Evaluating automated titre score as an alternative to continuous flow analysis for the prediction of passive anti‐D in pregnancy

Summary Objectives To evaluate the potential of the automated titre score (TS) as an alternative method to continuous flow analysis (CFA) for the prediction of the nature of anti‐D in pregnancy. Background The 2016 revised British Society for Haematology (BSH) antenatal guidelines recommended a measurement of anti‐D concentration by CFA to ensure the detection of potential immune anti‐D. Due to high referral costs and resource pressures, uptake has been challenging for hospital laboratories. Serious Hazards of transfusion (SHOT) data have previously shown that this has contributed to missed antenatal follow ups for women with immune anti‐D and neonates affected by haemolytic disease of the fetus/newborn. Methods/Materials In this multicentre comparative study, samples referred for CFA quantification were also tested by an ORTHO VISION automated anti‐D indirect antiglobulin test (IAT) serial dilution and then converted to TS. CFA results and history of anti‐D prophylaxis were used to categorise samples as passive or immune, with the aim of determining a potential TS cut‐off for CFA referral of at risk patients. Results Five UK National Health Service (NHS) trusts generated a total of 196 anti‐D TS results, of which 128 were classified as passive and 68 as immune. Diagnostic testing of CFA and TS values indicated a TS cut‐off of 35 to assist in distinguishing the nature of anti‐D. Using this cut‐off, 175 (89%) results were correctly assigned into the passive or immune range, giving a specificity of 92.19% and a negative predictive value of 91.47%. Conclusion TS in conjunction with clinical and anti‐D prophylaxis history can be used as a viable and cost‐effective alternative to CFA in a hospital laboratory setting.


| INTRODUCTION
Close collaboration between the blood transfusion laboratory and obstetric teams is critical to identify the presence of maternal red cell antibodies (1% of pregnancies) 1 and to monitor these levels throughout the antenatal period. Historically, anti-D was the most common cause of haemolytic disease of the fetus and newborn (HDFN), accounting for 18%-27% of all cases. 2 The advent of anti-D prophylaxis in the 1960s dramatically reduced the incidence of sensitisation and production of immune anti-D. Hence, the number of deaths from anti-D related HDFN cases reduced from 46/100000 to 1.6/100000, with a further reduction seen after the introduction of antenatal prophylaxis. 2 The anti-D prophylaxis programme continues to be hugely successful; however, sensitisation still occurs in 500 ples containing anti-D by either CFA or a method that has been extensively validated against CFA. 5 However, the referral of these samples presents hospitals with many challenges, including high referral costs and resource pressures for both the hospital and the reference lab, as well as long turnaround times for obtaining results.
Due to the issues discussed, many transfusion laboratories have been unable to implement the 2016 guidelines, meaning that patients are still potentially at risk.
In a climate of austerity, hospitals must develop strategies to provide the best care in a cost-effective manner. Some blood transfusion automated systems have the ability to perform automated titrations. Currently, reporting this method as an endpoint titre is considered to be semi-quantitative, does not accurately represent the clinical picture and correlates poorly with the severity prediction of HDFN. 7 The adoption of a titre score (TS), however, provides a more quantitative result that takes into account the strength of the reaction and the avidity of the antibody and is thought to better correlate with risk. [7][8][9] The aim of the first phase of this study was to assess if TS determined by automated ORTHO BioVue column agglutination technology (CAT) is a comparable alternative to the existing CFA for the categorisation of the nature of anti-D (prophylactic or immune).   Note: TP All patient titre scores > 35 assumed immune, FP All patient titre scores > 35 assumed prophylactic, TN All patient titre scores ≤ 35 assumed prophylactic, FN All patient titre scores ≤ 35 assumed immune.

| MATERIALS AND METHODS
reviewing Figure 1, we were unable to demonstrate a linear correlation between the TS and the concentration of anti-D in IU/ml. However, there appear to be emerging risk groups that will be further analysed as part of an ongoing study. Such comparable clinical decision and risk correlations have already been observed between CFA anti-D concentrations and anti-D titres in other studies. 11 Manual titration methods, both tube and column agglutination indirect antiglobulin test (IAT), have been associated with inter-laboratory variation due to the preparation of the reagents and serial dilutions, as well as the visual interpretation of the end result. 12 However, ORTHO BioVue Column Agglutination has been shown to be an appropriate replacement for tube IAT in antibody titration 13  A decision-making algorithm should accompany any implementation of this method to aid interpretation and clinical decision-making.
An example algorithm has been included in Appendix 1.

| CONCLUSION
In this study, the ORTHO VISION fully automated platform has provided the ability to standardise the TS methodology across multiple sites and systems, thereby removing variability inherent to manual titration techniques.
Using a TS cut-off value of >35, there was no additional clinical risk when facilitating conformity to BSH guidelines, thereby reducing the number of samples referred for CFA quantification and supporting the prevention of incidents related to the mis-categorisation of the nature of anti-D, as noted in the 2012 SHOT report. 6 Some hospitals involved in this study are currently in the process of implementing this method, which will be used to support decisionmaking algorithms for appropriate referral for CFA. This could be beneficial to the obstetric department by reducing turnaround times, offering potential financial savings and improving patient care.
The next phase of the study will involve gathering further data on women with immune anti-D, correlating the automated TS with CFA and clinical risk categories and outcomes. This will include interlaboratory variation studies.