This document summarizes a study comparing different sample processing methods for analyzing B lymphocyte subpopulations through flow cytometry. Fifty patients were analyzed using four sampling methods that differed in processing time, substances used, and storage type. Reliability was assessed using Cronbach's alpha and intraclass correlation coefficients, comparing each method to the gold standard first method. The results showed very high reliability and correlation for all three alternative methods compared to the standard. Therefore, different sample processing methods could be suitable for integrating flow cytometry in clinical practice for autoimmune diseases.

1. 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).
alejandrogaleno@hotmail.com

2. Apologies for my English level…

3. 1. Introduction.
2. Material and Methods
3. Results
4. Conclusions

4. 1. Introduction  Flow cytometry is a technique that allows the study of
lymphocyte subpopulations.
2. Material and Methods
 The B lymphocyte subpopulations according to the
3. Results Freiburg, Paris and EuroClass Classifications are divided
into: Double Negative (DN), Naive, Unswitched memory y
4. Conclusions Switched memory.
 Another possible classification is the B mature:
Bm1, Bm2, Bm2´ (preGC), PB, Bm3+4, early Bm5 y late Bm5.

5. THE FREIBURG, PARIS AND EUROCLASS CLASSIFICATIONS.

6. B MATURE (Bm) CLASSIFICATIONS.

7. 1. Introduction
2. Material and Methods
3. Results Our goal is
to analyze the reliability of other sample processings, equally
4. Conclusions effective in studying B lymphocyte subpopulations, using flow
cytometry.

8. 1. Introduction.  50 patients (25 female/25 male).
2. Material and Methods  None of the patients showed leukocyte pathology
(leukopenia or leukocytosis).
3. Results
 In our study we have compared four methods of sampling
4. Conclusions (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.

10. 1. Introduction.  50 patients (25 female/25 male).
2. Material and Methods  None of the patients showed leukocyte pathology
(leukopenia or leukocytosis).
3. Results
 In our study we have compared four methods of sampling
4. Conclusions (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.

11. Alfa de Cronbach Intraclass correlation
1. Introduction. coefficient.
EUROClass Classifications (Comparing
2. Material and Methods 0.997 0.995
procedure 2 to procedure 1)
EUROClass Classifications (Comparing
3. Results procedure 3 to procedure 1)
0.998 0.998
EUROClass Classifications (Comparing
0.990 0.996
4. Conclusions procedure 4 to procedure 1)
B mature Classifications (Comparing
0.997 0.996
procedure 2 to procedure 1)
B mature Classifications (Comparing
0.998 0.995
procedure 3 to procedure 1)
B mature Classifications (Comparing
0.990 0.991
procedure 4 to procedure 1)
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.

12. 1. Introduction. With all this, one can observe that in all cases, the three procedures
used have shown a very high reliability and correlation when these
2. Material and Methods are compared with the procedure "gold standard“ (procedure 1).
3. Results So, having the possibility of carrying out flow cytometry studies with
different sample processings, perhaps more suited to routine clinical
4. Conclusions practice, is important for the integration and use of this technique in
patients with autoimmune diseases.

13. 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.

15. Thanks…