Presentación 8 th autoinmunity congress_granada_2012_

STUDY ON TYPE B LYMPHOCYTE SUBPOPULATIONS THROUGH FLOW

CYTOMETRY.

Comparative analysis between different sample processings.

Alejandro Muñoz Jiménez.Rheumatology Unit.

Valme University Hospital (Seville, Spain).

[email protected]

Apologies for my English level…

1. Introduction.

2. Material and Methods

3. Results

4. Conclusions

1. Introduction

2. Material and Methods

3. Results

4. Conclusions

Flow cytometry is a technique that allows the study of lymphocyte subpopulations.

The B lymphocyte subpopulations according to the Freiburg, Paris and EuroClass Classifications are divided into: Double Negative (DN), Naive, Unswitched memory y Switched memory.

Another possible classification is the B mature: Bm1, Bm2, Bm2´ (preGC), PB, Bm3+4, early Bm5 y late Bm5.

THE FREIBURG, PARIS AND EUROCLASS CLASSIFICATIONS.

B MATURE (Bm) CLASSIFICATIONS.

1. Introduction

2. Material and Methods

3. Results

4. Conclusions

Our goal is to analyze the reliability of other sample processings, equally effective in studying B lymphocyte subpopulations, using flow

cytometry.

50 patients (25 female/25 male).

None of the patients showed leukocyte pathology (leukopenia or leukocytosis).

In our study we have compared four methods of sampling (fresh blood). The several procedures differ in the time spent, substances used and types of storage ❶. The cytometer, laboratory personnel and software used has been the same.

To assess reliability, Cronbach's alpha and correlation intraclass coefficient have been used, always comparing the process under study with the first of them (procedure 1=gold standard).

The fluorochromes used are: CD19, IgD, CD38 y CD27.

1. Introduction.

2. Material and Methods

3. Results

4. Conclusions

50 patients (25 female/25 male).

None of the patients showed leukocyte pathology (leukopenia or leukocytosis).

In our study we have compared four methods of sampling (fresh blood). The several procedures differ in the time spent, substances used and types of storage ❶. The cytometer, laboratory personnel and software used has been the same.

To assess reliability, Cronbach's alpha and correlation intraclass coefficient have been used, always comparing the process under study with the first of them (procedure 1=gold standard).

The fluorochromes used are: CD19, IgD, CD38 y CD27.

1. Introduction.

2. Material and Methods

3. Results

4. Conclusions

1. Introduction.

2. Material and Methods

3. Results

4. Conclusions

Alfa de Cronbach Intraclass correlation coefficient.

EUROClass Classifications (Comparing procedure 2 to procedure 1) 0.997 0.995

EUROClass Classifications (Comparing procedure 3 to procedure 1) 0.998 0.998

EUROClass Classifications (Comparing procedure 4 to procedure 1) 0.990 0.996

B mature Classifications (Comparing procedure 2 to procedure 1)

0.997 0.996

B mature Classifications (Comparing procedure 3 to procedure 1)

0.998 0.995

B mature Classifications (Comparing procedure 4 to procedure 1)

0.990 0.991

In the Cronbach's alpha a value above 0.8 is sufficient to ensure the reliability of the scale

In the intraclass correlation coefficient a value exceeding 0.9 indicates very good correlation.

1. Introduction.

2. Material and Methods

3. Results

4. Conclusions

With all this, one can observe that in all cases, the three procedures used have shown a very high reliability and correlation when these are compared with the procedure "gold standard“ (procedure 1).

So, having the possibility of carrying out flow cytometry studies with different sample processings, perhaps more suited to routine clinical practice, is important for the integration and use of this technique in patients with autoimmune diseases.

Acknowledgements

Rheumatology Unit. Valme University Hospital (Seville, Spain). Dr. J.L. Marenco and S. Rodríguez.

Biostatistics Group. Valme University Hospital (Sevilla, Spain).

Dra. Loreto Carmona. University Professor of the University Camilo José Cela.

Thanks…