Abstract

  Renal transplantation is the most successful treatment option for chronic renal failure patients. But success of this depends on HLA crossmatching between the renal recipient and donor. In this test, RBCs and platelets are removed and only donor lymphocytes are incubated with recipient’s serum and the complement and percentage of dead lymphocytes is documented. More than 5% of dead cells indicates a positive test and is a contraindication for renal transplantation. For HLA crossmatching, platelets must be removed before lymphocytes mixed with serum. Removal of platelets for crossmatching by traditional method is a very expensive procedure till now as commercially available thrombin is very costly, which increases the financial burden of the patients. Therefore, we tried to develop a cost effective technique to remove platelets by density gradient centrifugation in patients and donors visiting the Nephrology department of Kasturba Hospital, Manipal. After removing plasma and red blood cells, cell suspension in MCT vials is centrifuged @ 1000 rpm for 5 minutes using microcentrifuge. After this, supernatant is discarded and cell pellet is resuspended with buffer and again centrifuged @ 1000 rpm for 5 minutes. This step is repeated 4 or 5 times until the cell suspension is completely cleared off platelets. A total of 96 HLA crossmatching tests done on renal transplant patients and donors visiting Kasturba Hospital, Manipal. Out of this, two cases showed 5% platelets even after 5 times centrifugation. When we centrifuged the cell suspension @ 1000 rpm for 5 minutes, less denser platelets remained in supernatant and more denser lymphocytes sediment to the bottom. When this is repeated 4 or 5 times, all the platelets are removed. This is a very cost effective technique compared to commercial thrombin used earlier, which has reduced the cost of HLA crossmatching.

Keywords: HLA Crossmatch; MCT Vials; CRF

References

1. Tinckam KJ. “Basic histocompatibility testing methods”. In: Chandraker A, editor. Core concepts in renal transplantation”. New York: Springer Science + Business Media, LLC (2012): 21-42.
2. Ganguly P and Sonnichsen WJ. “A simple method for the isolation of blood platelets”. Journal of Clinical Pathology 26 (1973): 635-637.
3. Imandt L., et al. “An improved method for preparing platelet rich plasma”. Thrombosis Research 11 (1977): 429-432.
4. Levy-Tolendano S., et al. “Isolation and function of platelets. II. A new method using total blood:metrizamide gradient centrifugation”. Nouvelle Revue Française D'hématologie 16 (1976): 367-380.
5. Wilson RBJ and Graham JM. “Isolation of platelets from human blood using freeflow electrophoresis”. Clinica Chimica Acta 159 (1986): 211-217.
6. Ford TC., et al. “A new. rapid. one-step method for the isolation of platelets from human blood”. Clinica Chimica Acta 192 (1990): 15-120.
7. Hoffman M., et al. “A rapid method to isolate platelets from human blood by density gradient centrifugation”. American Journal of Clinical Pathology 5 (1992): 531-533.

Citation

Citation: Murali Adiga. “Cost Effective Technique of Removal of Platelets for HLA Crossmatching by Density Gradient Centrifugation". Acta Scientific Gastrointestinal Disorders 3.10 (2020): 17-19.

Copyright

Copyright: © 2020 Murali Adiga. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.