Laboratory Diagnosis for WBC Pathology

Transcription

1 CLINICAL PATHOLOGY Dr. Araceli Jacoba WBC Parameters WBC Count Differential count Absolute count Leukocyte Alkaline Phosphatase (LAP) Special stains of WBC Cluster designation Types of WBC Counts WBC Count Total # of WBC / mm 3 Differential Count Percentage of each type of WBC in relation to the total count Absolute WBC Count Number of specific WBC type/mm 3 Differential count x Total WBC count Differential Count NORMAL VALUE % ABSOLUTE VALUE Segmenters 40-60% 2-7 T Lymphocytes 20-40% 1-4 T Eosinophils 1-4% 350 Basophils 0-2% 200 Monocytes 3-8% 500 CAUSES OF NEUTROPHILIA Neutrophil count >700 Infection Toxins Drugs and chemicals Pb, Hg Metabolic DM, uremia Tissue destruction and necrosis MI Burns, surgical operation Fx, Ca Physical and emotional stimuli Heat/cold Exercise Pain Anger Hematologic disorders hemorrhage CAUSES OF NEUTROPENIA Neutrophil count <2,000 Increase destruction / decrease survival Infection Drugs Hypersplenism, Ig Ineffective granulocytopoesis Megaloblastic anemia, MDS Chemicals Abx, Anti-Ca drugs Aplastic anemia, metastatic Ca Inherited stem cell disorders Determinants to the degree of Neutrophilia Type of affecting organism Staph, GC, strep - N Salmonella, TB slight neutrophilia Virulence of the organism Amount of tissue involved Proportional to neutrophilia Hematologic reactivity Children > adults Weak rxn: neonates, elderly and debilitated patients react with N Steroid therapy Lymphocytes NORMAL VALUE: 7000 children 9000 infants 4000 adults Conditions that lymphocytes Acute viral infection : IM, mumps, hepatitis, measles Chronic infection : TB, Sy, Pertusis Leukemias and lymphomas Lymphocytes in: Thymic hypoplasia Radition Steroid therapy LE Renal failure Monocytes NORMAL VALUES: 750 children 500 adults Conditions that cause monocytosis Infections: TB, Ty, Rickettsia, Trypanosoma Others: UC, Sarcoidosis, Collagen Dis, Lymphoma

2 Eosinophils Conditions that cause Eosinophilia (>350/ l) Allergy Parasitic infection Foreign protein and protein breakdown Leukemia Decreased levels in steroid intake Basophils Conditions that cause Basophilia (>200/ l) CML Myeloproliferative disorder Others: UC, urticaria Subject to diurnal variation i.e. in am in pm Atypical Lymphocytes >20% IM, viral infection : hepa, Coxsackie, CMV <20% Viral infection: varicella, mumps, herpes, flu Radiation Toxic Pb PBS CHANGES IN INFECTION Bacterial Leukocytosis - neutrophilia Shift to the left - stabs and myelocytes Toxic granules, Dohle bodies, vacuolations Viral Leukocytosis or leukopenia Atypical lymphocytes Toxic Granules Evaluating WBC in Smears Number Increased in infection, leukemia Decreased in aplastic anemia, chemothx Nuclear lobulations Cytoplasmic granules Toxic granules, Dohle bodies Cell maturity Mature vs immature (blasts) Alder Reilly Anomaly Abnormal cytoplasmic granules Resemble toxic granules Not related to infection Present in all WBC Hereditary anomaly in healthy persons Mucopolysaccharidosis Stab Döhle Body

3 May Hegglin Anomaly Abnormal cytoplasmic granules Pale blue inclusions similar to Dohle bodies Assd with thrombocytopenia bleeding Presence of lymphoblasts in PBS ALL or CLL Lymphoma with spillage to the circulation Pelger Hüet Abnormality Abnormal nuclear lobulations Hereditary condition characterized by decreased segmentation Pseudo Pelger Hüet nuclei - MDS, Leukemia I. Types of Leukemia Acute lymphocytic Leukemia (ALL) Chronic lymphocytic Leukemia (CLL) Acute Myelogenous Leukemia (AML) Chronic Myelogenous Leukemia (CML) Other types: Hairy cell Leukemia Prolymphocytic Leukemia Large granular lymphocytic Leukemia Adult T cell Leukemia 1. Reactive 2. Leu w/ low blasts ACUTE VS CHRONIC LEUKEMIA Acute Chronic Course Rapidly fatal Long duration (several yrs) Onset Abrupt & stormy Insidious Age Younger patients Older patients WBC >10,000 >50,000 count Blasts >20% of WBC <20% of WBC Origin Pre B/T cells Peripheral B/T cells Signs and Symptoms Due to depressed BM activity, i.e.anemia thrombocytopenia Non-specific like fever, weight loss Acute lymphocytic leukemia Pathophysiology Block in maturation due to mutation of genes required for Bcell development Large blasts cells dont develop into the smaller mature forms Age: children < 15 years old Peak age= 4 years old time where lymphocytes is maximal in the BM Remission is very very good Prognosis 95% of children with complete remission 5 year SR - >75% Chromosomal abnormalities (90%) Hyperploidy, hypoploidy Translocations: t(12;21) or t(4:11) Prevents progression to maturity Philadelphia chromosome t(9:22) FAB Classification L1 homogenous small lymphocytes

4 L2 heterogenous grp of small & large lympho L3 large lymphocytes Immunochemistry Pre T cells: (20%) CD1, CD2, CD5, CD7 Pre B cells (80%) : CD 10, CD 19 Signs and Symptoms Symptoms due to marrow failure Mass effects Enlargement of spleen and liver, LN Bone pains- marrow expansion bone resorption Testicular enlargement only ALL will produce testicular enlargement CNS Haemorrhages from decreased platelets, microvascular occlusions, meningeal involvement Chronic Lymphocytic Leukemia Involvement of the BM present in almost all cases LN BM blood (~CLL like picture) Indistinguishable from SLL Age: elderly, >50 y/o, male (2:1) Sxs: asxtic nonspecific or mild: enlarged liver, LN Course: indolent, survival long (4-10 yrs) transformation to large B cell (10%) ~ Richter Syndr Cx: Ig, dev autoantibodies to RBC & platelets Immunophenotype: + CD19, CD20, CD 23, CD5 Histo: small round lymphocytes with smudge cells Hairy Cell Leukemia Predominantly a disease of middle aged Caucasian males (older males) Special form Low grade chronic B cell leukemia Leukemic cells have fine hairlike projections on the RBC surface that are best recognized under the phase contrast microscope Hairy cells have round, oblong or reniform nuclei Modest amounts of pale blue cytoplasm often with thread-like or bleb-like extensions Number of circulating cells is highly variable Massive splenomegaly Other abnormalities: Pancytopenia Most of the cells are seen proliferating in the bone marrow Very few spill into the blood Leukocytosis is not common Laboratory: + tartarate resistant acid phosphate (TRAP) Only hairy cells will give positive result II. Myeloid Disorders Acute Myelogenous Leukemia Myelodysplastic Syndrome Chronic Myeloproliferative Disorders Chronic Myelogenous Leukemia (CML) Polycythemia vera Essential thrombocytosis Myelofibrosis Acute Myelogenous Leukemia (AML) Laboratory Diagnosis Elevated WBC count Some may have values below 10,000 20% will have values above 100,000 Platelet counts and RBC counts are low >20% blast cells Special stains Nonspecific esterase monoblasts Myeloperoxidase- myeloblasts PAS- lymphoblasts M0 AML Minimal 2-3% Neg marker for Differentiation myeloblasts, neg peroxidase,+ myeloid Ag M1 AML w/o Differentiation M2 w/ maturation 30- M3 Acute promyelocytic M4 Myelo-mono Leukemia M5 Acute Monocytic Leu M6 Acute Erythro Leukemia M7 Acute Mega- Karyocytic Leu 20% Very immature, >3% peroxidase +, no granules or Auer rods Full myeloid maturation, 40% Auer rods, t (8;21) 5-10% Hypergranular promyelocytes, many Auer rods, DIC, t (15:17) 5-20% Myelocytes & monocytes present, monoblasts Inv (16) 10% + monoblasts (peroxidase neg, nonspecific esterase +) monocytes many, organomegally 5% Dysplastic erythroid precursors 1% Megakaryocyte react with platelet specific AB; myelofibrosis

5 Classification of AML M0-2 all myeloid M2 Granules in cytoplasm, +for myeloperoxidase M3 Predominant cell is promyelocyte DIC most common complication Monitor coagulation patterns! M4 Monocyte and myelocyte M5 Secondary to immature monocytes M6 Secondary to immature RBC M7 Immature platelets peroxidase Lymphoblast + for PAS M3 M3-block in RARA gene such that in giving RA, there will be development of the lymphocyte Myeloblasts VS Lymphoblasts AML ALL 1. Peripheral smear Auer Rods Present None No. of nucleoli Cytoplasm Abundant Scanty Cyto granules Abundant None 2. Cytochemistry PAS Negative Positive Myeloperoxidase Positive Negative TdT Positive Positive Diagnosis of AML is based on finding that myeloid blasts make up more than 20% of the cells in marrow Myeloblasts Have delicate nuclear chromatin 2 to 4 nucleoli More voluminous cytoplasm than lymphoblasts Cytoplasm often contains fine, aurophilic, peroxidise-positive granules Presence of Auer Rods Auer Rods Distinctive red-staining peroxidasepositive structures Represent abnormal azurophilic granule Present in many cases Particularly numerous in AML associated with the t(15;17) (acute promyelocytic leukemia) Presence is taken to be definitive evidence of myeloid differentiation Monoblasts Have folded or lobulated nuclei Lack auer rods Peroxidase negative Nonspecific esterase positive In some AMLs, exhibit megakaryocytic differentiation which is often accompanied by marrow fibrosis caused by the release of fibrogenic cytokines

6 Chronic Myelogenous Leukemia Disease primarily of adults between ages 25 to 60 years. Peak incidence- 40 to 50 years old Neoplastic transformation of pluripotent granulocytic stem cell with no block in maturation Presence of a distinctive molecular abnormality Translocation involving the BCR gene on chromosome 22 and ABL gene on chromosome 9 t(9:22) Philadelphia chromosome (90%) Therapy: BCR-ABL inhibitor (imatinib) Clinical features: Onset is insidious Mild to moderate anemia &hypermetabolism Due to increased cell turnover Lead to easy fatigability, wakness, weight loss and anorexia Slow progression After a variable period averaging 3 years, approximately 50% of patients enter an accelerated phase Increasing anemia & thrombocytopenia Striking peripheral blood basophilia Terminates in a picture resembling acute leukemia (blast crisis) Death from accelerated or blastic crisis Laboratory Diagnosis Elevated WBC counts - > 100,000 Absent alkaline phosphatase Philadelphia chromosome 90% PBS: thrombocytosis (50%) Leukocyte Alkaline Phosphatase Used to differentiate CML from leukemoid reaction Increased in In conditions with increased neutrophils Pregnancy Hodgkins Decreased in CML Myelodysplastic Syndrome (MDS) Group of clonal stem cell disorders characterized by maturation defects associated with ineffective hematopoiesis and an increased risk of transformation to AML (10-40%) Deletions and monosomy of 5 and 7 Bone marrow is partly or wholly replaced by the clonal progeny of a mutant multipotent stem cell that retains the capacity to differentiate into red cells, granulocytes and platelets In an ineffective and disordered fashion Disturbances usually manifest as peripheral blood cytopenias All forms can transform to AML Occurs most frequently and rapidly in t-mds Age: >60 years old Symptoms Presents with weakness, infections and haemorrhages all due to pancytopenia Survival Rate: 9-29 mo for MDS 4-8 mo for t-mds Therapy: Bone marrow transplant for younger patients Most characteristic finding is disordered (dysplastic) differentiation affecting all nonlymphoid lineages Erythroid, granulocytic, monocytic, megakaryocytic Cellular abnormalities Ringed sideroblasts Erythroblasts with iron-laden mitochondria visible as perinuclear granules in Prussian blue-stained aspirates or biopsies Megaloblastoid maturation Resembling that seen in vitamin B12 and and folate deficiency Megaloblastoid erythropoiesis Nuclear budding abnormality Nuclei with misshapen, often polyploid outlines Neutrophils often contain decreased numbers of secondary granules, toxic granulations, and/or Döhle bodies Pseudo-Pelger-Hüet Cells Neutrophils with only 2 nuclear lobes Neutrophils may even be seen that completely lack nuclear segmentation Pseudo-Pelger- Hüet Cells

7 Agranular Myelocytes Pawn ball megakaryocytes Megakaryocytes with single nuclear lobes or multiple separate nuclei Dysplastic Megakaryocytes Myeloblasts may be increased but make up <20% of the overall marrow cellularity Peripheral blood often contains pseudo- Pelger-Hüet cells, giant platelets, macrocytes, poikilocytes and a relative or absolute monocytosis Bone marrow is hypercellular < 5% normocellular or hypocellular PBS: cytopenia Essential Thrombocytosis Increased proliferation and production are largely confined to the megakaryocytic elements Most patients having platelet counts >600,000 per mm 3 Diagnosis of Exclusion Since all chronic MPDs can be associated with thrombocytosis Features that are characteristic of other MPDs are absent Bears some resemblance to polycythemia vera Cells of the megakaryocytic series have a diminished requirement for growth factors Major Clinical features Bleeding and thrombosis Dysfunction of platelets derived from the neoplastic clone is a contributing factor PBS= giant platelets, counts >600T Normal Essential Thrombocytosis Multiple Myeloma Most common among the plasma cells gammopathies Age: elderly ( y/o) Pathology Increase in Ig called the M protein or M component 55% - increased IgG 25% - increased immunoglobulin other than IgG (i.e. IgA, IgM, IgE, IgD) 20% - increased Bence Jones protein, seen in the urine of patients with MM Multifocal destructive bone lesion throughout the skeletal system. Common sites affected are: Vertebral column 66& Ribs 44% Skull 41% Pelvis 28% Femur 24% X-ray: soap bubble appearance Bone marrow biopsy: Replacement of marrow cells with plasma cells, Russels bodies Laboratory Diagnosis Urine: positive for Bence Jones protein Increased ESR because the hyperglobulin leads to rouleaux Serum Ig levels: >3mg/dL M-protein spike in blood or urine Increase IL-6 Increase serum calcium Classification of Lymphoma LCA CD45 T Cell CD1, CD5, CD4/CD8, CD3 B Cell CD 10- germinal center B CD19, CD20- pre B, mature B CD23- mature B IgM- plasma cells Monocyte/ CD11, CD13, CD14, CD15, CD33, Macrophage CD64 NK Cell CD16, CD56 Stem cell CD 34 Hodgkins CD 15, CD30 CD 45 Common to a lymphomatous tumor To differentiate lymphomatous from a non lymphomatous Non Hodgkins Lymphoma B cell CD 10, 19, 20, Ig

8 T cell CD 1, 3, 4, 5, 8 Hodgkins Lymphoma B cell markers plus CD 15, 30 B cell Lymphoma Small Lymphocytic Lymphoma/CLL (B+T) Follicular Lymphoma Diffuse large B-Cell lymphoma Burkitts Lymphoma Mantle Cell Lymphoma (B+T) Hairy cell Leukemia Multiple Myeloma T Cell Lymphoma Adult T cell Lymphoma NK T Mycosis Fungoides / Sezary Syndrome Terminal Deoxynucleotidyl Transferase (TdT) Marker for Immature B/T cells Acute Lymphoblastic Lymphoma / Leu (pre T) Adolescent male Mediastinal (thymic) rapidly enlarging symptomatic mass - 70% of cases Aggressive Acute Lymphocytic Leu / Lymphoma (Pre B) Children : <15 y/o, peaks at 4 y/o Responsive to treatment Reference: Dr. Jacoba s powerpoint Robbin s 7 th edition My notes last year on WBC Pathology