Normal Hematolymphoid Tissues
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Normal histology – Hematolymphoid tissues
Basic Hematopathology Course, TMH, June12-13, 2010
Dr. Sumeet Gujral,Associate Professor, Department of PathologyTata Memorial Hospital, [email protected]
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Hematolymphoid tissues
• Peripheral blood• Bone marrow• Lymph node• Spleen• Thymus• Waldeyer’s ring• Elsewhere
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Peripheral Blood Smear
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Peripheral blood
CellsRBCs, PlateletsWBCs
Plasma: whole blood minuscells
Serum: whole blood minus cells and the clotting factors
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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
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Approach to peripheral blood smear examination
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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)
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• Do at least 200 WBC count, and record any abnormal morphology of RBCs, WBCs, and platelets
• Look for parasites
Oil immersion
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Purplish pink
Light pale pink
Greyish pink
Purplish blue
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Pale blue
Sky blue
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Chromatin
Purplish blue
Sky blue
Pale blue
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Granular cells
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Round cells
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Hypersegmented polymorph
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??
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??
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Downey cells
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?
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Poor quality smears
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Delayed staining
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Stain deposits
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Drying artefacts
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Platelet clumps
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PBS as part of the Medical Record
Preserve and store
Indian J Pathol Microbiol. 2010 Jan-Mar;53(1):68-74
Importance of PBS examination
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Bone marrow preparationsaspirate
touch
trephine
clot
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Normal bone marrow
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Bone Marrow Aspirate
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Myeloid precursors
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2
31
4
5
1
2
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Promyelocyte
Neutrophil
Metamyelocyte
Myelocyte
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Promyelocyte may be larger than a blast and cytoplasm contains large black or purple granules. Nucleoli may be present
Promyelocyte
Blast
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Promyelocyte may be larger than a blast and cytoplasm contains large black or purple granules. Nucleoli may be present
Myelocyte
Metamyelocyte
Promyelocyte
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Promyelocyte may be larger than a blast and cytoplasm contains large black or purple granules. Nucleoli may be present
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Monocytic precursors
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Promyelocyte
Promonocyte
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Lymphoid precursors
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Lymphoblasts
Lymphocytes
Hematogones
Hematogones
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Erythroid precursors
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Erythroblasts
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Megaloblast
Colony
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??
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Platelet precursors
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Platelet precursors
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Platelet clumps -pseudothrombocytopenia
Anand M et alIndian J Pathol Microbiol. 2005 Jul;48(3):425-6
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??
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Normalcells
Dyspoieticcells
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NormalDyspoietic
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Normal Dyspoietic
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Normal Dyspoietic
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Blasts
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Acute Leukemia>20% blasts in peripheral blood or bone marrow
What are blasts?
Morphology
Exceptions Small sizeGranular blastsAbnormal promyelocytes – in AMLM3 Promonocytes – in AMLM5
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Guess
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Guess
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Clusters in bone marrow
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Bone Marrow Biopsy
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Indications of BM Biopsy
PUOStorage diseases
Aplastic anemiaDry tap MyelofibrosisMyelodysplastic syndromeStaging of lymphomas
Acute leukemia
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Procedure and processing
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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
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Cellularity
Cells: Fat cells, Hematopoietic cells (trilineage hematopoiesis), Megakaryocytes, Blasts, Others
Fibrosis, granulomas, tumor
Low power examination
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Cellularity
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Aspirate and Biopsy are complementary
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Bone marrow in a 40-year-old
Types of cells
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Types of cells
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Regenerating bone marrow
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Myeloid ++++
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Routine sections
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Hemorrhagic bone marrow biopsy
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Pediatric bone marrow biopsy
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Adequate bone marrow biopsy
Large subcortical area not truly representing overall hematopoietic activity
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Fragmanted BM biopsy
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ALCL
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Crushing artefacts - FL
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IHC may be useful
Follicular lymphoma
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Mantle cell lymphoma
IHC may be useful
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Good trephine - Joint responsibility
• Physician doing the biopsy (anesthetist)• OT Nurse • Technologist• Pathologist• Administrators• Vendors• Patients
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BM and lymphomas
Staging marrows
Diagnostic marrows
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Different patterns in lymphomas
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Patterns
• Diffuse• Interstitial• Nodular• Patchy• Intrasinusoidal
• Paratrabecular
• Focal non paratrabecular• Focal paratrabecular• Intrasinusoidal• Diffuse, interstitial• Diffuse, solid
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Focal paratrabecular
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Diffuse patternALL
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Diffuse patternCLL
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Interstitial- Exclusively: BL, LL, HCL
- Combined focal and interstitial: SLL, LPL, MCL, ALCL
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DLBCL – patch
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SLL – MixedNodule + Interstitial
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NodularAll MZL SLL, FL, HD
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CD20
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Nodule
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Nodule
CD138
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Hodgkin’s disease
Nodular, diffuse, patchy
Biopsy and aspirate are complementary
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CHD - Nodule
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CD3
PTCL – NOS, Patch / nodule
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ALCL - intrasinusoidal
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Blastic lymphoma versus
Burkitt’s lymphoma
Acute Leukemia Do we need bone marrow biopsy ??
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Burkitt’s lymphoma
ALL
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ALL
CD34
Tdt Mic2
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AML Non M3 AML M3
B-cell ALL
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Granulomas in HD
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Reactive lymphoid proliferations in bone marrow
1. Lymphoid aggregates
2. Hematogones
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Benign Lymphoid Aggregates1. 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
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Benign Lymphoid Aggregates
Young age – collagen vascular diseasesOld age
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CD3
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Hematogonesmedium sized lymphoid cells,scant cytoplasm, round to irregular nuclei, dense homogeneous chromatin no - very small nucleoli
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Parasites (in PB/BM)
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MP with satellitism
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Exflagellated microgametes of Plasmodium vivax
Tembhare P et al. Indian J Pathol Microbiol, 2009
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Microfilaria
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Parvovirus - BM
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Parvovirus - BM
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Lymph nodes
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Lymph node is a dynamic structure
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Primary folliclesIgM+ IgD+
Secondary follicles, IgD-
Mantle zoneIgM+ IgD+
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T
T
T
T
T
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Differentiation of B cells during their passage through the germinal center
Secondary B blasts
Fdcmacrophages
CCFdc
macrophages
CBFdc,
macrophages
Primary B blasts
Plasma cells Memory B cellsMantle zoneIgM+ IgD+
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T zone proliferation
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Identify
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Identify
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CD3
CD20
IHCs in a normal node
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CD23
Mib1
bcl2
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Various cells
Immunoblasts
CentroblastsPlasma cells
Centrocytes
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Common causes of lymphadenopathy
• Infections
• Malignancies
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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
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Benign lymphadenopathyInfections
viral (EBV, HIV, CMV), bacterial, parasitic,
Autoimmune disorders, Drug hypersensitivity reactions,
Kikuchi’s disease, Castleman’s disease, SHML, Kimura’s disease, PTGC, Toxoplasmosis
Dermatopathic lymphadenitis
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LN: Other patterns (other than granulomas)
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PTGCRTGCWierd looking
nodules
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Follicular hyperplasia versus
Follicular lymphoma
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Which one is a lymphoma?
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FL Grade 1 / MCL Follicular hyperplasia
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FL Grade 1 / MCL Follicular hyperplasia
bcl2
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FL (Grade 2) Follicular hyperplasia
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FL (Grade 3) Reactive
Mimic
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Gold standard
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Avoid FNAC / FSdiagnosis of lymphomas
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Spleen - Organ of Mystery
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Spleen
2 x 1.5 x 0.2 cm, immediate processing (may /may not wait for fixation)
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Congestedcords
Sinus
Red pulp
Trabeculae
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PALS
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SMZL
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Others
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Thymus
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CD3
Tdt
Thymus
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Tonsil
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Tonsil: Plasma cell rich lesion
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Conclude
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A. Myeloid neoplasmsB. Precursor lymphoid neoplasmsC. Mature B cell neoplasmsD. Mature T- and NK- cell neoplasmsE. Hodgkin lymphomaF. Immunodeficiency associated LPDG. Histiocytic and dendritic cell neoplasms
So many subtypes,Different treatment options
2008 WHO classification of Hematolymphoid Neoplasms
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• 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
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Staff, Residents and Colleagues at Hematopathology Laboratory and Department of Pathology, TMH