PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.
-
Upload
mark-arbon -
Category
Documents
-
view
215 -
download
0
Transcript of PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.
![Page 1: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/1.jpg)
PANEL DISCUSSION – INDIAN PERSPECTIVE
A. Das Gupta, MD, PhD
Super Religare Laboratories
![Page 2: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/2.jpg)
Points for discussion
• QA
• Tests offered by SRL
• Practical issues
• Potential areas of collaboration
![Page 3: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/3.jpg)
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
![Page 4: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/4.jpg)
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
![Page 5: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/5.jpg)
Strategy for Two-colour Pan LeucoGating-CD4 Count
![Page 6: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/6.jpg)
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
![Page 7: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/7.jpg)
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
![Page 8: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/8.jpg)
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
![Page 9: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/9.jpg)
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
![Page 10: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/10.jpg)
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
![Page 11: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/11.jpg)
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
![Page 12: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/12.jpg)
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
![Page 13: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/13.jpg)
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
![Page 14: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/14.jpg)
Thank you
![Page 15: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/15.jpg)
![Page 16: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/16.jpg)
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
![Page 17: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/17.jpg)
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.
![Page 18: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/18.jpg)
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.
![Page 19: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/19.jpg)
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.
![Page 20: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/20.jpg)
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.
![Page 21: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/21.jpg)
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
![Page 22: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/22.jpg)
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%)
![Page 23: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/23.jpg)
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
![Page 24: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/24.jpg)
• 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
![Page 25: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/25.jpg)
• 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.
![Page 26: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/26.jpg)
Something more…..
![Page 27: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/27.jpg)
Panel Discussion – Some Issues in Lymphocyte Subset
Enumeration…
A. Das Gupta, M. Ramani, V. Mehrotra and
A. VazifdarSuper Religare Laboratories, India
![Page 28: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/28.jpg)
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
![Page 29: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/29.jpg)
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
![Page 30: PANEL DISCUSSION – INDIAN PERSPECTIVE A.Das Gupta, MD, PhD Super Religare Laboratories.](https://reader038.fdocuments.us/reader038/viewer/2022110304/551c01c1550346ad4f8b4c74/html5/thumbnails/30.jpg)
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