STUDY DESIGN AND METHODS:
Using data from prior publications, actual risk calculations (ARCs) were made to predict the risk of eliminating the 37°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°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°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°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 ,OOO units. With respect to the 37°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°C reading. This represents a decrease of 20 percent in the number of samples requiring antibody identification annually. CONCLUSIONS: Eliminating the 37°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. n 1992, we reported that approximately 20 percent o f antibodies detected solely by reading for agglutination and hemolysis after 37°C incubation have Rh, K, I or Jk specificity.' Because we encountered approximately 25 patients with such antibodies annually, we elected at that time t o retain the 37°C reading as part o f our screening procedure for unexpected antibodies. This test tube procedure is based o n the low-ionic-strength saline (LISS) technique o f L o w and Messeter' and utilizes two reagent red cell (RBC) samples, 10-minute incubation at 37"C, and anti-IgG.
STUDY DESIGN AND METHODS:
We revisited our 1992 decision following a recent evaluation o f the gel test (ID-Micro Typing System, Ortho-Clinical Diagnostics, Raritan, NJ).3 In this evaluation, the test failed to detect many examples o f unwanted cold-reactive autoagglutinins and alloagglutinins found b y LISS. These antibodies are considered "unwanted" because their presence rarely requires the selection o f antigen-negative units for transfusion. However, the gel test missed three anti-K1 and one anti-Jk" that reacted predominantly as agglutinins in LISS. By virtue o f their specificity, we have considered these antibodies potentially significant, or "wanted," with respect to the transfusion o f antigen-positive RBCs. The term "wanted" is used here because the presence of such antibodies requires antigen-negative units to be selected for transfusion, and the units should also be compatible by the indirect antiglobulin t e~h n i q u e .~ Since other popular test tube methods, namely polyethylene glycol (PEG)5 and LISS additives, also missed agglutinins o f wanted spe~ificities,~ we questioned the true ABBREVIATIONS: ARCs = actual risk calculations; DAT = direct antiblobulin test; IAT-Xh4 = indirect antiglobulin crossmatch; LISS = low-ionic-strength saline; PEG = polyethylene glycol; RBCW = red cells(s1.
STUDY DESIGN AND METHODS:
relevance of these antibodies. Our 1992 data revealed that many examples of wanted agglutinins were from patients who were immunosuppressed, either by disease or treatment.' Evidence for immune suppression was also found among patients with wanted agglutinins that were not detected by the gel test.3 Further, we are unaware of published reports of hemolytic transfusion reactions despite the widespread use of PEG and the gel test techniques, neither of which includes a separate reading for direct agglutination after 37°C incubation. Accordingly, we eliminated the 37°C reading from our LISS procedure. Before making such a procedural change, we used our 1992 data to calculate the associated risk. We compared this risk to other risks we are taking because of past changes made to our pretransfusion testing protocols-namely, elimination of the pretransfusion direct antiglobulin test (DAT)6 and elimination of the routine antiglobulin crossmatch (IAT-XM1.7
STUDY DESIGN AND METHODS:
In this report, we present the results of these comparisons as well as the results of laboratory and clinical monitors designed to determine the effect of eliminating the 370C reading on workload and patient care.
MATERIALS AND METHODS
Actual risk calculations (ARCS) used the equations n x 5 x percent antigen-positive x 0.34 or n x 5 x percent antigenpositive x 0.80. For both equations, n equals wanted antibodies missed, and 5 represents the average number at our institution of units given per patient transfused. The percentage of antigen-positive units will vary with antibody specificity; the actual antigen frequencies used in the calculations were those corresponding to the antibodies missed following test elimination, as shown in our earlier studies.'~~,~ Cases involving anti-D were excluded, because the patients would normally receive Rh-negative blood. The factor of 0.34 represents the percentage of "typed and screened" patients who are subsequently transfused at our institution and is used to evaluate the risk of eliminating the DAT and the 37°C reading. A factor of 0.80 represents the percentage of "crossmatched" patients at our institution who are transfused and is used to evaluate the risk of eliminating the routine IAT-XM. Both factors were obtained by review of activity reports for 1 month. All calculations were adjusted to current annual workload volumes of 30,000 pretransfusion tests, 29,000 RBC transfusions, and 46,000 crossmatches. Confidence intervals for proportions were calculated according to Gardner and Altman.8 To monitor the impact of eliminating the 37°C reading on a prospective basis, we looked at the following information: l) antibody detection data, including the percentage of samples with a positive antibody screen and the nature of antibodies encountered in antibody identification studies; 2) the number of patients who presented with newly formed antibodies of potential significance following recent transfusion, whether or not there was clinical evidence of a hemolytic transfusion reaction, and whether or not these antibodies would have been detected earlier if a 37°C reading had been included; and 3) the number of transfusion reactions reported by clinicians to the transfusion service and the outcome of our investigation into these reactions. The latter investigations were performed in accordance with requirements ofAmerican Association of Blood Banks Standard KZ.000.4 For patients with a positive DAT or free serum hemoglobin after transfusion, screening tests that included a reading for agglutination and hemolysis after 37°C incubation were performed on prereaction and postreaction samples.
MATERIALS AND METHODS
We gathered these data for two study periods, for 6 months after elimination of the 37°C reading (1996-97) and for the same dates of the precedingyear (1995-961, when a 37°C readingwas included in our test protocol. In addition, for patients with newly formed antibodies who had been transfused within the preceding month, we reviewed hematologic and biochemical data to obtain evidence for antibody-mediated hemolysis. When previously submitted samples were available for testing, we performed a 37°C reading retrospectively using selected (antigen-negative; single-and double-dose-positive) reagent RBCs.
MATERIALS AND METHODS
For the 1995-96 study period, LISS tests for routine pretransfusion antibody detection were performed as described el~ewhere.~ These tests were modified for the 1996-97 study period by eliminating the centrifugation and reading for direct agglutination steps prior to washing for antiglobulin testing. Technologists were instructed to observe the size of the centrifuged RBC button as an indication of in vitro hemolysis. Reactions were graded as described el~ewhere.~
RESULTS
The results ofARCs (Table 1) reveal that one patient per year is likelyreceiving 2 units of antigen-positive blood, because we no longer perform a pretransfusion DAT/autocontrol. The number of patients at risk is higher when an immediate-spin crossmatch is used instead of an IAT-XM. Eight patients per year are likely exposed to 2 units of antigenpositive blood by the performance of an abbreviated crossmatch when unexpected antibodies are absent. By eliminating the 37°C reading, we calculate that nine patients are placed at risk of receiving 1 or 2 units of incompatible blood. When calculated in terms of the current number of RBC units transfused annually (approx. 29,000), the risk of transfusing incompatible blood following test elimination is approximately 1 in 13,000,l in 2,000, and 1 in 2,400 units transfused for eliminating the DAT, IAT-XM, and 37°C reading, respectively. The cumulative risk associated with elimination of all three tests is approximately 1 in 1,000 units transfused. Table 2 shows the differences in our activities for the two study periods. For the 6 months of 1996-97, our 
7
The percentage of incompatible units will vary with antibody specificity; the actual antigen frequencies used in the calculations were those corresponding to the antibodies missed following test elimination, as shown in our earlier s t u d i e~.~,~,~ ** Undefined lower limit of CIS for incompatible units transfused is zero. [608, 7561s [4.3, 4.91 [231, 2921 [54.4, 69.21 [137, 1871 [32.4, 44.11 (481, 5691 [3.2, 3.81 [234, 2981 [71.7, 91.01 [46, 761 [14.0, 23.31 Number of positive screening tests does not equal total (wanted + unwanted) of antibody identification studies performed. Patients whose current antibody screen and crossmatch results are consistent with a recently performed identification study are not subjected to repeat investigation. t Number of identification studies that revealed findings not considered important with respect to transfusion of antigen-positive blood. Finding rarely requires selection of antigen-negative blood for transfusion. * Number of identification studies that revealed at least one antibody that is considered potentially significant with respect to transfusion of antigen-positive blood. Presence requires selection of antigen-negative units and an IAT-XM. There is no significant difference in the numbers of wanted antibodies encountered either before or after elimination of the 37°C reading (Table  3) . However, had we not performed a 37°C reading in the 6-month period of 1995-96, we would have missed 13 "37°C only" antibodies of wanted specificities. This observation is consistent with our 1992 study, when we found approximately 25 patients with such antibodies per year.' Table 3 also shows the unwanted antibodies detected in both study periods. There was a dramatic reduction in the number of unwanted antibodies identified following elimination of the 37°C reading; this reduction equates to some 200 samples annually. The second part of our study looked at patients in whom we detected newly formed antibodies of potential significance and who had been transfused within the preceding month. We found 31 such antibodies in 25 patients in the 1996-97 study period. We evaluated these 25 patients for clinical evidence of immune hemolysis and compared our findings with those from 26 patients with newly formed antibodies encountered in the 1995-96 study period. While we found six cases of possible transfusion-related immune hemolysis in the 1996-97 study period versus four in 1995-96, this increase is not significant, given the total number of units transfused, which is in the order of 15,000 units for each study period.
7
For all 25 patients with newly formed antibodies of potential significance from the 1996-97 study period, we evaluated any available samples workload increase was such that we performed 5 percent more pretransfusion tests than in the same period of 1995-96. However, 4.6 percent of samples had positive tests for unexpected antibodies in 1995-96, compared to a 3.6-percent positive rate after elimination of the 37°C reading. Consequently, about 100 more samples required antibody identification studies in the 6 months of 1995-96 than in the same period of the following year. To a large extent, this difference is a reflection of the number of unwanted antibodies that are no longer detected following elimination of the 37°C reading. submitted prior to antibody ascertainment for direct agglutinating activity at 37°C. Retrospective testing of available, previously submitted samples from 17 patients revealed six antibodies reactive at 37°C (range f to 2+7. Onlyone ofthese (l+s anti-E) was associatedwith clinical evidence ofimmunemediated hemolysis.
7
Finally, in our review of transfusion reactions reported by clinicians, the number of reactions in the study periods did not differ significantly. No cases of transfusion-induced immune hemolysis were identified through our investigation of these reports. Moreover, we encountered no 37°C-only agglutinins in prereaction or postreaction samples from patients with a positive DAT or free serum hemoglobin after transfusion.
DISCUSSION
Approaches to pretransfusion testing have undergone considerable revision in the past two decades. Prompted by reductions in operating budgets for clinical laboratories that started with reforms to health-care reimbursement, practices that yield a large number of unwanted positive tests are being abandoned, as are those generating data not applied to patient management.I0 Past testing strategies are being replaced with streamlined protocols that facilitate timely, cost-effective provision of blood and blood products. Nevertheless, while modifymg testing protocols, it is important to maintain quality patient care and adhere to regulatory requirements.' Retrospective review of our experience with the pretransfusion DAT led us to eliminate this test in June 1984.6
DISCUSSION
At the same time, there was a national interest in eliminating the IAT-XM for all patients except those known to have unexpected antibodies. We replaced the routine IAT-XM with an immediate-spin crossmatch in 1986. As shown in Table 1 , each of these modifications to pretransfusion testing is associated with a risk of transfusing serologically incompatible blood to a few patients per year. However, this risk has proven acceptable, given the benefits accrued by not having to investigate large numbers of unwanted positive tests.
DISCUSSION
In 1992, we reported the results of a 3-year retrospective review of the incidence of potentially significant antibodies detected solely by reading for agglutination and hemolysis following 37°C incubation.' This 1992 study was prompted by further fiscal pressures to streamline test protocols and by the emergence of technologies that do not include a 37°C reading.5s'2*'3 Our 1992 study revealed that 21.7 percent of antibodies detected solely by reading for agglutination after 37°C incubation have Rh, K, or Jk specificity.' Because we encountered approximately 25 patients with such antibodies annually, we concluded that "elimination of the 37°C reading for agglutination and hemolysis should not be undertaken lightly." Moreover, we elected at that time to retain the 37°C reading as part of our screening procedure for unexpected antibodies.
DISCUSSION
We revisited this 1992 decision following our gel test evaluation3 The gel test failed to detect, in addition to many examples of unwanted agglutinins, four examples of potentially significant alloagglutinins (3 K1 and 1 Jka). All were from immunosuppressed patients and were nonreactive by PEG and LISS-additive procedures. These findings, and the absence of reported hemolytic reactions associated with the widespread use of gel technology, prompted us to eliminate the 37°C reading from our pretransfusion antibody detection procedure. This modification is permitted according to current American Association of Blood Banks StandardsP We estimate the risk of transfusing serologically incompatible blood associated with elimination of the 37°C reading to be somewhat less (1:2400 vs. 1:2000 units transfused) than that associated with eliminating the routine IAT-XM. This risk analysis pertains only to LISS tests performed in accordance with the procedure of Low and Messeter.2 Moreover, since the risks associated with elimination of the pretransfusion DAT/ autocontrol, IAT-XM, and the 37°C reading are cumulative, we calculate that approximately 1 in 1000 units transfused in our institution are incompatible because of to antibodies we no longer detect.
DISCUSSION
The risk associated with other methods that do not include a 37°C reading may or may not be comparable to our data. Alkhashan et al.14 performed LISS tests on 5027 samples that were nonreactive by PEG-IAT and found four 37+, IAT-Rh antibodies. These findings are in accord with the thirteen 37+, IAT-wanted antibodies we found among the 14,286 samples tested in the 1995-96 study period (see Tables 2 and 3) . Further, Rolih et aI.l5 found that five of eight 37+, IAT-LISS-reactive wanted antibodies, representing 0.2 percent of 2460 antibody-containing samples tested, were nonreactive by a solid-phase adherence assay. In contrast, from a re-evaluation of our gel test data,3 four of six 37+, IAT-wanted antibodies detected by LISS were nonreactive by gel technology, representing 2.8 percent of 144 antibodycontaining samples tested. However, our data are difficult to compare with those of Rolih et a1.15 because they evaluated only antibody-containing samples, the method by which their antibodies were ascertained is not defined, and their incidence of 37+, IAT-wanted antibodies (0.33% of 2460 antibody-containing samples) was different from that of our 1992 study (2.9% of 3560 antibody-containing samples).
DISCUSSION
Despite the above statistical differences, we conclude that the actual risk of eliminating the 37°C reading from antibody detection tests is minimal, as evidenced by the fact that we observed no significance increase i n the incidence or severity of hemolytic transfusion reactions following such a modification t o our LISS procedure. Elimination of the 37°C reading avoids discovery of a significant number of unwanted antibodies that otherwise would have prompted time-consuming, costly studies that cause delay to patient care. In our institution, this equates to a reduction of antibody identification workload by 20 percent, or approximately 200 samples, annually.

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