Complying with CAP TRM.31450:
Both CMS (42 CFR 493.1281) and the College of American Pathologists (TRM.31450 & COM.04250) require laboratories that use more than one method of testing a given analyte to have a system in place to compare those methods. Laboratories are required to check those methods against each other twice a year.
In a recent study, conducted at Moses H. Cone Memorial Hospital, the ALBAcheck® Competency Testing Kit from Quotient was incorporated into their comparability of methods protocol. The kit was shown to be a simple and convenient solution to complying with these requirements. To see the full poster (click here).
Are Quotient antigen profiles available to me if I use Electronic Antigen Profile software?
Yes. Antigen Profiles for Quotient Antibody Screening Cells, Antibody Identification Panels and papain-treated Antibody Identification Panels are available on all the electronic antigen profile programs.
Currently, Quotient sends the antigen profiles to Antigen Plus, RC-Aid, and Antibody Check as soon as our quality control processes are complete. This is well before the reagent red cells are available to ship out to customers. The electronic antigen profile companies then have time to enter the data into their systems. When you receive your red blood cell shipment, the information is loaded and ready for use in preparing panels for selected cell testing. If you are not familiar with the Electronic Antigen Profile companies, links to their sites are below. These programs are extremely popular with blood bank staff members. Supporters extol the software programs for their ease of use and increase in productivity / time-savings for staff as well as the accuracy of the selected cell panels created.
What does it mean when the package insert says “No U.S. Standard of Potency”?
First, let's discuss what is meant by the phrase, “US Standard of Potency”. The FDA has very strict guidelines that all manufacturers must meet when preparing blood grouping reagents. Whether the reagents are monoclonal or polyclonal, the FDA outlines exactly what is expected of reagents. In the FDA Docket 845-0181, the FDA outlines potency titer values (strength of reaction at a stated dilution), specificity results (including specific cells to test), and other details of testing that must be performed on each lot of antisera, such as Anti-A or Anti-B, that a manufacturer prepares for the US market. This docket covers ABO and Rh Blood Grouping Reagents, as well as Rare Blood Grouping Reagents. For reagents prepared under these guidelines, the package insert will state “Meets FDA potency requirements.”
“No U.S. Standard of Potency” is found on the package insert for Quotient Anti-A1, as a bullet on the top left section of the insert. Quotient Anti-A1 is a lectin, and there is no US Standard of Potency for lectins. In the Code of Federal Regulations, there is a US Standard of Potency for Anti-A1, but it only applies if the Anti-A1 is prepared from human plasma or human-derived monoclonal antibodies. For lectins, there is no potency standard. Although the statement may be found in a different location of the insert, if you look closely, you will see that the package inserts for all Anti-A1 lectins sold in the US have this statement.
“No U.S. Standard of Potency” will also be found on the package inserts of Reagent Red Cells. Perhaps the future will hold a time when manufacturers are able to grow red cells, but for now, reagent red cells are prepared using blood from humans. Molecular studies are reinforcing what many have always suspected: no two human beings are alike. So there is no FDA Standard of Potency for reagent red cells. This does not mean that the FDA has no guidelines or requirements. Section 21 CFR 660, Subpart D outlines donor suitability, collection of blood, testing of the source material, processing guidelines and labeling. There are also requirements that dictate the samples and paperwork that manufacturers must submit to the FDA with each lot of final product.
“No U.S. Standard of Potency” certainly does not mean that there is no US standard of quality!
When would I use the ALBAclone® Advanced Partial RhD Typing Kit?
The Advanced Partial RhD Typing Kit is particularly useful in those patients whose RhD type appears by standard typing techniques as D+ or Weak D+, and who demonstrate Anti-D, yet their Anti-D does not react with their own red cells.
Is it difficult to use?
The Advanced Partial RhD Typing Kit is not hard to use. Essentially, you run a series of anti-D tests – and record them on the profile sheet included with the package. You then compare the reaction pattern you obtained with the patterns on the profile sheet to determine the category of partial D.
ALBAclone® Advanced Partial RhD Typing Kit. For research use only in the United States.
Testing with the Advanced Partial D test is quick. Obtaining a pattern with the Partial D kit allows the laboratory to determine that the patient is a Partial D, and capable of producing a clinically significant Anti-D. The need to transfuse RhD negative blood is certain. Patient care is improved. The Advanced Partial D is more cost effective than molecular sequencing, which is often necessary to determine the single nucleotide changes that distinguish Partial D types.
Reactivity noted during research testing should be confirmed using a licensed / cleared test. The test results from a reactive Albaclone Advanced Parial RhD Typing Kit suggest that a portion of the RhD antigen is not expressed. Individuals who have an altered D epitope or have partial D red cells are capable of raising clinically significant alloantibodies directed to the portion of the D antigen that is altered or missing.
Why is saline so important? Is one type of saline better than another?
Way back in 1986, an article was published by Bruce, M. et.al. entitled “A serious source of error in antiglobulin testing.” Transfusion, 1986; 26:177-181. In their study, the authors evaluated 26 saline solutions from multiple manufacturers, obtained from 10 blood banks. They discovered a pH range of 4.8 – 8.4 in these 26 saline solutions.
In the abstract, the authors stated that:
“The investigation of a failure of proficiency showed that certain saline solutions are inappropriate for use in blood group serology tests. In particular, it was found that solutions of unexpectedly low pH…. could severely compromise the sensitivity of the antiglobulin test when used as wash solutions. The observed loss of sensitivity ranged from a reduction in titration score to a complete failure in the detection of clinically significant blood group antibodies.” Some examples of Anti-D, Anti-S, Anti-s, Anti-Fya, Anti-Jka, Anti-Mia and Anti-Vw failed to react or reacted weakly, when AHG testing was performed using saline with pH of less than 6.5. Again, from the abstract, they concluded that “improved standardization and sensitivity (in serological tests) could be achieved by using phosphate-buffered saline pH 7.0-7.2... It is recommended that unbuffered saline solutions of pH less than 6.0 should not be used for serological testing.”
Again, in 1993, Susan Rolih et.al. published “Antibody detection errors due to acidic or unbuffered saline” in Immunohematology, 1993, Vol. 9, pg. 15-18. This study set out to replicate the findings from 1986, using unbuffered and buffered saline, and to determine if pH had an effect on solid phase or hemagglutination-based antibody detection tests. The pH range studied was 5.5 – 8.0. In their conclusion, they wrote, “We also support the suggestion of these authors that saline solutions used in antibody detection tests be considered as important as the potentiating media, reagent red cell phenotype, or antiglobulin reagent employed. Thus the pH of saline should be strictly controlled at 7.0 – 7.5 when either solid phase testing or hemagglutination testing is performed.”
What is the science behind variable reactivity at different pH?
Red cells have a negative charge at pH = 7.0. Antibody molecules have a weakly positive charge at pH of 7.0 - 7.5. So attraction between antigens and antibodies during the first stage of agglutination / sensitization is enhanced at this pH.
The equilibrium of antibody association / disassociation is well known. This knowledge is utilized in the preparation of acid eluates. The lower pH of the acid causes the disassociation of antibody from antigen, so that the previously bound antibody is captured in the eluate. The same effect occurred in the studies mentioned above. They saw that when a very low pH saline (pH 4.8) was used as a wash solution in antiglobulin testing, the antibody was eluted and simply washed away, resulting in a reduction in titration score or complete failure to detect antibody presence.
What is the difference in Blood Bank Saline that is currently on the market? And when might I use the different types of saline?
There are two types of 0.85% - 0.9% NaCl solutions marketed to blood banks today. One is called Isotonic Blood Bank Saline; the other is Phosphate Buffered Blood Bank Saline. The primary difference between them is pH and whether that pH is achieved by buffers. Isotonic Blood Bank Saline usually has a pH range within 6.0 – 7.5; Phosphate Buffered Saline is buffered to maintain a tighter pH, within a range of 7.0 – 7.5.
Technologists who use automated testing may recognize the importance of saline to the operation of their equipment. Some instruments require that the operator purchase unbuffered isotonic blood bank saline, to which they add a packet of buffers. This ensures that the pH of the saline is within a strictly controlled range where antibody antigen association best occurs for that instrument’s testing technique.
Another example of special pH requirements is the Quotient Anti-M reagent, which calls for unbuffered saline. In this case, the monoclonal anti-M reacts optimally at pH 8.5 and is extremely sensitive to pH. Because unbuffered isotonic blood bank saline has no phosphate buffers, it does not affect the pH of the reagent. Phosphate buffered saline solutions contain phosphate which acts to adjust (buffer) the saline solution with a goal of maintaining the pH in the range of 7.0 - 7.5. This adjustment would adversely affect the Quotient Anti-M’s optimal pH of 8.5.