On a much higher than reported incidence of anti-c in R1R1 patients with anti-E.

A previous study involving tube IATs, untreated RBCs, and a low ionic-strength additive reagent revealed that approximately one-third of R(1)R(1) patients with anti-E have a concomitant anti-c. However, the current study finds a much higher incidence of anti-c in such patients, using gel technology in conjunction with ficin-pretreated RBCs. Results of antibody identification studies and transfusion records of 82 R(1)R(1) patients with anti-E were reviewed. Serologic test methods included a LISS wash solution for tube IATs (15 min at 37 degrees C, anti-IgG), ficin-tube IATs (30 min at 37 degrees C, anti-IgG + anti-C3), and gel IATs (untreated or ficin-treated RBCs or both, anti-IgG gels). LISS-tube or gel IATs with untreated RBCs revealed anti-c in 32 patients with anti-E. When gel-IAT and ficin-pretreated RBCs were used, 21 additional patients with anti-E were found to have anti-c. In samples from 26 R(1)R(1) patients with anti-E, anti-c was not demonstrable by ficin-gel IATs, and in 3 cases, the ficin-gel tests were inconclusive. In five cases in which E- RBCs not tested for c antigen were transfused to patients found by ficin-gel IAT to be without anti-c, all subsequently performed crossmatches with E-, c-untested RBCs were compatible. The incidence of anti-c in R(1)R(1) patients with anti-E in this study was 32 of 82 (39%) with untreated RBCs and 53 of 82 (65%) when the ficin gel data were included. The latter is significantly higher than the 32 percent incidence previously reported (p = 0.0001). Accordingly, all patients at our facility with an Rh antibody are now tested for those additional Rh antibodies they can make, as predicted from their Rh phenotype. The data from this study strongly support the selection of R(1)R(1) RBCs for all c- patients with anti-E.

Reactivity of FDA-approved anti-D reagents with partial D red blood cells.

Individuals whose RBCs are characterized as having a partial D phenotype may make anti-D if exposed to normal D+ RBCs; thus it is desirable that they be typed as D- should they require blood transfusion or Rh immune globulin (RhIG) prophylaxis. Further, use of different anti-D reagents by blood centers and transfusion services can account for FDA-reportable errors. For this study, anti- D reagents for use in tube tests were obtained from three U.S. manufacturers. They included three examples of IgM monoclonal anti-D blended with monoclonal IgG anti-D, one IgM monoclonal anti-D blended with polyclonal IgG anti-D, and two reagents formulated with human anti-D in a high-protein diluent. One anti- D formulated for use by gel column technology was also tested. Direct agglutination tests by tube or gel were strongly positive (scores 9-12), with partial D RBCs of types DII, DIIIa, DIIIb, and DIVa. No reagent anti-D caused direct agglutination of DVI type 1, DVI type 2, or DFR phenotype RBCs. One tube anti-D reagent formulated with an IgM monoclonal anti-D plus a polyclonal IgG anti-D failed to cause direct agglutination of DVa, DBT, and R(0)(Har) RBCs, while DVa RBCs reacted weakly with two high-protein reagents formulated with human IgG anti-D. In contrast, the anti-D used by gel column technology was strongly reactive (score 11) with DVa, DBT, and R(0)(Har) RBCs. The single monoclonal IgM-polyclonal IgG blended anti-D and the two high-protein reagents were also the only reagents that failed to react with R(0)(Har) RBCs by the IAT. Elimination of the test for weak D on all patient samples, using currently available FDA-licensed reagents, will ensure that partial D category VI (DVI) patients will type as D- for the purpose of RhIG prophylaxis and blood transfusion. However, RBCs of other partial D phenotypes will be classified as D+ in direct agglutination tests with some, if not all, currently available reagents. Testing donors for weak expression of D continues to be required, albeit that Rh alloimmunization by RBCs with a weak or partial D phenotype is uncommon. Further, because of differences in performance characteristics among FDA-approved reagents, conflicts between donor center D typing and transfusion service confirmatory test results are inevitable.

Molecular defects underlying the Kell null phenotype.

Expression of the Kell blood group system is dependent on two proteins, Kell and XK, that are linked by a single disulfide bond. Kell, a type II membrane glycoprotein, is a zinc endopeptidase, while XK, which has 10 transmembrane domains, is a putative membrane transporter. A rare phenotype termed Kell null (Ko) is characterized by the absence of Kell protein and Kell antigens from the red cell membrane and diminished amounts of XK protein. We determined the molecular basis of eight unrelated persons with Ko phenotypes by sequencing the coding and the intron-exon splice regions of KEL and, in some cases, analysis of mRNA transcripts and expression of mutants on the cell surface of transfected cells. Six subjects were homozygous: four with premature stop codons, one with a 5' splice site mutation, G to A, in intron 3, and one with an amino acid substitution (S676N) in exon 18. Two Ko persons with premature stop codons had identical mutations in exon 4 (R128Stop), another had a different mutation in exon 4 (C83Stop), and the fourth had a stop codon in exon 9 (Q348Stop). Two Ko persons were heterozygous for two mutations. One had a 5' splice site mutation (G to A) in intron 3 of one allele that caused aberrant splicing and exon skipping, and the other allele had an amino acid substitution in exon 10 (S363N). The other heterozygote had the same amino acid substitution in exon 10 (S363N) in one allele and a premature stop codon in exon 6 (R192Stop) in the other allele. The S363N and S676N mutants, expressed in 293T cells, were retained in a pre-Golgi compartment and were not transported to the cell surface, indicating that these mutations inhibit trafficking. We conclude that several different molecular defects cause the Kell null phenotype.

The first example of a paraben-dependent antibody to an Rh protein.

BACKGROUND: Parabens are added to a commercial LISS (C-LISS) to retard microbial growth. Paraben-dependent anti-Jk(a) has been detected by the use of C-LISS. CASE REPORT: Serum from a D+ woman reacted in antiglobulin tests with RBCs stored (2-4 hours, 22-25 degrees C) in C-LISS (Löw and Messeter formulation, Immucor). Freshly prepared C-LISS-suspended RBCs did not react; nor did RBCs stored in LISS-additive reagents, PEG, saline, or homemade LISS. RESULTS: Studies using C-LISS-stored RBCs revealed an antibody that reacted with D+ and rrV+ RBCs, but not with r'r, r"r, or rrV-VS- RBCs. All partial D RBC phenotypes tested reacted, as did D+LW-, rGr, r"Gr, r(y)r, r'(s)rV+VS+, and r'(s)rV-VS+ RBCs. The active ingredient in C-LISS was propylparaben. Other LISS ingredients were not required; saline solutions of propylparaben, ethylparaben, methyl salicylate, 2-phenoxyethanol, and butylparaben were active. Methylparaben and methyl-m-hydroxybenzoate were inactive. Reactivity to C-LISS-stored RBCs could not be inhibited by propylparaben. Reactivity with D+V- and D-V+VS+ RBCs was not separable by adsorption-elution. CONCLUSIONS: This antibody likely detects a neoantigen formed between active compounds and RBC membranes. Review of the structure of active compounds suggests that proximity between methyl and hydroxyl groups is important for binding with RBC membranes. The role of RhD is unclear; no single portion of RhD protein appears to be implicated.

PEG adsorption of autoantibodies causes loss of concomitant alloantibody.

Use of polyethylene glycol (PEG) to promote adsorption of autoantibodies is reported to give good recovery of concomitant alloantibodies. In initial experiments, PEG and ZZAP (Ficin and DTT) adsorption procedures were compared for removal of autoantibody and recovery of alloantibody. Postadsorption studies (n = 11) were performed and hemagglutination scores compared. In subsequent studies, equal volumes of alloantibody containing sera, PEG, and antigen-negative red blood cells (RBCs) were used in twofold adsorption experiments. Saline was substituted for PEG for control purposes. Postadsorption titers and immunoglobulin levels were determined. Autoantibodies were completely removed by both methods (n = 5); better by PEG (n = 3); better by ZZAP (n = 1); and not adsorbed (n = 1), and partially adsorbed by both (n = 1). Alloantibody recovery was comparable in three cases (E, K, Jka) but weaker by at least one reaction grade in four (K,E, Jka, and antibody to low-frequency antigen). The latter anti-Jka reacted 1+ with Jk(a+b+) RBCs after ZZAP adsorption but was nonreactive with the same RBCs following PEG adsorption. Titers of six alloantibodies adsorbed with antigen-negative RBCs in PEG were markedly weaker (range 2 to 8) compared to saline controls (range 4 to 32). IgG levels for PEG adsorbed (range 128 to 243 mg/dL) were 50% lower than controls (range 265 to 505 mg/dL). Although PEG adsorption is effective in removing autoantibody, the precipitation of immunoglobulin by PEG may result in failure to detect underlying alloantibody.

Revisiting the issue: can the reading for serologic reactivity following 37 degrees C incubation be omitted?

BACKGROUND: Omitting the 37 degrees C reading from screening tests for unexpected antibodies results in failure to detect some Rh, K, and Jk agglutinins of potential significance (wanted positives). However, this measure avoids unwanted positive tests due to cold agglutinins. STUDY DESIGN AND METHODS: Using data from prior publications, actual risk calculations (ARCs) were made to predict the risk of eliminating the 37 degrees C reading, pretransfusion direct antiglobulin test (DAT), and routine indirect antiglobulin crossmatch (IAT-XM). ARCs used the equation: wanted positives missed x 0.34 (or 0.80) x 5 x percent antigen-positive, where 0.34 = percent of patients transfused (ARCs for 37 degrees C reading and DAT); 0.80 = percent of crossmatched patients transfused (ARCs for IAT-XM); 5 = average number of units transfused. Following elimination of the 37 degrees C reading, the impact of this change on patient care was monitored. Antibody detection and identification data and transfusion reaction reports for 6 months after the change were reviewed. Recently transfused patients with new antibodies were evaluated for immune hemolysis by review of clinical and laboratory data. The findings were compared with those from the same dates of the preceding year. RESULTS: The risk of transfusing incompatible blood by eliminating the DAT, IAT-XM, and 37 degrees C reading is approximately 1:13,000, 1:2,000, and 1:2,400 units transfused, respectively. The cumulative risk from eliminating all three tests is approximately. 1 :1,000 units. With respect to the 37 degrees C reading, there were no differences between the pre-change and post-change study periods in the incidence of reported transfusion reactions or cases of immune hemolysis associated with newly formed antibodies. However, unwanted positive tests decreased from 162 to 61 following elimination of the 37 degrees C reading. This represents a decrease of 20 percent in the number of samples requiring antibody identification annually. CONCLUSIONS: Eliminating the 37 degrees C reading from pretransfusion antibody screening tests imposes less risk than omitting the routine IAT-XM, and it avoids the time and costs of evaluating unwanted positive tests, thus reducing expenditures and delays in patient care.

Requirements for the electronic crossmatch.

With the integration of laboratory information systems into transfusion services, it is now possible to develop standard operating procedures (SOPs) for an electronic crossmatch (EXM) to replace the immediate-spin crossmatch for detecting ABO incompatibility between the blood sample submitted for pre-transfusion testing and the donor unit selected for transfusion. Essential to the safety of an EXM are requirements that: 1) the computer contains logic to prevent assignment and release of ABO incompatible blood; 2) no clinically significant antibodies are detected in the recipient's serum/plasma and there is no record of previous detection of such antibodies; 3) there are concordant results of at least two determinations of the recipient's ABO type on record, one of which is from a current sample; 4) critical elements of the system have been validated on-site; and, 5) there are mechanisms to verify the correct entry of data prior to release of blood. EXM procedures are in use in at least 10 North American facilities, Scandinavia, Hong Kong and Australia. Some provide blood at remote sites lacking laboratory services. Experience has shown that a combination of properly programmed computer software and carefully developed SOPs can provide a safe and efficient means of detecting ABO incompatibility without performing a serological crossmatch.

The gel test: use in the identification of unexpected antibodies to blood group antigens.

The IgG GEL test was compared with the LISS tube test (Löw and Messeter's low-ionic-strength saline) for antibody identification. The suitability of red blood cells (RBCs) pretreated with ficin, dithiothreitol (DTT), or chloroquine diphosphate (CDP) also was assessed for use in the GEL test. In addition, time-in-motion studies were performed comparing GEL (12 panels per batch) with polyethylene glycol (PEG) tube tests (3 panels per batch). In 57 antibody identification studies, there were 63 GEL+ LISS+, 2 GEL+ LISS-, and 6 GEL-LISS+ antibodies. Among the GEL+ LISS+ antibodies were 19 that yielded stronger reactions in GEL than in LISS; by virtue of their specificity, 14 of these are considered potentially significant: D, 5 E, 2 e, 2 Jka, 2 S, K, and Fya. There were 38 antibodies that yielded equivalent results by both methods, including 31 that are considered potentially significant. Of six antibodies with significantly greater reactivity in LISS, there were three anti-Rh and three that are considered harmless with respect to transfusion management. The two GEL+ LISS- antibodies (anti-Jkb) were potentially significant. GEL- LISS+ reactions involved only harmless antibodies. Of the 50 antibodies of potential significance, GEL yielded equivalent or superior results in 47 (94%) instances. Additionally, GEL failed to detect 6 of 21 harmless antibodies. Expected results were obtained with normal serum or plasma and antibodies of known specificity in tests with RBCs treated with ficin, DTT, or CDP. Hands-on-time required for each GEL panel was 2 to 21/2 minutes compared with 12 minutes for PEG. These data document the suitability of GEL for use in antibody identification studies.

The gel test: sensitivity and specificity for unexpected antibodies to blood group antigens.

The recently FDA-licensed anti-IgG gel test for pretransfusion antibody detection requires crossover validation before implementation. Six hundred coded samples sent for routine pretransfusion tests were used to compare GEL (ID-MTS, Ortho Diagnostic Systems Inc., Raritan, NJ) with Löw and Messeter's low-ionic-strength saline (LISS). There were 456 GEL-LISS-, 97 GEL+LISS+, 45 GEL-LISS+, and 2 GEL+LISS- tests. The 144 positive tests involved 157 antibodies; 67 of these (cold auto, anti-M, -Le, etc.) were considered harmless with respect to transfusion management. GEL-LISS+ tests included seven samples containing potentially significant antibodies (assumed from specificity): anti-K(4), -Jka, -Fyb, and -S. Two potentially significant antibodies (anti- C and -D) were GEL+LISS-. Sensitivity and specificity for potentially significant antibodies were 92% and 96% for GEL, and 98% and 90% for LISS, respectively. The seven GEL-LISS+ samples associated with potentially significant antibodies were from six patients. Five of these antibodies, all detected in immune-suppressed patients, reacted predominantly as agglutinins in LISS. None of these seven antibodies were detected reliably by polyethylene glycol and LISS-additive tube methods. In light of the immune status of the patients with GEL-LISS+ agglutinins with specificity normally considered potentially significant, and because other valid methods did not detect these antibodies, their clinical importance is questionable. Excluding these questionable antibodies, GEL has the same sensitivity and better specificity than LISS. GEL is a valid method for pretransfusion antibody detection.