Validation of column agglutination technology for blood group alloantibody titration

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1 Validation of column agglutination technology for blood group alloantibody titration Melanie J Adriaansen and Holly E Perry Abstract Objective: To compare the performance of column agglutination technology (CAT) with the indirect antiglobulin test (IAT) tube method for antenatal titration of blood group alloantibodies. Methods: 42 plasma samples from pregnant women with positive blood group antibody screens were tested. On each sample, titrations were performed in parallel using the IAT tube method and the CAT method. The results were graded, recorded and compared for sensitivity by applying a binomial distribution to determine if the CAT titre was higher or lower than IAT tube titre. Difference in sensitivity was assessed by specific antibody type using the Fisher s exact test. Results: Of the 42 samples tested 16 (38%) had identical titres for the two methods. Of the 26 CAT titres that did not correspond with their IAT tube titre 17 (65%) were higher by CAT and 9 (35%) were lower by CAT. This was not considered significant by binomial test (p = 0.169), indicating no significant difference in sensitivity between CAT and IAT tube. 34 (81%) of the CAT titres were identical to, or fell within a 1 titre range of the IAT tube titre. Conclusion: The results from this study indicate that the performance of antenatal titrations by column agglutination technology (CAT) is at least as sensitive as the performance by the indirect antiglobulin test (IAT) tube method. CAT showed greater sensitivity than IAT tube when titrating Anti-c less sensitivity than IAT tube when titrating Anti-K. The CAT method also appears to be more sensitive for detecting samples that require referral to the obstetrician. Further studies on titration method and the critical titre value are needed. Key words: antibody titration, antenatal, blood group alloantibody, column agglutination technology oedema (3). Antibody titration plays a critical role in monitoring women at risk of HDFN and helps to determine the outcome of at least 300 affected pregnancies in New Zealand each year (Ministry of Health, written communication, July 2012). The traditional IAT tube method that is used for antenatal titrations is time intensive, prone to technical error, requires experienced staff to read results and gives only one chance to read the reaction without disturbance to the cell button. Furthermore, this method demonstrates low reproducibility (4) and variable sensitivity (1) and has been difficult to standardise between different laboratories (1, 5). Most areas of red cell serology, such as antibody screening and cross-matching, have readily adopted column agglutination technology (CAT) because of its technical simplicity (6) and increased sensitivity (7). Furthermore, CAT provides a stable end point for testing and has lent itself to automation in blood banking. However, the conversion of titrations to the CAT method has been slow and many laboratories continue to perform antenatal titrations using the IAT tube method. The aim of this study was to compare the performance of the CAT method with the IAT tube method for antibody titrations in the antenatal setting. Materials and methods The cohort consisted of 42 plasma samples from 23 pregnant women sent to North Shore Hospital Blood Bank between May 2010 and March Ethical approval was not required because samples were tested only where titration was clinically requested. All 23 women had a positive antibody screen and together showed six different alloantibodies active in vitro at 37 C (Table 1). Table 1. Specificity of alloantibodies present in cohort. NZ J Med Lab Sci 2013; 67: Antibody Number titred Introduction Titrations in blood banking are used primarily for semiquantitatively measuring the levels of alloantibodies in pregnant women. The results are critical in determining if a mother is referred to an obstetrician for invasive procedures that will diagnose haemolytic disease of the fetus and newborn (HDFN). The traditional method for the measurement of antenatal titrations is the indirect antiglobulin test (IAT) tube method, although this method has repeatedly demonstrated imprecision in its results (1, 2). Since the early 1990s many blood banking serology tests have changed to column agglutination technology (CAT) platforms but titrations have been slow to make the transition, especially in New Zealand. Approximately 1% of pregnant women have red cell alloantibodies that are implicated in HDFN (3). When paternal inheritance of a corresponding antigen occurs in the fetus then severe anaemia can develop and the fetus may respond by extending erythropoiesis to the liver and spleen, resulting in organ enlargement, portal hypertension and generalized Anti-c 4 Anti-D 7 Anti-D, probable prophylactic 2 Anti-E 9 Anti-Fy a 7 Anti-K 12 Anti-M 1 Total 42 IAT tube and CAT method were performed on specimens in parallel as described below. Where possible, the patient s current sample was also tested by IAT tube method in parallel with the patient s previous sample (from the same pregnancy). The results of the previous sample were then compared and acted as a control to ensure consistency between different scientists performing the titrations. The patient s previous sample was frozen at C between tests.

2 Preparation of dilutions Master serial two fold dilutions (neat 1 in 2048) were prepared for each sample according to the Australia and New Zealand Society of Blood Transfusion guidelines (8). Dilutions were continued to a titre of 2048 with the aim of allowing for at least two negative tubes beyond the titre end-point. One sample had not reached end point at 1 in 2048 and was run to a dilution of 1 in IAT Tube method 200μL of each master dilution and 50μL of 3% titration indicator cells (New Zealand Blood Service Reference Laboratory) was transferred to a clean glass tube. All indicator cells were R 1 R 2 (presumed genotype). The tubes were gently agitated and then incubated for 30 minutes at 37 0 C. Cells were washed three times using phosphate buffered saline (PBS) (Gibco, Life Technologies) and after the final wash the supernatant was completely decanted. Two drops of polyspecific antihuman globulin (AHG) reagent (Immucor Inc.) were added to the dry cell button. The tubes were mixed gently and centrifuged at 447g for 10 seconds. Tubes were then read for agglutination from the highest dilution to the lowest dilution and graded using the standard 0-12 grading scale (9). Coombs Control cells were added to all negative results to validate the reactions. The titre was reported as the reciprocal of the highest dilution at which at least grade 5 agglutination was observed (9). CAT method For this method 50μL of 0.8% R 1 R 2 titration Indicator cells (NZBS Reference Laboratory) and 25μL of each master dilution were added to individual wells in a Diamed (now Biorad) Liss/ Coombs card. Cards were incubated for 15 minutes at 37 0 C and then centrifuged for 10 minutes at 85g. Cards were graded using 0-4 grading scale. Confirmation reagent was added to all negative results to validate the reactions. The titre was determined as the reciprocal of the highest dilution at which grade 1 agglutination was observed (1). Laboratory results Further laboratory results (direct antiglobulin test and bilirubin) were assessed and reported for five babies whose maternal alloantibody titre by IAT tube was less than 16, but equal to 16 by CAT. Statistical analysis In all cases results from the current sample were used for analysis. For the titres that gave different results between the two methods, a simple binomial test was used to see if the results were significantly different by either method. The difference in the number of titres (IAT minus CAT), was recorded for each sample. As these differences were found to be approximately normally distributed (Figure 1) a simple linear model could be used. This model was used to test 1) if the difference between the 2 methods was significant; 2) if the observed differences were significantly different for each alloantibody; and 3) if the observed differences for any alloantibody type were different to the combination of the others. Results The end point of the titrations performed by IAT tube and CAT are presented in Table 2 and Figure 1. In all pregnancies where a previous sample was run in parallel with a current sample the results of the previous sample did not differ by more than one titre. That is, in no cases did the control fail to ensure consistency between tests. Of the 42 samples tested 16 (38%) had identical results for the IAT tube method and CAT method. Thirty-four (81%) of the CAT titres were identical or fell within a one titre range of the IAT tube titre. No plasma had greater than a three titre difference when comparing methods. These results are summarised in Figure 1. Table 2. Comparison of titers using IAT tube CAT Test Column agglutination technology (CAT) titre tube titer 0* 1** n= n=3 n= n=1 - n=5 n=1 n=3 n= n=2 - n=1 n= n=1 n=1 n=4 n=2 n= n= n= n= n= * 0 indicates that there was no reaction and the sample is considered negative for that antibody ** 1 indicates neat serum

3 Figure 1. Comparison of CAT titre with IAT tube titre. Of the 26 CAT titres that were not identical to their IAT tube titre 17 (65%) were higher by CAT method and 9 (35%) were lower. This was not statistically significant by binomial test (p = 0.169). Statistically, higher values are as likely as lower values; if a CAT titre does not correspond exactly to its IAT titre then it is just as likely to fall higher or lower. The highlighted data in Table 2 demonstrates samples that would have been referred to the obstetrician by CAT but not by IAT tube titres. These samples are further examined in Table 4. Results analysed by antibody specificity are presented in Table 3. The difference in titres for Anti-D, -E and -Fy a are not statistically significant between CAT and IAT tube methods (p values >0.05). For Anti-c, CAT shows significantly higher titres (p < 0.001). By contrast, the samples with Anti-K gave lower titres by the CAT method but this difference was not statistically significant (p = 0.091). However, the difference between CAT and tube when comparing Anti-K to the other antibody specificities was significant (p = 0.003). Table 4 shows the direct antiglobulin test (DAT) results and the total bilirubin results for five newborns that had an IAT tube titre of less than 16 and a CAT titre of 16. Of these five samples, all of the newborns had positive direct antiglobulin tests (DAT) and two out of the three that were tested had elevated bilirubin levels, indicating possible HDFN. Table 3. Comparison of the CAT titre to its corresponding tube titre by alloantibody type using the Fisher s exact test Alloantibody Titre difference (CAT titre subtracted from IAT tube titre) p value 1 p value D D_PP* E Fy a K ** M # NA NA <0.001** <0.001** * D_PP probable passive Anti-D ** p<0.005 # sample number too small for analysis p value 1 :Testing if the observed differences were significant for each alloantibody p value 2 : Testing if the observed differences for any alloantibody type were different to the combination of the others

4 Table 4. Analysis of five newborn samples that met obstetric referral criteria by CAT method but did not meet referral criteria by IAT tube method Sample Number Alloantibody titrated in mother Anti-D Anti-E Anti-D Anti-Fy a Anti-c Antibody eluted from baby s cells Anti-D Anti-E Anti-D Anti-Fy a Anti-c DAT result Positive (3+) Positive (grade Positive (grade Positive (4+) Positive (1+) not known) not known) Total Bilirubin (mmol/l) 362 Not tested Not tested Total Bilirubin Reference Range (mmol/l)* <300 <140 <200 Discussion The presence of red cell alloantibodies in a mother s serum indicates that a fetus should be monitored for possible HDFN. Initial monitoring is performed by antenatal titrations to determine if the titre of the alloantibody is increasing or is greater than or equal to 16. If so, then the pregnancy is referred to the obstetrician for further investigation (8). Given the clinical importance of antenatal titrations, a reliable and accurate method must be used. This study compared the indirect antiglobulin test (IAT) tube method with column agglutination technology (CAT). The results from this investigation indicate that the performance of antenatal titrations by column agglutination technology (CAT) is at least as good as the performance by the indirect antiglobulin test (IAT) tube method. Although less than half of samples (38%) showed identical results between the two methods, the majority of CAT titres (81%) were within a one titre range of the IAT tube result and would therefore have the same clinical interpretation. While CAT titres were slightly higher on average, this difference was not statistically significant. Therefore overall CAT was at least as sensitive as the IAT method. The CAT method and IAT test tube method were compared by antibody type to determine if a specific alloantibody gave higher or lower titres by either method. CAT showed greater sensitivity for Anti-c. That is, more Anti-c were higher by the CAT method than would be expected from random variation alone. This is consistent with the previous findings that CAT is more sensitive for detection of Rh antibodies (5, 11). The increased sensitivity for Anti-c found in this study might be attributed to technical bias. As only four samples were titrated, further investigation is needed to draw a conclusion. Other than Anti-c the relationship between CAT and IAT test tube methods is not dependent on alloantibody type. However further investigation is needed for K alloantibodies, in light of the different titre distribution. CAT titres demonstrated up to a three titre difference when compared with the IAT test tube results as shown in Figure 1. This is not an unexpected finding. Antenatal titrations performed by all methods frequently have inconsistent results and this has been demonstrated in quality control programmes internationally. For example, the June 2012 RCPA (Royal College of Pathologists Australia) titrations survey (12) observed a range of six titres from its submissions by blood banks (where there is only one target value). Similarly, the 2011 UK NEQAS (United Kingdom National External Quality Assessment Scheme) demonstrated a range of eight titres (2). This disparity in results is often attributed to a lack of standardisation, a problem that could be reduced by column agglutination technology. CAT uses specific volumes of red blood cells and reagents that are dispensed by pipettes, and CAT is also technically simpler, with washing steps eliminated and stable, well-defined end points produced. Standardisation of laboratory tests is necessary to produce results that are reliable, comparable and have strong medical value (10). AuBuchon et al. found that the use of the CAT method for antenatal titrations shows a trend towards decreased variance when compared with the IAT tube method (1). This provides a strong motivator to look at the adoption of CAT for antenatal titrations with the hope of standardising the test. As well as the variance found in titration results, another aspect of controversy is the significance of the critical titre. The critical titre is the titre that indicates when a mother should be referred to an obstetrician for further investigation. The current critical titre in New Zealand and Australia is 16, although in the United Kingdom, the critical titre is 32. The level of the titre does not necessarily correspond to the severity of HDFN and this is especially true in cases of Anti-K (4). It is up to the obstetrician to decide how they treat the pregnancy. A change to CAT as standard titration method may result in more referrals and therefore more invasive procedures to monitor fetuses at risk of HDFN and because of this the critical titre may need to be reviewed over time. In this study, five plasmas showed a titre of 16 by CAT but less than 16 by tube IAT. Of these five samples, all of the neonates showed a positive direct antiglobulin test (DAT) with the mother s respective alloantibody eluted from the newborn s red cells. Three of the neonates were also tested for bilirubin and two demonstrated elevated bilirubin levels indicating possible cases of HDFN. This suggests that CAT may have a higher sensitivity than the IAT test tube method for detecting samples that require referral. Retrospective studies would need to be done with clinical teams to establish whether intervention in these cases was warranted or not. It may be that the guide cut off value of 16 for referral would be reviewed for appropriateness. In no case was the tube titre of 16 or greater in tube IAT less than 16 in CAT. Consequently, using our current guide of 16, no fetus would be at risk of non-referral for further monitoring if CAT was adopted. Consideration of a change in grading scale cut-offs from the 0-12 scale to the 0-4 scale would also be needed for performing titrations by CAT. A major limitation of this study is that reproducibility was not assessed. A control sample of known antibody concentration ideally should be tested repeatedly by both methods to determine the spread of results. The degree of variation between CAT and IAT tube method could then be compared and hopefully progression towards the standardisation of antenatal titrations could be achieved. It is also acknowledged that low ionic strength saline used in CAT diluents, versus normal ionic strength in IAT tube, is a possible confounding variable in this study. Acknowledgements We would like to thank the following people for their support and knowledge and especially for their participation with the practical work: Phillip Shepherd, Lee-Ann Weiss, and all staff at North Shore Hospital Blood Bank. We would also like to thank Robin Hankin (Auckland University of Technology) and the editorial staff of NZIMLS journal for their assistance with the statistical

5 Author information Melanie J Adriaansen, BMLS, Medical Laboratory Scientist 1 Holly E Perry, MAppSc (Hons), Senior Lecturer 2 1 North Shore Hospital General Laboratory, Waitemata District Health Board 2 Bachelor of Medical Laboratory Science Programme, Auckland University of Technology Corresponding author: Melanie J Adriaansen, Waitemata DHB, North Shore Hospital General Laboratory, Private Bag 93503, Takapuna 0740, Auckland, New Zealand. References 1. AuBuchon JP, de Wildt-Eggen J, Dumont JL; Biomedical Excellence for Safer Transfusion Collaborative; Transfusion Medicine Resource Committee of the College of American Pathologists. Reducing the variation in performance of antibody titrations. Vox Sang 2008; 95: UK National External Quality Assessment Scheme for Blood Transfusion Laboratory Practice. Report of titration exercise and questionnaire (11E8) Distributed September 2011 UK and Republic of Ireland. Watford, UK: UKNEQAS; 2011: Howard H, Martlew V, McFadyen I, Clarke C, Duguid J, Bromilow I, Eggington J. Consequences for fetus and neonate of maternal red cell allo-immunisation. Arch Dis Child Fetal Neonatal 1998; 78: F62-F Bruce M, Chapman JF, Duguid J, Kelsey P, Knowles S, Murphy M, Williamson L. Addendum for guidelines for blood grouping and red cell antibody testing during pregnancy. BCSH Transfusion Task Force. Transfus Med 1999; 9: Novaretti MC, Jens E, Pagliarini T, Bonifàcio SL, Dorlhiac- Llacer PE, Chamone DA. Comparison of conventional tube test with diamed gel microcolumn assay for Anti-D titration. 2003; 25: Klein HG, Anstee DJ. Mollison s Blood Transfusion in Clinical Medicine (11th Edition). Malden, MA: Blackwell Publishing; Chapter 8, Blood Grouping Techniques; p. 299, Weisbach V, Ziener A, Zimmermann R, Glaser A, Zingsem J, Eckstein R. Comparison of the performance of four microtube column agglutination systems in the detection of red cell alloantibodies. Transfusion 1999; 39: The Australian & New Zealand Society of Blood Transfusion Inc. Guidelines for Blood Grouping & Antibody Screening in the Antenatal & Perinatal Setting. 2nd Edition. Sydney: ANZSBT; pp. 9. NZBlood. Antibody Titration (Antenatal), National 136M112, 5 th Version [Method] Tate J, Panteghini M. Standardisation - the theory and the practice. 2007; 28: Bromilow IM, Adams KE, Hope J, Eggington, JA, Duguid JK. Evaluation of the ID-gel test for antibody screening and identification. Transfus Med 1991; 1: Royal College of Pathologists Quality Assurance Programs Pty. Limited. AA Anti-D Titre Module. RCPA Transfusion QAP; 2012: p.3. Copyright: 2013 The authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

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