenopathy

Leukemic counterpart of small lymphocytic

Lymphoma

Lab diagnostics

Absolute lymphocytosis (> 4000/mm3)

Smudge cells

Variable anemia, neutropenia, and thrombocytopenia

Hypercellular bone marrow with lymphocytic infiltrates

Hypogammaglobulinemia

Elevated serum LDH

Chronic Myelocytic Leukemia (CML)

15-20 % of all leukemias

Young or middle-aged patients

Asymptomatic, fatigue, abdominal fullness, early satiety, weight loss, anorexia

Splenomegaly, bone pain, bone tenderness

Proliferation of myeloid cells in blood and bone marrow (mostly myelocytes polys)

Slow progression (3 yr survival without Rx)

Ph 1 chromosome in > 95 %

Bone Marrow in CML

Hypercellular bone marrow

High Myeloid : Erythroid Ratio

Myeloid hyperplasia

Relatively few blast cells

Mostly mature neutrophils

Increased basophils and eosinophils

Increased megakaryocytes

Multiple Myeloma

Multiple myeloma, also known as plasma cell myeloma or Kahler's disease, is a cancer of plasma cells, a type of white blood cell normally responsible for producing antibodies. In multiple myeloma, collections of abnormal plasma cells accumulate in the bone marrow, where they interfere with the production of normal blood cells. Most cases of myeloma also feature the production of a paraprotein an abnormal antibody which can cause kidney problems. Bone lesions and hypercalcemia (high calcium levels) are also often encountered.

Myeloma is diagnosed with blood tests (serum protein electrophoresis, serum free kappa/lambda light chain assay), bone marrow examination, urine protein electrophoresis, and X-rays of commonly involved bones.

The presence of unexplained anemia, kidney dysfunction, a high erythrocyte sedimentation rate (ESR), lytic bone lesions, elevated beta-2 microglobulin, and/or a high serum protein (especially raised globulins or immunoglobulin) may prompt further testing. The globulin level may be normal in established disease. A doctor will request protein electrophoresis of the blood and urine, which might show the presence of a paraprotein (monoclonal protein, or M protein) band, with or without reduction of the other (normal) immunoglobulins. One type of paraprotein is the Bence Jones protein which is a urinary paraprotein composed of free light chains. Quantitative measurements of the paraprotein are necessary to establish a diagnosis and to monitor the disease. The paraprotein is an abnormal immunoglobulin produced by the tumor clone. Very rarely, the myeloma is nonsecretory (not producing immunoglobulins).

In theory, multiple myeloma can produce all classes of immunoglobulin, but IgG paraproteins are most common, followed by IgA and IgM. IgD and IgE myeloma are very rare. In addition, light and or heavy chains (the building blocks of antibodies) may be secreted in isolation: κ- or λ-light chains or any of the five types of heavy chains (α-, γ-, δ-, ε- or μ-heavy chains).

Additional findings include: a raised calcium (when osteoclasts are breaking down bone, releasing calcium into the bloodstream), raised serum creatinine due to reduced renal function, which is mainly due to casts of paraprotein deposition in the kidney, although the cast may also contain complete immunoglobulins and albumin.

Cancer of plasma cells

Disease of older men and women (> 60 years)

Produce abundant useless monoclonal Ig (paraprotein, M-protein)

Decreased normal Ig, infections occur

Lytic bone lesions, bone pain, pathologic fractures, hypercalcemia

Tumorous masses of plasma cells (spine, skull, ribs, pelvis)

Renal failure may develop

Investigation of a patient with suspected myeloma should include the screening tests, followed by further tests to confirm the diagnosis. Electrophoresis of serum and concentrated urine should be performed, followed by immunofixation to confirm and type any M-protein present. Immunofixation and SFLC assessment are indicated in patients where there is a strong suspicion of myeloma but in whom routine serum protein electrophoresis is negative.

Quantification of serum M-protein should be performed by densitometry of the monoclonal peak on electrophoresis; immunochemical measurement of total

immunoglobulin (Ig) isotype level can also be used and is particularly useful for IgA and IgD M-proteins. Quantification of urinary total protein and light chain excretion can be performed directly on a 24-hour urine collection or calculated on a random urine sample in relation to the urine creatinine.

Quantification of SFLC levels is an additional tool for the assessment of light chain production. The serum tests are particularly useful for diagnosis and

monitoring of light chain only myeloma and patients with oligosecretory / non-secretory disease and in requests for which urine has not been sent to the laboratory. In renal impairment the half life and thus serum concentration of SFLC can increase ten fold and there is often an increased kappa: lambda ratio. A diagnosis of myeloma should be confirmed by bone marrow (BM) assessment. It is recommended that an adequate trephine biopsy of at least 20 mm in length be obtained in all patients as it provides a better assessment of the extent of marrow infiltration than aspirate smears. It is recommended that a diagnosis of myeloma be confirmed by the demonstration of an aberrant plasma cell phenotype and / or monoclonality. Plasma cell phenotyping may be performed by flow cytometry and / or immunohistochemistry on trephine sections. The European Myeloma Network have provided practical guidance on the optimal methods for flow cytometry.

Lymphogranulomatosis

Lymphogranulomatosis is a malignant tumor of lymphoid tissue with lesion of lymph nodes and organs, characterized by growth of the giant cells called Reed-Sternberg- Beresovsky cells, large and small atypical cells (Hodgkin,s cells) and inflammatory infiltration.

Hodgkin's lymphoma, also known as Hodgkin lymphoma and previously known as Hodgkin's disease, is a type of lymphoma, which is a cancer originating from white blood cells called lymphocytes.

Patients with Hodgkin's lymphoma may present with the following symptoms:

                    Lymph nodes: the most common symptom of Hodgkin's is the painless enlargement of one or more lymph nodes, or lymphadenopathy. The nodes may also feel rubbery and swollen when examined. The nodes of the neck and shoulders (cervical and supraclavicular) are most frequently involved (8090 % of the time, on average). The lymph nodes of the chest are often affected, and these may be noticed on a chest radiograph.

                    Itchy skin

                    Night sweats

                    Unexplained weight loss

                    Splenomegaly: enlargement of the spleen occurs in about 30 % of people with Hodgkin's lymphoma. The enlargement, however, is seldom massive and the size of the spleen may fluctuate during the course of treatment.

                    Hepatomegaly: enlargement of the liver, due to liver involvement, is present in about 5 % of cases.

                    Hepatosplenomegaly: the enlargement of both the liver and spleen caused by the same disease.

                    Red-coloured patches on the skin, easy bleeding and petechiae

 due to low platelet count (as a result of bone marrow infiltration, increased trapping in the spleen etc. i.e. decreased production, increased removal)

                    Systemic symptoms: about one-third of patients with Hodgkin's disease may also present with systemic symptoms, including low-grade fever; night sweats; unexplained weight loss of at least 10 % of the patient's total body mass in six months or less, itchy skin (pruritus) due to increased levels of eosinophils in the bloodstream; or fatigue (lassitude).

                    Cyclical fever: patients may also present with a cyclical high-grade fever known as the Pel-Ebstein fever or more simply "P-E fever". However, there is debate as to whether or not the P-E fever truly exist

Definitive diagnosis is by lymph node biopsy (usually excisional biopsy with microscopic examination). Blood tests are also performed to assess function of major organs and to assess safety for chemotherapy.

Bone Marrow Biopsy

Occasionally, a disease of the blood cell system cannot be diagnosed and classified on the basis of the blood count alone and a bone marrow biopsy is indicated. In such cases it is more important to perform this biopsy competently and produce good smears for evaluation than to be able to interpret the bone marrow cytology yourself.

Although the bone marrow cytology findings from the aspirate are sufficient or even preferable for most hematological questions, it is regarded as good practice to obtain a sample for bone marrow histology at the same time, since with improved instruments the procedure has become less stressful, and complementary cytological and histological data are then available from the start. After deep local anesthesia of the dorsal spine and a small skin incision, a histology cylinder at least 1.5cm long is obtained using a sharp hollowneedle (Yamshidi). A Klima and Rossegger cytology needle is then placed through the same subcutaneous channel but at a slightly different site from the earlier insertion point on the spine and gently pushed through the compacta. The mandrel is pulled out and a 5- to 10-ml syringe body with 0.5ml citrate or EDTA (heparin is used only for cytogenetics) is attached to the needle. The patient should be warned that there will be a painful drawing sensation during aspiration, which cannot be avoided. The barrel is then slowly pulled, and if the procedure is successful, blood from the bone marrow fills the syringe. The syringe body is separated from the needle and the mandrel reintroduced. The bone marrow aspirate is transferred from the syringe to a Petri dish. When the dish is gently shaken, small, pinhead-sized bone marrow spicules will be seen lying on the bottom. A smear, similar to a blood smear, can be prepared on a slide directly from the remaining contents of the syringe. If the first aspirate has obtained material, the needle is removed and a light compression bandage is applied. If the aspirate for cytology contains no bone marrow fragments (punctiosicca, dry tap), an attempt may be made to obtain a cytology smear from the (as yet unfixed) histology cylinder by rolling it carefully on the slide, but this seldom produces optimal results.

The preparation of the precious bone marrow material demands special care. One or two bone marrow spicules are pushed to the outer edge of the Petri dish, using the mandrel from the sternal needle, a needle, or a wooden rod with a beveled tip, and transferred to a fat-free microscopy slide, on which they are gently pushed to and fro by the needle along the length of the slide in a meandering line. This helps the analyzing technician to make a differentiatial count. It should be noted that too

much blood in the bone marrow sample will impede the semiquantitative analysis. In addition to this type of smear, squash preparations should also be prepared from the bone marrow material for selective staining. To do this, a few small pieces of bone marrow are placed on a slide and covered by a second slide. The two slides are lightly pressed and slid against each other, then separated. The smears are allowed to air-dry and some are incubated with panoptic Pappenheim staining solution (see previous text). Smears being sent to a diagnostic laboratory (wrapped individually and shipped as fragile goods) are better left unstained. Fresh smears of peripheral blood should accompany the shipment of each set of samples.

Cell composition in the bone marrow: normal values (%)

Median

Values (J. Boll)

Median values and normal

Range (K. Rohr)

Red cell series

Proerythrocytes

1

Macroblasts (basophilic erythroblasts)

3

3,5

0,5 7,5

Normoblasts (poly- and orthochromic erythroblasts)

16

19

7 40

Neutrophil series

Myeloblasts

2.7

1

0,5 5

Promyelocytes

9.5

3

0 7,5

Myelocytes

14

15

5 25

Metamyelocytes

10.5

15

5 20

Band neutrophils

9.8

15

5 25

Segmented neutrophils

17.5

7

0,5 15

Small cell series

Eosinophilic granulocytes

5

3

17

Basophilic granulocytes

1

0,5

0 1

Monocytes

2

2

0,5 3

Lymphocytes

6

7,5

2,5 15

Plasma cells

1,5

1

0,5 3

Megakaryocytes

Cell densities vary widely, 0.52 per view field during screening at low magnification.

Bone Marrow: Medullary Stroma Cells

1.    Fibroblastic reticular cells form a firm but elastic matrix in which the blood-forming cells reside, and are therefore rarely found in the bone marrow aspirate or cytological smear. When present, they are most likely to appear as dense cell groups with long fiber-forming cytoplasmic processes and small nuclei. Iron staining shows them up as a group of reticular cells which, like macrophages, have the potential to store iron. If they become the prominent cell population in the bone marrow, an aplastic or toxic medullary disorder must be considered.

2.    Reticular histiocytes (not yet active in phagocytosis) are identical to phagocytic macrophages and are the main storage cells for tissue bound iron. Because of their small nuclei and easy-flowing cytoplasm, they are noticeable after panoptic staining only when they contain obvious entities such as lipids or pigments.

3.    Osteoblasts are large cells with wide, eccentric nuclei. They differ from plasma cells in that the cytoplasm has no perinuclear lighter space (cell center) and stains a cloudy grayish-blue. As they are normally rare in bone marrow, increased presence of osteoblasts in the marrow may indicate metastasizing tumor cells (from another location).

4.    Osteoclasts are multinucleated syncytia with wide layers of grayish-blue stained cytoplasm, which often displays delicate azurophilic granulation. They are normally extremely rare in aspirates, and when they are found it is usually under the same conditions as osteoblasts. They are distinguished from megakaryocytes by their round and regular nuclei and by their lack of thrombocyte buds.

Lymph Node Biopsy

These procedures, less invasive than bone marrow biopsy, are a simple and often diagnostically sufficient method for lymph node enlargement or other intumescences. The unanesthetized, disinfected skin is sterilized and pulled taut over the node. A no. 1 needle on a syringe with good suction is pushed through the skin into the lymph node tissue. Tissue is aspirated from several locations, changing the angle of the needle slightly after each collection, and suction maintained while the needle is withdrawn into the subcutis. Aspiration ceases and the syringe is removed without suction. The biopsy harvest, which is in the needle, is extruded onto a microscopy slide and smeared out without force or pressure using a cover glass (spreader slide). Staining is done as described previously for blood smears.

Normal lymphadenogram

Cells

Contents, %

Lymphoid reticular cells

0-0,8

Plasma cells

0,2-0,6

Macrophages

0,1-0,5

Lipophages

0-0,1

Lymphoblasts

0,1-0,5

Prolymphocytes

65-80

Lymphocytes

20-35

Neutrophils

3-10

Eosinophils

0-0,5