Comparison of three low-ionic diluents for dilution and storage of reagent A(1) and B cells for testing in gel technology.

Currently, ABO serum grouping performed by gel technology employs a red cell diluent containing EDTA (MTS Diluent 2 Plus) that does not permit extended storage of the red cell suspensions. A diluent currently used for suspension and long-term storage of reagent red cells for antibody detection and identification (Ortho 0.8% Red Cell Diluent) was evaluated for use with A(1) and B cells. Because this diluent does not contain EDTA, testing was limited to EDTA samples. As a comparison, a Micro Typing Systems (MTS) diluent not containing EDTA (MTS Diluent 2) was also tested. MTS-suspended red cells were maintained for 24 hours and compared with Ortho-Clinical Diagnostics 0.8% suspended red cells maintained for 7 days. ABO serum grouping was performed on 144 EDTA plasma samples using all three cell suspensions. Acceptable results were noted in all aspects of testing.

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.

Survey on pretransfusion testing.

BACKGROUND: Hospitals and blood centers throughout the United States use a variety of reagents and methods to perform pretransfusion testing. A survey was developed to determine the reagents and methods in use and their relative prevalence in different work settings. STUDY DESIGN AND METHODS: A national survey on pretransfusion testing was conducted. Surveys were distributed to state and regional blood bank associations, which then distributed them to hospitals and blood centers within their region. In most instances, the blood centers distributed the survey to the local hospitals. Completed surveys were returned to the authors for review, and all information was entered into a database for analysis. RESULTS: Analysis of the data shows that the majority of blood banks use monoclonal reagents for ABO testing and monoclonal-polyclonal blended reagents for Rh testing. The data show that anti-IgG and polyclonal antihuman globulin reagents are used almost equally for antibody screening (detection) tests and that most blood banks use a three-cell antibody-screening test. Slightly more than 50 percent of hospitals use an immediate-spin crossmatch in the absence of unexpected antibodies. CONCLUSION: A number of approved reagents and methods are used by blood bank laboratories for pretransfusion testing. Facility size (number of beds) and type tend to influence the choice of methods and reagents employed. This survey provides an opportunity for blood bank laboratories to compare their current practices with those of their peers.

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.

Implementation of gel column technology, including comparative testing of Ortho ID-MTS with standard polyethylene glycol tube tests.

With the intent to increase laboratory efficiency and according to the Clinical Laboratory Improvement Act of 1988 (CLIA '88), a parallel testing program comparing traditional tube technology with the gel system technology was undertaken. Test tube indirect antiglobulin tests were performed using polyethylene glycol (PEG) as the antibody enhancement medium. Gel (GEL) column technology used the ID-Micro Typing System, using predispensed anti-IgG and low-ionic- strength saline for antibody enhancement. Tests were performed as described in the manufacturer's guidelines and the current edition of the Technical Manual of the American Association of Blood Banks. Testing included antibody detection, antibody identification, direct antiglobulin tests (DATs), antigen phenotyping (K, Fya, Fyb, S, and s), and elution studies. These procedures were evaluated for sensitivity, specificity, and efficiency. Sixty-six samples that had been tested for antibody activity by PEG tube techniques were evaluated by GEL. These samples included 49 that were nonreactive and 17 with a positive antibody detection test. Within the latter were 19 antibodies, 17 with specificities considered to be clinically significant and 2 usually considered clinically insignificant for red cell transfusion. GEL was nonreactive with the 49 PEG negative samples as well as with the 2 samples containing insignificant antibody. All 17 antibodies of probable clinical significance were detected. Antibody identification studies were performed on these latter samples, with GEL results consistent with PEG tube results in all cases. Concordant results were obtained with 10 of 10 DATs (7 negative, 3 positive), all 77 antigen phenotyping tests (37 negative, 40 positive), and the 6 parallel elution studies (4 negative, 2 positive). GEL testing was found to be comparable or better when compared with PEG tube testing in all procedures evaluated.

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.

Electronic verification of donor-recipient compatibility: the computer crossmatch.

BACKGROUND: This article describes standard operating procedures (SOPs) for a computer crossmatch to replace the immediate-spin crossmatch for ABO incompatibility between patient blood samples submitted for pretransfusion testing and the blood component selected for transfusion. These SOPs were developed following recent changes to the Standards for Blood Banks and Transfusion Services of the American Association of Blood Banks (AABB). STUDY DESIGN AND METHODS: SOPs were developed, utilizing currently available software, for pretransfusion testing. The SOP for donor unit processing entails bar code entry of the unit number, component name, and ABO/Rh type; computer entry and interpretation of serologic reactions; warning of discrepancies between bar code-entered blood type and result interpretation; and quarantine of the donor unit in such instances. The SOP for patient sample testing requires bar code entry of specimen accession number, which accesses patient demographics; computer entry and interpretation of ABO/Rh tests; repeat blood typing at the time of crossmatch if only one patient blood type is on record; and warning if there are nonconcordant current and historical blood types. The computer crossmatch SOP requires bar code entry of specimen accession and donor unit numbers; release of group O red cells pending resolution of discrepancies; and immediate-spin crossmatch during computer downtime. Tables validated on-site prompt warning messages and prevent both computer crossmatch and release if blood components of the wrong ABO type are selected. RESULTS: These SOPs meet the requirements of the 15th edition of the AABB Standards. Projected annual time savings at this institution are > 100,000 workload recording units. Further benefits include reduced patient sample volume requirements, less handling of biohazardous material, and elimination of unwanted positive or negative reactions associated with the immediate-spin crossmatch. Release of incompatible blood components when the wrong patient blood type is on record is addressed by requiring the use of group O red cells in the absence of two concordant blood types, one of which must be from a current sample. CONCLUSION: A combination of existing computer programs and carefully developed SOPs can provide a safe and efficient means of detecting donor-recipient incompatibility without performance of serologic crossmatch.

Cefotetan-induced immune hemolytic anemia.

Immune hemolytic anemia due to a drug-adsorption mechanism has been described primarily in patients receiving penicillins and first-generation cephalosporins. We describe a patient who developed anemia while receiving intravenous cefotetan. Cefotetan-dependent antibodies were detected in the patient's serum and in an eluate prepared from his red blood cells. The eluate also reacted weakly with red blood cells in the absence of cefotetan, suggesting the concomitant formation of warm-reactive autoantibodies. These observations, in conjunction with clinical and laboratory evidence of extravascular hemolysis, are consistent with drug-induced hemolytic anemia, possibly involving both drug-adsorption and autoantibody formation mechanisms. This case emphasizes the need for increased awareness of hemolytic reactions to all cephalosporins.

Anti-i causing acute hemolysis following a negative immediate-spin crossmatch.

A unique case of acute hemolysis following transfusion of red cells (RBCs) that were found compatible by immediate-spin (IS) crossmatch technique is reported. Screening tests for unexpected antibodies, using low-ionic-strength saline (LISS), 10 minutes' incubation at 37 degrees C, and anti-IgG, were nonreactive; however, 1 transfused unit was found crossmatch incompatible by indirect antiglobulin technique (IAT). An anti-i (titer 512 at 4 degrees C) that was not an autoantibody was identified in the patient's serum. Unlike the incriminated donor RBCs, most I+ RBCs did not react by LISS-IAT. Variable reactivity was seen with ficin-treated I+ RBCs, and there was marked hemolysis of iadult and icord RBCs. In marked contrast, dominant Lu(a-b-) RBCs, with reduced expression of i, did not react by any test method; nor did autologous I+, Lu(b+) RBCs. The in vivo clinical significance of this anti-i was confirmed by monocyte monolayer assay and RBC survival studies. The patient's i antigen may have been altered, by either chemotherapy or disease, and lacked part of the i antigen-mosaic. Her antibody was directed at epitopes of i that were absent from her RBCs. Those i epitopes missing from her RBCs are also absent on dominant Lu(a-b-) RBCs. This anti-i represents a unique cause of an acute hemolytic transfusion reaction. It also represents a case of acute immune-mediated hemolysis following transfusion of IS crossmatch-compatible blood when screening tests for unexpected antibodies are nonreactive. Because of the rarity of such cases (less than 1/200,000 RBC units transfused), modifications to pretransfusion testing protocols are not proposed.

Anti-Yta does not cause hemolytic disease of the newborn.

Anti-Yta has been reported to cause hemolytic disease of the newborn. We present a pregnancy complicated by anti-Yta alloimmunization. Based on reassuring monocyte monolayer assay results, the pregnancy was followed without invasive testing. The infant subsequently had a benign course in the nursery. We conclude that anti-Yta did not cause hemolytic disease of the newborn.

Can the reading for serologic reactivity following 37 degrees C incubation be omitted?

The need to detect antibodies that agglutinate and/or hemolyze red cells (RBCs) directly at 37 degrees C, but do not react in subsequently performed indirect antiglobulin tests (IATs), is of concern relative to the streamlining and automation of antibody detection methods. To determine incidence and significance of such reactions, data from 87,480 tests, which used low-ionic-strength saline, 10-minute incubation at 37 degrees C, and anti-IgG, were analyzed for unexpected antibodies. There were 3590 positive tests, of which 475 showed reactions at 37 degrees C but not in subsequently performed IATs (37 + IAT-). Of these, 196 reactions were due to autoantibodies or other factors usually considered insignificant with respect to the survival of transfused incompatible RBCs, 176 were due to alloantibodies of questionable clinical significance (M, Lea, P1, etc.), and 103 were associated with alloantibodies of potential clinical significance (63 E, 27 K, 5 Jka, 4 D, 3 cE, and 1 C). This latter reaction was seen in 72 patients, with two 37 + IAT-antibodies occurring in each of 3 patients. Of the 75 potentially significant 37 + IAT-antibodies, 57 were seen in patients recently exposed to homologous RBCs, 13 in patients with a history of transfusion and/or pregnancy, and 5 in patients with no known exposure to homologous RBCs. IAT reactivity was observed in subsequent samples with 27 of these antibodies. The predictive value of a 37 + IAT-test was 21.7 percent for a potentially significant antibody. The incidence was 0.12 percent of all tests for unexpected antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Percutaneous umbilical blood sampling and umbilical vein transfusions. Rapid serologic differentiation of fetal blood from maternal blood.

Percutaneous umbilical blood samples (PUBS), obtained under ultrasound guidance, are used for prenatal diagnosis and management of hemolytic disease of the newborn (HDN) and other fetal disorders. Rapid testing at the time of sampling is vital to distinguish fetal from maternal blood. Blood typing was performed by slide technique in the treatment room during 38 procedures on 25 patients. Anti-I was used to test 50 presumed PUBS; venous I-positive maternal blood was tested in parallel. Because anti-I cannot detect fetal blood after umbilical vein transfusion (UVT) of I-positive donor blood, ABO and Rh blood typing reagents were used to test 29 samples when maternal and fetal or donor blood groups differed. Monoclonal reagents were used for optimal detection of weak AB antigens in fetal blood. Avid, chemically modified anti-D was used for Rh typing. Blood typing showed 27 (34%) of 79 samples to be maternal blood. Fetal blood was obtained in 8 of 10 cases investigated for fetal disorder and in 16 cases of potential HDN (anti-D, 5; -CD, 5; -cE, 2; -K, 2; -c; -E). The absence of HDN (antigen-negative fetus) was determined in 4 cases. UVT afforded live birth of 9 of 10 infants with HDN and was not indicated in two cases.

Discrepancies in reverse ABO typing due to prozone. How safe is the immediate-spin crossmatch?

Three group O sera manifesting prozone in reverse ABO tests are reported. All were implicated in erroneous blood typing results. One sample failed to react with A1 red cells (RBCs) in immediate-spin (IS) tests, had anti-A and -B titers of 8192 and 2048, respectively, by indirect antiglobulin technique (IAT), and was from a diabetic patient; the parenteral administration of A substance present in porcine insulin is a possible cause of hyperimmunity in this case. The second sample was from the recipient of a single unit of group B fresh-frozen plasma; the serum anti-A and -B titers were 10,240 by IAT, but only weak reactions with A1 and B RBCs were noted in routine IS reverse typing tests; the hyperimmunity in the patient concerned was likely due to crossreacting anti-A, B stimulated by B-active glycoproteins and/or glycolipids in the transfused plasma. The third serum also had anti-A and anti-B IAT titers of 10,240 but did not react with A1 and B RBCs by IS; the hyperimmunity in this case may be related to sepsis from intestinal flora carrying A- and/or B-like antigens. These antibodies lysed A1 and/or B RBCs in tests incubated at room temperature (RT) and strongly agglutinated those RBCs by IS when diluted 10-fold with saline. The absence of the prozone phenomenon in tests with RBCs suspended in diluents containing EDTA is consistent with the previously published mechanism for anti-A prozone: namely, the steric hindrance of agglutination by the C1 component of human complement.(ABSTRACT TRUNCATED AT 250 WORDS)

False-positive results with chemically modified anti-D do not indicate a need to use a separate, immunologically inert Rh control reagent.

Thirteen of 1450 (0.9%) Rh-negative samples submitted for pretransfusion or prenatal testing gave weak false-positive reactions in immediate-spin (IS) tests with chemically modified anti-D (CM-D). Only seven reacted in control tests with anti-A and/or -B. The other six samples could have been considered Rh positive; however, such a conclusion is inappropriate, since stronger (greater than or equal to 3+) reactions are expected in IS tests with CM-D and the vast majority of true Rh-positive blood samples. Moreover, three of the six false-positive CM-D reactions associated with negative control tests were from patients previously found to be Rh negative. The other three samples gave 1+ to 2+ reactions with CM-D and did not react with anti-A and/or -B; these erroneous CM-D reactions were recognized by confirmatory tests with high-protein anti-D and Rh control reagents that were performed because previous typing results were not on file. Such confirmation, as well as careful grading and proper interpretation of serologic reactions, serves to prevent erroneous Rh typing without the use of a separate, immunologically inert Rh control reagent.

Donath-Landsteiner hemolytic anemia due to an anti-Pr-like biphasic hemolysin.

Anemia, hyperbilirubinemia, and reticulocytosis subsequent to viral infection were present in a 32-year-old woman. The direct antiglobulin test was negative, and no unexpected antibodies were detected in pretransfusion tests. Rosettes of red cells (RBCs) around neutrophils were observed in peripheral blood smears, and a Donath-Landsteiner (D-L) test was positive. However, the patient did not show the classic features of paroxysmal cold hemoglobinuria (PCH). There was no hemoglobinuria, and in vivo hemolysis was not precipitated by cold. The D-L antibody was IgG, but classic anti-P specificity was not apparent. Rather, protease- or neuraminidase-treated RBCs, as well as certain sialic acid deficient RBCs of uncommon MN phenotypes, were not hemolyzed in D-L tests. Further, D-L antibody activity could be inhibited by MN sialoglycoprotein. These data support a diagnosis of chronic D-L hemolytic anemia, caused by an anti-Pr-like biphasic hemolysin.

The evaluation of a positive direct antiglobulin test (autocontrol) in pretransfusion testing revisited.

Direct antiglobulin tests (DATs) using anti-IgG were performed on 65,049 blood samples from prospective transfusion recipients; 3570 tests (5.49%) were positive. Using criteria published previously (primarily excluding patients not transfused within the preceding 14 days), 778 samples from other than neonatal patients were selected for further evaluation. Eluates that did not react were obtained on 518 (66.6%) of these samples. Warm-reactive autoantibodies were apparent in 192 eluates, while 16 contained drug-related antibodies, anti-A or anti-B from prior transfusion with ABO mismatched blood components, or anti-D passively acquired from immune serum globulin. Fifty-two eluates contained alloantibodies; however, in only six of these cases did the corresponding serum lack unexpected alloantibodies, as determined by routine pretransfusion studies. Three additional weakly reactive clinically significant alloantibodies were detected solely through additional serum tests performed on DAT-positive samples. On the basis of these findings, the DAT had a low predictive value when used to detect the early manifestations of an immune response to recently transfused red cells. Elimination of the autocontrol from routine pretransfusion testing, therefore, carries minimal risk to patients yet will undoubtedly contribute to the containment of health care costs. Moreover, the risk is lower than that associated with the elimination of the antiglobulin crossmatch.