Research article

EFFECT OF RESTING TIME ON PERIPHERAL BLOOD MONONUCLEAR CELL YIELDS

Inna Narayani1*, Rasmaya Niruri1, Nyoman Mantik Astawa2

1 Faculty of Mathematics and Natural Sciences Udayana University 2 Faculty of Veterinary Sciences Udayana University

* Corresponding author : [email protected]

ABSTRACT

Cryopreservation of PBMC (peripheral blood mononuclear cells) was done to preserve and analyze the number of PBMC derived from blood samples which come in at different time. The batch analysis was performed at the same time in order to reduce variations in results.. The analysis on the cells numbers carried out after 1, 3, 6, 12, and 24 hours. Heparinized whole blood was collected from healthy subject by venipuncture, and stored at room temperature. Blood is processed by centrifugation in Ficoll density gradient following the established method of Balai Besar Veteriner Denpasar. Buffy coat layer was collected and washed twice with HBSS (Hank's balanced salt solution) and was counted in Turk’s solution. The cells were then dissolved in 1 ml of cold freezing medium containing 10% DMSO and 50% FBS (fetal bovine serum) and stored overnight at -80°C before storage in liquid nitrogen vessel for few weeks. The samples rapidly thawed in a water bath at 37°C and washed twice with PBS (phosphate buffered saline). The cells were stored in 4°C PBS and counted in Turk’s solution after 1, 3, 6, 12, and 24 hours. The results obtained were varied with a declining trend.

Keywords: buffy coat, cell count, , PBMC, resting time

INTRODUCTION

PBMC are live cells which easily damage, some of which start dying immediately after isolation from whole blood. The PBMC has to be analyzed immediately following isolation at the site where they were obtained. The blood requires to be processed and the cells tested one sample at a time as the patient become available, irrespective of the time of the day or week (Olson et al., 2011) .When the analysis are performed at different laboratory or hospital, transport and storage of clinical specimens become important variables that may affect PBMC viability and function. (Bull, M. et al., 2007, Olson et al., and 2011Kutscher, S. et al., 2013) The effect of resting time during storage and transport of peripheral blood on quantity represents the focus of this study.

MATERIAL AND METHODS

The study was approved by Sanglah Hospital – Faculty of Medicine, Udayana University ethics committee. The study was conducted at Balai BesarVeteriner (BBVet) Denpasar Bali on period of June – August, 2015.

PBMC processing method

Blood Collection

Venous blood samples were collected from one healthy volunteer at Faculty of Medicine Udayana University by venipuncture in sodium-heparin tubes. The tubes were kept at room temperature and transported

EFFECT OF RESTING TIME ON PERIPHERAL BLOOD MONONUCLEAR CELL YIELDS | Inna Narayani, Rasmaya Niruri, and

Nyoman Mantik Astawa

immediately to Balai Besar Veteriner laboratory for separation of PBMCs.

Blood Processing

Blood was processed by centrifugation in Ficoll density (3000 rpm or 1.400 g) for 5 minutes in refrigerated swing rotor centrifuge. Buffy coat layer was collected and washed twice with HBSS (Hank's balanced salt solution). The cells were counted under the microscope in Turk’s solution and divided into 3 vials for group A, B, C.

Cryopreservation

The cells were then put in cryovials and dissolved in 1 ml of cold freezing medium containing 10% DMSO (dimethylsulphoxide) and 50% FBS (fetal bovine serum) and stored overnight at -80°C. The next day the cells

were stored in liquid nitrogen vessel for few weeks.

Thawing

As the samples take out from liquid nitrogen, the cells rapidly thawed in a water bath at 37°C and immediately washed twice with warmed PBS (phosphate buffered saline). The cells were stored in 4°C PBS counted in Turk’s solution after 1, 3, 6, 12, and 24 hours.

RESULTS AND DISCUSSION

The numbers of cell after cryopreserved and thawing were counted, the data was shown on Tabel 1.

Tabel. 1 . Number of Cells After Thawing at Different Resting Time

Resting Time (hour)

0

1           3           6          12          24

Group

Mean Value Cell Numbers (x 10 3 cells /µl)

A               904

B                1705

C                1160

884        945        920        720        535

1270       1490       1004        620        515

1381       1280        910        910        540

There were a decrease of cell numbers after one hour resting at 4°C in PBS followed by a slightly increase in number after 3 hr. As the resting time continued to 6, 12 and 24 hr, the cells tend to decrease. Under ideal conditions, the PBMC can be tested immediately following isolation at the site where they were obtained (CPTP, 2015). Many times, however, this is unfeasible for

large multi-center clinical studies (Wunch, M., et. al., 2015). When the analysis is performed at multiple laboratories, transport and storage of clinical specimens become important variables that may affect lymphocyte viability and function ing blood and tissue specimens. The effect of temperature during storage and shipment of peripheral blood on subsequent processing, recovery, and function of

INTERNATIONAL JOURNAL OF BIOSCIENCE AND BIOTECHOLOGY • VOL. IIi NO. 2 • APRIL 2016     ISSN: 9 772302 257 000

ISSN ONLINE: 9 772303 337 008

lymphocytes are important. (Olson et al., 2011). Incubation of PBMC at room temperature for 1 hour did not affect the percentage of apoptotic cells. However, incubation at 4 degrees C for 24 hours resulted in the low percentage of apoptotic cells (Bergman,M. et al. 1985).

ACKNOWLEDGMENT

This research was Hibah Penelitian Dosen Muda 2015, funded by DIPA BLU Udayana University.

REFERENCES

Bergman M, H. Bessler , H .Salman, M.

Djaldetti. 1985. Relationship between temperature and apoptosis of human peripheral blood mononuclear cells. J Immunol Methods.;80 (1):39-43.

Bull, M., D.Lee, J.Stucky, Ya-Lin Chiu, A.Rubin,     H.Horton,     and M.

J.McElrath. 2007.

Defining Blood Processing Parameters for Optimal Detection of Cryopreserved Antigen-Specific Responses for HIV Vaccine Trials. J Immunol Methods. 322(1-2): 57–69.

Centre Physiopathologie de Toulouse-Purpan (CPTP). 2010. PBMC isolation and cryopreservation (version 2015 03 05)

Kutscher.S., C.J. Dembek,     S.Deckert,

C.Russo,     N.Körber. J.R. Bogner,

F.Geisler, A.Umgelter, M.Neuenhahn, J.Albrecht, A.Cosma, U.Protzer, T.Bauer. 2013. Overnight Resting of PBMC Changes Functional Signatures of Antigen Specific T- Cell Responses: Impact for Immune Monitoring within Clinical Trials. Plos One vol.8, issue 10

Mallone,R., S. I. Mannering, B. M. Brooks-Worrell, I. Durinovic-Belló, C. M. Cilio, F. S. Wong and N. C. Schloot . 2010. Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes  Society.

Clinical and Experimental Immunology, 163: 33–49

Olson, W.C., M.E Smolkin, E.M Farris, R.J Fink, A.R Czarkowski, J.H Fink,

K.A. Chianese-Bullock, C.L Slingluff Jr. 2011. Shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function. Journal of Translational Medicine 2011, 9:26

Wunsch. M., R. Caspell, S. Kuerten, P. V. Lehmann and S. Sundararaman. 2015. Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay. Cells, 4, 40-55

90 • asia oceania bioscience and biotechnology consortium