1. Multicolor Immunophenotyping
Standardization and
Applications
Dr Shanaz Khodaiji
Consultant Hematology
P.D.Hinduja National Hospital
Mumbai
Multicolor Imunophenotyping

2. History of FC at the PDHNH & MRC
FACSCalibur in 1995
FACSCanto II in 2008
CD4 and CD8 counts
Detailed Lymphocyte Subset
analysis
HLA-B27
Immunophenotyping of Acute
Leukemias and CLPDs
Platelet antigen studies 
Multicolor Imunophenotyping

3. Calibration using 7-colour setup beads
Prior to sample acquisition, the FC is set up using 7-Colour
setup beads. Controls need to address 3 issues
Detectors, Thresholds, and Spectral Overlap.
Beads are used
To adjust Detector Voltages: this ensures that fluorescence brightness is correct for stained cells in each detector.
Adjust the signal for events displayed in plots by changing detector voltages. Higher voltages amplify the signal. Lower voltages decrease the signal.
BD FACSCanto clinical software recalculates spectral overlap when you change detector voltages.
Multicolor Imunophenotyping

4. BD FACS 7-colour setup beads
Adjusting Thresholds
A threshold sets a channel number below which events will not be processed. Use threshold to filter out unwanted events. You can set one or more thresholds at a time, and choose whether any one or all need to be met.
Adjusting Spectral Overlap
Set fluorescence compensation. Fluorochromes emit light over a range of wavelengths. During cytometer setup, fluorescence spillover is automatically determined and corrected. If necessary, you can use the spectral overlap controls to make manual adjustments.
Multicolor Imunophenotyping

5. Monitor daily instrument performance
The LJ Chart for PMT Voltages and Blue and Red laser current are to be reviewed on a monthly basis to monitor Cytometer performance and see shifts or trends in parameters as they occur. The results are to be signed by the consultant and filed.
For the HLA B27 and Lymphocyte subset analysis the Lyse/No wash setup is used and for leukemia/lymphoma Immunophenotyping a lyse/wash setup is used.
Multicolor Imunophenotyping

6. Cytometer setup report
Multicolor Imunophenotyping

7. Multicolor Imunophenotyping

8. Multicolor Imunophenotyping
Optimization – why?
Optimizing Cytometer Settings
When you performed cytometer QC, voltage settings were adjusted to set each parameter at a target value. These settings might not be appropriate for the stained samples you plan to analyze. Before recording data, adjust FSC, SSC, and threshold settings; gate on the population of interest (such as lymphocytes) and adjust voltages to optimize fluorescence signal.
Multicolor Imunophenotyping

9. Reference range for Lymphocyte subsets
CD4 Ab/%
CD4Ab/%
CD8 Ab/%
CD3 Ab/%
CD19 Ab/%
CD16+56
CD16+56 Ab/%
Mean
790/ 34
838/ 35
821/ 35
642/ 27
1612/70
1590/67
353/ 15
419/ 17
Minimum
355/ 14
401/ 23
245/ 13
243/ 13
760/ 49
796/ 49
125
133/7
115/6
Maximum
1960/62
1451/48
1876
/62
1206/41
2787 /84
2679/80
707
714/28
1009/37
Multicolor Imunophenotyping

10. Multicolor Imunophenotyping
Reference ranges in children Surg Cdr Gaurav Narula, Dr Shanaz Khodaiji, Col M.S. Bindra
Age Group
CD4
Range (Mean)
CD8
CD3
CD 16/56
CD 19
Range (Mean
Cord Blood
(1707)
(836)
(2859)
---
0-6 mo
(2932)
(1544)
(3631)
(15.8)
20- 77*
(405)
7-12 mo
(2427)
(1133)
(2994)
(47.6) *
(790)
13-36 mo
(2029)
(1269)
(2506)
(103.5)
(563)
37-60 mo
(1977)
(1373)
(2590)
(556) *
(741)
Multicolor Imunophenotyping

11. Comparative study of CD 4 counts (x 103/ml)
Age
Our study (mean)
Kotylo et al
Denny T et al#
Cord blood
(1707) *
0-6m
(2932)
7-12m
(2427)
13-36m
(2029)
37-60m
(1977)
Multicolor Imunophenotyping

12. Multicolor Imunophenotyping
Role of flow cytometric evaluation of lymphocyte subsets in predicting acute rejection episodes in renal transplant pts A. Dasgupta, S.Khodaiji et al
Aim of the study
To study short term results of renal transplant using low dose (1 ml/day) anti CD3 monoclonal antibody induction
to examine role of flow cytometrically determined lymphocyte counts in predicting ARE
Conclusion
Low dose Mab significantly decreases the CD3 count & CD4:CD8 ratio
FC allows monitoring of lympho subsets even at very low counts
AREs tend to be milder and later with use of Mab induction
CD3 counts< 10% and CD4:CD8 < 0.11 of baseline decrease the risk of ARE
Multicolor Imunophenotyping

13. Multicolor Imunophenotyping
Flow cytometric analysis helps in the diagnosis of dense granule deficiency and qualitative deficiency of GPIIB/IIIA in platelets
Sheeba Abraham & Amar Das Gupta
To correlate aggregometry & flow cytometry
findings
To evaluate the role of FC in diagnosis PFD
Multicolor Imunophenotyping

14. Results and observations
Flow cytometry helped in distinguishing between quantitative
(5 cases) and qualitative (1 case) defects of platelet
GpIIb/IIIa in GT patients
In patients with bleeding diathesis but normal
aggregation response, the diagnosis of SPD could be
made from flow cytometric demonstration of reduced
mepacrine uptake
Reduced P-selectin (CD62P) expression by flow cytometry
was seen as an isolated defect in 6 patients with near
normal aggregation response
FC analysis of platelet structure & function supplements
information obtained by aggregometry and helps in further
diagnosis & sub classification of platelet function disorders
Multicolor Imunophenotyping

15. Multicolor Imunophenotyping
Flow cytometric estimation of CD41, CD61 for GT
Multicolor Imunophenotyping

16. Multicolor Imunophenotyping
Comparison of platelet counts by Sysmex XE-2100 (I and O) and LH 750 with the International Flow Reference method in thrombocytopenic patients
Multicolor Imunophenotyping

17. Multicolor Imunophenotyping
Analysis of Sysmex reported, Sysmex Impedence, Sysmex Optical and LH750 Methods with the IRM at Different Transfusion Thresholds
Multicolor Imunophenotyping

18. Multicolor Imunophenotyping
PNH project
Standardisation of Six colour Flowcytometry assay for the diagnosis and monitoring of Paroxysmal Nocturnal Hemoglobinuria using FLAER, as per 2010 International Guidelines
FLAER
CD24 PE
CD45 Per-CP
CD33 PECy7
CD15 APC
CD14 APC- Cy7
PNH marker
Neutro
Gating marker
Mono
RBCs- CD55/CD59/CD235a
Multicolor Imunophenotyping

19. Multicolor Imunophenotyping
MRD for B ALL
MRD assay Standardisation based on the COG protocol
Normal samples/Non B ALL post induction Bone marrows /Staging marrows shall be used for making normal templates
Dilutional studies shall be done for linearity assessment and establishing cutoffs
FITC PE
PerCP-Cy5.5
PE CY7
APC
APC-H7
Tube 1
CD20
CD10
CD38
CD19
CD58
CD45
Tube 2
CD9
CD13+33
CD34
Tube 3
SYTO 16
CD33
CD3
CD71
Tube 4
CD15
CD24
Multicolor Imunophenotyping

20. Multicolor Imunophenotyping
Comparison of FC analysis on LNs with histopathologic study to diagnose NHL
Why should we do it?
FCM facilitates the analysis of cells within discrete subpopulations defined and selected (gated) based on other parameters, allowing a valid and reliable diagnosis, especially in NHLs and enabling their subclassification.
FCM is faster than the histopathologic examination, allowing for therapeutic decisions to be made quickly.
Allows a clear-cut correlation of multiple measurements (antigen expressions, DNA content, light scatter) in individual cells.
Multicolor Imunophenotyping

21. Thank You and seminars Training programs Research projects ILCP CAP
Conducted workshops
and seminars
Training programs
Research projects
ILCP
CAP