PANEL DISCUSSION – INDIAN PERSPECTIVE

A. Das Gupta, MD, PhD

Super Religare Laboratories

Points for discussion

• QA

• Tests offered by SRL

• Practical issues

• Potential areas of collaboration

TESTS OFFERED BY SRL

• Lymphocyte subset enumeration (T, B & NK) (2-colour, 3-colour, 4-colour)

• Leukemia/lymphoma immunophenotyping• PNH diagnosis by RBC analysis• HLA B27 typing• Zap 70• DNA/Cell cycle analysis

ISSUESGeneral issues• Cost – Instrument upgrading (multicolour assays), Reagent cost,

“Ideal” (often large) panels

• Validation

• Trained manpower

• Distant testing

Specific issues• DNA/Cell cycle analysis

Review indications (Hematolymphoid, Solid tumours)

Standardize assay

• MDS diagnosis

Need for each lab to develop its own data on normal antigenic

profile of hematopoietic cells

Strategy for Two-colour Pan LeucoGating-CD4 Count

TETRACHROME METHOD

PL

G M

ET

HO

D

0 500 1000 15000

500

1000

1500

R² = 0.93

Correlation of CD4 lymphocyte counts by Tetrachrome and PLG-CD4 reagent

Conclusion 1

CD4 lymphocyte counts obtained by PLG-CD4 (2-colour) reagent (one-third cost) showed a high degree of correlation with those obtained by more expensive Tetrachrome (4-colour) reagent

Stability of CD4 counts in EDTA

PLG-CD4 Stability ( EDTA )

0

200

400

600

800

1000

1200

1400

0 HOUR 24 HOUR 48 HOURS 72 HOURS 96 HOURS

Stability of CD4 counts in heparin

PLG-CD4 Stability ( Heparin )

0

200

400

600

800

1000

1200

1400

0 HOUR 24 HOUR 48 HOURS 72 HOURS 96 HOURS

Conclusion 2

• Suitability of anticoagulants For two colour CD4 enumeration using

Pan LeucoGating (PLG) strategy heparin was found to be as good an anticoagulant for distant testing as EDTA (48 hrs).

• Allowable time since collection Forty-eight hrs in EDTA and Heparin

Correlation between CD4 counts obtained by using full and half volume

reagents

0 100 200 300 400 500 600 700 8000

100

200

300

400

500

600

700

800R² = 0.99

Correlation between Half and Full volume

Half volume

Ful

l vo

lum

e

Conclusion 3

Excellent correlation was observed between CD4 lymphocyte subset counts obtained by using the recommended volume of reagents and those obtained by half the recommended volume

POTENTIAL AREAS OF COLLABORATION

• Zap 70 assay standardization• Immunodeficiency panels• Wider and more organized PT programme• Sharing of patient data and other

information• Role of premier (teaching) institutions and

reference laboratories

Thank you

Proposed Scoring System for Immunophenotypic Distinction between

Acute Myelogenous Leukemia with Thymic (T) Markers and T-Acute

Lymphoblastic Leukemia with Myeloid Markers

A. Das Gupta, M. Ramani, A. Vazifdar and V. Mehrotra

Super Religare Laboratories, India

Background● A commonly observed example of expression of trans-

lineage markers by blast cells in AL is the expression of T lymphoid markers by myeloblasts in AML (20-40%) [AML(T)] Conversely, 20-30% of T-ALL cases express myeloid markers [T-ALL(M)].

● An accurate distinction between these two entities using commonly applied primary antibody panels is a significant challenge.

● Even the use of an advanced software (Infinicyt) and EuroFlow ALOT that includes cytoplasmic markers was not able to correctly classify >3% (5/157) cases of AL. All these cases had an overlapping myeloid and T-lymphoid phenotype.

APS (Automated Population Separation) : Unsupervised classification of the different leukemias by principal component analysis (PCA). Based on the ALOT combination, all typical cases of AL (152/157) can be recognized with the new approach. No case was misclassified.

Five atypical cases of AL clustered together either in an area comprised between AML and T-ALL cases, or at the bottom of the 2 clusters. There was no equivalent B/AML overlap.

Therefore, there is a need for identifying immunophenotypic features of blast cells in these two conditions and develop a method/system that will allow distinction between these two entities.

Four-Colour Primary Antibody Panel Used

Myeloid markersCD13, CD33, CD117

Lymphoid markersCD3, CD5, CD7, CD10,

CD19, CD20, CD22

Non-lineage markers CD34, HLA-DR

Cytoplasmic markerscMPO, cCD3, cCD79a

Results

Total leukemia cases 1874

AML 791 (42%)

Classical AML 647 (82%)

AML(T) 144 (18%)

ALL(B+T) 1071 (57%)

T-ALL 128 (12%)

Classical T-ALL 85 ( 66%)

T-ALL(M) 43 (34%)

COMPARISON OF EXPRESSION OF LINEAGE-ASSOCIATED MARKERS IN AML(T) AND IN T-ALL(M)

CD markers expression

AML(T)

(144)

T-ALL(M)

(43)

Scoring System favoring myeloblasts

CD45 Dim Normal -

CD5 4(0.03%) 28(65%) Negative=2

CD10 2 (01%) 9 (21%) Negative=1

CD13 131(91%) 8(19%) Positive=1

CD117 127 (88%) 18 (42%) Positive=0.5

HLA-DR 135 (94%) 10 (23%) Positive=1

• All cases of T-ALL with or without myeloid marker expression had a score of =/<3.5

• All cases of AML with or without T-lymphoid markers had a score =/>4.5

• Cases of true mixed lineage leukemia had a score between 3.5-4.5

• We have applied the scoring system in a prospective manner to test its power in predicting the correct diagnosis.

• We are refining the scoring system further by adopting statistical methods.

Something more…..

Panel Discussion – Some Issues in Lymphocyte Subset

Enumeration…

A. Das Gupta, M. Ramani, V. Mehrotra and

A. VazifdarSuper Religare Laboratories, India

Issues in Lymphocyte Subset Enumeration

Cost-related Issues

• Cost-effective CD4+ lymphocyte subset enumeration (two colour reagent vs 3-4 colour reagent)

• Use of lower volume of reagents per assay

Issues of distance testing

Transit time in distant testing scenario – allowable time from sample collection and suitability of anticoagulants

Issues in Lymphocyte Subset Enumeration

Cost-related Issues

• Cost-effective CD4+ lymphocyte subset enumeration (two colour reagent vs 3-4 colour reagent)

• Use of lower volume of reagents

Issues of distance testing

Transit time in distant testing scenario – allowable time from sample collection and suitability of anticoagulants

Issues in Lymphocyte Subset Enumeration

Cost-related Issues

• Cost-effective CD4+ lymphocyte subset enumeration (two colour reagent vs 3-4 colour reagent)

• Use of lower volume of reagents

Issues of distance testing

Transit time in distant testing scenario – allowable time from sample collection and suitability of anticoagulants