TMH, J une12-13, 2010 Dr. Sumeet Gujral, Associate Professor, Department of Pathology Tata Memorial Hospital, Mumbai s_gujral@hotmail.com Hematolymphoid tissues Peripheral blood Bone marrow Lymph node Spleen Thymus Waldeyers ring Elsewhere Peripheral Blood Smear Peripheral blood Cells RBCs, Platelets WBCs Plasma: whole blood minus cells Serum: whole blood minus cells and the clotting factors Steps for preparation of smears Finger prick, fresh blood with no anticoagulant added EDTA - anticoagulated blood: Film should be made within 2-4 hours (storage artifacts) Heparinized blood to be avoided Approach to peripheral blood smear examination To evaluate the quality, approximate number of WBCs and platelets - WBC count in cells/ml on PBS is low power x 3000, - Platelet counts in cells/ml on PBS is oil immersion x 20,000 Detect rouleaux formation, platelet clumps, and leukocyte clumps and other abnormalities. Select an optimal area for evaluation at higher power Low power (10X) Do at least 200 WBC count, and record any abnormal morphology of RBCs, WBCs, and platelets Look for parasites Oil immersion Purplish pink Light pale pink Greyish pink Purplish blue Pale blue Sky blue Chromatin Purplish blue Sky blue Pale blue Granular cells Round cells Hypersegmented polymorph ?? ?? Downey cells ? Poor quality smears Delayed staining Stain deposits Drying artefacts Platelet clumps PBS as part of the Medical Record Preserve and store Indian J Pathol Microbiol. 2010 J an-Mar;53(1):68-74 Importance of PBS examination Bone marrow preparations aspirate touch trephine clot Normal bone marrow Bone Marrow Aspirate Myeloid precursors 2 3 1 4 5 1 2 Promyelocyte Neutrophil Metamyelocyte Myelocyte Promyelocyte may be larger than a blast and cytoplasm contains large black or purple granules. Nucleoli may be present Promyelocyte Blast Promyelocyte may be larger than a blast and cytoplasm contains large black or purple granules. Nucleoli may be present Myelocyte Metamyelocyte Promyelocyte Promyelocyte may be larger than a blast and cytoplasm contains large black or purple granules. Nucleoli may be present Monocytic precursors Promyelocyte Promonocyte Lymphoid precursors Lymphoblasts Lymphocytes Hematogones Hematogones Erythroid precursors Erythroblasts Megaloblast Colony ?? Platelet precursors Platelet precursors Platelet clumps - pseudothrombocytopenia Anand M et al Indian J Pathol Microbiol. 2005 J ul;48(3):425-6 ?? Normal cells Dyspoietic cells Normal Dyspoietic Normal Dyspoietic Normal Dyspoietic Blasts Acute Leukemia >20% blasts in peripheral blood or bone marrow What are blasts? Morphology Exceptions Small size Granular blasts Abnormal promyelocytes in AMLM3 Promonocytes in AMLM5 Guess Guess Clusters in bone marrow Bone Marrow Biopsy Indications of BM Biopsy PUO Storage diseases Aplastic anemia Dry tap Myelofibrosis Myelodysplastic syndrome Staging of lymphomas Acute leukemia Procedure and processing Trephine (Hammersmith Protocol) Fixative (AZF) Decalcifying agent (10% FA and 5% formaldehyde) 2-3 micron thick section Immunohistochemistry Adequate biopsy Both aspirate and imprints with the biopsy (>1.6 cm) Ideally, reporting of trephine biopsy sections should be done by an individual who is competent in both histopathology and hematology Cellularity Cells: Fat cells, Hematopoietic cells (trilineage hematopoiesis), Megakaryocytes, Blasts, Others Fibrosis, granulomas, tumor Low power examination Cellularity Aspirate and Biopsy are complementary Bone marrow in a 40-year-old Types of cells Types of cells Regenerating bone marrow Myeloid ++++ Routine sections Hemorrhagic bone marrow biopsy Pediatric bone marrow biopsy Adequate bone marrow biopsy Large subcortical area not truly representing overall hematopoietic activity Fragmanted BM biopsy ALCL Crushing artefacts - FL IHC may be useful Follicular lymphoma Mantle cell lymphoma IHC may be useful Good trephine - J oint responsibility Physician doing the biopsy (anesthetist) OT Nurse Technologist Pathologist Administrators Vendors Patients BM and lymphomas Staging marrows Diagnostic marrows Different patterns in lymphomas Patterns Diffuse Interstitial Nodular Patchy Intrasinusoidal Paratrabecular Focal non paratrabecular Focal paratrabecular Intrasinusoidal Diffuse, interstitial Diffuse, solid Focal paratrabecular Diffuse pattern ALL Diffuse pattern CLL Interstitial - Exclusively: BL, LL, HCL - Combined focal and interstitial: SLL, LPL, MCL, ALCL DLBCL patch SLL Mixed Nodule + Interstitial Nodular All MZL SLL, FL, HD CD20 Nodule Nodule CD138 Hodgkins disease Nodular, diffuse, patchy Biopsy and aspirate are complementary CHD - Nodule CD3 PTCL NOS, Patch / nodule ALCL - intrasinusoidal Blastic lymphoma versus Burkitts lymphoma Acute Leukemia Do we need bone marrow biopsy ?? Burkitts lymphoma ALL ALL CD34 Tdt Mic2 AML Non M3 AML M3 B-cell ALL Granulomas in HD Reactive lymphoid proliferations in bone marrow 1. Lymphoid aggregates 2. Hematogones Benign Lymphoid Aggregates 1. Distribution - Usually perivascular, intertrabecular 2. Number/size - Few in number, small in size 3. Circumscription - Well circumscribed (except in AIDS) 4. Cell composition - Mature looking cells, Heterogeneous cell population consisting of small to large sized lymphocytes, plasma cells, histiocytes 5. Germinal centres - seen in drug related or in autoimmune diseases 6. IHC - T cells predominate Benign Lymphoid Aggregates Young age collagen vascular diseases Old age CD3 Hematogones medium sized lymphoid cells, scant cytoplasm, round to irregular nuclei, dense homogeneous chromatin no - very small nucleoli Parasites (in PB/BM) MP with satellitism Exflagellated microgametes of Plasmodium vivax Tembhare P et al. Indian J Pathol Microbiol, 2009 Microfilaria Parvovirus - BM Parvovirus - BM Lymph nodes Lymph node is a dynamic structure Primary follicles IgM+IgD+ Secondary follicles, IgD- Mantle zone IgM+IgD+ T T T T T Differentiation of B cells during their passage through the germinal center Secondary B blasts Fdc macrophages CC Fdc macrophages CB Fdc, macrophages Primary B blasts Plasma cells Memory B cells Mantle zone IgM+IgD+ T zone proliferation Identify Identify CD3 CD20 IHCs in a normal node CD23 Mib1 bcl2 Various cells Immunoblasts Centroblasts Plasma cells Centrocytes Common causes of lymphadenopathy Infections Malignancies Warning signs of lymphadenopathy suggestive of a malignant etiology include - size >2 cm in size, - duration >2 month, - location - supraclavicular, and - generalized lymphadenopathy with hepatosplenomegaly or B-symptoms. VIP Syndrome Benign lymphadenopathy Infections viral (EBV, HIV, CMV), bacterial, parasitic, Autoimmune disorders, Drug hypersensitivity reactions, Kikuchis disease, Castlemans disease, SHML, Kimuras disease, PTGC, Toxoplasmosis Dermatopathic lymphadenitis LN: Other patterns (other than granulomas) PTGC RTGC Wierd looking nodules Follicular hyperplasia versus Follicular lymphoma Which one is a lymphoma? FL Grade 1 / MCL Follicular hyperplasia FL Grade 1 / MCL Follicular hyperplasia bcl2 FL (Grade 2) Follicular hyperplasia FL (Grade 3) Reactive Mimic Gold standard Avoid FNAC / FS diagnosis of lymphomas Spleen - Organ of Mystery Spleen 2 x 1.5 x 0.2 cm, immediate processing (may /may not wait for fixation) Congested cords Sinus Red pulp Trabeculae PALS SMZL Others Thymus CD3 Tdt Thymus Tonsil Tonsil: Plasma cell rich lesion Conclude A. Myeloid neoplasms B. Precursor lymphoid neoplasms C. Mature B cell neoplasms D. Mature T- and NK- cell neoplasms E. Hodgkin lymphoma F. Immunodeficiency associated LPD G. Histiocytic and dendritic cell neoplasms So many subtypes, Different treatment options 2008 WHO classification of Hematolymphoid Neoplasms Optimal tissue fixation, processing followed by a thin, well stained (Haematoxylin and Eosin) section is most important for lymphoma diagnosis. Lack of trained hematopathologists, inadequate sampling of the tissue and improper processing of the specimen Second opinion and multidisciplinary clinic NHL, follicular type, grade 1 Staff, Residents and Colleagues at Hematopathology Laboratory and Department of Pathology, TMH s_gujral@hotmail.com