Bone marrow pathology

October 2013

BHS Educational Course

• Technical aspects

• Interpretation of BMB

Introduction: technical aspects of

histology

• Identification of a disease process or a

diagnosis

• By tissue analysis

• Tissue preparation to allow

microscopic evaluation

• Knowledge of normal histology and

disease processes

BM biopsy: tissue preparation

• Fixation: buffered formalin, B5 fixative, (Bouin)

• Decalcification:

acid decalcifiers: formic acid (1-2 days),

rapid decalcifier (1 hour)

chelating decalcifiers: EDTA (2-4 days)

electrolysis: Sakura TDETM 30 decalcifier

system (1h ½)

ultrasonic decalcification in EDTA: (2 hours)

• Embedding: paraffin, (“plastic” methylmetacrylate)

New!

Fixation for at least

6 hours

Decalcification

90 min

Overnight automated

processing

Embedding in paraffin cold plate

H&E immunohistochemistry

microtome

Hot plate

tissue

AG

PRIMARY MOUSE ANTISERUM

chromogen

BMB

• Indications: diagnosis, staging, response to treatment & follow-up

• Intrinsic BM diseases MDS/AML

MPD

MDS/MPD

Plasma cell proliferations

Lymphomas

Mast cell proliferation

…

• Infections (opportunistic infections in eg. HIV)

• Vitamin deficiencies

• Metastatic disease

• …

The pathological report

• Representativity: length + composition of BMB

• Cellularity: estimation of % cells versus fat cells

cave hypoplastic AML /MDS

• Hematopoietic cell types

architecture / distribution

maturation

dysplastic features

• Other relevant information

lymfoid infiltrates

stroma , blood vessels, .. (eg amyloid)

presence of granulomas

fibrosis

Histologic Interpretation of BMB: morphology

Pattern recognition: morphological important

clues

- Cellularity

- Focal lesions

- Fibrosis

- Dysplasia

- Disease specific clues

Age and bone marrow cellularity

Mean and 95% range in 177 cases of sudden death

Anterior Iliac crest specimens decalcified and paraffin-embedded

Hartsock et al. Am J Clin Pathol 1965; 43: 326-31

Quantification of bone marrow fibrosis

0 No reticulin fibres demonstrable

1 Occasional fine individual fibres and foci of a fine fibre network

2 Fine fibre network throughout most of the section; no coarse fibres

3 Diffuse fibre network with scattered thick coarse fibres but no mature collagen

4 Diffuse often coarse fibre network with areas of collagenization

Diagnostic clues

• Predominant cell type

• Necrosis

• Gelatinous transformation

• Granulomas

• Lymphoid infiltrates

• Blast count (CD34 , CD117, …)

• …

Histologic Interpretation of BMB:

immunohistochemistry

• Hematological marker: LCA

• “Myeloid markers”: MPO, CD15, CD34, CD68,

CD117

• “Lymphocytic markers”: CD3, CD5, CD20,

CD10, CD21, CD23, CD30,CD138, kappa, lambda …

• Erythroid marker: GlycophorinA

• “Megakaryocytic marker”: FVIII, LAT1,

CD61

« the hypercellular marrow »

• Correlation with age

• Predominant cell type

lymfoid lymphoma

plasma cell myeloma

myeloid

MKC

erythroid cells

blasts

panmyelosis: 3 series

….

• Neoplastic vs reactive

dysplasia

N°cells

presence of disease related features: clustering, fibrosis,

….

« the hypocellular marrow »

• Correlation with age

• Presence of dysplastic features?

hypoplastic MDS

• Increase of blasts? hypoplastic AML

• Hypoplasia / aplasia: reactive

lymphocytes and plasma cells

Pattern recognition

• The hypercellular marrow

myeloid: MDS – AML – MDS/MPD

MPD: CML- PRV

leukemoid reaction

lymphoid: reactive infiltrates, lymphoma

plasmacells: MM

mastcells: mastocytosis

• The hypocellular marrow

aplasia – MDS – AML

• The fibrotic marrow (reticulin / collagen)

MPD (IMF) – AML(M7) - MDS/MPD- mastocytosis – lymphoma – metastatic carcinoma- auto-immune MF – HIV myelopathy-…

Pattern recognition

• The necrotic marrow

ALL – neuroblastoma- DIC/antiphospholipiden syndr – medication

or chemoR/ effect - sickle cell anemia- GvHD- severe infections-

…

• The “dysplastic” marrow

MDS – HIV – vit deficiencies – BM regeneration – chronic

alcoholism – heavy metals – infections – auto-immunity –

paraneoplastic

• The granulomatous marrow

infections (viral-fungal-bacterial-toxo), Hodgkin and non-Hodgkin

lymphomas, CML, AML, ALL, MDS, carcinomas, drugs, auto-

immune disorders, foreign bodies, .

Dysplasia/ dysmyelopoiesis

Dysgranulopoiesis: INCOMPLETE MATURATION / LACK IN GRANULOCYTES (LEFT SHIFT)

PRESENCE OF “IMMATURE CELLS” AWAY FROM THE BONEY TRABECULAE

ABNORMALLY LOCATED IMMATURE PRECURSORS (ALIP)

Dyserythropoiesis: ABSENCE OF MATURATION WITHIN THE ERYTHRON (SYNCHRONISATION)

PREDOMINANTLY LATE STAGE MATURATION CELLS

MITOTIC FIGURES

MEGALOBLASTOID FEATURES (MAINLY IN RARS)

Dysmegakaryopoiesis: SMALL MEGAKARYOCYTES

HYPO OR HYPERLOBULATION OF THE NUCLEUS

MONOLOBULAR MEGAKARYOCYTES OF ALL SIZES

NO CLUSTERING OF MEGAKARYOCYTES

NON-LOBULATED NUCLEI RING NUCLEI

MULTIPLE NUCLEI

NAKED NUCLEI

Left shifted granulopoiesis

MEGALOBLASTOID RED SERIES

DYSMEGAKARYPOIESIS

ABNORMALLY LOCATED IMMATURE PRECURSORS OR ALIP

VERY SMALL, ROUND TO OVAL CELLS OCCURRING AS

SINGLE CELLS OR IN SMALL CLUSTERS AWAY FROM

THE BONEY TRABECULAE

HAVING ALMOST NO CYTOPLASM

THIN BUT WELL DEMARCATED NUCLEAR MEMBRANE

FINE NUCLEAR CHROMATINE AND SMALL BUT INCONSPICUOUS

NUCLEOLUS

TO BE COMPARED WITH MYELOBLASTS RESIDING

ALONG BONE TRABECULAE

MOSTLY CD34 POSITIVE

ALIP

CD34

Myeloproliferative features

• High cellularity (100% in CML)

• Fibrosis (in IMF)

• Clustering of MKC (PV, ET, IMF)

• Small MKC in CML

• Very large MKC (PV, ET, IMF)

Identification of blasts

• « round » immature cells

• Dd blasts versus precursors

• CD34

• (CD117)

• Other markers: CD68, MPO

Staging • Carcinomas

Staging & Follow-up

• Lymphomas: Hodgkin & non-Hodgkin

lymphomas

What does the clinician

expect from the pathologist?

• 1) is the BM infiltrated by lymphoma?

• 2) what is the (sub)type of lymphoma?

• 3) additional information on

hematopoiesis, stromal alterations,

therapy-effect, …

Bone marrow involvement by lymphoma

• Often focal involvement

• Ideally bilateral iliac crest BM biopsy

• Ideally step sections of BM biopsy

• Sometimes extensive fibrosis

• Some lymphomas can easily be missed by

cytology and flow, due to the low number of

neoplastic cells (e.g. Hodgkin, THRBCL)

Advantages of bone marrow biopsy:

• overall assessment of neoplastic infiltration: Pattern of involvement - extent of involvement - morphology/cytology of neoplastic cells

May give diagnostic or prognostic clues

• Overall assessment of hematopoiesis and stromal component: bone marrow fibrosis, necrosis, T-cell infiltration after treatment for FL with Rituximab, mast cells in LPL, erythrocyte extravasation in HCL,…

• Immunohistochemistry

“a reliable identification of the subtype of lymphoma can be made in

a significant number of cases”

Disadvantages of BM biopsy

• IHC: some antibodies (e.g. CD103,

CD25) do not work on paraffin

embedded material

• Kappa/lambda: difficult to detect

clonality in B-cells, easy for plasma

cells

Pattern of involvement by

lymphoma:

• Focal non-trabecular

• Focal trabecular

• Diffuse interstitial

• Diffuse solid

• Intrasinusoidal/intravascular

Bone marrow involvement by B-cell lymphoma

NHL type Incidence Pattern

B-ALL/LBL 40-60% Patchy, interstitial

CLL/ SLL 45-75% Nodular, non-paratrabecular

LPL 75-90% Vaguely nodular, non-paratrabecular

MCL 50-80% Nodular, para + non-paratrabecular

FL 40-70% paratrabecular

Splenic MZL ~100% Nodular, non-paratrabecular and

intrasinusoidal (usually focal and in

combination with other infiltration

patterns)

DLBCL 8-35% Nodular, diffuse

THRBCL 0-62% Nodular, diffuse

Intravascular BCL 10-20% intrasinusoidal

Burkitt 20-35% Interstitial (diffuse)

Hairy cell leukemia ~100% Interstitial diffuse

Large paratrabecular

infiltrate

Diagnostic bone marrow biopsy: Reticulin stain

CD20 CD10

Diagnosis: follicular lymphoma

Diffuse interstitial infiltration pattern

“hairy” cells: pale cytoplasm

Reticulin fibrosis, extravasated rbc

Associated hematopoietic alterations

-Hyperplastic (megaloblastoid) RCS

-Dysplastic MKC

-Hypoplastic myelopoiesis

Hairy cell leukemia

Erythroid

hyperplasia

Extravasated

RBC

Interstitial hairy cells

CD20

Diagnostic bone marrow biopsy CD20

CD20 Diagnosis: DLBCL

Diagnostic bone marrow biopsy CD15

CD30 Diagnosis: classical HL

Pitfalls

• Hematogones vs ALL/BL

• Reactive vs neoplastic infiltrates

• Therapy effect: post-Rituximab T-cell

infiltrates

Cytologic/Morphologic assessment

• Discordant morphology: most often DLBCL

in LN , small B-cell infiltration in BM

• Transformation: Richter transformation in

CLL, transformation to DLBCL

• Morphological variant: MCL, blastoid

variant

Fibrosis in

lymphoproliferative diseases • “Any lymphoproliferation – from benign lymphocytic

aggregates to replacement of the BM in CLL- is accompanied by varying degrees of increase in reticulin fibers, particularly within nodules, when present.”

Current Diagnostic pathology 1997:4:36-44

Most frequently:

• Hairy cell leukemia

• Multiple myeloma

• Classical Hodgkin’s lymphoma

• DLBCL

• THRBCL

Cave: underestimation of BM infiltration on aspirate and flow or even discordant results with bone marrow biopsy

In conclusion: bone marrow examination

• Indication: diagnosis, staging, response to

treatment & follow-up

• Material: BM aspiration smears, core biopsy (min

length 2cm) , touch imprints, clot sections and PB

smears

• Methods: cytology, histology,

immunohistochemistry, flow cytometry,

karyotyping, (F)ISH, PCR

Ideally: combined approach

“BM involvement in NHL: increased diagnostic sensitivity by combination of

immunocytology, cytomorphology and trephine histology” Br J Haematol

1992

References • Guidelines for subtyping small B-cell lymphomas in bone marrow

biopsies. Henrique, Achten, Maes et al. Virchows Arch, 1999, 435:

549-558

• Bone marrow manifestations of infections and systemic diseases

observed in the bone marrow trephine biopsy. Diebold, Molina,

Camilleri-Broët et al. Histopathology 2000, 37: 199-211

• The bone marrow trephine biopsy: a review of normal histology.

Brown, Gatter. Histopathology 1993,22:411-422

• Hodgkin and non-Hodgkin lymphoma involving bone marrow.

Viswanatha, Foucar. Seminars in diagnostic pathology, 2003,20: 196-

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