Lab Diagnosis of Chronic lymphoproliferative disorders (CLPD);Flowcytometric Evaluation Basavatarakam Indo-American Cancer Hospital and Research Institute

1. Dr. Siddartha. K
Moderator: Dr. Faiq Ahmed
Dr. Vishal Rao
Basavatarakam Indo-American Cancer Hospital and Research
Institute

2. • Definition
• WHO Classification of hematolymphoid malignancy
• Lymphoproliferative disorders
• Lab diagnosis
• Flowcytometry
• Limitations and
• Take home message

3. • Are set of disorders characterized by abnormal proliferation of
lymphocytes into monoclonal population (lymphocytosis).

9. • Chronic lymphocytic leukemia/ small lymphocytic lymphoma,
• B-cell prolymphocytic leukemia,
• Follicular lymphoma,
• Mantle cell lymphoma,
• Marginal zone lymphoma,
• Diffuse large B cell lymphoma,
• Hairy cell leukemia and its variant,
• Lymphoplasmacytic lymphoma

10.  T cell prolymphocytic leukemia,
 T cell large granular lymphocytic leukemia,
 Adult T cell leukemia/lymphoma,
 Hepatosplenic T cell lymphoma,
 Sezary syndrome.
 Angioimmunoblastic T cell lymphoma
 Anaplastic large cell lymphoma; ALK +ve and –ve.

11. • Chronic Iymphoproliferative disorder of NK cells
• Aggressive NK cell lymphoma

12. • S. Lactate dehydrogenase
• β2 microglobulin
• Microscopic lymph node examination
• Immunphenotyping

13. Biochemical Parameters
• S.Lactate dehydrogenase
levels are increased in patients with lymphoproliferative
disorders.
• Serum β2 microglobulin
levels are increased in pts with lymphoproliferative disorders.

14. Histopathological Examination

15. • Small cells with scanty cytoplasm -- CLL, MCL, MZL, low grade FL
• Small cells with moderate cytoplasm – HCL, MZL
• Small cells with eccentric nucleus and lymphoplasmacytic cells –
LPL, MZL
• Mixed small cells, cleaved cells and large blastic cells – FL, CLL,
CLL/PLL, MCL
• Large cells with or with out vacuoles – high grade FL, DLBCL, BL,
B-PLL, blastic variant of MCL.

16. Immunophenotyping
Immunohistochemistry
Flowcytometry

17. • Immunohistochemistry
CD20

18. Flow cytometry (FCM) has been used in the analysis of human
lymphomas since the late 1970s
Flow cytometery is the methodology used to detect cell surface
antigens using monoclonal antibodies conjugated with different
fluorochromes.

19. • A light source.
• Fluid lines and controls to direct a liquid stream containing particles
through the focused light beam.
• An electronic network for detecting the light signals coming from
the particles as they pass through the light beam and then
converting the signals to numbers that are proportional to light
intensity.
• A computer for recording the numbers derived from the electronic
detectors and then analyzing them.

20.  The specimen must be in a
monodisperse suspension.
 In this, isotonic fluid is forced
under pressure where a fluid
column with laminar flow and a
high flow rate is generated (so-
called sheath fluid).
 The sample is introduced into the
flow cell in the center of the sheath
fluid, creating a coaxial stream so
that they are presented to the light
beam one at a time.

21. • The emitted light is focused by a lens onto fiber optic cables and
transmitted to octagonal detectors.
• The sensors convert the photons to electrical impulses that are
proportional to the number of photons received and to the number
of fluorochrome molecules bound to the cell.

22. • Gating - isolate a specific group of cytometric events from a large
set.
• Selectively visualize the cells of interest while eliminating results
from unwanted particles e.g. dead cells and debris.
• To separate b/w
 Neoplastic and non neoplastic
 Lymphoid and non lymphoid populations

23. • To assess the clonality
• For diagnosis
• Staging of lymphomas
• Evaluation of Prognostic markers
• Detection of target molecules for therapies
• Detection of minimal residual disease

24. Samples for flowcytometry
• Peripheral blood
• Bone marrow
• Body cavity fluids
• Needle aspirates
• Biopsies of lymph node, spleen and skin
(Peritoneal,Pleural,CSF)

25. CD5
CD19
CD23
CD10
CD20
CD103
FMC 7
Kappa
Lambda
CD38
CD56
IgG
CD4
CD8
IgG/IgG/45/19
10/23/45/19
22/5/-/19
κ/λ/20/19
FMC7/103/-/19
HLADR/34/45/19
38/3/-/19
56/4/8/19

26. Identification of abnormal mature B-lymphoid cells.
a. Immunoglobulin light chain restriction
b. Aberrant antigen expression

27. Immunoglobulin Light Chain Restriction

28. CD5 expression in B cells.
 Normally CD5 is seen in T cells but not in B cells.
 But CD5 expression over B cells is aberrantly expressed - neoplastic
proliferation.
Presence of antigens not normally expressed by B cells.
 Aberrant expression of myeloid antigens like CD13, CD33 is found(
less frequently).
 Eg. Lymphoplasmacytic lymphoma

29. Abnormal expression of antigens not typically in a subset of B cells
belonging to a distinct biologic compartment.
Bcl-2 is absent in germinal center B cells, but increased expression is
seen in follicular lymphoma and some DLBCL.
Alteration in intensity of staining for B lineage – associated antigens.
• Dim – CD20, CD22 and CD79a – CLL
Moderate – CD20, – MCL
Bright – CD20, CD22 – HCL

30. Lymphoid cells
Clonal lymphoid
cells
B- Cells T -Cells

31. Clonal B Lymphoid
cells
CD5 +
CD10-
CD5 +
CD10+
CD5 -
CD10+
CD5-
CD10-
CLL/SLL
MCL
LPL
DLBCL
PLL
DLBCL
FL
MCL
CLL/SLL
BL
FL
DLBL
BL
HCL
MZL/SMZL
LPL
HCL/Variant
DLBL
FL
MCL

33. Neoplasm composed of monomorphic small, round to
irregular B lymphocytes in the PB, bone marrow, spleen
and lymph nodes, admixed with prolymphocytes and
paraimmunoblasts forming proliferation centers in tissue
infiltrates.
• Antigen experienced B-cell
• CD20,CD22,CD79b,surface IgM/IgD -- weak
• CD23 and CD5 – mod positive
• FMC7 Negative
CD38 & ZAP 70 prognostic markers

35. • CD5 neg
• CD23 neg
• FMC7 Positive
• CD 11c Positive
• CD79b positive
• S Ig strongly positive

36. • Monomorphic small to medium-sized lymphoid cells with irregular
nuclear contours and CCND1 translocation.
• Peripheral B-cell of inner mantle zone.
• M/c site - LN; the spleen and BM with or without PB involvement.
• CD5 +ve, CD10 neg.( +ve CD10 in aberrant)
• CD19, CD20, CD22, CD79a +ve.
• Intense surface IgM/lgD (λ > κ).
• CD23 weak or neg
• FMC7 +ve and CD43 neg.

37. • Cyclin D1 -- in all the cases
• Cyclin D1 negative MCL show positivity for Cyclin D2 or Cyclin D3.
• t(1 1.14)

40. • Neoplasm of B prolymphocytes affecting the PB, BM and spleen.
• CD5 -- 20-30% +ve.
• CD23 – 10-20% +ve.
• IgM/IgD strong +ve
• CD 19, 20, 22, 79a, 79b, FMC7 +ve.
• ZAP-70 and CD38 are expressed in 57% and 46% of the cases
respectively.

42. • Neoplasm of small B lymphocytes, plasmacytoid lymphocytes, and
plasma cells usually involving BM, lymph nodes and spleen.
• IgM, IgG +ve and rarely IgA.
• But IgD will be neg.
• CD138 +ve
• CD5
• CD10
• CD23
• CD103
CD19
CD20
CD22
CD79a
-ve
+ve

43. • Indolent neoplasm of small mature B lymphoid cells with oval
nuclei and abundant cytoplasm with "hairy" projections involving
PB and diffusely infiltrating the BM and splenic red pulp.
• Activated memory B-Cell .
• CD5 & CD10 – neg .
• Bright expression of CD20, CD22 and CD11c.
• CD103, CD25, CD123, T-bet, Annexin A1, DBA.44, FMC-7 will be +ve

44. • CD5 & CD10 – neg
• Annexin 1 -- negative
• CD123, CD25 – negative
• DBA.44, Pan-B-cell antigens, CD11c, IgG, CD 103 and FMC7 -- pos

45. • Lymphoma cells may be found in the peripheral blood as villous
lymphocytes.
• IgM/IgD +ve
• CD20, CD79a  +ve
• CD5, 10, 23, 43 are negative
• Annexin 1, cyclin D1 and CD103 are neg

47. Germinal center B cell
• FL predominantly involves LN, spleen, BM, PB and Waldeyer ring.
• Usually CD5 neg and CD10 positive.
• B- cell Ag- CD 19,20,22, CD79a  +ve
• BCL-2 & BCL-6 +ve
• Grade 3B cases may lack CD 10, but retain BCL6 expression.
• CD10 expression is often stronger in the follicles than in
interfollicular neoplastic cells .
• May be absent in the interfollicular component, as well as in areas
of marginal zone differentiation,

48. IRF4/MUM1 is typically absent in FL.
BCL2 protein is expressed by a variable proportion of the neoplastic
cells in 85-90% of cases of grade 1 and grade 2 FL, but only 50% of
grade 3 FL using standard antibodies.

49. • Peripheral B-cells of either germinal centre or post
germinal centre (activated B-cell ) origin.
• CD 19, 20, 22, CD79a-- +ve
• Surface and/or cytoplasmic immunoglobulin (lgM>lgG>lgA) can be
demonstrated in 50-75% of cases.
• CD30 - especially in the anaplastic variant.
• CD5 +ve in 10% of cases.
• CD 10 +ve - (30-60)%
• BCL6 +ve – (60-90)%
• MUM1 +ve – (35-65)%

50. • Subgrouping DLBCL, NOS by immunophenotyping

51. Clonal T Lymphoid
cells
CD4 + CD8-
CD4 +
CD8+
CD4 –
CD8+
CD4- CD8-
T-PLL
ATLL
SEZARY Syn
ALCL
AITCL
PTCL-NOS
T-PLL
ATLL
T-LGL
PTCL-NOS
T-LGL
NK-LGL
HSTCL
SEZARY syn
EATCL
HSTCL
Aggressive NK
Cell Leukemia

53. • Aggressive T-Cell leukemia – small to medium size prolymphocytes.
• Cytoplasmic protrusions or blebs.
• HTLV-1 negative.
• CD1a, TdT –neg, TIA -1 neg
• Mem CD3 – weak
• CD4 + CD8 -ve --- 60%
• CD4 + CD8 +ve --- 25%
• CD4 - CD8 -ve --- 15%
• CD7 positive
• CD25 variable
CD52(CAMPATH-1 Ag) dense positive
http://www.bloodjournal.org/content/bloodjournal/12
0/3/538/F2.large.jpg?sso-checked=true
Protrusion

54. • CD4 + CD8 –ve ( rarely both positive)
• CD 25 Strong staining
• CD2, CD3, CD5 positive
• CD7 negative
• Large transformed cells + CD30 (ALK Neg)
• HTLV-1 positive

55. • CD4 + CD8 –ve
• CD2+, CD3+ , TCRβ+ and CD5+.
• CD7 & CD 26 - negative,
• CD 25 variable staining
Erythroderma,
Generalized LAP
Sezary cells

56. Mature T-Cell Leukemias Including T-Prolymphocytic Leukemia, Adult T
Cell Leukemia/Lymphoma, and Sézary Syndrome
Kathryn Foucar, MD

57. • PB involvement seen in small cell variant .
• CD4 + CD8 –ve.
• Pan T cell marker (CD3) – neg.
• CD2 frequently +ve but CD5 and CD7 are negative.
• CD 30 strong positive in large cells (weak in small and medium)
• CD13, CD15, CD33 (aberrant myeloid markers +ve).
• t(2 ;5)/ NPM-ALK will be positive

58. • CD4 Follicular Helper T Cell
• CD4 + CD8 –ve
• Pan T cell marker CD2, CD3, CD5 – positive.
• Decreased expression of CD3 and CD7.
• CD10, BCL6, CXCL13, PD-1 +ve (Normal T helper cells).

59. • PB is sometimes involved, but leukemic phase is uncommon.
• CD4 + CD8 –ve ( uncommon both +ve and both neg).
• Aberrant loss of CD7 & CD5.
• TCR β chain is expressed.
• CD 30 +ve.
• Campath 1 Ag +ve in 40%.

61. • Persistent increase in the no. of PB large granular lymphocytes
(2-20 ×109/L).
• Sustained immune stimulation.
• Interstitial or intrasinusoidal infiltrate is seen.
• CD4 neg, CD8 + ve.
• CD3, TCR αβ +ve.
• CD16 & CD57 +ve ( few are +ve for CD56).
• Abnormally diminished or lost expression of CD5 and / or CD7 is common.
Uncommon Variants
• CD4 +ve TCR αβ +ve,
• CD4 +ve TCR γδ +ve.

62. • Persistent increase in the no. of PB NK cells (≥ 2 ×109/L) with out
identified cause.
• CD4 neg, CD8 + ve.
• Surface CD3 is neg but Cyt CD3 is often +ve.
• Diminished or lost expression of CD2, CD7 and CD57.
• CD16 +ve, CD56 weak positive.

64. • Cytotoxic T cells usually of γδ TCR type.
• 20% of HSTL arise in the setting of chr. Immuno supression.
• CD4 neg, CD8 neg ( rarely CD8 +ve)
• CD3 +ve
• Complete absence of CD5
• CD16, CD56 +ve , CD57 neg
• TIA-1 pos.
• γδ TCR +ve
• Isochromosome 7q is seen.

65. • A systemic neoplastic proliferation of NK-cells almost always
associated with Epstein-Barr virus and an aggressive clinical course.
• The most commonly involved sites are PB, BM, liver and spleen.
• CD4 neg, CD8 neg
• CD2+, surface CD3-. CD3 ε+, CD56+
• CD16 is frequently positive, but CD57 is negative
• CD11b may be expressed

67.  Main drawback is, it requires fresh and unfixed tissue for analysis.
 When reactive cells are more than the neoplastic cells .
 Dilution of sample.
 Cannot grade the Follicular lymphoma.
 Cost .

68. • Though morphology is the gold standard for diagnosing
lymphoproliferative disorder, however Immunophenotyping is
essential for subtyping
• Targeted therapy is possible
• Genotypic studies when needed can be done

69. • WHO Classification of tumours of haematopoietic and lymphoid
tissues , 4th edition.
• Ioachims lymphnode pathology 4th edition.
• Wintrobes text book of clinical hematology.