TRALI risk-reduction strategies have been widely implemented by blood centers and often involve testing female apheresis donors, or certain subsets of female apheresis donors based on assessing pregnancy history, for the presence of HLA Ab, which is thought to be a major cause of TRALI7,8,17
. Blood centers performing HLA Ab testing by Luminex technology have implemented a variety of cutoff levels for HLA Ab assays11
. Our study provides data impacting the operational issue of how to potentially reduce the risk of TRALI by screening for HLA antibodies while maintaining an adequate supply of blood products. This analysis is not intended to recommend specific cutoffs. Rather, the data presented here should enable blood centers to set their own parity and assay cutoffs based on how much donation loss they can tolerate. Selecting a cutoff requires one to balance enhanced safety with potential donation loss. Ideally, clinical data would be evaluated in a prospective study to determine the levels (e.g., end-point dilution titers, NBG ratios, Optical Densities (OD)) and breadth of HLA reactivities in plasma from HLA-Ab-reactive donations implicated in TRALI cases to support establishing definitive cutoffs to achieve optimal safety. Although no such prospective data exist, support for the premise of our analysis is provided by a recent Japanese retrospective study of recipients with transfusion reactions.18
This study showed that donor HLA antibody strength as determined by both ELISA and flow cytometry single antigen bead assays, when combined with reactivity to the recipient’s cognate antigen, correlated with the occurrence of TRALI.
Our analyses demonstrate that samples yielding higher signals on a Luminex based screening assay are associated with a broader array of antigen specificities, compared with samples that yielded lower signals. Donations with a larger number of Ab specificities tended to have a greater probability of finding cognate antigens in randomly selected potential recipients; this was seen by the average CPRA values increasing as the NBG ratios increased (overall) and also as the number of SABs increased (). Since the majority of donations in the very high screen category (>5SD) had large numbers of SABs (), this suggests that donations with very high HLA NBG ratio include those donations that present the greatest risk to potential recipients. Consequently, establishing higher HLA Ab screening assay cutoffs may result in removing most of the donations that are associated with the high risk for TRALI, while minimizing donor/product loss.
In examining the relationship between assay signal strength and Ab titer, titration plots showed a linear relationship between the log dilution and the log NBG ratio values within the NBG ratio range of 2.2 and 100. These data support previous findings obtained in the organ transplant setting by Mizutani et al15
showing that HLA Ab titer was directly associated with values from Luminex assays. This titration work provides data to allow one to estimate how diluting (reducing) the plasma in blood components for transfusion can impact HLA antibody screening values and consequently the levels of HLA Abs infused into patients. For example, a 3-fold dilution resulted in a 1.84-fold reduction in the CL-I and 2.10-fold reduction in the CL-II NBG ratios. A 3-fold reduction of plasma in an apheresis platelet component may be achieved by use of platelet additive solutions. Although platelet additive solutions are used in some European countries, there are no available hemovigilance data to indicate whether their use reduces the incidence of TRALI.19,20
Imposing a strategy of screening all females and applying higher cutoffs would impact detection of donations with large numbers of CL-I specificities more so than donations with large numbers of CL-II specificities. For example, 98% and 95% of those with 4 or more CL-II SABs were detected at the >4SD and >5SD cutoffs, respectively (Table 4). However, moving from the >4SD cutoff to the >5SD cutoff had a bigger impact on CL-I SAB detection. At the >4SD cutoff, 85.0% of those with 4 or more CL-I SABs were detected; this detection rate dropped to less than 50% of those with 4 or more CL-I SABs when the >5SD screening cutoff was applied. The pattern described here for all females was similar in an analysis that was restricted to ever pregnant females (not shown).
Imposing a TRALI risk reduction strategy of screening females by pregnancy also would reduce detection of donations with reactivity to large numbers of SABs. The biggest impact on detection of donations with reactivity to four or more SABs was observed between 2 or more pregnancies and 3 or more pregnancies, where detection dropped by approximately one third (average reduction of 28%; range of 14% decrease for CL-I at >5SD cutoff to 32% decrease for CL-I at >3SD cutoff; ). Moving from the 3 or more pregnancies to 4 or more pregnancies categories also resulted in a large decrease in detection of donations with large numbers of SABs, with detection rates declining between 1.9 and 2.0-fold. When both extreme TRALI reduction strategies were combined, detection of donations with large numbers of reactive SABs was further reduced; i.e. at the highest cutoff presented (>5SD) and largest pregnancy restriction (4 or more pregnancies), only 15% of all donations with 4 or more CL-I SABs and 29% of all donations with 4 or more CL-II SABs were detected.
By applying more stringent TRALI risk-reduction strategies, such as screening females with four or more pregnancies and/or higher HLA Ab screening assay reactivity thresholds, centers can expect to lose a smaller number of donations. While the most stringent criteria decreased the loss of donations to less than 1%, using those criteria also caused the percentage of HLA Ab reactive donations detected to decrease by more than 6.5-fold compared with a strategy of screening ever pregnant females and implementing the lowest cutoff (>3SD) presented (). Additionally, using the most stringent criteria decreased the detection rate for donations with high numbers of CL-I and CL-II SABs. Therefore, imposing higher screening assay cutoffs and pregnancy criteria may allow loss of fewer donations, but in doing so, one needs to carefully consider whether the potential reduction in detection of HLA Ab reactive donations, particularly those with higher titers and larger numbers of SAB specificities, is acceptable.
Combining different HLA Ab assay cutoff levels with strategies of triaging female donors to be tested based on the number of reported pregnancies results in wide variations in the number of tests performed and the number of HLA Ab reactive donors detected. Although statistical approaches such as receiver operator curves (ROC) could be applied to generating a HLA Ab assay NBG cutoff that would be predicted to be “most effective”, the “optimal” NBG cutoff value with the desired sensitivity for detecting HLA antibodies that cause TRALI remains unknown. Hashimoto, et. al.18
used the approach of ROC analysis and determined a range of potential optimal cutoffs (2.0 to 6.5) for an ELISA HLA Ab assay; those results were based on testing a small number of donations that went to recipients diagnosed with TRALI (n=32) or other nonhemolytic transfusion reactions (n=36). However, currently, there is no reliable clinical case information to be able to make this assessment for the assays used in our study. In the absence of such data, a local hemovigilance program that monitors the occurrence of TRALI cases in hospitals served by the blood center, when feasible, could assist the center in assessing the effectiveness of their intervention strategy.
This study provides blood centers information to help formulate informed decisions on implementation of pregnancy and assay cut-offs as a potential means to minimize the risk of TRALI while maintaining an adequate platelet supply. Several blood centers began their risk reduction programs using high cutoff values (such as >5SD) as well as a high number of pregnancies (such as 4+ pregnancies) in order to minimize donor and donation loss14
. However, as these centers successfully compensate for the loss of plateletpheresis donors and platelet inventories become stable after the introduction of a given screening strategy, they may wish to identify a larger number of HLA antibody donors (thereby theoretically increasing their TRALI risk reduction) by lowering either the pregnancy cutoff or the assay cutoff values. Our data show that the strategy of decreasing the number of pregnancies that trigger testing and retaining a high NBG cutoff threshold, would be the a more effective way of detecting donations associated with higher titers and broader specificities than would the alternative approach of decreasing the assay cutoff and retaining the current pregnancy triage criteria.
There are some limitations to the estimates of donation loss presented in this manuscript. First, in projecting donation loss, we assumed a donor and donation gender and parity distribution that is comparable to the REDS-II centers16
. Second, the donation loss estimates presented here may be overestimating the rate of component loss since it is likely that females make fewer annual apheresis donations than do males and are also less likely to have their plateletpheresis split into two or three transfusable apheresis units. Thirdly, these estimates of donation loss from current blood donors do not reflect the loss of a similar percentage of donations from newly recruited donors if centers continue to recruit female apheresis donors. If newly recruited parous female donors were to be recruited in a similar proportion, then similar rates of future donation loss would be expected.
It is possible that potential TRALI risk for recipients may be more accurately linked to the average overall PRA of the transfused component than to the number of SAB specificities. The average calculated PRA for different groups of donors, grouped by NBG ratio and by number of SAB specificities is shown in . The overall calculated PRA for donors classified as HLA antibody positive at each screening cutoff level can be assessed by multiplying the proportion of donors with the designated number of SAB specificities at each cutoff (derived from ) by the PRA for that number of SAB specificities (derived from ). shows that the largest proportion of donors with high numbers of SAB specificities were found in the group that was reactive at >4 SD for Class I (High or Very High in ) and >5 for Class II (Very High).. shows that this group of donors also had the highest average PRAs calculated. When combined, these two observations clearly demonstrate that the largest proportion of risk for potential cognate antigen-antibody interactions in recipients is found in donors that are screened as positive at the higher cutoff levels.
A limitation of the information in is that samples demonstrating antibodies against HLA-C and HLA-DP specificities (approximately 30% for the group with >4SAB but <10% for the other groups) were excluded because those specificities could not be entered into the CPRA calculation. These donors would be expected to have higher CPRA values on average because the majority of them also had antibody specificities against HLA-A, B, DR and/or DQ specificities. Donors with HLA-C and DP specificities were predominantly within the groups with >4 SAB specificities at the higher cutoff values. It should also be pointed out that a fraction (approximately 12.5%) of donors that screened above NBG 2.2 demonstrated antibodies against both Class I and Class II specificities, which would also raise the CPRA above the levels shown in . These were also predominantly within the >4 SAB groups at higher cutoff values. Finally, the PRAs included in the Table are calculated values rather than values obtained from in – vitro PRA assays. Although these limitations suggest that the number of SAB specificities may not be a direct surrogate for measured PRA, our analysis nevertheless establishes a correlation between number of SAB specificities and the likelihood of a cognate antigen-antibody match.
The three screening cutoffs examined in this analysis are not meant to be definitive numbers to be used by centers using Luminex technology. Rather, they should be viewed as illustrative to help describe the impact of implementing moderate, high, or very high cutoffs for HLA Ab screening assays. Different HLA Ab screening assays will require different cutoffs to yield comparable performance13
, in terms of detection of HLA Ab positive donations and donor loss, to those reported here. Additionally, sample type (plasma or serum)21
and lot-to-lot variation can alter assay signal strength. Furthermore, the results presented here are based on the REDS-II LAPS cohort. While LAPS included donors from across the country, an individual blood center’s own donor population may vary from the demographics of LAPS donors10
. Centers are therefore encouraged to determine their own cutoffs using their own population of non-alloexposed donors and their chosen HLA Ab assay.