Bone marrow pathology

Educational course, Belgian Society of Hematology

18 Nov 2017

Dr. J. Somja, CHU Sart Tilman, Liège, joan.somja@chu.ulg.ac.be

 Learning objectives
When ?

Bone
Why ? marrow What ?
examination

How ?
 I. When ?

Indications for BM examination

 Indications for BM examination
• Haematological abnormalities that cannot be
explained by available clinical and laboratory
data.
• A good indication is essential for an accurate
diagnosis
• 3 main indications :
– Diagnostic purposes
– Staging for malignant diseases
– Monitoring

Riley et al., JCLA, 2004

 II. What?

What specimens to collect ?

(Bone marrow
Particle clot)
BONE MARROW

Bone marrow aspirate Bacteriology

Bone marrow trephine biopsy
if necessary

Smear Preparation + Heparin +EDTA Heparin

FFPE
2-10 days
Molecular Morpho, IHC,
Flow biology Cytogenetics FISH, (clonal
cytometr (clonal days-weeks expansion, NGS)
y expansion,
NGS, …) Karyotyping BM biopsy
MGG Cyto- FISH (hours)
Days-Weeks FISH touch imprint
cytology chemistr If necessary
(hours) y (hours) (days) (hours)
Usefull if dry tap
 BM aspirate or trephine biopsy ?
BM ASPIRATE
• Quick results
• Fine cytological detail
• Enumeration of marrow
cellular elements
• Wider cytochemical stains
can be used
• Ideal for flow cytometry,
cytogenetics/molecular
studies
BM BIOPSY
• Complete assessement of
cellularity and architecture
• More sensitive for focal
lesions
• Allows grading of fibrosis
• Use of IHC
• Useful for assessment of
AA, metastasis, some
infections
 BM aspirate or trephine biopsy ?
A thorough bone marrow examination includes
both BM aspiration and trephine biopsy.

– MDS: BM aspirate >>> BM biopsy

– MPN: BM aspirate = BM biopsy
– MPN/MDS BM aspirate > BM biopsy
– AML: BM aspirate >>> BM biopsy
– NHL: BM aspirate << BM biopsy
– HL: BM aspirate <<< BM biopsy
– MM: BM aspirate < BM biopsy
– Carcinoma: BM aspirate << BM biopsy
 III. How?
How to collect these specimens ?

1. Anatomic sites
2. Collection procedure
3. Adequacy
 Anatomic sites
• Crest of the posterior superior iliac spine
– Preferred site
• Sternum
– Experienced operator
– Only marrow aspiration !
– Not in MM
• Anterior superior iliac spine
– Rarely performed
• Anterior tibial plateau
– Very young children
Riley et al., JCLA, 2004
 Collection procedure
BM aspirate adequacy
• Adequate aspirate
– 6-12 slides
– Bone marrow particles !
– Not crushed
– Not too thick
– Not clotted
– Allow to dry quickly
– Dry tapes represent 2-7% of the cases
– If an adequate aspirate has not been possible,
considerations should be given to preparing touch imprints
of the core biopsy prior to placing it in fixative
BM Aspirate smears

Riley et al., JCLA, 2004

Touch imprints of the core biopsy

Riley et al., JCLA, 2004

Touch imprints of the core biopsy
Be gentle, be delicate !
Do NOT CRUSH !

Riley et al., JCLA, 2004

 Collection procedure
BM trephine biopsy adequacy
• 11-gauge needle AT LEAST
• If osteopenic, a 8-gauge needle allows the collection of
an intact core biopsy with minimal crush artifact
• 13-gauge biopsy needle for paediatric patients

• Adequate core biopsy,

– At least 1.6 cm to 2 cm long
• Prior to fixation
• Exclusive of cortical bone, cartilage, or periosteum
• Free of crush artifact or interstitial hemorrhage or fragmentation
Riley et al., JCLA, 2004
 I. Why?
Understanding of the BM examination reports
Shemes like this should not have any
secrets …
BM trephine : Generalities

Jaffe, Hematopathology, second edition, 2017

 Erythropoïesis Granulopoïesis

Courtesy of Pr. Tassin and Dr. Keutgens Jaffe, Hematopathology, second edition, 2017
 Mégacaryocytes Other?
• Monocytes
• Macrophages
• Plasma cells
• Lymphoid cells
• Mast cells
• Oestoclasts
• Bone
• Iron
• …

Courtesy of Pr. Tassin and Dr. Keutgens Jaffe, Hematopathology, second edition, 2017
 Immunohistochemistry
• Erythroid: GlycophorinA, LMO2, CD71
• Myeloid: MPO
• Megacaryocytes: CD61, Factor VIII
• Blasts: CD34, CD117, CD33
– CD34+ cells are rare in normal marrow
– CD34 does not equal blast
• not all blasts are CD34+
• not all CD34+ cells are blasts
– Not all AML’s are CD34+
– CD34 is not lineage specific
• Mastocytes: Tryptase, CD117, CD25, CD2
• Plasma cells: CD138, IgKappa, IgLambda
• Lymphocytes: CD20, CD3, CD30, …
• …
 Cellularity
100-age = expected cellularity for age

Severe AA is characterized by a markedly hypocellular bone marrow (<25% of normal for

age or 25 to 50% of normal with <30% hematopoietic cells) accompanied by two of the
following: granulocytes <0.5 × 109/L; platelets <20 × 109/L; or corrected reticulocyte
count <1%
Riley et al., JCLA, 2009 Jaffe, Hematopathology, second edition, 2017
Bone marrow (neoplastic) pathology
WHO 2016 Myeloid Neoplasms
WHO 2016 Myeloid Neoplasms
 1. Myeloproliferative neoplasms
• Clonal hematopoietic disorders

• Characterized by proliferation of cells of one or more of the

myeloid lineages; erythroid, granulocytic, or megakaryocytic

• Initially, the proliferation in the bone marrow is effective and

associated with maturation of the neoplastic cells

• Leads to increased numbers of mature granulocytes, red blood

cells (RBCs), and platelets in the peripheral blood

• Splenomegaly and hepatomegaly are common and caused by the

sequestration of excess blood cells, extramedullary hematopoiesis
or both in these organs.
 Chronic Myeloid Leukaemia, BCR-ABL1+
• Blood findings
– Leukocytosis
– Plts N or ↗
– Often anaemia
– Spectrum of maturing granulocytes with a « myelocyte bulge »
– Blasts usually <2% if WBC
– Absolute basophilia
– No significant dysplasia
• BM findings
– Hypercellularity
– Increased M:E ratio
– Blasts usually <5%, always <10%
– Widening of maturing granulocytes with myelocyte bulge but no dysplasia
– Megs N or ↗ in number, with « dwarf » morphology
– Reticulin fibers N to moderately increased
• Genetics
– 100% have Phi chromosome or BCR-ABL1 fusion gene
Courtesy of Pr. Tassin and Dr. Keutgens
Jaffe, 2017
Jaffe, 2017
Jaffe, 2017
Jaffe, 2017
Jaffe, 2017
Essential Thrombocytaemia

From major mixed

to the 4th to minor

Jaffe, 2017
Jaffe, 2017
Possible cause of Thrombocytosis
• Secondary
– Infection
– Inflammation and AI diseases
– Blood loss, hemorrhage
– Chronic iron deficiency
– Post-splenectomy
– Hyposplenism
– Trauma (brain injury)
– Post-surgical procedures
– Neoplasms (Non-hemato and non-myeloid hemato)
– BM regeneration, rebound, following chemotherapy
• Myeloid neoplasm related
– MPN
– CML, BCR-ABL1+
– PV
– ET
– AML with t(3;3)(q21.3;q26.2) or inv(3)(q21.3q26.2)
– MDS with isolated del(5q) abnormaly
– MDS/MPN-RS-T
 Polycythaemia Vera

Was >18.5 g/dL Was >16.5 g/dL

From minor to
major criteria

Endogenous erythroid colony formation in vitro

Jaffe, 2017
Jaffe, 2017
Jaffe, 2017
Primary Myelofibrosis
Jaffe, 2017
 Diagnostic criteria of distinctive value regarding WHO-defined ET (left) versus early-
prefibrotic stage of PMF (right), including standardized morphologic features (Table 1
contains more details), allowing the generation of characteristic histologic BM patterns

Jürgen Thiele et al. Blood 2011;117:5710-5718

©2011 by American Society of Hematology

Jaffe, 2017
Jaffe, 2017
Jaffe, 2017
Bone marrow fibrosis
European consensus on
grading bone marrow
fibrosis and assessment
of cellularity. J.Thiele et
al. Haematologica
2005; 90:1128-1132
Standard grading
Bauermeister 1971; Manoharan et al. 1979
Jaffe, 2017
 2. Myelodysplastic syndromes
Definition
• Sustained unexplained anemia, neutropenia or
thrombocyopenia (Hb<10g/dL; Abs. Neutrophil count <1.8 x109 or platelets <100 x109/L)

• And at least one of the following

– Dysplastic morphology in erythroid cells, granulocytes or
megacaryocytes, affecting at least 10% of the cells of at least
one of these lineages

– Acquired conal MDS-associated cytogenetic abnormality in

hematopoïetic cells and absence of de novo AML-defining
cytogenetic abnormalities t(15;17), inv (16)/t(16;16) or t(8;21)

– Increased blasts (at least 5% of marrow cells) not attributable to

exogenous GF administration or transient marrow recovery
 Acquired conal MDS-associated
cytogenetic abnormality

Del(20q), +8 and –Y abnormalities, although common findings in MDS, are not considered
MDS defining and cannot in isolation be used to make a diagnosis of MDS
 Dysplastic Erythropoeisis
• Nuclear
– Nuclear budding
– Internuclear bridgking
– Karyorrhexis
– Multinuclearity
– Nuclear hyperlobation
– Megaloblastic changes
• Cytoplasmic
– Ring sideroblasts
– Vacuolisation
– PAS positivity

Jaffe, Hematopathology, 2017

Cantù Rajnoldi et al., Ann Hematol, 2005
 Ring Sideroblasts
• 3 types of sideroblasts
– Type 1 : <5 siderotic granules
• Reactive ring sideroblasts
– Type 2 :>5 siderotic granules but no perinuclear
distribution
– Alcohol (plasma cell iron)
– Type 3 : Ring sideroblasts : 5 or more perinuclear
granules in a perinuclear distribution, surrounding
– Zinc
the nucleus or at least 1/3 of the nuclear
circonference
– Pyridoxine deficiency
– Drugs (isoniazid, cycloserine)

• MPN and MDS/MPN

– PMF, ET
– MDS/MPN-RS-T

• Hereditary
– Hereditary sideroblastic
anaemia

Mufti et al., Haematologica, 2008

Dysplastic Myelopoiesis
• Small or unusually large
size
• Nuclear hypolobation
(pseudo-Pelger-Huët;
pelgeroid)
• Irregular
hypersegmentation
• Decreased granules;
agranularity
• Pseudo-Chédiak-Higashi
granules
• Auer rods

Jaffe, Hematopathology, 2017

Cantù Rajnoldi et al., Ann Hematol, 2005
Dysplastic Megacaryotes
• Micromegacaryocytes
• Nuclear hypolobulation
• Multinucleation
– Normal megacaryocytes
are uninucleate with
lobulated nuclei

Jaffe, Hematopathology, 2017

Cantù Rajnoldi et al., Ann Hematol, 2005
Hasserjian, XIII EBMWG , 2017
 MDS : Pitfalls
Non-MDS conditions associated with
cytopaenia and >10% dysplasia
• Drugs/toxins
– Recent (<6 m) chemotherapy
– Heavy alcohol intake
• Metabolic deficiencies: B12, folate, copper
• « Stress erythropoiesis » due to haemoglobinopathy or
acquired/congenital haemolytic aenemia
• Infections (HIV, HepC, …)
• Autoimmune diseases
• Concurrent neoplasm
– Infiltrating marrow (especially MM and HCL)
– Rare paraneoplastic dysplasia for remote tumour

Castello A et al., Haematologica, 1992

 MDS : WHO update 2016
Cytogenetivs by
Dysplastic RS as % of marrow BM and PB conventional karyotype
Cytopaenias
lineage erythroid elements blasts analysis

MDS with single lineage dysplasia Any, unless fulfills all

1 1 or 2 <5% BM
(MDS-SLD) criteria for del(5q)
MDS with multilineage dysplasia Any, unless fulfills all
(MDS-MLD) 2 or 3 1-3 <5% BM criteria for del(5q)

MDS-RS with single lineage ≥15%/≥5% and Any, unless fulfills all
dysplasia (MDS-RS-SLD) 1 1 or 2 SF3B1 mutation BM criteria for del(5q)

MDS-RS with multilineage Any, unless fulfills all

≥15%/≥5%and
dysplasia (MDS-RS-MLD) 2 or 3 1-3 BM criteria for del(5q)
SF3B1 mutation
Del(5q) alone or with
one additional
MDS with isolated del(5q) 1-3 1-2 None or any BM
abnormality except -7 or
del(7q)
BM 5-9% or PB
MDS-EB-1 0-3 1-3 None or any 2-4% Any
No auer rods
BM 10-19% or
MDS-EB-2 0-3 1-3 None or any PB 5-19% or Any
Auer rods
MDS, unclassifiable (MDS-U)

BM, no Auer
-with 1% blood blasts 1-3 1-3 None or any Any
rods
-with single lineage dysplasia and
1 3 None or any BM Any
pancytopenia
-based on defining cytogenetic MDS-defining
0 1-3 <15% BM
abnormality abnormality
 MDS :
Recurrent somatic genetic mutations

-Not sufficient to diagnose MDS in a

cytopenic patient (CHIP)!
-May support an MDS diagnosis
suspected by other observations

Genomic architecture of MDS. (A) Frequency of driver mutations identified in the sequencing
screen or by cytogenetics in the cohort of 738 patients, broken down by MDS subtype.

Elli Papaemmanuil et al. Blood 2013;122:3616-3627

©2013 by American Society of Hematology

 Indolent Myeloid Haematopoietic
Disorders
ICUS IDUS CHIP CCUS MDS
Somatic
mutation - - +/- +/- +/-

Clonal
karyotypic - - +/- +/- +/-
abnomality
Marrow
- + - - +
dysplasia
Cytopaenia + - - + +

ICUS : idiopathic cytopaenia of unknown significance

IDUS: idiopathic dysplasia of unknow significance •Heterogeneous group
CHIP: clonal hematopoeisis of indeterminate potential
CCUS : Clonal cytopaenia of unknown significance •Can evolve into MDS or AML
•Frequent monitoring of blood count recommended

Valent et al., Am J Cancer Res, 2011

 5. Acute Myeloid Leukaemia
• Heterogeneous group of diseases
• Clonal proliferations of immature, non-lymphoid, bone marrow–
derived cells
• Most often involve the bone marrow and peripheral blood
• May present in extramedullary tissues
• Aggressive clinical course.
• Diagnostic on the basis of a minimum blast cell count in bone
marrow (>20%) or peripheral blood
• Several specific AML types are now defined without regard to blast
cell count
– Acute myeloid leukemia with t(8;21)(q22;q22.1); RUNX1-RUNX1T1
– Acute myeloid leukemia with inv(16)(p13.1q22) or
t(16;16)(p13.1;q22); CBFB-MYH11
– Acute promyelocytic leukemia with PML-RARA
 Still a role for morphology in the
diagnosis of AML?
• Blasts (in all cases) should be counted the old fashion way, not
based on flow cytometry !
• May allow establishment of a quick diagnosis
– Especially important in the diagnosis of acute
promyelocytic leukemia so therapy can be started
• Leads to the ability to review all of hematopoiesis
– AML with MDS related changes
» Previous MDS
» Dyspoiesis > 50% of the cells in 2 cell lines
» Cytogenetic MDS abnormalities
• Exclude relevant differential diagnoses
• Clues to the diagnoses of AML with recurrent genetic
anomalies can be obtained by evaluating morphology
Jaffe, 2017
AML with MDS-related changes

Jaffe, 2017
 Survival data.

Olga K. Weinberg et al. Blood 2009;113:1906-1908

©2009 by American Society of Hematology

Jaffe, 2017
 CONCLUSION
• Integration of clinical, morphologic,
immunophenotypic, genetic, and other
biologic features is mandatory to define
specific disease entities
• The relative contribution of each feature
varies, depending on the case
• Make your
cytologists/pathologists/geneticians good ! by
providing them relevant clinical informations
and optimal samples.
 BIBLIOGRAPHY
• Elaine S. Jaffe et al., Hematopathology, second edition,
2017,
• Bain B., Bone Marrow Pathology 4th ed. 2009
• The WHO classification of tumors of the hematopoietic
and lymphoid tissues. 2016 revision. Arber et al. Blood
2016; 127(20):2391-2405
• Swerdlow SH, Campo E, Harris NL, et al, eds. WHO
Classification of Tumours of Haematopoietic and
Lymphoid Tissues. Revised 4th ed. Lyon, France: IARC
Press; 2017.
• XIII EBMWG International Course and Workshop on
Bone marrow Pathology, Utrecht, 2017