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ORIGINAL ARTICLE  
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Hemolysin test as a tool to screen high-titer Group O platelet apheresis donors: A prospective study


1 Department of Transfusion Medicine, Malabar Cancer Centre, Thalassery, Kerala, India
2 Department of Transfusion Medicine, Goa Medical College, Bambolim, Goa, India
3 Department of Oncopathology, Malabar Cancer Centre, Thalassery, Kerala, India

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Date of Submission18-Feb-2022
Date of Decision23-Apr-2022
Date of Acceptance22-May-2022
Date of Web Publication26-Sep-2022
 

   Abstract 

BACKGROUND: Minor ABO-incompatible apheresis platelet transfusion poses a risk of hemolytic transfusion reactions in non-Group O recipients when donor's plasma possesses unusual high titers for anti-A and anti-B. The aim was to determine whether the hemolysin test can be used as a screening tool to predict high-titer Group O platelet apheresis donors.
METHODS: A prospective study, with Group O platelet donor's samples, was tested for hemolysin test and antibody titration test in parallel. Antibody titration was also performed on products suspended in platelet additive solution (PAS). Hemolysin test was assessed for diagnostic accuracy against antibody titration. Chi-square test and Mann–Whitney U-test were used to determine the relationship between the hemolysin test and antibody titration.
RESULTS: Among 107 Group O platelet donations, median anti-A and anti-B titers in donors were 32 (8–128) and 32 (4–256), respectively. High titer (≥128) for ABO antibodies was seen in 18% of donations, whereas hemolysin test was positive in 69% of donations. Hemolysin test results differ significantly with antibody titration results (P = 0.03). Hemolysin test had higher sensitivity (89%) with a strong negative predictive value (94%). None of the products suspended in PAS had high-titer antibodies.
CONCLUSION: Adopting hemolysin test as a screening tool may label a large number of units (69%) unsuitable for ABO-incompatible platelet transfusion. Alternatively identifying donors with high antibody titer or positive hemolysin test and selectively suspending their product in PAS may be a cost-effective approach and certainly prevent high-titer antibodies in the product.

Keywords: Antibody titration, apheresis, hemolysin, hemolytic transfusion reaction


How to cite this URL:
Murugesan M, Augustine M, Velayudhan SC, Darussalam T, Nayanar SK. Hemolysin test as a tool to screen high-titer Group O platelet apheresis donors: A prospective study. Asian J Transfus Sci [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.ajts.org/preprintarticle.asp?id=356880



   Introduction Top


ABO identical platelets are the principal and preferred choice for transfusions. However, ABO-incompatible platelet transfusions are acceptable when platelet inventories are insufficient. Even with increasing use of apheresis platelets, ABO-incompatible transfusions are inevitable.[1] A high titer of anti-A and anti-B is found in 10%–26% of Group O blood donors.[2] Hence, minor ABO-incompatible platelet transfusion poses a risk of hemolytic transfusion reaction (HTR) in non-Group O recipients when transfused with Group O donor's plasma containing an unusually high titer of anti-A and anti-B antibodies.[3]

The hemolysis due to ABO incompatibility can be severe and often life threatening even though the incidence is low (approximately 50 in 10 lakhs).[4] Hence, to limit the hemolysis due to ABO-incompatible apheresis platelet transfusion, centers adopt methods such as volume reduction of Group O apheresis platelet units before ABO-incompatible transfusion or screen Group O donors for high titers anti-A and/or anti-B, or hemolysin test or suspending platelets in platelet additive solutions (PASs).[1]

Among these methods, high-titer determination in Group O donors which was evaluated in larger studies had shown variable benefits in preventing HTR from donor plasma.[5],[6] There is a paucity of literature from India about the use of hemolysin tests to screen high-risk apheresis donors with the potential to cause passive hemolysis. The main objective of the study was to determine the incidence of positive hemolysin test in Group O platelet apheresis donors and to determine the sensitivity, specificity, positive predictive value, and negative predictive value of hemolysin test by comparing with ABO antibody titration method. The study aims to determine whether hemolysin test can be used as a screening tool in Group O plateletpheresis donors to predict the risk of high-titer ABO antibodies and also whether PAS can help reducing the ABO antibody levels in platelet units prepared from donors with positive hemolysin test or donors with high-titer anti-A or anti-B.


   Methods Top


This was a prospective study performed on Group O blood donors eligible for donating platelets by apheresis. The study was initiated after institutional review board and ethical committee approval. Group O blood donors eligible to donate platelets by apheresis as per institutional guidelines and consented to participate in the study were included. Assuming that 26% of the Group O platelet donors have high-titer ABO from India; the study required a sample size of 65 donors for estimating the expected proportion with 10% absolute precision and 95% confidence.[2] Operational definition for high titer was when anti-A and/or anti-B levels ≥128 dilutions at the saline phase in conventional tube technique (CTT).[3]

Group O blood donors with informed consent were chosen to donate platelets by Trima Accel or Spectra Optia (Terumo BCT, Lakewood, USA) apheresis equipment. The platelet units were collected and suspended either in donor plasma or PAS. Donors, who chose to return to donate within an interval of 2 weeks to 6 months, were also included for a paired crossover with plasma or PAS during subsequent donation based on the previous donation. Blood samples collected for routine testing during the apheresis procedure were tested for hemolysin test and antibody titration test in parallel on the same day.

Hemolysin test

Fresh donor serum (2 volumes) in duplicate and freshly prepared in-house pooled A and B cells (1 volume) were incubated at 37°C for 45 min. Tubes were examined for the presence or absence of hemolysis macroscopically. The presence of hemolysis was taken as a positive hemolysin test.

Donor antibody titration

Anti-A and anti-B titration was performed in the CTT as described in AABB Technical Manual.[7] The donor plasma was serially diluted up to 1024 dilutions in duplicate. Freshly prepared in-house A and B cells were added to the respective tubes. Only immunoglobulin M (IgM) titers were determined by saline technique. The results were interpreted as the highest titer in which 1+ macroscopic agglutination reaction is identified.

Antibody titration in apheresis units

Antibody titration was performed from the supernatant of apheresis platelets suspended in PAS during quality control on day 1 of collection. Antibody titration was not repeated for apheresis units suspended in plasma, where donor antibody titer was taken as reference.

The technical staff performing the tests was trained adequately before the start of the study. The test results were verified by a second technical staff to minimize the interpretation bias. A difference in one dilution between the staff was acceptable and the highest value was taken as antibody titer. A positive hemolysin test was considered, if at least one among two of the technical staff reported the hemolysin test reaction as positive.

Statistical analysis

Descriptive statistics such as proportions and median (range) were used. A Chi-square test and Mann–Whitney U-test were used to determine the relationship of the hemolysin test and donor with high-titer antibody. Diagnostic accuracy parameters such as sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the hemolysin test in accordance with the Standards for Reporting of Diagnostic Accuracy Studies.[8] Wilcoxon signed-rank test was used to study the donors with repeat donations. Spearman correlation test was used to study the relationship of ABO antibodies between donor samples and antibody titers in the PAS apheresis platelet units. All analysis was performed using the SPSS software version 20. P < 0.05 was considered statistically significant.


   Results Top


A total of 107 Group O platelet donors were evaluated using both antibody titration and hemolysin test. The median anti-A and anti-B titers in donors were 32 (8–128) and 32 (4–256), respectively. High titer (≥128) for ABO antibodies was seen in 18% of donations [[Figure 1] - anti-A: 6% and anti-B: 17%]. Hemolysin test was positive in 69% of donations [[Figure 2] - 55% with anti-A and 62% with anti-B].
Figure 1: Bar diagram displaying the distribution of anti-A and anti-B titer in predonation sample of Group O platelet donors

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Figure 2: Bar diagram displaying the distribution of positive hemolysin among anti-A and anti-B in predonation sample of Group O platelet donors

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[Table 1] displays the relationship between donor antibody titer and hemolysin test results. Among the high-titer donor samples, hemolysin test was positive in 90% of samples. However, in samples with low titer, hemolysin test was negative only in 36% of samples. The donor hemolysin test results differ significantly when antibody titration results are classified as high and low titers (P = 0.03).
Table 1: Relationship between donor ABO antibody titer (high vs. low) and hemolysin test in Group O platelet apheresis donors (n=107)

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[Figure 3] displays the median antibody titer levels between donors, with and without hemolysis. The median anti-A titer was high among positive hemolysin test 64 (8–128) than negative hemolysin test 32 (8–128) (P = 0.049). Similarly, the median anti-B titer was 64 (8–256) and 32 (4–128) for donors with positive and negative hemolysin tests, respectively (P = 0.001).
Figure 3: Box-whisker plot displaying the relationship between hemolysin test and median ABO titers in predonation sample of Group O platelet donors

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Hemolysin test had 89% sensitivity with very low specificity (36%) in predicting high antibody titer. Furthermore, the hemolysin test had a stronger negative predictive value (94%) with low positive predictive value (24%). Sixteen donors returned to donate again during the study period. In paired sample analysis, there was no difference in ABO antibody titer values (P = 0.118) and hemolysin test results (P = 1.000) among their donations.

About 24.5% of products suspended in plasma had high titer, when donor antibody titer was taken as reference. A total of 58 products suspended in PAS were analyzed. There was a two (1–5)-fold reduction in antibody titer with the addition of PAS in the product relative to donor antibody titer and none of the products suspended in PAS had high titer when tested. There was a strong correlation between donor antibody titer and product PAS titer (for anti-A, r = 0.73, and anti-B, r = 0.74). [Table 2] shows that when donors were grouped as high and low titers in their predonation samples; there was a significant difference in product antibody titer levels for anti-A (P = 0.021) and anti-B (P = 0.011). However, when the donors were categorized based on hemolysin test, the product anti-A (P = 0.262) and anti-B (P = 0.730) titers did not vary significantly.
Table 2: Comparing the relationship between ABO antibody titer and hemolysin test against apheresis platelets suspended in PAS among Group O platelet donors (n=58)

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   Discussion Top


The goal of the study was to estimate the frequency of positive hemolysin test among Group O apheresis platelet donors and to determine the correlation between ABO antibody titration and hemolysin test. Our study revealed a high incidence (69%) of positive hemolysin test. However, the hemolysin test results did not correlate well with the standard ABO antibody titration method by CTT.

The prevalence of ABO-incompatible platelet transfusions varies widely with the United States, reporting that approximately 10%–40% of all platelet transfusions to be incompatible.[1] During the transfusion, approximately 200 ml of plasma in apheresis platelets gets diluted in recipient's blood volume.[6]

The incidence of HTR often goes unrecognized in ABO-incompatible platelet transfusion. There is an increased risk of HTR if platelet component contains a high ABO antibody titer or in a recipient in whom there is absence of soluble A or B blood group antigens in their plasma which neutralize anti-A or anti-B from the donor's plasma.[9] The U. S. Food and Drug Administration reported six cases of fatal HTR from 2005 to 2015 due to ABO-incompatible platelet transfusions.[9] HTR estimates range from 1:2500 to 1:9000 even before any screening for high-titer ABO antibodies was implemented.[5]

Minimizing the risk of HTRs in ABO-incompatible platelet transfusions has been a topic of interest recently. Strategies adopted to reduce the risk comprises plasma reduction of ABO-incompatible platelets or screening of donor plasma for high titers by performing anti-A and/or anti-B titration or assays for qualitative hemolysin or use of PAS for storage.[5],[9],[10]

Volume reduction of ABO-incompatible platelets is a time-consuming laborious process that may contain 15%–33% of lesser platelets with impaired function with an expiration of 4 h.[9] A case of HTR had been reported in one patient despite transfusion of volume-reduced apheresis platelet.[5] This induced the centers to adapt to methods to screen all platelet donors for high-titer ABO antibodies. A platelet component exceeding a set threshold is labeled as high titer and is excluded from out-of-group transfusions, if not volume-reduced or suspended in PAS.[9]

Among the methods for ABO titration available, CTT is the oldest and most standardized method with certain limitations. Published literature used critical titer when IgM titer is between 64 and 100 in tube technique or column agglutination (gel) technique, and immunoglobulin G (IgG) titer is between 200 and 512.[5],[10] In the United Kingdom, authors adopted using a single dilution cutoff of 1 in 100 by automated direct agglutination, whereas Scotland used a dilution cutoff of 1 in 50 for screening for high-titer anti-A and/or anti-B in the products.[5]

Considering an antibody titer cutoff level of ≥128 by tube method which is used in our center, 18% of Type O platelet units were labeled as high titer, which was comparable (14.2%) with a study by Landim et al.[3] Josephson et al. observed 30%–40% of samples as high titers among 100 Group O apheresis donors when IgM and IgG titers were defined as a minimum of 64 and at least 256, respectively.[1] Moreover, Karafin et al. observed 26% of samples of Group O apheresis platelets having titers > 256 when tested by gel method.[6]

In the present study, hemolysin test was positive in 69% of donations and the median donor antibody titer (anti-A, P = 0.049 and anti-B, P = 0.001) varied significantly when compared between the hemolysin test results [Figure 3]. While Landim et al. observed 38.6% of donor samples having either partial or total hemolysis, the median antibody levels in donors did not differ much between the groups (P = 0.733).[3]

The high-titer anti-B was more than twice prevalent than anti-A in the present study [[Figure 1]: 17% vs. 6%]. Similarly, hemolysin-positive anti-B was greater than hemolysin-positive anti-A [[Figure 2]: 62% vs. 55%]. There was no linear relationship between the high-titer antibody and the prevalence of hemolysin, as lysis was observed even at titers as low as 8. Furthermore, high titers of anti-A and anti-B were seen in two donors among negative hemolysin test results [Table 1]. A similar study by Olawumi and Olatunji observed that hemolysin-positive anti-B was twice common than hemolysin-positive anti-A, whereas the titer of hemolysin-positive anti-A was higher than hemolysin-positive anti-B. The anti-A and anti-B titers and their relationship with the risk of hemolysis were not linear.[11] Titers above 64 and titers above 128 were not correlated to the risk of hemolysis by hemolysin test (P = 0.454 and 0.677), respectively, in another study.[3] There was no relationship between donor age and the prevalence of hemolysin test, which was also observed by Kannan et al.[11]

Approximately two-third reduction in IgM and IgG titers of donor sample was observed when platelets were suspended in PAS, consistent with the 65% replacement of plasma with PAS as in published literature.[12] In the present study, there was a median of two-fold reduction in antibody titer of donor sample relative to platelet product suspended in PAS. None of the units prepared from donors with high titer or positive hemolysin test resulted in high-titer products when products were suspended in PAS.

The drawbacks with the antibody titration are the choice of IgM or IgG for detection with defining critical titer threshold, testing method, serial dilution versus single predetermined dilution, and interlaboratory variation.[5],[13] With the two titration methods, tube titer showed higher values than that found with gel method with 60% discrepancy between the two.[1] However, Josephson et al. demonstrated that titers were consistently 1–2 dilutions higher by the gel method than the tube method.[1] Pandey et al. demonstrated that gel method showed higher ability in detecting ABO antibody than the manual tube method and automated method (solid phase) showed even lower ability than the manual tube method.[13] Similarly, hemolysin test is also affected by interpretation bias and shall only be performed manually. By automation of antibody titer and implementing a cutoff level of 100, less than 15% of O group, ABO incompatible platelet transfusions would be labeled as high risk and hence reducing the wastage.[3] Hence, interpreting ABO antibody titer results to be interpreted with caution, taking into consideration the detection method used.[13]

Sprogøe et al. serially observed the least interpersonal variation in anti-A and anti-B titers over time among healthy volunteers.[14] In the present study, there was no difference in ABO antibody titer values (P = 0.118) and hemolysin test results (P = 1.000) among the repeat donations. Hence, it can be interpreted that repeat antibody titration or hemolysin test on the same donor is not necessary if donations are made at longer intervals with no potential immunizing events between the donations.[14]

Diagnostic accuracy studies are at risk of bias, unlike other clinical studies. The risk of bias and concerns about the applicability should be considered when performing diagnostic accuracy studies.[8] In the present study, with a high negative predictive value, the absence of hemolysin safely rules out the presence of high titers among Group O platelet donors. However, adopting the hemolysin test as a routine screening tool may label a large number of donations unsuitable for ABO-incompatible transfusions. Major limitations of the present study was a single center study. Prospective multicenter approach with uniformity in testing methodology, threshold for critical titer may help draw valid recommendations on hemolysin test in identifying high titer group O donors.


   Conclusion Top


Adopting hemolysin test as a screening tool may label a large number of units unsuitable for ABO-incompatible platelet transfusion. Alternatively identifying donors with high antibody titer or positive hemolysin test and selectively suspending their product in PAS may be a cost-effective approach and certainly prevent high-titer antibodies in the product.

Acknowledgment

The authors would like to acknowledge the staff of the blood bank.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors Mohandoss Murugesan, Merline Augustine, Shibin Cherukunnuparambil Velayudhan, Thahira Darussalam, and Sangeetha K Nayanar declare that they have no competing interests. All authors have reviewed and approved the manuscript.



 
   References Top

1.
Josephson CD, Castillejo MI, Grima K, Hillyer CD. ABO-mismatched platelet transfusions: Strategies to mitigate patient exposure to naturally occurring hemolytic antibodies. Transfus Apher Sci 2010;42:83-8.  Back to cited text no. 1
    
2.
Mathai J, Sindhu PN, Sulochana PV, Sathyabhama S. Haemolysin test for characterization of immune ABO antibodies. Indian J Med Res 2003;118:125-8.  Back to cited text no. 2
    
3.
Landim CS, Gomes FC, Zeza BM, Mendrone-Júnior A, Dinardo CL. Prophylactic strategies for acute hemolysis secondary to plasma-incompatible platelet transfusions: Correlation between qualitative hemolysin test and isohemagglutinin titration. Rev Bras Hematol Hemoter 2015;37:217-22.  Back to cited text no. 3
    
4.
Menis M, Izurieta HS, Anderson SA, Kropp G, Holness L, Gibbs J, et al. Outpatient transfusions and occurrence of serious noninfectious transfusion-related complications among US elderly, 2007-2008: Utility of large administrative databases in blood safety research. Transfusion 2012;52:1968-76.  Back to cited text no. 4
    
5.
Quillen K, Sheldon SL, Daniel-Johnson JA, Lee-Stroka AH, Flegel WA. A practical strategy to reduce the risk of passive hemolysis by screening plateletpheresis donors for high-titer ABO antibodies. Transfusion 2011;51:92-6.  Back to cited text no. 5
    
6.
Karafin MS, Blagg L, Tobian AA, King KE, Ness PM, Savage WJ. ABO antibody titers are not predictive of hemolytic reactions due to plasma-incompatible platelet transfusions. Transfusion 2012;52:2087-93.  Back to cited text no. 6
    
7.
Cohn CS, Delaney M, Johnson ST, Katz LM, editors. METHOD 3-15. Antibody Titration Procedure. Technical Manual, Methods Append. 20th ed. Bethesda, Maryland: AABB; 2020.  Back to cited text no. 7
    
8.
Cohen JF, Korevaar DA, Altman DG, Bruns DE, Gatsonis CA, Hooft L, et al. STARD 2015 guidelines for reporting diagnostic accuracy studies: Explanation and elaboration. BMJ Open 2016;6:e012799.  Back to cited text no. 8
    
9.
Tynuv M, Flegel WA. Quality improvement with platelet additive solution for safer out-of-group platelet transfusions. Immunohematology 2019;35:108-15.  Back to cited text no. 9
    
10.
Castilho L. ABO isohemagglutinin titration or hemolysin test: What should we do to reduce the risk of passive hemolysis? Rev Bras Hematol Hemoter 2015;37:215-6.  Back to cited text no. 10
    
11.
Kannan S, Kulkarni R, Basavarajegowda A. Prevalence of high titered anti-A and anti-B antibodies among O blood group individuals and their associated factors. Glob J Transfus Med 2020;5:187-91.  Back to cited text no. 11
  [Full text]  
12.
Pagano MB, Katchatag BL, Khoobyari S, Van Gerwen M, Sen N, Rebecca Haley N, et al. Evaluating safety and cost-effectiveness of platelets stored in additive solution (PAS-F) as a hemolysis risk mitigation strategy. Transfusion 2019;59:1246-51.  Back to cited text no. 12
    
13.
Pandey P, Setya D, Ranjan S, Singh MK, Singh MK. Comparison of ABO isoagglutinin titres by three different methods in group O blood donors. ISBT Sci Ser 2021;16:76-84.  Back to cited text no. 13
    
14.
Sprogøe U, Yazer MH, Rasmussen MH, Antonsen B, Bistrup C, Assing K. Minimal variation in anti-A and -B titers among healthy volunteers over time: Implications for the use of out-of-group blood components. J Trauma Acute Care Surg 2017;82:S87-90.  Back to cited text no. 14
    

Top
Correspondence Address:
Mohandoss Murugesan,
Department of Transfusion Medicine, Malabar Cancer Centre, Thalassery, Kerala
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajts.ajts_26_22



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