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Impact of Uniform Methods on Interlaboratory Antibody Titration Variability: Antibody Titration and Uniform Methods.

Antibody titration (ABT) is a semiquantitative method used to detect the reactivity of antibodies present in the patient's plasma.1 Antibody titration is used prenatally to screen for risk of hemolytic disease of the fetus and newborn (HDFN), hemolytic reactions, or assessment in solid organ or hematopoietic stem cell transplant. To assess risk for HDFN, if the mother has a clinically significant alloantibody, ABT is performed. When the antibody and the titer strength are identified, ABT is periodically performed throughout pregnancy, whereby plasma samples are compared in parallel with the previously frozen samples to determine increase in titer strength. With the advent of middle cerebral artery Doppler assessment, the reliance on titers alone to predict fetal well-being has decreased. (2) However, each laboratory must still define a critical titer for which further fetal assessment with Doppler ultrasonography or amniocentesis needs to be initiated. Antibody titration is also commonly used for screening blood products, particularly platelets and plasma. To decrease the risk of hemolytic transfusion reactions due to passive anti-A/anti-B antibodies, the titer of group O products is determined and those with high titers (typically >1:100) are labeled and used for group O individuals only. (3) In addition, ABT has a role in preventing graft rejection for ABO-incompatible solid organ transplants of heart, liver, and lung as well as in delaying erythroid engraftment after hematopoietic progenitor cell transplants. (1,4,5)

Laboratories have multiple variables to determine in their ABT procedures, including platform, diluent, incubation time, strength of reading cutoff, and testing phase. (6) Traditionally, ABT is carried out in a test tube. However, gel methods appear to be more sensitive and less dependent on test performer, so many laboratories are switching to the gel method using saline as the diluent. (7) The AABB Technical Manual's method, which is well adopted for non-ABO antibodies, uses saline, 60 minutes incubation at 37[degrees]C, and anti-immunoglobulin G (IgG) with the tube technology. (8) Variation is also seen in the cutoff strength of the reaction to determine the titer, such as weak plus (w+) versus one plus (1+). With the increased sensitivity of gel versus tube, the strength of titer reading is usually 1+ reactivity in gel versus w+ reactivity in tube. (9) Since patients often seek care at different institutions with varying standard operating procedures for ABT, titer strength reported could have a wide range, which could inadvertently subject patients to high-risk procedures. (10,11) Hence, it is critical not only to identify the appropriate antibody and its exact strength, but also to make sure test methodology is well defined to get reproducible results in different settings. Hence, the College of American Pathologists (CAP) and Biomedical Excellence for Safer Transfusion (BEST) Committee in 2008 introduced a well-defined standardized procedure (uniform method) based on the findings of a pivotal study that showed significant reduction in interlaboratory titer variability. (9) To assess the impact of introduction of uniform methods for ABT, we analyzed data from consecutive CAP proficiency testing (PT) conducted post uniform method introduction.

METHODS

CAP PT survey was conducted biannually (survey A and B) post uniform procedure announcement (period: 2009-2013, 10 surveys) for anti-A and anti-D antibody titration in 2008. (9) Laboratories reported their results based on their primary testing method, as per PT standards. Each survey contained distributions for 2 separate sera (anti-A and anti-D) and a group A1 RhD-positive (D+) red cells for laboratories to perform antibody titration. For anti-A, titers were performed both at immediate spin (IS) and/or room temperature (RT) and anti-human globulin (AHG) phase, and for anti-D, at AHG phase either by using uniform or other methods as per laboratories' primary standard operating procedure. Hence, for anti-A, titers were reported with uniform tube RT (UT RT), uniform tube AHG (UT AHG), other tube immediate spin (OT IS), other tube room temperature (OT RT), and other tube AHG (OT AHG) methods with tube platform, and uniform gel (UG) or other gel (OG) methods with gel platform. For anti-D, titers were always performed in the AHG phase with uniform or other methods by using tube (UT, OT) or gel (UG, OG) platform. In addition to reporting anti-D OT method with immunoglobulin G (IgG) AHG reagent, OT methods were also performed with 6% albumin diluent (OT 6% alb), 22% albumin diluent (OT 22% alb), polyspecific AHG (OT poly AHG) reagents, and miscellaneous other techniques (OT others). A detailed description of uniform methods is available in Tables 1 and 2. Results of PT were submitted by using a standardized form, which collected information on the laboratory's methodology and titer results. For simplistic interpretation, instead of mentioning individual-year PT results separately, the median number of tests performed with different methods/ platforms (tube or gel) and their median range (minimum to maximum) are highlighted in the Results section. To give primacy to the original data, percentages (%) are presented in parenthesis next to the raw numbers from which they are derived. However, in analyzing the strength of titer results, mode (mode defined as the titer with highest frequency of responses), rather than median, was calculated. To incorporate all titer values and simplify the interpretation of wide range of titers, mode [+ or -] 1 dilution, mode [+ or -] 2 dilutions, and greater than mode [+ or -] 2 dilutions were also calculated. Finally, since titration results with different methods had varying response rates (number of titer results reported per method), for final titer variance reduction analysis, data from the top 3 or 4 methods reported for anti-A and anti-D only were included for statistical comparisons. Hence, although initially intended, owing to low response rate, titer variance reduction analysis between UG and UT or UG and OG for anti-A, and OG and UG for anti-D, was not performed.

Statistical Analysis

Results were grouped according to antibody (anti-A versus anti-D) and platform (tube or gel) and for anti-A separately in the IS/RT and the AHG phases. While analyzing for ABT variance between uniform and other methods for anti-A, UT versus OT methods were compared at both the RT and AHG phases. For anti-D, since all the methods were performed at AHG phase, statistical significance for titer variance was calculated separately for different platforms, that is, UT versus OT and UT versus UG. After analyzing all titer values, visual outlying titers were removed to prevent bias to the variability estimates. Finally, all reported titers were converted to log2 and the standard deviation and variance was calculated for each of the methods (log2 default value for a titer of 1 was 0.1). Also, in determining titer variance reduction, only data from 2010-2013 were included in analysis, since the 2009 surveys permitted laboratories to report results with multiple attempts of testing and hence were deemed not suitable to check for variance for that particular year. The primary statistical test performed was the variance ratio test (F test) to compare the significance of the ratio of the variance estimates of the 2 methods.

RESULTS

Anti-A Antibody Titer

Method.--The median for anti-A test results was 574 (range, 509-682) over a 5-year study period. Among these test results, uniform methods were reported more frequently than other methods both with tube and gel platforms (UT RT, 147.5 [range, 119-159] versus OT RT, 95 [range, 73117]; UT AHG, 143.5 [range, 134-150] versus OT AHG, 97 [range, 82-116]; UG RT, 19.5 [range, 12-24] and UG AHG, 24.0 [range, 21-27] versus OG, 12 [range, 10-15]). Only a minority of responders chose to report anti-A separately at IS phase (32; range, 15-71) and with polyspecific AHG reagent (10.5; range, 10-14) in the OT methods (Table 3).

Distribution of Titers.--Based on Method.--With the uniform methods in both RT and AHG phase, most titers were reported either at mode or at mode 6 1 dilution. Specifically, at mode, the titer values reported by laboratories had a wide range for different phases, which varied from 38.8% to 52.1% (UTRT, 612 of 1440 [42.5%]; UT AHG, 561 of 1427 [39.3%]; UG RT, 76 of 196 [38.8%]; UG AHG, 125 of 240 [52.1%]). Similarly, at mode 6 1 dilution (UT RT, 537 of 1440 [37.3%]; UT AHG, 667 of 1427 [46.7%]; UG RT, 57 of 196 [29.1%]; UG AHG, 95 of 240 [39.6%]), mode [+ or -] dilutions (UT RT, 196 of 1440 [13.6%]; UT AHG, 162 of 1427 [11.3%]; UG RT, 21 of 196 [10.7%]; UG AHG, 19 of 240 [7.9%]), and at greater than mode 6 2 dilutions (UT RT, 95 of 1440 [6.6%]; UT AHG, 37 of 1427 [2.6%]; UG RT, 42 of 196 [21.4%]; UG AHG, 1 of 240 [0.4%]), numerous titer values falling outside the range of mode were reported. With the use of other methods too, most titers were reported either at mode or at mode 6 1 dilution. Specifically, at mode, a wide range (38.7% to 59.1%) of titers was reported by both the tube and gel platforms (OT IS, 186 of 387 [48.1%]; OT RT, 449 of 961 [46.7%]; OT AHG, 386 of 998 [38.7%]; OG AHG, 65 of 110 [59.1%]). The remainder of titer values falling outside the range of mode was captured at mode [+ or -] dilution (OT IS, 158 of 387 [40.8%]; OT RT, 397 of 961 [41.3%]; OT AHG, 457 of 998 [45.8%]; OG AHG, 38 of 110 [34.6%]), mode [+ or -] dilutions (OTIS, 41 of 387 [10.6%]; OTRT, 103 of 961 [10.7%]; OT AHG, 133 of 998 [13.3%]; OG AHG, 6 of 110 [5.4%]), and greater than mode [+ or -] dilutions (OT IS, 2 of 387 [0.5%]; OT RT, 12 of 961 [1.2%]; OT AHG, 22 of 998 [2.2%]; OG AHG, 1 of 110 [0.9%]) by different platforms (Figure 1).

Based on Platform.--Cumulative analysis of titers at mode and (mode [+ or -] dilution) with the gel platform showed that more than 90% of all titers with the rare exception of UG RT were captured (UG RT, 133 of 196 [67.9%]; OG AHG, 103 of 110 [93.6%]; UG AHG, 220 of 240 [91.7%]). However, with the tube platform, greater than 80% but less than 90% of all titers were captured within the cumulative end point comprising values reported at both mode and mode [+ or -] dilution, with the exception of UT RT method (OT IS, 344 of 387 [88.9%]; OT RT, 846 of 961 [88.0%]; UT AHG, 1228 of 1427 [86.1%]; OT AHG, 843 of 998 [84.5%]; UT RT, 1149 of 1440 [79.8%]) (Figure 1).

Based on Phase.--When analyzing the range of distribution of titers at different phases individually, OT IS phase captured 344 of 387 titers (88.9%) at mode + (mode [+ or -] dilution). At the RT phase also most of the titers were detected at mode + (mode [+ or -]dilution), but with a wide range of results based on platform and method applied (UT RT, 1149 of 1440 [79.8%]; OT RT, 846 of 961 [88.0%]; UG RT, 133 of 196 [67.9%]) (Figure 1). However, at the AHG phase, irrespective of the method used (uniform or other), greater than 80% of titers were consistently reported at mode + (mode [+ or -]dilution) (UT AHG, 1228 of 1427 [86.1%]; OT AHG, 843 of 998 [84.5%]; UG AHG, 220 of 240 [91.7%]; OG AHG, 103 of 110 [93.6%]) (Figure 1).

Variance.--For the years 2011 (Survey A), 2012 (Survey A), and 2013 (Survey B), a total of 3, 1, and 2 outlier titer values reported in the respective PT were removed before analysis. In the cumulative analysis, at RT, 0 of 8 PT surveys comparing UT versus OT methods (0%) showed a statistically significant reduction in titer variance. In fact, 6 of 8 (75.0%) showed statistically significant higher variance. However, at AHG phase, 1 of 8 surveys comparing UT versus OT (12.5%) showed statistically significant reduction in anti-A titer variance and none had statistically significant higher variance (Figure 2; Table 4).

Anti-D Antibody Titer

Method.--The median for anti-D titers reported over the 5-year period was 1100 (range, 1059-1211) (Table 3). Between uniform and other methods with tube platform, the latter was reported more frequently (OT, 451 [range, 431-465]; UT, 404 [range, 382-462]). In contrast, with the gel platform, most laboratories opted to report with uniform methods over other methods (UG, 137 [range, 121-153]; OG, 15 [range, 10-19]). Apart from using IgG AHG reagent, other methods were also reported with 6% albumin diluent (57.5; range, 50-79), 22% albumin diluent (18.5; range, 14-28), and with poly-specific AHG reagents (23; range, 19-30) by different laboratories (Table 3).

Distribution of Titers.--Based on Methods.--Most titers were reported either at mode or mode 6 1 dilution with uniform method. Specifically, at mode, greater than 40% of all titer results were captured by different platforms with uniform method (UT, 1800 of 4109 [43.8%]; UG, 729 of 1368 [53.3%]). Similarly, at mode 6 1 dilution (UT, 1823 of 4109 [44.4%]; UG, 522 of 1368 [38.2%]), mode [+ or -] dilutions (UT, 414 of 4109 [10.0%]; UG, 97 of 1368 [7.1%]), and greater than mode [+ or -] dilutions (UT, 72 of 4109 [1.7%]; UG, 20 of 1368 [1.5%]), a wide range of other titer values falling outside the range of mode were reported with different platforms, using uniform methods. With the application of other methods, again most titers were reported either at mode or mode [+ or -] dilution. Specifically, at mode, both the tube and gel platforms captured approximately 39.8% to 54.7% of all titers reported (OT, 1834 of 4495 [40.8%]; OT6% alb, 258 of 604 [42.7%]; OT22% alb, 93 of 193 [48.2%]; OT poly AHG, 94 of 236 [39.8%]; OG, 64 of 117 [54.7%]). The remainder of titer values was distributed at different dilution range from mode (mode 6 1 dilution: OT, 2132 of 4495 [47.4%], OT6% alb, 264 of 604 [43.7%], OT22% alb, 74 of 193 [38.3%], OT poly AHG, 101 of 236 [42.8%], OG, 46 of 117 [39.3%]; mode [+ or -] dilution: OT, 444 of 4495 [9.8%], OT 6% alb, 71 of 604 [11.7%], OT 22% alb, 19 of 193 [9.8%], OT poly AHG, 35 of 236 [14.8%], OG, 6 of 117 [5.1%]; and greater than mode [+ or -] dilutions: OT, 85 of 4495 [1.9%], OT 6% alb, 11 of 604 [1.8%], OT22% alb, 7 of 193 [3.6%], OT-poly AHG, 6 of 236 [2.5%], OG, 1 of 117 [0.9%]) by other methods.

Based on Platform.--Gel platform methods had greater than 90% of titers captured with the end points comprising mode and (mode [+ or -] dilution) (OG, 110 of 117 [94.0%]; UG, 1251 of 1368 [91.5%]) (Figure 1). Tube platform with both UT and OT methods consistently captured greater than 80% but less than 90% of anti-D titers cumulatively at mode and within 1 dilution range from mode (OT, 3966 of 4495 [88.2%]; UT, 3623 of 4109 [88.2%]; OT22% alb, 167 of 193 [86.5%]; OT6% alb, 522 of 604 [86.4%]; OT poly AHG, 195 of 236 [82.6%]).

Variance.--In the cumulative analysis, none (0 of 8) of the PT surveys comparing UT versus OT and UT versus UG methods showed statistically significant reduction in titer variance (Figure 2; Table 4). However, for 5 of 8 PT surveys (62.5%), UT had statistically higher variance than UG and for 1 of 8 surveys (12.5%), UT had statistically higher variance than OT (Figure 2; Table 4).

DISCUSSION

Significant variation in titer reporting between different laboratories has been reported previously. (12) The BEST/CAP Transfusion Medicine Resource Committee in 2008 published the uniform method to be incorporated in clinical practice to decrease interlaboratory titer variation. Evaluating clinical practice via PT surveys for ABT our study demonstrates several key current trends. (1) Uniform methods (tube + gel platform) are practiced by approximately half of the laboratories and tube platform is used more frequently than gel platform (Table 3). (2) Assessing the phase at which anti-A antibody titer is frequently reported, our findings suggest that an almost equal number of participants report at RT or AHG phase, irrespective of the method (uniform or other) or platform (tube or gel) chosen (Table 3). (3) Gel platform appears to be more sensitive than tube platform to detect antibodies at mode + (mode [+ or -] 1 dilutions) (>90% of titers versus >80% but <90%) consistently (Figure 1). (4) Comparing the 3 or 4 most commonly performed techniques for anti-A and anti-D, application of uniform methods did not show statistically significant interlaboratory reduction in titer variance consistently, except on rare occasion (Table 4).

In the pivotal study reported by AuBuchon et al, (9) reduced titer variability was noted with a w+ end point and not with 1+ end point with UT method. The same end points were used in our study but with different outcomes (Tables 1 through 4). In comparison to the study by AuBuchon et al, (9) which had 19 laboratories participating (14 from the United States), our study had a higher number of participants. It is highly likely that with a higher number of participants, consistently reproducing the success of uniform method could be a difficult task for several reasons. First, although the uniform method provides clear technical notes for performing ABT procedures, grading the strength of agglutination (ie, distinguishing between w+ and 1+) is done manually. Based on the expertise of individuals from a diverse pool of laboratories participating in PT surveys, interpreting titration end points, and thereby antibody strength, could be subject to variations (operator bias). Prior studies have shown that even in a single laboratory, titer reports can substantially vary, giving credence to operator-dependent-bias hypothesis as one of the factors contributing to variance. (13) Second, the equipment (centrifuge machines, speed/duration of centrifugation, test tube size, diluents, etc) used for the ABT process between different institutions participating in PT surveys could be different, thereby introducing additional bias (laboratory or circumstantial bias). Finally, for both anti-A and anti-D, the use of uniform methods in several PTs showed an increase in titer variance, rather than reduction in variance. Although our study did not specifically explore the reasons for this discrepancy, variation in ABT results is likely to be multifactorial. Hence, further research to identify additional factors, which work either individually or in tandem with the currently known variables to influence ABT, needs to be prioritized.

Another key observation in our study is the platform (tube > gel) used to do ABT. This preference may reflect the fact that because despite many advantages, such as increased automation, retrospective supervisory review, and decreased reliance on manpower, using gel technique is not without disadvantages. (14) The absence of robust evidence linking gel ABT levels with clinical outcomes could be limiting more widespread gel technique incorporation. (1,15) Within its limitations (not adequately powered), both our study and that of AuBuchon et al (9) have shown an association between the use of the gel method and a high proportion of laboratories capturing the same antibody strength (mode) (Figure 1). Thus, this platform holds promise to replace tube platform as an effective alternative with improved precision. To facilitate this transition, these findings need to be reconfirmed in well-designed future clinical studies adequately powered for statistical and clinical relevance. (16)

An almost equal number of laboratories in our study reported anti-A titers either at RT or AHG phase, highlighting the wide range of practice across the country in choosing the most appropriate phase for ABT (Table 3). Before red cell transfusions, laboratories could choose from IS, RT, or AHG phase for antibody identification, irrespective of the platform used, and if necessary they could perform subsequent ABT with any of the 3 phases. (17) Although it is desirable to have all 3 phases of testing done before every transfusion, it is often not practical considering the cost of reagents, labor, and need for efficiency. Also, the data in this field are not consistent, with several studies showing feasibility with different phases (IS versus AHG), irrespective of the platform chosen. (18,19) The merits of antibody identification and titration with different phases are also debated in the solid organ transplant setting, where performing ABO-incompatible transplants are challenging. Based on a validation performed with a small cohort of ABO-incompatible kidney transplants performed at Johns Hopkins Hospital, the conduct of IS/RT ABT is considered redundant and favors AHG phase alone, which could potentially help in faster turnaround times for testing. (20,21) Similar observations in large-scale studies to identify appropriate phase of ABT for other solid organ and stem cell transplant are currently lacking. Hence, until a strong consensus evolves, laboratories might have to continue to report anti-A titers on the basis of their standard operating procedure or clinicians' preference. For anti-D, all titers were reported at AHG phase. Since HDFN and other clinically significant hemolytic events associated with anti-D predominantly involve IgG (best detected with anti-globulin), identifying the phase to determine anti-D titers was not the focus of our study. Rather, tube and gel platform were compared with or without uniform methods to determine if titer variability could be reduced, which our study failed to demonstrate with uniform methods. Thus, determining the titer of the old frozen plasma concurrently with the most recent plasma sample for anti-D with a common platform (tube or gel) should continue to be a common clinical practice in managing HDFN.

Limitations

First, there were fewer anti-A titer results reported than anti-D titer results and even among them, fewer responses with gel platform were noted, thereby reducing the power of the study to determine statistical significance. Second, this study did not analyze the reactivity of other clinically significant alloantibodies (eg, Rh, Kell, Kid) implicated in hemolytic transfusion reactions and HDFN, as these are not included in this PT survey. Therefore, our data cannot be extrapolated to other alloantibodies.

Future Perspective

Since discrepancies and inconsistencies with the current methods for ABT persist despite implementation of uniform methods, exploring alternative novel techniques using enzyme-linked immunosorbent assay, flow cytometry, surface plasmon resonance, and Kodecytes (KODE Biotech, Auckland, New Zealand) in the near future could be prudent. (22-28)

CONCLUSIONS

Our study reflects current practice for ABT post uniform method introduction in different laboratories. Standardization of antibody titration techniques aimed at improving precision of results continues to remain an elusive and complex task despite implementing the uniform methods. Our understanding about the possible reasons for discrepancies in ABT reporting with the uniform methods is still incomplete and needs to be pursued further in future prospective large-scale studies. Future research should also focus on incorporating novel emerging technologies and mitigating discrepancies noted with current methods for antibody titration.

The authors would like to thank the members of the College of American Pathologists Transfusion Medicine Committee for reviewing the manuscript and providing their valuable input.

References

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(3.) Hill EA, Bryant BJ. Comparison of antibody titers in donor specimens and associated AS-1 leukoreduced donor units. Transfusion. 2014;54(6):1580-1584.

(4.) Schwartz J, Winters JL, Padmanabhan A, et al. Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Writing Committee of the American Society for Apheresis: the sixth special issue. J Clin Apher. 2013;28(3):145-284.

(5.) Booth GS, Gehrie EA, Bolan CD, Savani BN. Clinical guide to ABO-incompatible allogeneic stem cell transplantation. Biol Blood Marrow Transplant. 2013;19(8):1152-1158.

(6.) Reverberi R, Reverberi L. Factors affecting the antigen-antibody reaction. Blood Transfus. 2007;5(4):227-240.

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(8.) Judd WJ. Practice guidelines for prenatal and perinatal immunohematology, revisited. Transfusion. 2001;41(11):1445-1452.

(9.) AuBuchon JP, de Wildt-Eggen J, Dumont LJ;Biomedical Excellence for Safer Transfusion Collaborative;Transfusion Medicine Resource Committee of the College of American Pathologists. Reducing the variation in performance of antibody titrations. Arch Pathol Lab Med. 2008;132(7):1194-1201.

(10.) Kang SJ, Lim YA, Baik SY. Comparison of ABO antibody titers on the basis of the antibody detection method used. Ann Lab Med. 2014;34(4):300-306.

(11.) Cacciatore A, Rapiti S, Carrara S, et al. Obstetric management in Rh alloimmunizated pregnancy. J Prenat Med. 2009;3(2):25-27.

(12.) Kobayashi T, Saito K. A series of surveys on assay for anti-A/B antibody by Japanese ABO-incompatible Transplantation Committee. Xenotransplantation. 2006;13(2):136-140.

(13.) Judd WJ, Luban NL, Ness PM, Silberstein LE, Stroup M, Widmann FK. Prenatal and perinatal immunohematology: recommendations for serologic management of the fetus, newborn infant, and obstetric patient. Transfusion. 1990;30(2):175-183.

(14.) Cid J, Nogues N, Montero R, Hurtado M, Briega A, Parra R. Comparison of three microtube column agglutination systems for antibody screening: DG Gel, DiaMed-ID and Ortho BioVue. Transfus Med. 2006;16(2):131-136.

(15.) Novaretti MC, Silveira EJ, Filho EC, Dorlhiac-Llacer PE, Chamone DA. Comparison of tube and gel techniques for antibody identification. Immunohematology. 2000;16(4):138-141.

(16.) Kumlien G, Wilpert J, Safwenberg J, Tyden G. Comparing the tube and gel techniques for ABO antibody titration, as performed in three European centers. Transplantation. 2007;84(12 suppl):S17-S19.

(17.) Do you think that the cross match with donor red cells can be omitted when the serum of a patient has been tested for the presence of red cell alloantibodies with a cell panel? Vox Sang. 1982;43(3):1 51--168.

(18.) Havemann H, Lichtiger B. Identification of previous erythrocyte alloimmunization and the type and screen at a large cancer center: a 4-year retrospective review. Cancer. 1992;69(1):252-255.

(19.) Barrett RM. Detection of ABO incompatibility in the extended IgG gel crossmatch. Paper presented at: AABB; October 2013; Denver, CO.

(20.) Tobian AA, Shirey RS, Montgomery RA, Ness PM, King KE. The critical role of plasmapheresis in ABO-incompatible renal transplantation. Transfusion. 2008; 48(11):2453-2460.

(21.) Shirey RS, Cai W, Montgomery RA, Chhibber V, Ness PM, King KE. Streamlining ABO antibody titrations for monitoring ABO-incompatible kidney transplants. Transfusion. 2010;50(3):631-634.

(22.) Satoh A, Kawagishi N, Minegishi M, et al. Development of a novel ELISA for detection of anti-A and anti-B antibodies in recipients of ABO-incompatible living donor liver grafts. Tohoku J Exp Med. 2007;211(4):359-367.

(23.) Stussi G, Huggel K, Lutz HU, Schanz U, Rieben R, Seebach JD. Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method. Br J Haematol. 2005;130(6):954-963.

(24.) Valli PV, Puga Yung G, Fehr T, et al. Changes of circulating antibody levels induced by ABO antibody adsorption for ABO-incompatible kidney transplantation. Am J Transplant. 2009;9(5):1072-1080.

(25.) Kimura S, Yurugi K, Segawa H, et al. Rapid quantitation of immunoglobulin G antibodies specific for blood group antigens A and B by surface plasmon resonance. Transfusion. 2005;45(1):56-62.

(26.) Frame T, Carroll T, Korchagina E, Bovin N, Henry S. Synthetic glycolipid modification of red blood cell membranes. Transfusion. 2007;47(5):876-882.

(27.) Sundback M, Grufman P, Teller J, et al. Quantification of blood group A and B antibodies by flow cytometry using beads carrying A or B trisaccharides. Transplantation. 2007;84(12 suppl):S24-S26.

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Lohith S. Bachegowda, MD; Yan H. Cheng, MD; Thomas Long, MPH; Beth H. Shaz, MD

Accepted for publication April 20, 2016.

Published as an Early Online Release September 28, 2016. From the Department of Transfusion Medicine (Drs Bachegowda, Cheng, and Shaz), New York Blood Center, New York, New York; and Biostatistics, College of American Pathologists, Northfield, Illinois (Mr Long).

The authors have no relevant financial interest in the products or companies described in this article.

This research was presented at the American Association of Blood Banks Annual Meeting; October 2015; Anaheim, California.

Corresponding author: Beth H. Shaz, MD, New York Blood Center, 310 E 67th St, New York, NY 10065 (email: Bshaz@nybc.org).

Please Note: Illustration(s) are not available due to copyright restrictions.

Caption: Figure 1. Proportion of anti-A and anti-D antibody titers around mode. Abbreviations: AHG, anti-human globulin; IS, immediate spin; OG, other gel; OT, other tube; Poly, polyspecific; RT, room temperature; UG, uniform gel; UT, uniform tube.

Caption: Figure 2. Titer variance with different techniques. Abbreviations: AHG, anti-human globulin; OT, other tube; RT, room temperature; UG, uniform gel; UT, uniform tube.
Table 1. Proficiency Testing: Uniform Procedure
(Tube and Gel Methods) for Anti-A Antibody

  Anti-A Antibody

Summary of Uniform Procedure

Dilution of sample
  Volume: 1 mL
  Diluent: 0.9% NaCl, unbuffered
  Technique: Calibrated pipette;new tip for each dilution
Tube technique: Anti-A
  First reading: Defined UT RT
  Incubation: Room temperature for 30 [+ or -] 1 min and read
    without additional augmentation
  Second reading: Defined UTAHG
  Incubation: 37[degrees]C [+ or -] 1[degrees]C
    for 30 [+ or -] 1 min
  Washes: Four times with at least 10-fold the volume
    of red cell + sample
  Testing phase: Anti-IgG
  End point: w+, read macroscopically
Gel card technique: Anti-A
  Two separate cards:
    Gel only, incubated at room temperature for 15 [+ or -] 1
      min (defined UG RT)
    Anti-IgG, incubated at 37[degrees] [+ or -] 1[degrees]C for
      15 [+ or -] 1 min (defined UG AHG)
  End points: 1 +
Provision of reagent red cell
  For the purposes of ABT survey testing, participants
    should use the titer cell provided with the ABT kit, and
    not a red cell supplied by their laboratory as defined
    in the uniform procedure.

Abbreviations: ABT, antibody titration;AHG, anti-human globulin;
IgG, immunoglobulin G;NaCl, sodium chloride;RT, room temperature;
UG, uniform gel;UT, uniform tube;w+, weak plus.

Table 2. Proficiency Testing: Uniform Procedure
(Tube and Gel Methods) for Anti-D Antibody

  Anti-D Antibody

Summary of Uniform Procedure

Dilution of sample
  Volume: 1 mL
  Diluent: 0.9% NaCl, unbuffered
  Technique: Calibrated pipette;new tip for each dilution
Tube technique: Anti-D (defined UTAHG)
  Reagent red cell concentration: 3%-5% in 0.9% NaCl
  Volumes: 0.05 mL red cell suspension + 0.10 mL
    specimen, delivered by calibrated pipette
  Incubation: 37[degrees]C [+ or -] 1[degrees]C
    for 30 [+ or -] 1 min
  Washes: Four times with at least 10-fold the volume of
    red cell + sample
  Testing phase: Anti-IgG
  End point: w+, read macroscopically
Gel card technique: Anti-D (defined UG AHG)
  Card type: Anti-IgG
  Reagent red cell concentration: 0.80% in 0.9% NaCl or
  diluent specified by manufacturer
  Volumes: 0.05 mL red cell suspension + 0.025 mL
    specimen, delivered by calibrated pipette
  Incubation: 37[degrees]C [+ or -] 1[degrees]C
    for 15 [+ or -] 1 min
  End points: 1 +
Provision of reagent red cell
  For the purposes of ABT survey testing, participants
    should use the titer cell provided with the ABT kit, and
    not a red cell supplied by their laboratory as defined in
    the uniform procedure.

Abbreviations: ABT, antibody titration;AHG, anti-human
globulin; IgG, immunoglobulin G; NaCl, sodium chloride;
UG, uniform gel; UT, uniform tube; w+, weak plus.

Table 3. Distribution of Titers Performed With
Different Methods

                               Proficiency Test Year

                                        2009

                                  A             B

Anti-A
  Total No. of responses         672           682
  UT RT, No. (%)             148 (22.03)   150 (21.99)
  UT AHG, No. (%)            146 (21.73)   145 (21.26)
  UG RT, No. (%)              18 (2.68)     21 (3.08)
  UG AHG, No. (%)             27 (4.02)     24 (3.52)
  OT IS, No. (%)             71 (10.57)     68 (9.97)
  OT RT, No. (%)             117 (17.41)   116 (17.01)
  OT IgG AHG, No. (%)        111 (16.52)   116 (17.01)
  OT polyspecific AHG,        11 (1.64)     14 (2.05)
    No. (%)
  OG, No. (%)                    (a)        14 (2.05)

Anti-D (b)
  Total No. of responses        1211          1192
  UT, No. (%)                462 (38.15)   434 (36.41)
  UG, No. (%)                153 (12.63)   149 (12.50)
  OT AHG, No. (%)            449 (37.08)   458 (38.42)
  OT 6% Alb AHG, No. (%)      79 (6.52)     77 (6.46)
  OT 22% Alb AHG, No. (%)     28 (2.31)     26 (2.18)
  OT polyspecific AHG,        27 (2.23)     30 (2.52)
    No. (%)
  OG, No. (%)                 13 (1.07)     18 (1.51)

                               Proficiency Test Year

                                       2010

                                  A             B

Anti-A
  Total No. of responses         564           578
  UT RT, No. (%)             147 (26.06)   148 (25.61)
  UT AHG, No. (%)            138 (24.47)   144 (24.91)
  UG RT, No. (%)              19 (3.37)     18 (3.11)
  UG AHG, No. (%)             24 (4.26)     21 (3.63)
  OT IS, No. (%)              41 (7.27)     39 (6.75)
  OT RT, No. (%)             91 (16.13)    94 (16.26)
  OT IgG AHG, No. (%)        82 (14.54)    93 (16.09)
  OT polyspecific AHG,        11 (1.95)     10 (1.73)
    No. (%)
  OG, No. (%)                 11 (1.95)     11 (1.90)

Anti-D (b)
  Total No. of responses        1098          1104
  UT, No. (%)                411 (37.43)   402 (36.41)
  UG, No. (%)                121 (11.02)   125 (11.32)
  OT AHG, No. (%)            432 (39.34)   465 (42.12)
  OT 6% Alb AHG, No. (%)      63 (5.74)     56 (5.07)
  OT 22% Alb AHG, No. (%)     19 (1.73)     18 (1.63)
  OT polyspecific AHG,        23 (2.09)     23 (2.08)
    No. (%)
  OG, No. (%)                 19 (1.73)     15 (1.36)

                                Proficiency Test Year
                                        2011

                                  A             B

Anti-A
  Total No. of responses         509           570
  UT RT, No. (%)             119 (23.38)   144 (25.26)
  UT AHG, No. (%)            134 (26.33)   143 (25.09)
  UG RT, No. (%)              12 (2.36)     23 (4.04)
  UG AHG, No. (%)             22 (4.32)     23 (4.04)
  OT IS, No. (%)              15 (2.95)     34 (5.96)
  OT RT, No. (%)             73 (14.34)    97 (17.02)
  OT IgG AHG, No. (%)        113 (22.20)   94 (16.49)
  OT polyspecific AHG,        10 (1.96)        (a)
    No. (%)
  OG, No. (%)                 11 (2.16)     12 (2.11)

Anti-D (b)
  Total No. of responses        1109          1099
  UT, No. (%)                397 (35.80)   402 (36.58)
  UG, No. (%)                137 (12.35)   130 (11.83)
  OT AHG, No. (%)            458 (41.30)   448 (40.76)
  OT 6% Alb AHG, No. (%)      59 (5.32)     61 (5.55)
  OT 22% Alb AHG, No. (%)     16 (1.44)     17 (1.55)
  OT polyspecific AHG,        26 (2.34)     26 (2.37)
    No. (%)
  OG, No. (%)                 16 (1.44)     15 (1.36)

                               Proficiency Test Year
                                       2012

                                A              B

Anti-A                      Anti-A
  Total No. of responses     565               560
  UT RT, No. (%)            134 (23.72)   142 (25.36)
  UT AHG, No. (%)           137 (24.25)   140 (25.00)
  UG RT, No. (%)            24 (4.25)      22 (3.93)
  UG AHG, No. (%)           26 (4.60)      26 (4.64)
  OT IS, No. (%)            30 (5.31)      29 (5.18)
  OT RT, No. (%)            90 (15.93)    97 (17.32)
  OT IgG AHG, No. (%)       101 (17.88)   94 (16.79)
  OT polyspecific AHG,      10 (1.77)         (a)
    No. (%)                 13 (2.30)      10 (1.79)
  OG, No. (%)
Anti-D (b)
  Total No. of responses   1059             1078
  UT, No. (%)              382 (36.07)   415 (38.50)
  UG, No. (%)              132 (12.46)   137 (12.70)
  OT AHG, No. (%)          442 (41.74)   431 (39.98)
  OT 6% Alb AHG, No. (%)   52 (4.91)      55 (5.10)
  OT 22% Alb AHG, No. (%)  20 (1.89)      19 (1.76)
  OT polyspecific AHG,     20 (1.89)      21 (1.95)
    No. (%)  11 (1.04)         (a)
  OG, No. (%)

                                      Proficiency Test Year
                                             2013

                                A             B        Median, N (%)

Anti-AAnti-A
  Total No. of responses      584           578            574
  UT RT, No. (%)            159 (27.23)   149 (25.78)   147.5 (25.3)
  UT AHG, No. (%)           150 (25.68)   150 (25.95)   143.5 (25.0)
  UG RT, No. (%)             19 (3.25)     20 (3.46)     19.5 (3.3)
  UG AHG, No. (%)            24 (4.11)     23 (3.98)     24.0 (4.1)
  OT IS, No. (%)             30 (5.14)     30 (5.19)     32.0 (5.6)
  OT RT, No. (%)             90 (15.41)    96 (16.61)    95.0 (16.4)
  OT IgG AHG, No. (%)        97 (16.61)    97 (16.78)    97.0 (16.7)
  OT polyspecific AHG,          (a)           (a)        10.5 (1.9)
    No. (%)                   15 (2.57)    13 (2.25)     12.0 (2.1)
  OG, No. (%)
Anti-D (b)
  Total No. of responses      1101          1081          1100.0
  UT, No. (%)              398 (36.15)   406 (37.56)   404.0 (36.5)
  UG, No. (%)              145 (13.17)   139 (12.86)   137.0 (12.5)
  OT AHG, No. (%)          459 (41.69)   453 (41.91)   451.0 (41.0)
  OT 6% Alb AHG, No. (%)    52 (4.72)     50 (4.63)     57.5 (5.2)
  OT 22% Alb AHG, No. (%)   16 (1.45)     14 (1.30)     18.5 (1.7)
  OT polyspecific AHG,      21 (1.91)     19 (1.76)     23.0 (2.1)
    No. (%)   10 (0.91)        (a)        15.0 (1.4)
  OG, No. (%)

Abbreviations: AHG, anti-human globulin; Alb, albumin;IgG,
immunoglobulin G; IS, immediate spin;OG, other gel;OT, other
tube;RT, room temperature;UG, uniform gel;UT, uniform tube.

(a) Data included in the table only if greater than 10 titer
results were reported with individual method for every proficiency
test. However, in determining the total percentage of each titer
method performed, the denominator included values from methods with
10 titers or fewer reported per proficiency testing. If the third
decimal was 0.005 or greater, the value of titer was rounded to the
next higher second digit decimal. Similarly, when calculating the
median, if the second decimal value was 0.05 or greater, the value
was rounded to the next higher first digit decimal (examples: 0.026
is rounded as 0.03 and 0.17 is rounded as 0.2).

(b) For Anti-D, all methods were performed with IgG AHG phase, with
1 exception when polyspecific AHG was used.

Table 4. Titer Variance Between Uniform Methods
and Other Methods

Survey            Methods Compared (n)

Anti-A
  2010 A    UT RT (147) versus OT RT (91)
            UTAHG (138) versus OT AHG (82)
  2010 B    UT RT (148) versus OT RT (94)
            UTAHG (144) versus OT AHG (93)
  2011 A    UT RT (119) versus OT RT (73)
            UT AHG (134) versus OT AHG (113)
  2011 B    UT RT (144) versus OT RT (97)
            UTAHG (143) versus OT AHG (94)
  2012 A    UT RT (134) versus OT RT (90)
            UTAHG (137) versus OT AHG (101)
  2012 B    UT RT (142) versus OT RT (97)
            UTAHG (140) versus OT AHG (94)
  2013 A    UT RT (159) versus OT RT (90)
            UTAHG (150) versus OT AHG (97)
  2013 B    UT RT (149) versus OT RT (96)
            UTAHG (150) versus OT AHG (97)
Anti-D
  2010 A    UT (411) versus UG (121)
            UT (411) versus OT (432)
  2010 B    UT (402) versus UG (125)
            UT (402) versus OT (465)
  2011 A    UT (397) versus UG (137)
            UT (397) versus OT (458)
  2011 B    UT (402) versus UG (130)
            UT (402) versus OT (448)
  2012 A    UT (382) versus UG (132)
            UT (382) versus OT (442)
  2012 B    UT (415) versus UG (137)
            UT (415) versus OT (431)
  2013 A    UT (398) versus UG (145)
            UT (398) versus OT (459)
  2013 B    UT (406) versus UG (139)
            UT (406) versus OT (453)

Survey      Standard Deviation       Variance       P Value

Anti-A
  2010 A    1.36 versus 0.94     1.85 versus 0.88     .001
            1.13 versus 1.07     1.28 versus 1.14     .29
  2010 B    1.13 versus 0.81     1.28 versus 0.66     .001
            0.94 versus 1.07     0.88 versus 1.14     .09
  2011 A    0.53 versus 0.35     0.28 versus 0.12     .001
            1.09 versus 0.85     1.19 versus 0.72   .46 (a)
  2011 B    1.36 versus 1.17     1.85 versus 1.37     .06
            1.12 versus 1.01     1.25 versus 1.02     .29
  2012 A    1.08 versus 0.95     1.17 versus 0.90     .09
            1.02 versus 1.21     1.04 versus 1.46   .30 (a)
  2012 B    1.10 versus 0.92     1.21 versus 0.85     .03
            0.91 versus 1.08     0.83 versus 1.17     .03
  2013 A    1.27 versus 0.97     1.61 versus 0.94     .002
            1.07 versus 1.15     1.14 versus 1.32     .19
  2013 B    1.27 versus 0.94     1.61 versus 0.88   .048 (a)
            1.19 versus 1.07     1.42 versus 1.14     .13
Anti-D
  2010 A    1.14 versus 0.92     1.30 versus 0.85     .002
            1.14 versus 1.00     1.30 versus 1.00     .003
  2010 B    0.91 versus 0.66     0.83 versus 0.44     .001
            0.91 versus 0.93     0.83 versus 0.86     .40
  2011 A    0.85 versus 0.77     0.72 versus 0.59     .08
            0.85 versus 0.89     0.72 versus 0.79     .14
  2011 B    1.03 versus 1.06     1.06 versus 1.12     .30
            1.03 versus 1.08     1.04 versus 1.17     .14
  2012 A    0.91 versus 0.85     0.83 versus 0.72     .15
            0.91 versus 0.91     0.83 versus 0.83     .44
  2012 B    0.94 versus 0.82     0.88 versus 0.67     .03
            0.94 versus 0.99     0.86 versus 0.98     .14
  2013 A    0.98 versus 0.82     0.96 versus 0.67     .006
            0.98 versus 1.00     0.96 versus 1.00     .63
  2013 B    1.03 versus 0.83     1.06 versus 0.69     .002
            1.03 versus 1.07     1.04 versus 1.14     .19

Abbreviations: AHG, anti-human globulin; OT, other tube; RT,
room temperature; UG, uniform gel; UT, uniform tube.

(a) Outlier titers removed before analysis.
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Date:Jan 1, 2017
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