Prewarm Technique

Prewarm technique can be used to prevent cold-reactive alloantibodies or autoantibodies from reacting in the IAT phase. Specifically, prewarm technique prevents cold antibodies from binding complement at RT (as opposed to 37^o C ) and subsequently being detected by anti-C3 in the IAT by polyspecific AHG serum. This assumes that pretransfusion testing is done

using clotted specimens (C1qrs requires free Ca⁺⁺ ions; EDTA, for example, chelates Ca⁺⁺ and stops complement binding at C1q)
using polyspecific AHG containing both anti-IgG and anti-C3.

If these pretransfusion test conditions apply, an antibody-antigen reaction occurring at RT, before tests are incubated at 37^o C can be detected in the IAT phase. What happens is that the antibody reacts at RT and binds complement to C3. The clinically insignificant cold antibodies then elute off the red cells at 37^o C , but any C3 bound at RT will remain on the cells and be detected by the anti-C3 in polyspecific AHG serum.

Prewarm technique is somewhat controversial, because some transfusion services use it to determine the clinical significance of antibodies, something that it was never intended to do.

Method

The prewarm method is briefly outlined below. A detailed method can be found in the AABB Technical Manual and similar references.

Prewarm serum and red cells to 37^o C for approximately 10 minutes prior to doing the pretransfusion test.
Also prewarm a bottle of saline and a pipette to 37^o C .
Add serum to cells using a prewarmed pipette.
Ensure that the tests remain at 37^o C before they are read. This includes washing the antiglobulin tests using saline prewarmed to 37^o C and using a centrifuge kept inside a 37^o C dry-air incubator.

There is no need to identify cold antibodies solely reactive at RT or lower unless they cause an ABO discrepancy. In this case, once the antibody is identified and the ABO discrepancy is resolved, it is not necessary to antigen type donors. Crossmatch-compatible blood (using prewarm technique) can be issued.

Note: If clotted specimens are used for pretransfusion tests, to prevent detection of C3 that may be bound at RT by cold antibodies, rather than doing prewarm technique, some labs prevent the problem by using monospecific anti-IgG antiglobulin serum.

Enrichment activity #3

Read this Medline abstract of a 1997 paper by Burin des Roziers & Squalli on comparing elution methods that leave intact red cells for the purpose of antigen phenotyping. Based on the information, answer the following questions. E-mail responses to Pat.

Which three elution methods were compared?
Of 50 antibodies eluted from red cells, how many antibodies could not be successfully eluted by each method?
Which method did the authors determine to be the method of choice?
E-mail responses to the following questions to the class mailing list.
(a) Under which circumstances is prewarm technique used in your laboratory (or geographical area)?
(b) Does your laboratory have experience with prewarming away antibodies that are considered to be clinically significant? If so, how did it influence your laboratory's prewarm policy?
(c) Comment on whether you believe prewarm technique is potentially dangerous unless used wisely.
(a) Provide the elution method of choice (prior to antigen typing) done in your laboratory (or geographical area).
(b) Briefly discuss how well it performs the task of eluting antibodies and preserving antigens.
(c) Indicate approximately how often elutions of this type are performed.
(a) Provide the elution method of choice (prior to antibody identification) done in your laboratory (or geographical area).
(b) Indicate approximately how often elutions of this type are performed.
(a) For which purposes are antibody titrations done in your laboratory (or geographical area)?
(b) Which diluent is routinely used to prepare the titration (saline; 6% albumin; other)?
(c) Which protocols are used to minimize titration variables?
(a) Adsorptions are time-consuming methods. For which uses are adsorptions done in your laboratory (or geographical area)?
(b) Indicate approximately how often adsorptions are done in your laboratory (or geographical area).
Send a comment to the class mailing list in response to information about elutions, titrations, or adsorptions sent by another class member.
E-mail responses to Questions #9 and #10 to Pat.
And now, for something completely different, some calculations.
(a) A physician has ordered four red cell concentrates for a patient with anti-Fy^a and anti-e. What is the minimum number of donors that should be antigen phenotyped?
(b) A physician has ordered three red cell concentrates for a patient with anti-Jk^a , anti-K, and and anti-s. What is the minimum number of donors that should be antigen phenotyped?
ABO and Rh typing results on a patient are as follows:

anti-A anti-B A₁cells B cells high protein
anti-D Rh
control

-- 4+ 4+ -- 2+ 2+

(a) What is the patient's ABO group?
(b) What is the patient's Rh type?
(c) Explain the problem and its probable cause.
(c) What further tests, if any, are required?
(d) How would you antigen type such a patient for the Fy^a antigen using IAT-reactive typing sera ?

anti-A	anti-B	A₁cells	B cells	high protein anti-D	Rh control
--	4+	4+	--	2+	2+

Prewarm Technique