How Many Markers is

Enough?
Hematopathology
Emina Emilia Torlakovic, MD, PhD
College of Medicine
University of Saskatchewan
Saskatoon, SK, Canada

Kremer M, Quintanilla-Martinez L, Nahrig J, von Schilling C, Fend F.

Immunohistochemistry in bone marrow pathology: a useful adjunct
for morphologic diagnosis. Virchows Arch. 2005 Oct 18;:1-18

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IHC in BM pathology

 Acute leukemia (ALL and AML)

 Blasts estimates/counts
– MDS, MDS/MPN, MPN
 Malignant lymphoma
 Metastatic tumors
 Systemic mastocytosis
 Other

Acute Leukemia

 CD34, TdT, MPO, CD33, CD117, F8-ra,

CD61 (or CD42b), Hgb-A, Gly-C, Pax-
5, CD10, CD79a, CD20, CD45, CD3

 To consider: NPM1, CD68, CD63,

lysozyme, CD15, CD123, CD99, CD31

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MDS and MDS/MPN

 CD34, CD117, CD61 (or CD42b), MCT

 To consider: MPO, CD33, MCT, Hgb-A,

Parvovirus B19

Myeloproliferative Neoplasms
(MPN)

 CD34, MPO, CD61 (or CD42b or F8-

ra), PG-M1, Hgb-A

 To consider: CD33, TdT, CD3, CD20,

Pax-5

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CD34 Counts & Estimates

 (%) in 500
cell count
 > 3 pos. cells
 > 5 pos. cells

Mature B-cell Neoplasms

 CD3, CD20, CD10, CD5, cyclin D1,

CD23, Bcl-6

 To consider: CD79a, IgM, IgD, CD25,

Annexin-1, Bcl-2, k, l, CD138, CD72,
Bcl-3, ZAP70, CD43

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CD20 and CD3 are always
interpreted together

CD20 ≠ Pax-5 ≠ 79a

CD20 ≠ Pax-5

J Pathol

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Pax-5 in FL

CD20: Lymphoma

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Cyclin D1
MCL HCL MM

Mature T-cell Neoplasms

 CD2, CD3, CD4, CD5, CD7, CD8,

CD56, CD57, TIA-1, grenzyme B, EBV

 To consider: MUM1, CD45RO, Bcl-6,

CD30, ALK-1, perforin

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Hodgkin Lymphoma
 NLPHL: Pax-
Pax-5, CD20, CD3, Bcl-
Bcl-6, Oct-
Oct-2, CD57

 Classical:
 For presentation in the BM: CD30, CD15, CD3,
CD20, Pax-
Pax-5, MUM1
– To consider:
consider: EBV, PU.1, Oct2, BOB.1
 For staging when malignant cells present: CD30,
CD15
 For staging when abnormal cells not
morphologically obvious: CD3, Pax-
Pax-5, CD30

Plasma Cell Disorders

 CD138, k, l, CD45, CD56, CD20

 To consider: Ki-67, EBV, IgM, IgD,

IgA, IgG, IgE, Pax-5, CD117, cyclin
D1, HHV-8, EBER

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Histiocytic and Dendritic Cell
Neoplasms

 CD68, CD163, PU.1, CD45, CD21,

CD23, CD35, CD123, S-100, CD1a,
vimentin

 To consider: fascin, langerin,

lysozyme, TCL1

Mastocytosis

 MCT, CD25, CD117

 To consider: CD34, CD3, CD20

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Lymphoma

 WHO 2008 Classification

– Selection of markers depends on criteria

required for the classification

– Evidence based medicine

Basic Panel

 CD3, CD20, Bcl-2, CD138, kappa,

lambda

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Always Interpreted
Together

 
Lymphoplasmacytic
lymphoma

CD3

CD45RO-UCHL1≠CD3

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 Cytoplasmic CD3

•T-lymphoblastic
•T/NK lymphomas

T/HRBCL

CD3 CD8

CD8 CD20

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 CD20

CD20 CD21

CD75 CD79a

NLPHL

CD75 CD75

CD79a CD20

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CD57

Oct-2

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Ki-67 in
Hematopathology
 Burkitt lymphoma: “nearly 100%”
100%” (class I)I)
 Mantle cell lymphoma: > 40% adverse prognostic
indicator (class
(class II)
II)
 ALL (low – better prognosis): higher in adults than
in children and higher in T-
T-ALL than in B-
B-ALL; good
prognosis ALL <25% (class
(class II)
II)
 Very high in ALPS (class
(class I)
I)
 Low in most low-
low-grade LPD (class
(class I)
I)
 Plasmablastic lymphoma: minimum 75%, usually
>90% (class
(class I)
I)
 Other…
Other…

Cyclin D1 in Mantle Cell Lymphoma

Control

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 ALK-1 in ALCL with t(2;5)

WHAT IS YOUR DIAGNOSIS?

Pax-5

Skin Bx:
Blastic morphology,
TdT+, Pax-5+,
CD45-

Answer:
Merkel Cell Carcinoma
(CK20+)

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Critical B-Cell Markers

– CD5 – Pax-
Pax-5
– CD10 – CD138 & 
– CD20 – Bcl-
Bcl-2
– CD21 – Bcl-
Bcl-6
– Cyclin D1
– CD23
– Ki-
Ki-67
– CD43
– TdT
– CD45
– CD34
– CD79a
– EBV (EBER or LMP1)

Additional Important B-
Cell Markers
 HHV-8 (PEL, CD)
 CD30
 IgM
 IgG, IgD, IgA
 MUM1
 PU.1

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Critical T-Cell Markers

– CD56
– CD2, CD3, CD5, CD7
– ALK
– CD10, CD57, PD-
PD-1, CLCX-
CLCX-1
– TdT
– CD21
– CD34
– CD30
– CD4
– CD43
– CD8
– CD45, CD45RO
– TIA-
TIA-1
– Granzyme B/perforin
– EBV (EBER or LMP1)

Hodgkin Lymphoma

– CD3 and CD20

– CD15 and CD30
– Pax-
Pax-5, MUM1, and Oct-
Oct-2
– CD21 and CD23
– CD45
– Bcl-
Bcl-6, EMA, CD57

– EBV (EBER or LMP1)

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 CD20 (Membranous)

 Most commonly used pan-

pan-B antibody
 Some B-cell lymphomas are defined
by the lack of expression of CD20
(PLASMABLASTIC LYMPHOMA)
 Some myelomas are defined by
expression of CD20 (CD20+ MM)
 It is not necessarily negative in cHL
 Special attention to Rituximab TX

CD79a (Cytoplasmic)
 It is considered as a back-
back-up B cell antibody;
It is very valuable in providing additional
information
 Expressed at all stages of B-
B-cell differentiation
(best marker for lymphoplasmacytic
lymphoma)
 It is not lineage marker; Expressed in some
AML, and also some T- T-ALL
 Malignant plasma cells express CD79a variably
 Very unusual to see in cHL;
cHL; CD20 is much
more common than CD79a

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 Pax-5 and MUM1/IRF4
(Nuclear)

 Not really back-up markers

 Pax-5 widely expressed
 MUM1 very selectively expressed
(post-GC marker)
 Very useful for HL, ALL, CD20-
negative B-cell lymphomas

Kappa and Lambda

 Demonstrate cytoplasmic light chains in
plasma cells and some B-cell lymphomas
– MM, LPL, IBL, MZL

 Pitfalls
– Extremely difficult to optimize to detect surface
light chain and to reliably interpret results of
cytoplasmic light chains
– Consider in situ hybridization for light chain
mRNA: the sensitivity is the same but the
specificity is higher

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CD21, CD23 (Pan B)

 Mainly for detection of follicular dendritic cells

(FDCs)
FDCs)
– Follicular lymphoma (! needle core bx)
– AITCL (relationship to vascular structures)
– FLg3 vs. DLBCL
– cHL
– Follicular T-
T-cell lymphoma
– Dendritic cell tumors
 Chronic lymphocytic leukemia
 Rare in DLBCL

Bcl-2 (Cytoplasmic)
 CRITICAL MARKER FOR:
– FL vs. reactive follicular hyperplasia

 Buritt lymphoma vs, DLBCL (along with Ki-

Ki-
67)
– BL is Bcl-
Bcl-2 NEGATIVE!

 Pitfalls
– Negative in minority of FLs (just like CD10)
– Negative in primary cutaneous FL
– Colonizing marginal zone cells will be bcl-
bcl-2(+):
correlate with Bcl-
Bcl-6 and CD10 expression.

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 CD5 (Membranous)

 Essential in the panel for evaluation

of small B-cell lymphoprolifertive
disorders (CLL & MCL vs. MZL, LPL,
FL)
 CLL > MCL
 CD5+ DLBCL is very aggressive
disease
 Loss of CD5 in T-cell lymphomas

CD4 and CD8

(Membranous)
 Double positive in T-ALL
 Often mixed even in T-cell lymphomas
 Reactive CD8 cells can predominate
and can have very atypical
morphology (T/HRBCL)
 CD4 prim Ab may be difficult to
optimize in FF/PE tissue

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 CD56

 All nasal-type NK/T cell lymphomas

 Positive in aggressive NK-cell leukemia
 Positive in the rare blastic plasmacytoid
dendritic cell neoplasm (CD4+, CD56+
hematodermic neoplasm)
 Positive in MM
 Rarely positive in T-LGL

CD30
(Golgi, Cyotplasmic, Membranous)

 Typically used for ALCL, HL, CD20-

CD20-DLBCL,
 Also expressed on reactive immunoblasts both in
T and B immunoblasts
 Doesn’
Doesn’t work well in B5 fixed tissues; NOTE:
CD15 doesn’
doesn’t work so well in formalin-
formalin-fixed
tissues
 Embryonal Ca
 Many large cell lymphomas in subpopulation of
cells
 Often found together with MUM1 (ALCL, cHL)cHL)

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