Bone Marrow Interpretation for Hematologic Diagnosis

Introduction

Ways to approach a hematologic diagnosis: CBC Þ Leukocyte differential Þ microscopic blood smear Þ bone marrow aspirate Þ bone marrow biopsy.

Reasons to do a bone marrow biopsy include: anemia of unknown cause, leukopenia, leukocytosis with immature granulocytes and/or blasts in the blood, occurrence of unusual cells in blood (dwarf megakaryocytes, thrombocytopenia, and marked thrombocytosis.

Bone marrow sample is usually taken from the posterior iliac crest

Disorders in which a bone marrow aspirate may be diagnostic of a specific disease: Involvement of bone marrow by malignant lymphoma or Hodgkin’s disease, acute leukemia, chronic granulocytic leukemia, CLL, macroglobulinemia, multiple myeloma, aplastic anemia, myelofibrosis, myelophthistic anemia, fungal or mycobacterial involvement of marrow, osteoporosis, storage diseases, amyloidosis, mastocytosis, myelodysplastic disorders, megaloblastic anemia, iron deficiency anemia, and sideroblastic anemia.

Normal Bone Marrow

Cellularity

Hypocellular - excessive amounts of fat cells, multiple clear areas under the low power.

Normocellular - approximately 50-70% hematopoietic cells and 30-50% fat.

Hypercellular - 80-100% cells and little fat.

Megakaryocytes – large size and dark coloration under low power. Nuclei are multilobular rather than multinuclear (osteoclasts). Size and number of lobes is determined by the magnitude of the platelet count in the peripheral blood, in a negative feedback mechanism. The cytoplasm is dark and volumnious in mature megakaryocytes. Under higher power, cytoplasm contains multiple sized granules.

Erythroid Series - Proerythroblasts or pronormoblasts are the earliest recognizable erythroid precursors. Large cells with prominent nucleoli and deeply basophilic cytoplasm. Intermediate normoblasts display darkly stained aggregates of chromatin, and polychromatophilic cytoplasm, indicating hemoglobin.

Granulocytic Series

Primitive appearing mononuclear cells (blasts) are regarded as stem cells or precursors of other hematopoietic cells

Promyelocyte is the earliest recognizable granulocyte containing one or more nucleoli, basophilic cytoplasm with azurophilic granules or lysosomes. As it matures, nucleus becomes smaller and indented, and cytoplasm becomes more voluminous.

Myelocytes show round nuclei, and cytoplasm containing granules which stain yellow-orange in Wright’s stain

Metamyelocyte and Band – additional ß in nuclear size and Ý indentation

Neutrophils are the final maturational stage in development.

Cells of the eosinophilic series have a maturational sequence identical to the neutrophilic series.

Other cell types seen: plasma cells – small nucleus, basophilic cytoplasm; fibroblasts – spindle shaped, stromal cells, and macrophages – scavenger cells of bone marrow.

Pancytopenia

reduction in erythrocytes, granulocytes, and platelets in the peripheral blood

May be caused by drugs, aplastic anemia, hypersplenism (overactive spleen), infiltrative disorder of bone marrow, replacement of marrow by fibrosis, myelodysplastic disorder, refractory anemia, or leukemia.

Pancytopenia secondary to drug effect: virtually any drug, combination or drugs or volatile solvents can cause pancytopenia in susceptible individuals

frequently seen following chemotherapy
Bone marrow findings range from a maturation arrest of developing cells leading to reduction of mature cells in the blood, to hypoplasia and even aplasia of the marrow

Pancytopenia secondary to aplastic anemia: causes reduction in all formed elements of the blood

bone marrow is markedly hypocellular and consists largely of the supporting network of the marrow comprised of fibroblasts, connective tissue, scattered lymphocytes, mast cells, fibroblasts, and plasma cells, and fat cells
in severe cases, few if any normal hematopoietic cells are present

Pancytopenia secondary to hypersplenism: enlarged spleen sequesters and destroys formed elements of the blood, leading to isolated cytopenias, such as thrombocytopenia and/or anemia, or to pancytopenia

intense erythroid hyperplasia is seen on bone marrow biopsy as a compensatory mechanism for peripheral erythrocyte destruction by the enlarged spleen, a partial maturation block of cells of the neutrophilic granulocytic series at the band stage of development, and increased numbers of megakaryocytes as a compensatory phenomenon resulting from increased peripheral destruction of platelets by the enlarged spleen
diseases causing hypersplenism: Felty’s syndrome, congestive heart failure, cirrhosis of the liver with portal hypertension and splenomegaly, Gaucher’s disease, many others

Pancytopenia resulting from an infiltrative disorder of the bone marrow, such as lymphomatous involvement of the marrow, a plasma cell dyscrasia like multiple myeloma, or metastatic carcinoma: bone marrow is usually hypercellular and all three major cell lines are represent

myeloma cells appear pleomorphic (variable in size and shape) with prominent nucleoli and abundant basophilic cytoplasm
pale staining cells represent myeloma; Spoke of the wheel pattern

Hairy cell leukemia, B cell lymphoproliferative disorder often characterized by pancytopenia and "dry tap on bone marrow aspiration"

Metastatic carcinoma can stain with acid phosphatase stain.

Pancytopenia resulting from replacement of the marrow by fibrosis, as seen in advanced cases of myelofibrosis: due to replacement of normal marrow areas by fibrous connective tissue (hypersplenism plays a role too, see above)

bone marrow aspirates are usually not obtainable because of the extensive fibrosis, and a "dry tap" results. Imprints should always be made from the biopsy core in cases where "dry taps" occur
microscopically, the actual areas of fibrosis are patchy and scattered, along with increased numbers of megakaryocytes. As the disease advances, megakaryocytes decrease in number, and the extent of fibrosis increases and eventually replaces the normal marrow

The stain (silver impregnation) for reticulin is strongly positive

tailed poikilocytes (tear drop cells) are often found in the peripheral blood of patients with myelofibroisis
dense fibrosis often display proliferation of hematopoitic cells including megakaryocytes with distended nucleoli.

Pancytopenia resulting from an infiltrative disorder of the bone marrow as found in a myelodysplastic disorder such as refractory anemia with ring sideroblasts: bone marrow aspirate and biopsy are markedly hypercellular and demonstrate intense erythroid hyperplasia

Granulocytic cells like neutrophils are often decreased, and megakaryocytes are either increased or decreased and appear atypical
using the Prussian blue stain for the ferric (Fe 3+) hydroxide oxidation state, iron appears as bright blue dots surrounding the nuclei of intermediate normoblasts, a maturational state where hemoglobin synthesis is manifest microscopically by polychromasia. Ferric iron is deposited in the lamellae and cristae of mitochondria.
It has been postulated that the iron may act as a mitochondrial poison, causing reduction in activities of certain enzymes involved in hemoglobin synthesis, such as heme synthase and delta-ALAase. Macrophages are also found in marrow of patients with this disorder. Macrophages often contain hemosiderin and phagocytized erythrocytes, providing cytological evidence for ineffective erythropoiesis and accelerated intramedullary cell death.

Pancytopenia caused by refractory anemia with excess blasts, a myelodysplastic disorder: bone marrow shows intense erythroid hyperplasia, variable numbers of ring sideroblasts, and 20-30% myeloblasts (contain delicate nuclear chromatin strands, few chromatin aggregates, and prominent nucleoli with perinucleolar condensations of chromatin)

sometimes activity of myeloperoxidase can be identified in blasts from an aspirate of bone marrow

Sudan black B can be used to demonstrate granulocytic properties in some leukemic blasts

As the percentage accelerates, the number of blasts increases in the bone marrow. When the number of blasts reaches 20-30%, the disorder has been called refractory anemia with excess blasts in transformation – evolution into acute leukemia is imminent.

Pancytopenia secondary to an infiltrative disorder like acute leukemia: bone marrow is largely replaced by leukemic blasts (often resulting in pancytopenia); so many blasts that appear as large and small mononuclear cells with prominent nucleoli and irregular nuclear contours

to determine the type of chemotherapy, cytological type of leukemic blast be identified as precisely as possible; most of the time morphologic features are insufficient to determine the origin of the blast, so must utilize techniques that reveal properties of cytoplasmic constituents, such as granules or lysosomes, and techniques that reveal properties of the cell surface, or surface markers, as determined by flow cytometry

Cytochemical stains of potential diagnostic usefulness in bone marrow interpretation

PAS (periodic acid-Schiff)	A stain for glycogen in leukemic lymphoblasts and in malignant erythroblasts in erythroleukemia. ALL, erythroleukemia.
Myeloperoxidase	May be positive in leukemic myeloblasts and promyelocytes. Myeloblastic origin. AML
Sudan Black B	May be positive in leukemic myeloblasts and promyelocytes. Granulocytic origin.
Acid phosphatase	Unipolar in T-cell convoluted lymphoblasts. Tartrate-resistant activity in hairy cells in hairy cell leukemia. Myeloblastic origin.
Nonspecific esterase	Using alpha-naphthyl butyrate as substrate, intense activity that is fluoride sensitive in leukemic monocytes. AML (M4)

Flow cytometry using a panel of immunologic reagents such as CD4, CD13, and CD34, to name only a few, is essential for the full evaluation of leukemic blasts. Cytogenetic studies are also important in evaluating leukemic blasts in patients with acute leukemia, since certain types of acute leukemia are consistently identified with specific kinds of cytogenetic abnormalities.

Myelodysplastic Disorders and Acute Leukemia

As a part of the FAB classification of preleukemic disorders, it was recognized that bone marrows from afflicted individuals displayed a panmyelopathy, that is, a disturbance of all three major cell lines in the bone marrow, namely, erythroblastic, granulocytic, and megakaryocyte lines. As a result, the name MYELODYSPLASTIC DISORDERS was proposed, and became a useful concept for those dealing with patients having these conditions, and the likelihood of evolution into acute leukemia

Refractory anemia - disorder characterized by anemia refractory to or unresponsive to hematologic agents used alone or in combination

Peripheral Blood Smear: red blood cells with variation in size (anisocytosis) and in shape (poikilocytosis)

Bone Marrow: intense erythroid hyperplasia

ineffective erythropoiesis – red blood cell precursors (erythroblasts) proliferate at a high rate in the bone marrow while few actually mature into erythrocytes; intramedullary cell death occurs, leading to anemia

Risk of Developing Acute Leukemia: less than 5%.

Refractory anemia with ring sideroblasts

Perpheral Blood Smear: variability in size and shape of erythrocytes, as well as pancytopenia, or a reduction in all of the formed elements of the blood (erythrocytes, granulocytes, and platelets)

Bone Marrow: intense erythroid hyperplasia, and often contain morphologically abnormal megakaryocytes; Characteristically, erythroblasts containing hemoglobin as manifested by polychromasia contain mitochondria laden with ferritin, and arranged in a ring or necklace around the nucleus. Iron in these cells is visualized with the Prussian blue stain, and acidified solution of potassium nitroferricyanide. Ineffective erythropoiesis is present, ferritin values are unusually high, and patients are often transfusion dependent

Risk of Developing Acute Leukemia: 5-7%.

Refractory anemia with excess blasts – believed to represent acute leukemia in a slowly developing or indolent form

Peripheral Blood Smear: displays variation in size and shape of erythrocytes, and usually variable degrees of leukemic blasts

Bone Marrow: demonstrates intense erythroid hyperplasia, and usually less than 20% blasts

Risk of Developing Acute Leukemia: 70-90%.

Refractory anemia with excess blasts in transformation

Peripheral Blood Smear: variation in size and shape of erythrocytes, as well as variable degrees of leukemic blasts and thrombocytopenia

Bone Marrow: intense erythroid hyperplasia, and between 20-30% blasts

Risk of Developing Acute Leukemia: 100%; Evolution into acute leukemia is usually imminent.

Chronic myelomonocytic leukemia – regarded as an indolent form of myelomonocytic leukemia; evolution into acute leukemia occurs weeks to months after diagnosis.

Peripheral Blood Smear: monocytosis, variation in size and shape of erythrocytes, thrombocytopenia

Bone Marrow: contains greater than 30% monocytes, as well as atypical appearing erythroblasts and megakaryocytes.

NOTE: Many authors also believe that the evolution of myelodysplastic disorders into acute leukemia occurs in the following sequence: refractory anemia with ring sideroblasts Þ refractory anemia with excess blasts Þ refractory anemia with excess blasts in transformation Þ acute leukemia. Erythroleukemia may be interposed in this sequence, particularly when it is preceded by a pure erythroblastic disorder such as acute erythremic myelosis.

FAB classification of ACUTE LEUKEMIAS

Divided into acute lymphoblastic leukemias and acute myeloblastic leukemias and their variants.

For acute lymphoblastic leukemia:

L1 - lymphoblasts that are small and more or less uniform in size and shape.

L2 - considerable pleomorphism of blasts, and many contain prominent nucleoli.

L3 - Burkitt form of lymphoblastic leukemia, in which leukemic blasts show prominent cytoplasmic vacuoles that stain positively with Oil red 0, marked leukocytosis is present, and a mediastinal mass is often found.

In acute myeloblastic leukemias:

M1 - uniform appearing population of leukemic blasts that demonstrate little if any differentiation beyond the blast stage.

M2 - differentiation beyond the blast stage is found, and metamyelocytes, myelocytes, and promyelocytes occur in the bone marrow.

M3 - acute promyelocytic leukemia, in which leukemic blasts contain multiple Auer rods and granules, and a 15;17 chromosomal translocation occurs.

M4 - monocytoid blasts are seen along with monocytes. The stain for nonspecific esterase using alpha naphthyl butyrate as substrate is strongly positive.

M4 with increased eosinophils - monocytosis of the bone marrow, along with eosinophilia, and the occurrence of abnormal cells containing eosinophilic and basophilic granules in the same cell. In this variant, there is usually an inverted chromosome 16.

M5 - monocytosis occurs and the cells are strongly positive for fluoride sensitive nonspecific esterase.

M6 - erythroleukemia, and bone marrows contain a more or less equal admixture of abnormal appearing erythroblasts and myeloblasts. Erythroleukemia often evolves into acute myeloblastic or myelomonocytic leukemia.

For the diagnosis of acute leukemia, a number of diagnostic tools are usually required. These include microscopic evaluation of a Wright stained smear of bone marrow particles and peripheral blood, H and E stained sections of particles and bone marrow biopsy core, a variety of cytochemical stains including myeloperoxidase, Sudan black B, PAS (periodic acid-Schiff), and acid phosphatase (see table above). For most types of acute leukemia, flow cytometry and cytogenetic studies are mandatory.