Introduction. Previous works have demonstrated the significantly influence of the cryopreservation in the different subsets of lymphocytes respect to fresh cells(1) .

The alterations in the phenotype by the cryopreservation in lymphocytes of rheumatoid arthritis patients is unknown.

AimThe purpose of this study was assess the possible alterations caused by the cryopreservation in NK, NKT cells, T lymphocytes (TL) and B lymphocytes (BL) in healthy controls (HC) and patients with Rheumatoid arthritis (RA).To compare the immunophenotype of NK cells (CD3- CD56+), NKT cells (CD3+ CD56+), B Lymphocytes (CD19+) and memory/naïve subsets of T lymphocytes (CD3+) of fresh and cryopreservated cells in HC and patients with RA.

Material and Methods

In this study were included two patients with AR and two HC. PBMC was obtained by Ficoll-Hypaque. The characterization of immune system cells receptors was made by stain of direct immunofluorescence with monoclonal antibodies: CD3FITC, CD56PE, CD19PECy5.5, CD8APC, CD45RAFITC, CD62LPE, CD3PerCP and CD4APC. The cryopreservation was realized with DMSO at 10% with FCS and storage at -80ºC. The acquisition of data was performed in a FaCSCalibur flow cytometer. The naïve/memory subsets in TL were defined: Naïve CD45RA+CD62L+, central memory CD45RA-CD62L+, effector memory CD45RA+CD62L- and CD45RA-CD62L-. (Figure 1)

Results.

•In basal situation the patients with AR show increase in the frequency of total lymphocytes than HC. After cryopreservation the frequency of lymphocytes in HC is higher than AR patients. Before cryopreservation the frequency of total lymphocytes decrease in AR patients, and the other hand in HC patients increases these values (Fig 2. A).

•Also there are a increase of ThE(td) cells after cryopreservation in RA patients an HC. In basal condition there are a decrease of these cells respects to HC. (Fig 2B)

•Exits a decrease of naïve T helper cells before cryopreservation in RA patients and HC. In basal condition there are a increase of naïve subset in RA respect to HC (Fig 2.C).

Conclusion:The result of this study prove that manipulation of PBMC by cryopreservation affects significantly the subpopulations of lymphocytes in AR patients, specially the BL, ThEM, ThE(TD), Tc, Th naïve lymphocytes and NKT cells.

Bibliography: 1.Peripheral blood manipulation significantly affects the result of dendritic cell monitoring. (J.H.Gerrits et al, 2006).

2.. Boyum,A " Isolation of mononuclear cells and granulocytes from human blood ." Scand.J.Clin.Lab Invest. 21, Suppl. 97 : 77, 1968.

3. www.uv.es/cytomics/activacion.pdf

4.www.andrologyjournal.org/cgi/content/abstract/23/4/537

Acknowledgements:Martin Villarroel

CONDICIONES

CSDESCCSBASALARDESCARBASAL

Mea

n LI

NF

OC

ITO

S

60

50

40

30

52.09

43.14

32.49

53.25

CONDICIONES

CSDESCCSBASALARDESCARBASAL

Mea

n LI

NF

OC

ITO

S/T

CE

LL H

ELP

ER

/E(T

D)

40

30

20

10

0

25.14

34.30

6.42

27.17

CONDICIONES

CSDESCCSBASALARDESCARBASAL

Mea

n LI

NF

OC

ITO

S/TC

ELL

HE

LPER

/NA

IVE

80

70

60

50

40

30

20

10

72.47

23.67

47.46

40.07

Cryopreservation affects the different subsets of lymphocytes in Rheumatoid Arthritis patients and healthy control.

De las Heras Alonso.A, Diéguez Gónzalez.P, Errasquin Ordoñez.S, García García M.

Fig.2.A. Total lymphocytes in RA basal, RA thawed, HC basal and HC thawed. The number represents the media values of frequency.Fig. 2.B Lymphocytes T helper in RA basal, RA thawed, HC basal and HC thawed. The number represents the media values of frequency.Fig.2.C. Lymphocytes T naïve in in RA basal, RA thawed, HC basal and HC thawed. The number represents the media values of frequency.

Fig.1.Analysis of naïve/memory subsets of T lymphocytes CD8+.A. Thawed cells. B, Fresh cells.1. Selection of lymphocytes by SSC-FSC. 2. LTCD8+ on the upper right quadrant 3.

Falta definir las poblaciones restantes

A

B

1

1

2

2

3

3