Antibody stability in plasma/serum samples stored in direct contact with red cells

A colleague from the Kentucky/Southern Indiana area reports that a transfusion service in their community is switching from using clotted blood samples to using EDTA anti-coagulated samples for ABO/Rh determination, antibody screening, antibody identification, and crossmatch testing. The inquiring colleague's transfusion service uses EDTA anti-coagulated samples, which they separate so that the EDTA-plasma is stored in a different tube than the patient's red cells. However, the technologists at the neighboring hospital will be storing the EDTA-plasma in the SAME tube in direct contact with patient cells. The inquiring colleague wonders if others have experience with how long a patient's EDTA-plasma is stable and usable for antibody detection and identification testing, if the EDTA-plasma is stored in the same tube along in direct contact with red cells versus being stored in its own separate tube?

The Editor wishes to expand the discussion to colleagues who might have experience with how long a patient's serum is stable and usable if it is stored in the same tube with the patient's cells versus being stored apart from the patient's red cells in a separate tube.

ADDENDA Aug. 23, 2004

The following responses have been received.

1. A colleague reports that his institution switched to gel technology approximately two years ago. In anticipation of that change they started using EDTA tubes four years ago (it took two additional years to get budgetary approval for gel!). Since beginning to use EDTA tubes, they do not separate the EDTA-plasma from the red cells, to preserve storage space. He reports that in their experience EDTA-plasma antibody reactivity appears to remain detectable for at least two weeks of storage of EDTA-plasma in contact with red cells, which corresponds to the length of time they store samples for preadmission testing. Longer storage periods may be complicated by some degree of sample hemolysis.

ADDENDA Aug. 24, 2004

2. An immunohematologist in California reports that while he has no data to provide a definitive answer regarding stability of antibodies in EDTA-plasma, it has been his experience that most blood group antibodies are very stable, and so he does not think that red cell antibodies will be affected by storing (at either room temperature or 4°C) as either whole blood versus separated plasma. The major difference will be that after awhile the plasma from the whole blood will be tinged with hemoglobin which might interfere with visual observation of direct agglutination reactions (EDTA plasma cannot be used for hemolysin detection, so in contrast to serum, the presence of hemoglobin is irrelevant in this situation).

ADDENDA Aug. 25, 2004

3. In the opinion of W. John Judd, FIBMS, MIBiol, Professor of Immunohematology, University of Michigan Medical Center (attribution used with permission), there is a paucity of evidence concerning the use of stored samples for pretransfusion testing. Some guidance is to be found in the BCSH guidelines published in Transfusion Medicine 1996;6:273-283; with the first sentence as a disclaimer: 48 hours for EDTA whole blood at 18-25 C, one week at 4 C, and 6 months for separated serum or plasma at -30 C. While he acknowledges that he has not personally generated data on this issue, he does offer the following comments:

First, he is concerned that plasma being separated from the RBCs at one of the institutions. He states that there is clearly a potential for error in transferring the plasma to a secondary container. He asks "what data have both institutions generated to show that antibodies in separated plasma are stable and for how long, and why would antibodies deteriorate more readily in a whole blood sample than in one that has been separated?"

Second, he comments that we are guided by reagent manufacturers’ product circulars and regulatory/accrediting agencies. These product circulars dictate the nature (serum and/or plasma, type of anticoagulant) and storage requirements of specimens to be used for ABO/Rh, antibody detection and identification. AABB Standard 5.13.3.2 dictates the age of the sample used for detecting unexpected antibodies.

He adds that we are down to essentially two reagent manufacturers in the USA, so that a comparison of product circulars can readily be accomplished. He states "Manufacturer A states that serum or plasma used for reverse ABO tests can be stored at 2-8 C for up to 21 days. For detecting unexpected antibodies using gel column technology Manufacturer A’s RBC product circular states that serum should be used to “assure the presence of adequate complement and calcium.” However, use of antihuman globulin containing anti-C3 is no longer a requirement for pretransfusion antibody detection! Conflicting with this statement, Manufacturer A states in their RBC panel circular that serum or plasma can be used when performing antibody identification using gel column technology. Circulars for all of one product line from Manufacturer B state that: 1) samples should be stored at 1-10 C; 2) testing should be performed as soon after collection as possible; and, 3) antibodies may lose reactivity after a few days if kept at room temperature or following prolonged storage at 1-10 C. Circulars for the other product line from Manufacturer B permit 21 days storage at 1-8 C for reverse typing, and for antibody detection recommend testing as soon as possible after collection or storage at 1- 8 C. All manufacturers indicate that samples should be stored for no longer than is permitted by the relevant regulatory agencies. No manufacturer states that plasma/serum must be separated from the RBCs prior to storage. These differences in storage requirements undoubtedly relate to the extent and nature of information the manufacturers provided to the FDA when obtaining market approval, rather than reflecting a superiority of one product line over another."

He points out that AABB Standard 5.13.3.2 requires that samples used for detecting unexpected antibodies be obtained within 3 days of scheduled transfusion if the patient has been transfused or pregnant within the preceding 3 months, or if the history is uncertain or unavailable. In his experience, obtaining transfusion history is time-consuming and often unreliable. Therefore, except for same day admission surgery patients, his institution places a 3-day requirement on all samples submitted for pretranfusion testing. For same day admission patients they do AB0/Rh and antibody screen as soon after sample receipt as possible, usually within 24 hours of collection. Since they perform an electronic crossmatch when no unexpected antibodies are present currently or by history (if the latter, they do not qualify for their same day admission program), the time limit for samples from these patients is influenced by their ability to examine the plasma for hemolysis whenever a potential hemolytic reaction is reported. They need to subtract from this time the number of days they are required to keep a sample posttransfusion. Currently, samples from their same day admission patients can be collected within one month from scheduled surgery; testing is performed promptly upon sample receipt, and in the absence of a historical record, a second ABO/Rh type is performed on the same sample without undue delay to permit electronic crossmatching should transfusion be needed. In the absence of an electronic crossmatch process, he would recommend following the sample requirements outlined in the product circulars of A1 and B RBCs, which now appears to be 21 days. Of course, a validation study would be required if a change is being made!

Finally, he adds that in his opinion there is no correlation between sample requirements for ABO forward typing and those for reverse typing, and different specimen requirements may apply when using additives/enhancement media when testing for unexpected antibodies.

4. A colleague in Arizona reports that her hospital performs pretransfusion testing on EDTA-anticoagulated whole blood samples. These samples are maintained with the cells and plasma in the original tube and stored for up to three weeks. They have not seen any appreciable loss in titer in antibody tests repeated two and even three weeks later by a solid phase method. The stored blood samples do develop a zone of hemolysis above the red cells, but since hemolysis is not an endpoint with solid phase, that isn't a consideration for them.

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Ira A. Shulman, MD
CBBS e-Network Forum Editor & Moderator