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REACTIVE MORPHOLOGIC CHANGES IN MONOCYTES Morphology Associated with Thin and band-like, or segmentation of nucleus; increased Infection, recovery from bone marrow aplasia,...

REACTIVE MORPHOLOGIC CHANGES IN MONOCYTES Morphology Associated with Thin and band-like, or segmentation of nucleus; increased Infection, recovery from bone marrow aplasia, and granulocyte cytoplasmic volume and granulation, and/or evidence of monocyte colony stimulating factor (GM-CSF) administration phagocytic activity (cytoplasmic vacuolation, intracellular debris, and irregular cytoplasmic borders) MORPHOLOGIC CHANGES IN REACTIVE LYMPHOCYTES Heterogeneous population of various shapes and sizes Cells exhibit increased amount of variably basophilic cytoplasm Lymphocyte population exhibits variation in nuclear/cytoplasmic ratio and/or nuclear shape. Chromatin is usually clumped however some cells may demonstrate less mature (less clumped) pattern. Nucleoli may be visible. The cytoplasm may be indented by surrounding RBCs LEUKEMOID REACTION ▪ Refers to a reactive leukocytosis above 50 x 109/L with neutrophilia and a marked left shift (presence of immature ▪ neutrophilic forms) Leukemoid reactions are mostly a result of acute and chronic infection, metabolic disease, inflammation, or response to ▪ a malignancy It may be confused with CML LAB FINDINGS: a. Increase WBC count b. Left shift of the WBC (Presence of increase IMMATURE WBC in blood) c. Increase LAP scores d. Presence of toxic granulations and Dohle bodies in WBC LEUKEMOID REACTION CHRONIC MYELOGENOUS LEUKEMIA A response or reaction to infection or malignancy A form of malignancy in blood High WBC count High WBC count (malignant cells) Increase LAP DECREASE LAP Left shift Left shift Presence of Dohle bodies and toxic granules Presence of Auer rods ABSENCE OF AUER RODS PRESENCE OF PHILADELPHIA CHROMOSOME ABSENCE OF PHILADELPHIA CHROMOSOME MALIGNANT LEUKOCYTE DISORDERS LEUKEMIA Virchow was the first to recognize leukemia as a distinct clinical disorder between 1839 and 1845. He named this disorder leukemia because of the white appearance of the blood from patients with fever, weakness, and lymphadenopathy Abnormal, uncontrolled proliferation and accumulation of one or more of the hematopoietic cells Major symptoms of leukemia are fever, weight loss, and increased sweating. Enlargement of the liver, spleen and lymph nodes may occur more predominantly in chronic leukemias. The basic metabolic rate is often elevated, and there may be hemorrhagic tendencies. If marked thrombocytopenia is present. Bone pain from a large leukemia cell mass in the bone marrow is typical in the acute leukemias. Differentiating Luekemia From Other Malignant Leukocyte Disorders Leukemia A disease, usually of leukocytes, in the blood and bone marrow. Overproduction of various types of immature or mature leukocytes in the bone marrow and/ or peripheral blood Lymphoma General term for malignancy that starts in the lymph system, mainly the lymph nodes. Two main types of lymphomas are Hodgkin lymphoma and non-Hodgkin lymphoma Myeloma A form of cancer of the plasma cells. In myeloma, the cells overgrow, forming a mass or tumor that is located in the bone marrow. Classification Duration Acute leukemia = days to 6 months Subacute leukemia = 2 to 6 months Chronic leukemia = 1 or 2 years or more I.K Aytona Page 76  Number of white blood Aleukemic leukemia = WBC ct. 30 % blast 30% blast >30% granulocytic cells M3-ACUTE PROMYELOCYTIC LEUKEMIA >30% blast >10% granulocytic cells 30% or >50 % promyelocytes M4- ACUTE MYELOMONOCYTIC LEUKEMIA 20 to 80% monoblast M5b- ACUTE MONOBLASTIC LEUKEMIA WITH MATURATION >80% monocytic cells 30 % blast >50% erythrocytic precursors M7-ACUTE MEGAKARYOCYTIC LEUKEMIA >30% blast >30% megakaryocytic cells I.K Aytona Page 78  M1 FAB M1 is characterized by either a rapid or gradual onset that may resemble an acute infection. The patient may have a history of fever, infections, fatigue, and bleeding episodes. Physical examination may reveal tenderness of the bones, particularly the ribs and sternum; ulcerated mucous membranes; petechiae; and purpura. Additional physical findings may include hepatomegaly, splenomegaly, and lymphadenopathy. The outstanding feature of the peripheral blood smear and bone marrow is the predominance of myeloblasts. These blasts usually have a regular cytoplasmic outline and may contain slender, red-staining Auer rods in the cytoplasm. The nuclear chromatin is very fine and homogeneous associated with CHLOROMA = localized tumor masses consisting of myeloblasts. In these tumors, the presence of large quantities of the enzyme MPO produces a green appearance if the tissue is cut Hemorrhagic manifestations such as easy bruising, epistaxis, gingival bleeding, and M2 petechiae are common initial symptoms. Hepatomegaly, splenomegaly, and lymphadenopathy are seen infrequently Myeloblasts predominate on peripheral blood smears. The nuclei are usually round or oval with one or more prominent nucleoli and fi ne reticular chromatin. The cytoplasm is basophilic with a variable number of azurophilic granules. Auer rods are commonly seen Fatigue and symptoms of bleeding such as bruising, hematuria, and M3 petechiae are common. Hepatomegaly, splenomegaly, and lymphadenopathy are seen infrequently. Appears to be the most aggressive of acute leukemia with a severe bleeding tendency and a fatal course Promyelocytes are the predominating cell type. The promyelocytes may be hypergranular, microgranular, or hypogranular variations. Coarsely granular promyelocytes with dumbbell-shaped or bilobed nuclei may be seen. The nuclear chromatin is finely reticular and the cells often lack nucleoli M3 is characterized by a balanced reciprocal translocation between chromosomes 15 and 17, which results in the fusion between PML gene and retinoic acid receptor a (RARA) It is associated with DIC This form of leukemia may also be referred to as Naegeli type monocytic leukemia. Occurrence of this form of leukemia is uncommon in children and young adults. The highest frequency of occurrence is in adults older than 50 M4 years of age Symptoms of this form of leukemia are similar to those of other forms of acute leukemia. Pharyngitis may be observed. Gingival hyperplasia due to leukemic infiltration may be noted Associated with leukostasis together with M5 Leukostasis refers to a pathological finding of slightly dilated, thin-walled vessels filled with leukemic cells. The brain and lungs are the most commonly involved organs. Symptoms of leukostasis are headache, visual impairment, and shortness of breath. In FAB M4 proliferation of granulocytes and monocytes is characteristic. Also known as Schilling type M5 The onset of this form of leukemia is dramatic, with headaches and fevers being the chief complaints. Typical symptoms of monocytic leukemia additionally include fatigue, weight loss, and bleeding from the mouth or nose. Physical examination frequently reveals gingival (mouth and gums) hyperplasia, as in myelomonocytic leukemia; pallor; and skin lesions Monocytes and promonocytes constitute 25% to 75% of the nucleated cells. Blasts frequently have a muddy or smoggy gray-blue cytoplasm containing tiny granules, and pseudopods are common. The nucleus has a reticular granular chromatin pattern and may contain from one to five large nucleoli Acute erythroid leukemia. This form of leukemia, also referred to as erythemic myelosis or Di Guglielmo syndrome M6 represents a proliferation of both immature granulocytic and erythrocytic cells. Erythroblasts on blood smears typically have an irregular outline with a high nuclear-cytoplasmic ratio Blasts of myeloid origin may have Auer rods Promyelocytes may also be present as well as monocytes and promonocytes. I.K Aytona Page 79  M7 In this form of acute leukemia, 50% or more of the blasts are of megakaryocyte lineage. Organomegaly is infrequent except in children. Radiographic evidence of bone lytic lesions has been observed in children Immunophenotyping reveals that megakaryoblasts express one or more of the platelet glycoprotein: CD41 or CD61. Blasts are negative with anti-MPO antibody. CYTOCHEMISTRY / CYTOCHEMICAL STAINS Differentiating AML from ALL STAIN AML ALL MPO SBB TDT MPO reactions and Sudan black B reactions frequently PARALLEL to each other STAIN PURPOSE SPECIMEN Leukocyte alkaline Stains ALP, present in the neutrophil and to a small degree, in Fresh capillary blood phosphatase certain B cells. Alternatively, blood may be collected Helpful in differentiating chronic myelogenous from a using heparin as anticoagulant. leukemoid reaction or polycythemia vera Peroxidase Stains peroxidases present in the granulocytes and monocytes Fresh blood smears made from capillary Used to differentiate acute myelogenous and monocytic blood are recommended, or use of fresh leukemia from acute lymphocytic leukemia whole blood anticoagulated with EDTA or heparin Sudan Black B Stains lipids present in the monocytes and granulocytes Air dried bone marrow smears Used to differentiate acute myelogenous and myelomonocytic leukemias from acute lymphocytic leukemia Chloroacetate Stains esterases present in the granulocytes Air dried blood or bone marrow smears. esterase Used to differentiate granulocytic cells from monocytic cells Blood anticoagulated with EDTA or heparin may also be used Non specific esterase Stains esterases present in the monocytic cells, macrophages, Air-dried blood or bone marrow smears. megakaryocytes and platelets. Blood anticoagulated with EDTA or Used to differentiate monocytic leukemias from granulocytic heparin may also be used leukemias Periodic acid shiff Stain mucoproteins, glycoproteins, and high molecular weight Air dried blood or bone marrow smears carbohydrates normally present in almost all blood types except pronormoblast Used to help in the diagnosis of DiGugleilmo’s syndrome and may be an aid, when used in conjunction with other stains, to classify some acute leukemias Acid phosphatase Stains ACP present in the myelogenous cell, lymphocytes, Air-dried blood or bone marrow smears, plasma cells, monocytes and platelets or use blood anticoagulated with heparin Using L (+) tartaric acid, the stain is helpful in diagnosing hairy cell leukemia NAME TYPE CONSTITUENTS STAINED INTERPRETATION IMPORTANT INFORMATION Myeloperoxidase Enzyme Marker for primary granules of Peroxidase activity produces MPO enzyme auer rods dark brown granules in deteriorates cytoplasm of granulocytes and Stain should be done monocytes only on fresh specimens Sudan Black B Non-enzyme Marker for phospholipids and Dark purple black granules in Can be done on stored lipids neutrophil precursors specimens Lymphoblasts are negative Parallel MPO For interpretation Terminal Enzyme DNA polymerase Is present in 90% cases of Used to differentiate deoxyribonucleotidyl immunoperoxidase ALL AML to ALL transferase I.K Aytona Page 80  Periodic Acid Schiff Non-enzyme Marker for glycogen, Activity results in bright Lymphoblastic leukemia glycoproteins, mucoproteins fuchsia pink or magenta red shows blocky or chunky and high molecular weight Pattern of staining varies with pattern carbohydrates each cell type L1 and L2 block pattern Erythroblasts in M6 leukemia is positive Naphthol AS-D Enzyme Marker for mature and Enzyme activity results in Known as specific chloroacetate immature neutrophils and bright red granules in esterase Esterase mast cells cytoplasm of neutrophils, Stable enzyme that neutrophil precursors and lasts for months mast cells Alpha- napthyl Enzyme Marker for monocytes, Monocytic stain red-brown Known as nonspecific acetate esterase megakaryocytes, and plasma esterase cells Alpha- naphthyl Enzyme Identifying monocytes, Enzyme activity results in dark Known as nonspecific Butyrate esterase promonocytes, and red precipitates in cytoplasm esterase monoblasts Acid phosphatase Enzyme Present in all hematopoietic Activity is indicated by purple Cannot be stored cells and found on lysosomes to red granules Marker for hairy cell leukemia Tartrate resistant acid Enzyme Activity is indicated by purple Excellent marker for phosphatase to dark red granules in hairy cell leukemia cytoplasm Leukocyte alkaline Enzyme Neutrophils is the only 100 cell count is done. Used to differentiate Phosphatase (LAP) leukocyte that contain this Neutrophils are scored from CML from a leukamoid activity with no activity to 4 with a reaction large amount of activity Toluidine Blue Non-enzyme Binds with acid Strongly metachromatic Useful for recognition of mucopolysaccharides in blood mast cells and tissue cells basophils CELL RATING AMOUNT (%) SIZE OF GRANULES STAIN INTENSITY 0 none - None 1+ 50 small Faint to moderate 2+ 50 t0 80 small Moderate to strong 3+ 80 to 100 Medium to large Strong 4+ 100 Medium to large Brilliant LAP STAIN SCORING (Turgeon) LAP is an enzyme found in the membranes of secondary granules of neutrophils. To perform the LAP a blood film is incubated with a naphthol-phosphate substrate and diazo dye at an alkaline pH. The LAP enzyme hydrolyzes the substrate, and the liberated naphthol reacts with the dye, producing a colored precipitate on the granules. The slide is examined microscopically and 100 segmented neutrophils and bands are counted and rated from 0 to 41 based on the intensity of the staining. The LAP score is calculated by multiplying each score by the number of cells, and adding the products. For example, 5 cells with 4+ staining, 5 cells with 3+, 25 cells with 2+, 45 cells with 1+, and 20 cells with 0 staining calculates to a LAP score of 130 Because scoring is subjective, the mean score of two examiners is reported, and they should agree within 10%. Sample reference interval for the LAP score is 15 to 170, but every laboratory establishes its own. I.K Aytona Page 81  OTHER LYMPHOPROLIFERATIVE DISORDERS: LEUKEMIA, LYMPHOMA, NEOPLASM AND MYELOMA Hairy Cell leukemia Hairy cell leukemia is characterized by small B lymphocytes with abundant cytoplasm and fine (“hairy”) cytoplasmic projections. The postulated cell of origin is the peripheral B cell of post– germinal center stage (memory B cell). More common in males than in women Clinical findings: fatigue, anemia, leukocytopenia, thrombocytopenia, splenomegaly, and marrow fibrosis. Pancytopenia is common. Bleeding and infection can be present. The cytochemical features of HCL include a strong acid phosphatase reaction that is not inhibited by tartaric acid or tartrate-resistant acid phosphatase (TRAP) stain. Mantle cell Mantle cell lymphoma is a lymphoproliferative disorder characterized by medium-sized lymphoid Lymphoma cells with irregular nuclear outlines derived from the follicular mantle zone lymph nodes show a replacement of normal nodal architecture with a diffuse proliferation of monotonous, medium-sized lymphoid cells with irregular nuclear outlines Follicular Lymphoma Follicular lymphoma originates from germinal center B cells and in most cases recapitulates follicular architecture. Numerous closely spaced follicles replace the normal nodal architecture Burkitt Lymphoma Burkitt lymphoma is characterized by medium- sized, highly proliferating lymphoid cells with basophilic vacuolated cytoplasm The lymphoid proliferation is diffuse and at low magnification shows a prominent “starry sky” pattern imparted by numerous tangible body macrophages The WHO classification lists three variants of this lymphoma: endemic (occurring predominantly in Africa), sporadic, and immunodeficiency associated. Mycosis fungoides & Mycosis fungoides is the most common cutaneous lymphoma Sezary syndrome It affects T lymphocyte Sezary cells -composed of small to medium-sized lymphoid cells with irregular nuclear outlines (cerebriform nuclei). Sézary syndrome is by definition a disseminated disease with leukemic presentation and skin and lymph node involvement I.K Aytona Page 82  Hodgkin’s lymphoma Malignant lymphoma but differs in that the cells reacting to the neoplasm predominant rather than the neoplastic cells themselves Distinguished from other lymphomas by the presence of REED-STENBERG CELLS It is divided into 2 broad categories: CLASSICAL HODGKIN and NODULAR LYMPHOCYTE PREDOMINANT HODGKIN NODULAR LYMPHOCYTE PREDOMINANT HODGKIN= a B cell neoplasm composed of relatively rare neoplastic cells/ “Popcorn cells” that is scattered within the nodules of reactive lymphocytes Rye classification of Hodgkin = based to histologic appearance of the involved tissue from lymph node biopsy Ann Arbor classification of Hodgkin = Most widely used staging scheme, depends on histologic type and the extent of tissue involvement MYELOPROLIFERATIVE DISORDERS/NEOPLASMS The MPNs are interrelated clonal hematopoietic stem cell disorders characterized by excessive proliferation of one or more mature myeloid cell lines, for example, granulocytes, erythrocytes, megakaryocytes, or mast cells. Each MPN is characterized by the clonal expansion of one or more myeloid cell lines, but one cell line dominates. The MPNs have the propensity to transform into other MPNs or progress into acute leukemias (ALs). Myeloproliferation largely is due to hypersensitivity or independence of normal cytokine regulation resulting from genetic mutations that reduces cytokine levels through negative feedback systems normally induced by mature cells All of the MPNs involve dysregulation at the multipotent hematopoietic stem cell (CD34), with one or more of the following shared features: Cytogenetic abnormalities, Overproduction of one or more types of blood cells with, dominance of a transformed clone, Hypercellular marrow or marrow fibrosis, Thrombotic and/or hemorrhagic bleeding, Extramedullary hematopoiesis, Transformation to acute leukemia TYPES OR CATEGORIES Chronic Myelogenous Leukemia Stem cell disorder affecting the granulocytic, monocytic, erythrocytic, and megakaryocytic cell lines In 90% of the cases of this disease one arm of chromosome 22 is found to be translocated to chromosome 9(Philadelphia chromosome) Associated with BCR/ABL1 abnormality Patients with this disorder who are negative for the Philadelphia chromosome usually have a poorer prognosis Myelofibrosis with Myeloid Metaplasia/Primary myelofibrosis/Agnogenic myelofibrosis Characterized by fibrosis and granulocytic hyperplasia of the bone marrow, with granulocytic and megakaryocytic proliferation in the liver and spleen Associated with splenomegaly, and ineffective hematopoiesis (marrow hypercellularity) JAK2 V617F mutation is involved in the pathogenesis and is found in 65% of PMF patients Presence of Dacryocytes Essential thrombocythemia/ Primary thrombocytosis/ Hemorrhagic thrombocythaemia/ Idiopathic thrombocytosis Chronic MPD characterized by thrombocytosis in excess of 1000x109/L, with spontaneous aggregation of functionally abnormal platelets The JAK2 V617F mutation is found in 50% to 60% of ET patients and supports the diagnosis of ET The clinical manifestations of essential thrombocythemia are hemorrhage, platelet dysfunction, and thrombosis Polycythemia vera Characterized by an absolute increase in RBC, WBC and platelets The specific JAK2 mutation, JAK2 V617F, is detected in 90% to 97% of patients with PV Also known as the primary absolute polycythemia, polycythemia Rubra vera. Patients exhibit a RUDDY skin coloration due to increase RBC concentration and viscosity of the blood Increase RBC mass, Decrease EPO, increase RBC, WBC, and platelet count PATHOPHYSIOLOGICAL CLASSIFICATION OF POLYCYTHEMIA Relative polycythemia- Normal RBC mass, Increase hematocrit, Normal EPO a. Diminished plasma volume; dehydration, diarrhea, burns and shock b. Spurious polycythemia (Stress polycythemia: Gaisbock’s syndrome) Absolute Polycythemia a. Primary absolute- Polycythemia vera b. Secondary polycythemia with appropriately increase EPO. -Hypoxia, high altitudes, pulmonary disease, cyanotic heart disease, carboxyhemoglobinemia, high oxygen hemoglobinopathy, 2-3DPG deficiency I.K Aytona Page 83  c. Secondary polycythemia with inappropriately increase EPO -Neoplasms, acute hepatitis, hepatoma, renal carcinoma, post renal transplant, wilm’s tumor d. Genetic polycythemia / Congenital secondary Polycythemia -primary familial congenital polycythemia, and Chuvash polycythemia TYPE RBC MASS/COUNT HCT EPO LEVEL Relative polycythemia Normal Increase Primary absolute (PV) Increase Increase Secondary absolute Increase Increase Genetic polycythemia Increase Increase MYELODYSPLASTIC SYNDROMES(MDS) Group of disorders that result from clonal abnormalities of hematopoietic pluripotential stem cells. Characterized by a hypercellular bone marrow and abnormalities in the cellular maturation of the erythroid cells, granulocytes, and megakaryocytes. MDS are a group of acquired clonal hematologic disorders characterized by progressive cytopenias in the peripheral blood, reflecting defects in erythroid, myeloid, and/or megakaryocytic maturation Historically, this pattern of abnormalities was referred to as refractory anemia, smoldering leukemia, oligoblastic leukemia, or preleukemia The median age at diagnosis is 70 TRADITIONAL FAB COOPERATIVE GROUP CLASSIFICATION OF MDSs MDS Blasts in Blasts in BM Ringed Peripheral blood peripheral blood (%) sideroblasts Monocytes (%) (%) Refractory anemia (RA)