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Practical Accomplishment of the validation for Blood Groups Genotyping in KCBB

Practical Accomplishment of the validation for Blood Groups Genotyping in KCBB. Hana Al-Shatti Head of Molecular Laboratory Kuwait central blood Bank. Introduction.

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Practical Accomplishment of the validation for Blood Groups Genotyping in KCBB

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  1. Practical Accomplishment of the validation for Blood Groups Genotyping in KCBB Hana Al-Shatti Head of Molecular Laboratory Kuwait central blood Bank

  2. Introduction The Validation of reagents used for blood typing is a very well known issue in blood transfusion practice, it is usually done by following universal blood banks standards such as AABB standard

  3. Validating reagents for heamagglutination reactions is easier than that of molecular genotyping, as the later application interferes with machines, specific equipments as well as a very low volumes reagents. (ranges: 0.5µl – 100 µl)

  4. Test Description Bloodchip detects the genotype by analyzing fluorescence signals corresponding to hybridization rate between labeled amplified DNA fragments and probes attached to the surface of the chip glass. Blood chip test analyzes more than 130 polymorphisms determining the blood groups: ABO, Rh, Kell, Kidd, Duffy, MNS , Diego, Dombrock , Colton, Lutheran and HPA.

  5. Work Flow The DNA Sample is amplified in three different PCR reactions, After which it will be fragmented and labeled with two different flurophores. The labeled PCR products are applied to the surface of the microarray slide for hybridization. Finally the slides are scanned by microarray scanner.

  6. Work Flow 1 Amplification 3 Labelling 4 Hybridization 5 Results analysis 2 Fragmentation TdT+ Biotin-ddUTP or dCTP-Cy5 PCR mix 1, 2 , 3 DNAse + Alkaline Phosphatase 5ul DNA (20ng/ul) 10 ul 3 hours 30 minutes 3hours 30 minutes 45 minutes 60 minutes

  7. The Data analysis software generates the genotype results as well as the phenotype for each allelic variants of all blood groups Included in the software.

  8. The blood chip platform was installed by the supplier engineers and consisted of . • Pre PCR area • 1- DNA extraction machine. • 2- spectrophotometer. • 3- PCR mix preparations.(PCR cabinet) • (B) Post PCR area • 1- Thermal cycler. • 2- hybridization station. • 3- slides Scanner. • After installation, all installation qualification documents were signed by the project leader.

  9. what was done to validate Bloodchip?? Validation: the whole process of establishing documented evidence which provides a high degree of assurance that the performance of process (including equipments) will consistently produce a product that meet its pre-determined specifications and quality attributes.

  10. Validation procedure:- • 1) In Kuwait central Blood bank, we have started our validation process by forming the blood chip validation committee; a group of field experts, quality assurance staff, operational users, IT staff, engineering staff and the project leader. • This committee was approved by our medical director.

  11. 2) Operator's training was completed for technologists by the manufacturer’s experts in a period of 4 weeks in molecular lab of Kuwait central blood bank as well as one week in the manufacturer’s facility in Spain. • 3)The Designing of validation flow chart related to samples and genotyping results; a flow chart that shows the kind of samples to be tested during validation and the kind of tests to be performed . • The samples were chosen to cover donors and patients.

  12. Bloodchip validation Flow chart

  13. Internal validation Results : • genotyping and serological phenotyping results were 100% matching , ( except for some variants that were not comparable as these are not available as serological reagents) . • (2) the HPA genotyping results were also a 100 % matching when compared with the serology using pack 12 and HPA1 typing assays.

  14. (3)The test is consistent; the 4 different technologists performed the blood chip validation testing have had an identical results for all the samples.

  15. Externally: (1) we have participated in an external exercise with NIBSC (National Institute for Biological Standards and Control) in the United kingdom. This exercise is specially for human platelets antigen (HPA) typing. During our validation period we have participated in 3 exercises (exercise# 2010-B, 2011-A, 2011-B) each included 4 sample to be genotyped for HPA-1 ,-2,-3,-4,-5,-6 and -15. Results were very pleasant as we had a 100% correct genotyping.

  16. (2) The samples for two patients were sent to the platelet immunology reference lab in Cambridge, UK for HPA -1,-2,-3,-4,-15 for result confirmation. The results were completely matching to our Blood chip results. (3) 15 samples were sent to blood chip manufacturer facility ( progenika) in Spain to be tested in parallel to Kuwait blood chip testing, and the results were 100 % identical.

  17. The validation committee agreed that the test bloodchip is valid to be used as the blood groups genotyping test in Kuwait central blood bank and that was approved by our medical director .

  18. Performance Qualification Definition The last of the qualification processes and should only commence after successful completion of operational qualification .The output of the process being validated should demonstrate satisfactory performance over the full range of expected operating conditions.

  19. During 6 months testing following the validation of the bloodchip, (420 tests) 1) Contamination problem (once ). 2) Not valid results (14%). 3) No call results (9%).

  20. Conclusion: Validation process of Blood chip was not an easy task to do, we have actually succeeded in recruiting experts in the field, an advanced genotyping technology and a lot of specific requirements to Validate such an accurate testing. We were also able to overcome difficulties in the process of results confirmation and comparison with other institutes abroad for the purpose of full filling the validation requirements which satisfies our aspirations in:

  21. 1) Pushing up advancements of blood grouping in KCBB. • 2) Continuing our mission of earning more accreditations.

  22. Special thanks to my colleagues in the validation committee : 1) Dr.Reem Al-Radwan (Medical director of KCBB) 2) Ohood Al-Ayyadi (Lab department manager) 3) Alia Al-failakawi (Molecular lab staff) 4)Ohood Al-shaikh (Molecular lab staff) 5) Eng.Suleiman Qasem(ATC) 6) Dr.Rachid Al-hems (Progenika)

  23. Hana Al-Shatti

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