Standardization and Applications Multicolor Imunophenotyping Multicolor Immunophenotyping Dr Shanaz...

Standardization and

Applications

Multicolor Imunophenotyping

Multicolor Immunophenotyping

Dr Shanaz KhodaijiConsultant Hematology

P.D.Hinduja National HospitalMumbai

History of FC at the PDHNH & MRC

FACSCalibur in 1995FACSCanto II in 2008•CD4 and CD8 counts•Detailed Lymphocyte Subset analysis•HLA-B27 •Immunophenotyping of Acute Leukemias and CLPDs •Platelet antigen studies


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.


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.


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.



Cytometer setup report

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.


Reference range for Lymphocyte subsets

CD4 Ab/%

CD4Ab/%

CD8 Ab/%

CD8 Ab/%

CD3 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


Reference ranges in children Surg Cdr Gaurav Narula, Dr Shanaz Khodaiji, Col M.S. Bindra


Age GroupCD4

Range (Mean)

CD8

Range (Mean)

CD3

Range (Mean)

CD 16/56

Range (Mean)

CD 19

Range (Mean

Cord Blood

2209- 3205 (1707) 312- 1360 (836)3100- 5200

(2859)--- ---

0-6 mo 1516-4348

(2932)

970- 2118 (1544)

1767- 5495 (3631)

5.8- 35.8 (15.8)20- 77*

(405)

7-12 mo 1056- 3799

(2427)

541- 2807

(1133)

1278- 4710

(2994)

7.8- 89

(47.6)

25- 1852*

(790)

13-36 mo1113- 2946

(2029)

523- 2015

(1269)

1222- 3790

(2506)

54.5- 261.5

(103.5)

15- 2445

(563)

37-60 mo 839- 3115

(1977)

749- 1997

(1373)

1368- 3812

(2590)

53- 1160

(556)

4- 10997*

(741)

Age Our study (mean) Kotylo et al Denny T et al#

Cord blood 2209-3205 (1707) 2310-2990* 1460-5110

0-6m 1516-4348 (2932) 2800-3900 1690-4610

7-12m 1056-3799 (2427) 2600-3500 --------------

13-36m 1113-2946 (2029) 1200-2300 1020-3600

37-60m 839-3115 (1977) 560-2700 900-2860

Comparative study of CD 4 counts (x 103/ml)


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 AREConclusion• 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


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

Flow cytometry helped in distinguishing between quantitative (5 cases) and qualitative (1 case) defects of platelet GpIIb/IIIa in GT patients In 4 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

Results and observations


Flow cytometric estimation of CD41, CD61 for GT


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


Analysis of Sysmex reported, Sysmex Impedence, Sysmex Optical and LH750 Methods with the IRM

at Different Transfusion Thresholds


PNH projectStandardisation of Six colour Flowcytometry assay for the diagnosis and monitoring of Paroxysmal Nocturnal Hemoglobinuria using FLAER, as per 2010 International Guidelines

FLAER CD24PE

CD45 Per-CP

CD33 PECy7

CD15 APC

CD14 APC- Cy7

PNH marker

NeutroPNH marker

Gating marker

Mono Gating marker

Neutro Gating marker

MonoPNH marker

RBCs- CD55/CD59/CD235a


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 CD19 CD10 CD45

Tube 3 SYTO 16 CD33 CD3 CD19 CD71 CD45

Tube 4 CD15 CD24 CD20 CD19 CD10 CD45


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.


• Conducted workshops

and seminars

•Training programs

•Research projects

•ILCP

•CAP

Standardization and Applications Multicolor Imunophenotyping Multicolor Immunophenotyping Dr Shanaz...

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