Comparison of Platelet Antibody Screen, Crossmatching and HLA Antibody Testing in Patients Refractory to Platelet Transfusions

1. Introduction

Patients who are refractory to platelet transfusions present difficult diagnostic and management issues for the blood bank [1–2]. Causes of this are often nonimmune including fever, splenomegaly, bleeding, sepsis, and graft-versus-host disease [3–4]. In these patients, the transfusion service cannot offer products that might provide better results. In contrast, patients who are refractory to platelet transfusion due to an alloimmune/antibody mechanism may benefit from selected platelet units. Immune causes are most commonly due to antibodies against human leucocyte antigens (HLA), but antibodies against human platelet antigens are also implicated [5]. A variety of tests can be performed including platelet Ab screens, platelet crossmatching, and HLA Ab testing for patients with suspected alloimmune related platelet refractoriness. However, the relative utility of these tests is unclear, and their utility depends on individual patients [6]. Initial testing of these patients often involves either platelet antibody screening or HLA antibody testing. When these screening tests are positive, platelet crossmatch testing, or HLA-matched platelets can be considered. It has previously been shown that crossmatched units are most useful for mildly alloimmunized patients (defined as having 1–40% reactive antibody, in antiglobulin-augmented LCT assay), which is expected, since severe alloimmunization (>70% reactive antibody) should render it unlikely a compatible unit can be identified by crossmatch [6]. Hence, it is critical platelet antibody screens accurately identify the extent of alloimmunization in these patients as this serves as a precursor to crossmatch testing.

The Immucor Capture-P Ready Screen (platelet antibody screen) is a commonly used solid phase red cell adherence assay for identifying platelet antibodies. In March of 2013, this test was recalled due to issues with production that resulted in weak strength in the positive control and was attributed to poor performance of indicator red cells as they neared their expiration date. After the recall, positivity rates of the screen appeared to increase, and one preliminary report (abstract form only) attributed the increased sensitivity to improved indicator RBCs [7]. At our institution, the platelet antibody screen was generally the first test performed and crossmatch (Immucor) testing was performed at the discretion of the transfusion medicine provider on service. If the screen was completely or mostly negative, no crossmatch was performed as it was expected that nearly all units would be compatible. If the screen was broadly positive, crossmatch testing was often not performed as it was presumed that nearly all randomly tested units would be incompatible [6]. In these cases, HLA matched platelets would be requested if possible. Thus, decreases in the sensitivity of the screen could result in a failure to identify antibodies, while an overly analytically sensitive test could result in a failure to perform a platelet crossmatch and requesting HLA platelets which take longer to obtain and are more expensive. Likewise, this could also result in cases where a minimally immunized patient’s alloimmunization is over estimated and a crossmatch is unnecessarily performed. A systematic comparison of results with the platelet antibody screen compared to the platelet crossmatch and HLA antibody testing has not been performed. The primary purpose of this study was to analyze how the positivity rate of the Immucor Capture-P Ready Screen has changed over time and comparing it to that of Immucor platelet crossmatch testing and HLA antibody testing to determine if the platelet antibody screen testing should be performed.

2. Materials & Methods

2.1. Study Overview

This retrospective study was approved by the institutional review board (IRB#202204129) at our large academic medical center, which includes an active hematopoietic stem cell transplantation service and CarT cell therapy. Blood bank and electronic records of platelet screening and crossmatch results from 2010 to 2021 were entered into a spreadsheet for this analysis. For most of this time period, crossmatch testing was performed at the discretion of the transfusion medicine faculty on service and this determination was made after completion of the platelet antibody screen. In general, for completely negative screens, no crossmatch was performed (crossmatch performed on just 1 of 13 completely negative screens from 2010–2015). During this same period, no crossmatch was performed after 9 of 18 completely positive screens.

2.2. Platelet Antibody Screen and Crossmatch Results

Immucor Capture-P Ready Screen tests were performed per manufactures instructions. In brief, platelets of known antigen phenotype are bound to the surface of polystyrene plates and patient plasma is added and incubated to allow patient antibodies to bind the platelets if present. The wells are washed to remove unbound antibodies, and indicator red cells coated with anti-IgG are added to the wells to determine if antibody binding is present. In positive wells, red cells are dispersed in the well and this reactivity can be graded from to w+ to 4+ (no pellet) following the manufactures instructions. For negative results, red cells pool at the bottom of the well, forming a well demarcated pellet. The screen involves a positive and negative control and 13 test samples. Platelet crossmatch testing (Capture-P from Immucor) follows a similar protocol, except donor platelets of unknown antigen phenotype are added and bound to the wells prior to performance of the test. The number of test samples varies depending on availability of platelet units. For most tests, between 10 and 14 units are tested. Like the screen, positive wells can be graded from w+ to 4+.

All paper blood bank records of platelet antibody screens and platelet crossmatch testing from 2010 to 2021 were reviewed and results entered into a spreadsheet. During this period, a total of 151 patients had testing performed with 44 having only a screen performed, 9 having only a crossmatch performed, and 98 patients having both performed. 162 valid platelet antibody screens were performed on 142 patients, and 202 crossmatch tests were performed on 107 patients. For each test the number of positive and negative wells were recorded. Weak reactivity was the minimum required for the well to be considered positive. The positive control on a few screens failed to give strong reactivity, and these tests were considered invalid by the blood bank staff and were excluded from the analysis. To avoid multiple tests on the same patient, only patients’ first valid platelet antibody screen and/or first valid platelet crossmatch were included in the analysis. All paper blood bank records from this same period were entered into a spreadsheet and reviewed using the same minimum reactivity (weakly reactive) for a unit to be considered incompatible. The percentage of total reactive wells from all patients’ platelet antibody screen and crossmatch testing each year was calculated and analyzed to see if there was a change in positivity over time. To ensure that findings were robust to random yearly variability, the data were also analyzed across four-year grouped time-periods (2010–2013, 2014–2017, and 2018–2021).

2.3. Paired analysis

To control for variability between patients, an additional comparison was performed on patients who had their first platelet antibody screen and platelet crossmatch testing performed within 7 days of each other. For this comparison, results were dichotomized to either a positive or negative result, and tests were considered positive if greater than 50% of the wells were positive. The incidence of these in the periods before and after the recall in 2013 were determined and analyzed to determine whether the discordance rate of the tests changed before and after the recall.

2,4. HLA Antibody Testing

HLA antibody testing were also gathered from patients’ electronic medical records which was performed using solid phase bead assays [8]. When positive, most of the reports calculated and reported the panel-reactive antibody (PRA) which has been shown to identify patients at risk of being refractory to platelet transfusions [9]. The PRA was not consistently reported in years prior to 2018 so these results were only collected for the 2018–2021 period. Over this period, 18 patients were found in which HLA ab testing was performed within 7 days before and 28 days after a patient’s initial paired screen and crossmatch tests. For these 18 subjects, the mean and standard error of the mean were calculated for the PRA, screen, and crossmatch positivity rate.

2.5. Statistical analysis

The ICTS statistical core assisted with all statistical analyses. Logistic regression analysis, with time (yearly/4 year periods 2010–13;2014–17;2018–2021) serving as the independent variable, was used to determine the odds ratio, 95% confidence interval, and p-values for time’s effect on the positivity rates of the platelet antibody screen and crossmatch testing. Fisher’s Exact Test was used determine odds ratio and p-value for the difference in the discordance rate of paired tests before and after the recall in 2013 [10]. A Wilcoxon rank sum test was used to determine the p-values for the differences in mean positivity of the 18 subjects who had PRA, screen, and crossmatch results.

3. Results

3.1. Platelet Antibody Screen and Crossmatch Testing Over Time

Neither the platelet antibody screen nor the platelet crossmatch have “cutoff’s” that are routinely used to define a “positive” test result. Therefore, the results for each individual well were examined for each test with any level of reactivity (w+ to 4+) considered positive for that well. Using these criteria, the percentage of positive wells from patients’ first platelet antibody screen and first platelet crossmatch is calculated versus the year the test was performed. Between 2010-2012, the highest percentage of positive wells in any year was 65%. From 2017–2021, the lowest percent positive wells observed for any year was 90%. The percentage of positive wells in platelet antibody screens increased significantly (OR 1.40, 95% CI 1.35–1.46, p < 0.001) over time. In comparison, the percentage of positive/incompatible wells in crossmatch tests decreased slightly (OR=0.96, 95% CI 0.93–0.99, p=0.014) over this same period (Figure 1).

Figure 1: Increase in positivity of platelet antibody screens over time.

The percentage of positive wells in the first platelet antibody screen (Screen) and first crossmatch tests for each patient is shown as indicated. The numbers above the data points indicate the total number of tests performed each year. Reactivity rates of the platelet antibody screen increased significantly over time (OR=1.40, 95% CI 1.35–1.46, p < 0.001), while rates of the crossmatch testing slightly decreased (OR=0.96, 95% CI 0.93–0.99, p=0.014). Statistical analysis was performed as described in the Materials and Methods Section.

Given the recall event that occurred with this testing in 2013, the results before and after the recall were examined by looking at reactivity rates of the platelet antibody screen and crossmatch testing during four-year periods (2010–2013, 2014–2017, and 2018–2021), one before and 2 after the recall. The positive reactivity rate of the platelet antibody screen increased significantly after the recall while the positivity rate of the platelet crossmatch decreased (Figure 2). The overall reactivity rate of the screen increased between each of the periods. The logistic regression model found significant increases between 2010–2013 vs 2014–2017 (OR=3.60, 95% CI=2.80–4.65, p<0.001) and between 2010–2013 vs 2018–2021 (OR=14.6, 95% CI=10.5–20.8, p<0.001). Crossmatch results showed an overall decrease over this period, but slightly increased from 2014–2017 to 2018–2021. The logistic regression model showed a significant decrease between 2010–2013 vs 2014–2017 (OR=0.52, 95% CI=0.38–0.70, p<0.001) and 2010–2013 vs 2018–2021 (OR=0.70, 95% CI=0.53–0.91, p=0.008).

Figure 2: Reactivity rates of platelet antibody screens and crossmatch testing before and after the platelet antibody screen recall.

The percentage of reactive wells in all patients’ first platelet antibody screen (Screen) and crossmatch testing is grouped from 2010–2013 (before the recall) and from 2014–2017 and 2018–2021 (after the recall). Numbers above bars indicate total number of tests performed in each 4-year period. The reactivity rate of platelet antibody screens increased significantly after the recall (2010–13 vs 2014–17: OR=3.60, 95% CI=2.80–4.65, p<0.001; 2010–13 vs 2018–21: OR=14.6, 95% CI=10.5–20.8, p<0.001). The reactivity rate of platelet antibody crossmatch decreased significantly after the recall (2010–13 vs 2014–17: OR=0.52, 95% CI=0.38–0.70, p<0.001; 2010–13 vs 2018–21: OR=0.70, 95% CI=0.53–0.91, p=0.008). Statistical analysis was performed as described in the Materials and Methods Section.

3.2. Paired Data

An analysis using only data from paired samples (tested within 7 days of each other) was performed to assess agreement between the two testing methods. For this analysis all samples tested before the recall are compared to those samples tested after the recall. The cutoff for a positive screen or crossmatch was set at greater than 50% positive wells. The data from 2010–2013 found that 14 of 19 (74%) paired samples showed concordant results between the screen and the crossmatch (Table 1). Two patients had a positive crossmatch with a negative screen while three had a negative crossmatch with a positive screen. In contrast, the data from after the recall (2014–2021) showed concordant results in only 55% (35 of 64) of the pairs while 44% (28 of 64) of the pairs had positive screen results with a negative crossmatch while only 1 (1.6%) had a negative screen with a positive crossmatch. The discordance rate in 2010–2013 (5 discordant of 19, 26.3%) compared to that in 2014–2021 (29 discordant of 64, 45%) increased overall, but this relationship was not significant (OR=0.419, p=0.1867).

Table 1:

Paired Screen and Crossmatch results

		Crossmatch
2010–2013		Positive	Negative
Screen	Positive	11 (58%)	3 (16%)
	Negative	2 (11%)	3 (16%)
2014–2021		Positive	Negative
Screen	Positive	31 (48%)	28 (44%)
	Negative	1 (2%)	4 (7%)

Test considered positive if >50% of wells were positive

3.3. HLA Testing

HLA antibody testing and PRA determination is another common approach utilized in patients suspected of being refractory to platelet transfusions [9–11]. The PRA was compared to screen positivity and crossmatch positivity in 18 patients who had all three tests performed within 21 days of each other. The mean positivity for these 3 tests on these 18 patients shows the PRA is significantly lower than the platelet antibody screen and nearly matches the positivity of the crossmatch (Figure 3).

Figure 3: Positivity Rates of Platelet Antibody screen, HLA testing, and Crossmatch Testing from 2018–2021.

The mean positivity percentages of platelet antibody screens (screens), HLA antibody tests (reported as panel-reactive antibody (PRA)), and platelet crossmatch tests from 2018–2021 performed within 21 days of each other. The error bars represent the standard error of the mean. The average positivity of these screens was significantly higher than both the average PRA (p=0.0012) and the average crossmatch positivity (p=0.0056), while the average PRA and crossmatch positivity were not statistically different (p=0.5167). Statistical analysis was performed as described in the Materials and Methods Section.

4. Discussion

Following the recall of the platelet antibody screen in 2013, there was a perceived increase in the rate of positive platelet antibody screens in our patients. In some cases, crossmatch results following a positive screen were completely negative (data not shown). While no peer reviewed publications have examined the positivity rate for the platelet antibody screen over time, preliminary data from 2016 also reported an increase in the % of positive platelet antibody screens after 2013 [7]. Based on this report and on our anecdotal experience, a review of all platelet antibody test results from 2010–21 was initiated to determine whether objective data could be obtained to demonstrate that the performance of this test has changed over time and in particular whether these changes may be related to the recall of this test in 2013. Our study includes data from platelet crossmatch testing and HLA antibody testing which allows one to compare positivity rates of these different tests and determine if the increased positive reactivity rate could be explained by changes in the patient population.

The results reported here demonstrate that the positivity rate in the platelet antibody screen increased substantially with time, while crossmatch positivity showed a slight reduction over the course of the study. Moreover, from 2018–21 platelet antibody screen results were significantly more positive than both the PRA from HLA antibody testing and crossmatch results, while the PRA and crossmatch results did not differ. This change in the screen over time and greater consistency between PRA and crossmatch results suggests the analytic sensitivity of the screen has increased with time. In our experience, this change has reduced the clinical utility of the platelet antibody screen. This is exemplified in the analysis of the paired data, where 44% of these paired tests in the years following the recall demonstrated a positive screen (defined as >50% positive wells) while the cross match was negative. Since the recall in 2013, there have been multiple instances where the screen is 100% positive yet crossmatching was performed and crossmatch compatible units were identified and successfully transfused to patients (data not shown).

This data indicates that the Immucor Capture-P Ready Screen analytic sensitivity has increased to the point that nearly all patients are positive. In fact, from 2016–2021, 100% of the platelet antibody screens have shown some positivity with 93% of those showing greater than 50% positivity (data not shown). Given these results, we have determined there is little utility in performing a platelet antibody screen and instead go directly to platelet crossmatch testing in patients suspected of being refractory to platelet transfusion due to alloimmune mechanisms. A disadvantage of the platelet crossmatch is that it requires sufficient platelets in inventory to justify performing this test. Platelet inventory was particularly challenging during the COVID-19 pandemic. In our case, even in 2020, we had sufficient inventory to test 14 platelets (the maximum we routinely test) in 14 of the 15 tests performed.

The Capture-P solid phase platelet antibody screen has been in use at our facility for over 20 years and solid phase testing for platelet antibodies in patients suspected of platelet antibodies have been in use for over 40 years [12–14]. However, testing “negative” patients or normal donors to determine the specificity of the assay is not well documented. The product information sheet states that the specificity of each lot is determined using “sera shown to be free of antibodies” but no additional information about specificity is provided. Studies to look at specificity of the Platelet Ab screen in subjects who have never been pregnant or transfused could directly address the specificity of the platelet antibody screen.

In summary, this study looking at the positivity rates of the Immucor Capture-P Ready Screen and Immucor platelet crossmatch testing found the positivity rates of the screen have significantly increased over time, while the crossmatch positivity has slightly decreased over the study period. Over the last several years, the platelet antibody screen positive rate has approached 100%, and are on average much higher than that of HLA antibody testing and crossmatch testing. Given these results, we have found the platelet antibody screen has little clinical utility and rarely perform this testing and if inventory of platelets are sufficient we go directly to platelet crossmatching or HLA antibodyas the initial testing for patients suspected to be refractory due to alloimmune mediated factors.