1. PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories

2. Points for discussion QA Tests offered by SRL Practical issues Potential areas of collaboration

3. 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

4. ISSUES General 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

5. Strategy for Two-colour Pan LeucoGating-CD4 Count

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

7. 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

8. Stability of CD4 counts in EDTA

9. Stability of CD4 counts in heparin

10. 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

11. Correlation between CD4 counts obtained by using full and half volume reagents

12. 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

13. 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

14. Thank you

16. 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

17. 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.

18. 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.

19. 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.

20. 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.

21. Four-Colour Primary Antibody Panel Used Myeloid markers CD13, CD33, CD117 Lymphoid markers CD3, CD5, CD7, CD10, CD19, CD20, CD22 Non-lineage markersCD34, HLA-DR Cytoplasmic markers cMPO, cCD3, cCD79a

22. Results Total leukemia cases1874 AML 791 (42%) Classical AML647 (82%) AML(T)144 (18%) ALL(B+T)1071 (57%) T-ALL128 (12%) Classical T-ALL85 ( 66%) T-ALL(M)43 (34%)

23. 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 CD45DimNormal- CD54(0.03%)28(65%)Negative=2 CD102 (01%)9 (21%)Negative=1 CD13131(91%)8(19%)Positive=1 CD117127 (88%)18 (42%)Positive=0.5 HLA-DR135 (94%)10 (23%)Positive=1

24. 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

25. 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.

26. Something more…..

27. Panel Discussion – Some Issues in Lymphocyte Subset Enumeration… A. Das Gupta, M. Ramani, V. Mehrotra and A. Vazifdar Super Religare Laboratories, India

28. 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

29. 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

30. 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