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Posted: June 30, 2005

Addenda: July 7; Aug. 2, Oct. 28 & 31, 2005

 

Experience in using GEL methodology for extended RBC phenotyping

A colleague in India is interested in her laboratory performing red cell phenotyping for antigens other than ABO and D, using a GEL methodology. She wonders if other centers use the GEL methodology to perform extended red cell antigen phenotyping of patients and/or donors. If so, what has been the experience, and are there published data for review?


The following comments have been received.

ADDENDA July 7, 2005

  1. Daniel Kettler, Transfusion Services Supervisor at Washoe Medical Center in Reno, Nevada (attribution used with permission), reports that since 1996 his facility has been performing extended RBC antigen phenotyping using the GEL methodology. He and his colleagues presented their experience with Weak D testing and Rh phenotyping for C, E, c and e at the 1997 AABB meeting. Shortly afterwards they expanded antigen typing in GEL to Duffy, Kidd, Kell and Ss and have routinely supplied cellular blood components for transfusion to patients with the corresponding allo-antibody. They have also successfully used GEL technology to perform CAP Transfusion Medicine Survey J (A-C) since 1996.

ADDENDA Aug. 2, 2005

  1. A transfusion medicine physician and Quality Manager of a blood bank in Santander, Spain reports that since the year 2000, his blood bank has been using the GEL technology for red cell phenotyping, including routine D typing and Rh phenotyping (C,E,c,e). He reports that they have performed about 60,000 D typings and about 6,000 phenotypes without discovering any discrepancies with previous results. They also use the GEL to type other antigens including those in the Kell, Duffy, Kidd, MNS systems. They find the GEL technology to be fast and simple to perform, easy to read, and the results can be documented by taking a photo of the GEL card. It is easy to teach and train staff to do the test. He believes that the staff would not like to go back to tube typing. On the other hand, they must use the suppliers' materials and instructions. Sometimes they find it difficult to find positive and negative reagent red cells for quality control in each typing batch. In this case, they use cells from an antibody identification panel that the purchase from other supplier. Of course, sample identification errors and clerical mishaps and other problems common to blood banking are the same as with tube techniques.

  2. A colleague requests that the Transfusion Services Supervisor at Washoe Medical Center (see the July 7, 2005 posting above) provide some additional information regarding Rh, Duffy, Kidd, Kell and Ss phenotyping using MTS Rh typing cards and buffered gel cards. The inquiring colleague also wonders if the aforementioned typing routines were validated according to their respective package inserts, which antisera were used, and if a copy of the relevant procedures can be made available?

    In reply, we are told that at Washoe Medical Center the following procedures are followed (example SOPs are also provided below):

    Into Buffered Gel cards, they pipette ORTHO BioClone Anti-C, -E, -c, -e antisera manufactured for 5" incubation and centrifugation. Into anti-IgG Gel cards, they pipette Immucor/Gamma Anti-Jka/Jkb and ORTHO Anti-K, -Fya/-Fyb, -S and -s. Both Immucor and ORTHO antisera are manufactured for the indirect antiglobulin test.

    For the direct agglutination test, they prepare 0.8% red cells in MTS Diluent 2; dispense 50µL of test cells and 25µL of antiserum into a Buffered Gel card microtube; incubate for 5 minutes at room temperature; centrifuge 10 minutes in a Gel card centrifuge and read for agglutination.

    For the indirect antiglobulin test, they prepare 0.8% red cells in MTS Diluent 2; dispense 50µL of test cells and 25µL of antiserum into an Anti-IgG Gel card microtube; incubate for 15 minutes at 37C; centrifuge 10 minutes in a Gel card centrifuge and read for agglutination.

    All these antisera performed well with 12.5µL in initial testing, validation and implementation. Later they standardized on 25µL of antiserum because staff complained of difficultly pipetting 12.5uL. The validation testing listed below was done in 1996-1997.

    To validate Anti-Jka/Jkb, they tested 12 panel cells, 20 patients and 20 donor units by standard tube test and by Gel. Based on their validation studies, they decided to use Immucor/Gamma Anti-Jka/Jkb.

    To validate Anti-K, they tested 20 patients and 66 donor units by standard tube and by Gel. There were no discrepancies.

    To validate Anti-S/-s, they tested 20 patients and 20 donor units by standard tube and by Gel. There was one discrepant sample that tested negative by tube and 1+ by Gel with anti-s.

    To validate anti-Fya/-Fyb, they tested 20 patients and 20 donor units by standard tube and by Gel. There were no discrepancies.

    Over the past 8 years, while transfusing nearly 10,000 RBC units annually, they report not having detected any transfusion reactions in an alloimmunized patient due to the transfusion of a donor unit that was incorrectly typed negative by the Gel test using the above antisera and typing system. During the same time period, they reporting having successfully completed numerous CAP Set J proficiency testing episodes. They routinely perform the C,c,E,e ungraded antigen typing in Gel as well.

    Attached are their SOP's for antigen typings (pdf files).

ADDENDA Oct. 31, 2005

  1. Sheryl A. Kochman, Chief of the Devices Review Branch at CBER/OBRR/DBA (attribution used with permission) cautions that with regards to posting #3 of this discussion in which SOPs have been provided as examples: "It is of the utmost importance that colleagues reading information on this website know and recognize that the Gel test available in North America is not exactly the same as the one available in the rest of the world. It is crucial that individual facilities perform their own validation studies".

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