Normal histology – Hematolymphoid tissues

Basic Hematopathology Course, TMH, June12-13, 2010

Dr. Sumeet Gujral,Associate Professor, Department of PathologyTata Memorial Hospital, Mumbais_gujral@hotmail.com

Hematolymphoid tissues

• Peripheral blood• Bone marrow• Lymph node• Spleen• Thymus• Waldeyer’s ring• Elsewhere

Peripheral Blood Smear

Peripheral blood

CellsRBCs, PlateletsWBCs

Plasma: whole blood minuscells

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 Jan-Mar;53(1):68-74

Importance of PBS examination

Bone marrow preparationsaspirate

touch

trephine

clot

Normal bone marrow

Bone Marrow Aspirate

Myeloid precursors

2

31

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 alIndian J Pathol Microbiol. 2005 Jul;48(3):425-6

??

Normalcells

Dyspoieticcells

NormalDyspoietic

Normal Dyspoietic

Normal Dyspoietic

Blasts

Acute Leukemia>20% blasts in peripheral blood or bone marrow

What are blasts?

Morphology

Exceptions Small sizeGranular blastsAbnormal promyelocytes – in AMLM3 Promonocytes – in AMLM5

Guess

Guess

Clusters in bone marrow

Bone Marrow Biopsy

Indications of BM Biopsy

PUOStorage diseases

Aplastic anemiaDry tap MyelofibrosisMyelodysplastic syndromeStaging 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 - Joint 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 patternALL

Diffuse patternCLL

Interstitial- Exclusively: BL, LL, HCL

- Combined focal and interstitial: SLL, LPL, MCL, ALCL

DLBCL – patch

SLL – MixedNodule + Interstitial

NodularAll MZL SLL, FL, HD

CD20

Nodule

Nodule

CD138

Hodgkin’s disease

Nodular, diffuse, patchy

Biopsy and aspirate are complementary

CHD - Nodule

CD3

PTCL – NOS, Patch / nodule

ALCL - intrasinusoidal

Blastic lymphoma versus

Burkitt’s lymphoma

Acute Leukemia Do we need bone marrow biopsy ??

Burkitt’s 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 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

Benign Lymphoid Aggregates

Young age – collagen vascular diseasesOld age

CD3

Hematogonesmedium 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 folliclesIgM+ IgD+

Secondary follicles, IgD-

Mantle zoneIgM+ IgD+

T

T

T

T

T

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+

T zone proliferation

Identify

Identify

CD3

CD20

IHCs in a normal node

CD23

Mib1

bcl2

Various cells

Immunoblasts

CentroblastsPlasma 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 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

LN: Other patterns (other than granulomas)

PTGCRTGCWierd 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 / FSdiagnosis of lymphomas

Spleen - Organ of Mystery

Spleen

2 x 1.5 x 0.2 cm, immediate processing (may /may not wait for fixation)

Congestedcords

Sinus

Red pulp

Trabeculae

PALS

SMZL

Others

Thymus

CD3

Tdt

Thymus

Tonsil

Tonsil: Plasma cell rich lesion

Conclude

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

• 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