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.