INTRODUCTION
Accurate identification of the blood group of pre-transfusion blood specimens is essential for performing blood transfusions. Despite advances in profiling the molecular genetics of blood groups, a conventional serological grouping method is still widely used as a screening test in transfusion medicine [1-3]. As errors in blood grouping may lead to severe complications, such as a hemolytic transfusion reaction, and even death [4], several countries have developed individual standards for the quality assessment of blood grouping reagents to ensure safe and effective transfusion. In the United States of America, the standards are specified in the Code of Federal Regulations, (CFR) Title 21, Chapter I, Subchapter F. Biologics, part 660, subpart C [5]. In European Union (EU) countries, the ABO and Rh blood grouping reagents are classified as in vitro diagnostic medical devices, which comply with regulations stipulated in the common technical specifications for in vitro diagnostic medical devices (2009/108/EC) [6]. The Government of India provides guidance materials for quality control of ABO and Rh blood grouping reagents [7].
In Korea, as in the United States of America or EU, medical devices, including in vitro diagnostic reagents, are categorized into classes 1 to 4 according to the level of regulatory control required to ensure the safety and effectiveness of medical practices [8]. ABO and D blood grouping reagents are classified as class 4 medical devices, and a performance test report for the quality control of domestically produced or imported ABO and D blood grouping reagents is therefore required for the validation of a brand new reagent or a new batch of existing reagents, according to the approved and reviewed guidelines for ABO blood grouping reagents [9]. As outlined in Supplementary Table 1, the report should include the sensitivity, specificity, potency titer, and avidity of anti-A and -B sera with ABO subgroups, including A2 and A2B, and anti-D sera with weak-D and Rh D-negative groups [9]. However, the availability of qualified blood that can be used to test the quality of the reagents, and at the same time meet the national regulatory testing standards, remains an issue. The United States Food and Drug Administration (FDA) recommends using fresh or frozen red blood cells to prepare cell suspensions for testing blood grouping reagents [10]. However, from 2007 to 2009 in Korea, only 0.052% of 3,397,983 healthy blood donors had diverse ABO subgroup phenotypes, and the frequencies of A2 and A2B were only 0.002% and 0.015%, respectively [11]. In addition, unlike in Caucasians, the prevalence of D-negative and weak-D blood groups is relatively low in the Korean population (only 0.15% and 0.01%, respectively) [12]. Due to the rarity of certain ABO and D subgroups in Korea, and the Korean Blood Management Act, which prohibits the use of voluntary non-remunerated blood for a different purpose other than transfusion, many reagent companies in Korea have struggled to obtain qualified red blood samples for testing the quality of blood grouping reagents and submitting performance test reports.
To address this issue, in the present study we aimed to prepare standard cryopreserved RBC panels using a high-glycerol method to verify the quality of monoclonal ABO and D blood grouping reagents, and to evaluate the suitability of the panels for use as a standardized reference, in cooperation with the Ministry of Food and Drug Safety (MFDS) of Korea.
MATERIALS AND METHODS
1. Sample collection
All participants submitted written informed consent prior to the acquisition of whole blood samples from 61 eligible volunteers who had undergone several screening tests (ABO/Rh D blood grouping, complete blood count, routine chemistry, and serological tests for hepatitis B virus, hepatitis C virus, HIV, and syphilis) at Severance Hospital, Seoul, Korea in August 2018. The samples were collected into blood collection bags containing citrate phosphate dextrose adenine (CPDA)-1 anticoagulant solution. The collected whole blood units were from the following blood groups: A (N=10), B (N=10), O (N=20), AB (N=8), Rh D-positive (N=8), and Rh D-negative (N=10). Subsequently, RBC concentrate was produced from each whole blood unit using standard operating procedures. Whole blood units of weak-D (N=2) and ABO subgroups (N=8), including A2B (N=2), A2B3 (N=2), A1B3 (N=2), B3 (N=1), and A3B (N=1), were obtained from the Korean Red Cross Central Blood Center, Seoul, Korea, with the approval of the Institutional Review Board of the Ethics Committee of Korean Red Cross. Due to the unavailability of A2 subgroup donors in Korea, two units of A2 subgroup blood, produced by the Continental Services Group, Inc. (Miami, FL, USA), were imported by Mirr Scitech Corp. (Seoul, Korea). This study was approved by the Institutional Review Board and Ethics Committee of Severance Hospital (IRB No.: 4-2018-0143).
2. Cryopreservation and thawing of RBC panels
A modified version of a high-glycerol method that is generally used in clinical applications was used for cryopreservation of RBCs, as previously described [13-16]. Within 7 days of collection, the RBC concentrates were glycerolized using Glycerolyte 57 solution (Baxter Healthcare, Deerfield, IL, USA) to a final glycerol concentration of 40%. Aliquots of the glycerolized RBCs (1 mL) were dispensed into separate cryotubes (SPL Life Sciences, Gyeonggi-do, Korea) using an automatic dispenser (DOSE IT, Integra Biosciences, Tokyo, Japan), and the cryotubes were frozen and stored at −70°C in a controlled-rate cryo-freezer (Thermo Fisher Scientific, Waltham, MA, USA), which freezes cells by reducing the temperature by approximately 1°C per minute, with a continuous temperature monitoring system. In total, 400 sets of RBC panels (Supplementary Fig. 1A), composed of group A (N=5), B (N=5), O (N=10), AB (N=4), Rh D-positive (N=4), Rh D-negative (N=5), and weak-D (N=1), and 200 sets of RBC subgroup panels (Supplementary Fig. 1B), composed of A2, A2B, A2B3, A1B3, B3, and A2, A2B, A2B3, A1B3, A3B (N=1, each), were produced.
After 6 or 12 months of storage, the cryopreserved RBCs were thawed in a 37°C water bath for 2–3 minutes with gentle mixing, and then transferred into test tubes. One milliliter of 9% NaCl was then added dropwise into the test tubes, and the test tubes were incubated at room temperature for at least 1 minute. The thawed RBCs were centrifuged at 3,400 rpm (about 1,000-1,020×g) for 20 seconds, and the supernatant was removed. The process of mixing with 1 mL of 2.5% NaCl, centrifuging, and removing the supernatant was repeated several times until a clear supernatant was obtained. The thawed RBCs were finally stored at 4°C.
3. Evaluation of RBC panels
1) Quality of frozen RBC panels
To evaluate the stability of the ABO/D antigens in newly produced RBC panels before and after 6 or 12 months of cryopreservation, a traditional agglutination method was performed using both slides and tubes [1, 11]. The two anti-A, -B, and -D monoclonal reagents used for ABO/D grouping in the present study were Sihdia (Shinyang Chemicals, Seoul, Korea), designated as reagent 1, and Bioclone (Ortho Clinical Diagnostics, Raritan, NJ, USA), designated as reagent 2, as they were the most easily obtained reagents. All reagents and blood grouping tests were performed according to the manufacturer’s instructions.
The sensitivity and specificity of the RBC panels were evaluated to determine the presence or absence of antigen A, B, and/or D, using both the test tube and the slide methods. The potency titers were measured using both the test tube and the column agglutination technology methods by determining the reciprocal of the greatest dilution of the reagent that showed visible agglutination [3]. Additional inter-laboratory studies were performed to confirm the quality of the newly developed RBC panels, in terms of sensitivity, specificity, potency titer, and avidity time at 6 and 12 months after cryopreservation at the Catholic University of Korea Eunpyeong St. Mary’s Hospital, Seoul, Korea and the Asan Medical Center, Seoul, Korea.
2) Performance of two commercial blood grouping reagents
The diagnostic performances of blood grouping reagents 1 and 2 were evaluated using our post-thawed RBC panels, comprised of group A (N=2), B (N=2), O (N=1), AB (N=2), Rh D-positive (N=2), and Rh D-negative (N=1) samples, at 6 and 12 months after cryopreservation. The potency titers determined using the two selected reagents were compared with those determined using the WHO International Standard of blood grouping reagents (NIBSC codes for anti-A: 03/188, anti-B: 03/164, and anti-D: 99/836).
3) Effect of storing RBC panels in preservatives after thawing
The effect of Alsever’s solution (Sigma-Aldrich Inc., St. Louis, MO, USA) on the preservation of post-thawed RBCs was compared with that of normal saline. The cryopreserved RBC panels, including group A (N=2), B (N=2), AB (N=2), and Rh D-positive (N=2) samples stored for 12 months, were thawed and suspended in either Alsever’s solution or normal saline. The potency titers of the freeze-thawed RBCs stored at 4°C in the refrigerator before testing were then evaluated on the day of thawing, and at 1, 3, and 7 days after thawing.
4) Effect of transportation using different storage media and time on RBC panels
An accelerated deterioration test was performed for different transport times with either dry ice or ice packs as the transport media. The RBC panels that were cryopreserved for 6 months, comprising group A (N=2), B (N=2), AB (N=2), O (N=1), Rh D-positive (N=2), and Rh D-negative (N=1) samples, were placed in one of two styrofoam boxes, one filled with dry ice and the other filled with ice packs, mimicking the actual transportation conditions across Korea. After 6 and 24 hours of storage, the RBC panels were thawed, and their potency titers were measured and compared.
RESULTS
1. Quality evaluation of freeze-thawed RBC panels
Our newly developed RBC panels had a sensitivity of 100% and a specificity of 100% to the two selected anti-A, -B, and -D blood grouping reagents, regardless of the cryopreservation duration. All the group A and AB samples reacted with anti-A sera, and all the group B and AB samples reacted with anti-B sera. All D-positive samples agglutinated well with anti-D sera. Group B and O, group A and O, and Rh D-negative and weak-D samples did not react with anti-A, -B, or -D sera, respectively. The strength of agglutination ranged from +2 to +4, fulfilling the Korean batch release criteria outlined in the Supplementary Table 1 [9].
The time required for agglutination expressed in seconds for each blood type, also known as the avidity time, using the two selected reagents is shown in Table 1. The avidity time for each RBC panel also fulfilled the batch release criteria, and were therefore considered to be acceptable.
The potency titers of the RBC panels for each blood group before and after 6 and 12 months of cryopreservation according to the two selected blood grouping reagents are shown in Table 2. Changes in potency titers that would not satisfy the batch criteria listed in the Supplementary Table 1 were not observed in any of the blood groups tested.
An additional quality assessment of the 6 or 12 month-cryopreserved RBC panels was performed at two different laboratories in Korea using the two selected blood grouping reagents (data not shown). Although there were some inter-laboratory variations in potency titers, all the RBC panels demonstrated a sensitivity and specificity of 100% regardless of the test center, blood groups of thawed RBCs, blood grouping reagents used, and cryopreservation time. The avidity time measured was always less than 120 seconds, which is satisfactory according to the manufacturer’s instructions for blood grouping reagents.
2. Performance evaluation of selected blood grouping reagents in comparison to the WHO International Standard Reagents
The diagnostic performance of two selected blood grouping reagents in comparison with that of WHO standard reagents on the RBC panels at 6 and 12 months after cryopreservation is shown in Table 3. The potency titers of the RBC panels measured using the selected reagents were always equal to or higher than those measured using the WHO standardized reagents, suggesting promising diagnostic performance for the selected reagents.
3. Effect of using preservatives on ABO/D antigen stability
The potency titers of freeze-thawed RBC panels preserved either in Alsever’s solution or in normal saline on the day of thawing, and at 1, 3, and 7 days after thawing, were compared and are shown in Fig. 1. Some of the RBC panels preserved in both preservatives showed a slight decrease in potency titers over time, but not in more than 2 titers, except in one sample.
4. Effect of transportation conditions on ABO/D antigen stability
The potency titers of freeze-thawed RBC panels stored for 6 and 24 hours, either on dry ice or ice packs, were measured and are shown in Fig. 2. All the RBC panels showed a maximum decrease of 1 titer in potency titers, regardless of the transportation time and media used.
DISCUSSION
Since the discovery of ABO blood groups by Karl Landsteiner in the early 1900s, serological tests using agglutination have been the standard for ABO grouping and D typing [17, 18]. With the evolution of molecular genotyping technology in blood grouping systems, certain parts of the conventional serological methods have been replaced in limited clinical settings; however, serological methods are still widely used and are continuously improved for the accurate identification of blood groups [2, 3, 18, 19]. In conventional serological methods, performing quality assurance checks of blood grouping reagents before they are routinely used is extremely important. However, because of the limited number of RBC samples available for quality checks in Korea, further research is needed to identify high-quality testing alternatives. In 1950, it was first demonstrated that blood mixed with glycerol could be frozen, and then retained its viability after thawing [20]. Since then, several studies have been carried out to establish protocols for RBC cryopreservation, and cryopreserving RBC units for future use has been a routine procedure in blood banks worldwide [21]. To address the need for standardized RBC panels for controlling the quality of blood grouping reagents in Korea, in the present study we developed cryopreserved RBC panels using a high-glycerol method and evaluated their qualities for use as a standardized panel for assessing blood grouping reagents. Despite the FDA’s recommendation of using frozen red blood cells for testing blood grouping reagents [10], to the best of our knowledge, no other researchers elsewhere have carried out equivalent experiments.
In the present study, 400 sets of cryopreserved RBC panels comprising ABO/D groups, and 200 sets of cryopreserved RBC panels comprising various ABO subgroups, were prepared using a high-glycerol method. According to the Korean batch release criteria, the quality of these newly developed panels before and after 6 or 12 months of cryopreservation was evaluated and found to be satisfactory. These criteria included positive reactions with reagents bearing the corresponding antibodies, negative reactions with reagents lacking the corresponding antibodies, and shorter agglutination times compared to those specified in the manufacturer’s instructions. In addition, the measured potency titers were equal to, or higher than, those listed in the batch release criteria. Therefore, our newly developed RBC panels are suitable for use as a standardized reference panel for quality assurance of blood grouping reagents.
Even though several blood grouping reagents are available in Korea, we tested the diagnostic performance of two reagents that are more easily accessible in Korea than the WHO standardized reagents on our newly developed RBC panels. We verified the quality and performance of the selected reagents in terms of sensitivity, specificity, avidity, and potency titer. Based on these results, we propose that any available blood grouping reagent can be used on our RBC panels depending on the hospital conditions.
In the present study, we tested the effects of two preservatives, Alsever’s solution and normal saline, and the effects of transportation conditions on the stability of ABO/D antigens. There was no significant difference between the two preservatives based on the potency titers of the RBC panels cryopreserved for 12 months. However, using normal saline as a preservative increases the number of fragmented RBCs and causes changes in RBC morphology [22], and Alsever’s solution is therefore a better preservative for post-thawed RBC panels. The cryopreserved RBC panels stored on both ice packs and dry ice yielded similar potency titers, regardless of the transportation time, indicating the stability and safety of the RBC panels if transported within Korea, and the applicability of both media for transporting blood components. However, a temperature range from 1°C to 10°C is recommended for transporting frozen blood components, including frozen RBCs and fresh frozen plasma, which is safer and easier to achieve using an insulated packaging system with dry ice [23].
The main limitation of the present study was our inability to evaluate the stability of ABO/D antigens and the safety of cryopreserved RBC panels for longer than a year. However, several studies have shown that frozen RBCs stored at −80°C are safe and effective for 21 years [24] and up to 37 years [25]. Therefore, it is reasonable to believe that the quality of our newly developed RBC panels would be acceptably maintained for decades.
In conclusion, we successfully evaluated the performance of cryopreserved RBC panels that can be used as a standardized reference material for evaluating the quality of blood grouping reagents. We believe that these RBC panels will alleviate the issues faced by domestic reagent companies in Korea regarding the difficulty of obtaining blood samples for quality control of blood grouping reagents, and thus improve the quality of reagents for more accurate blood grouping in the near future (Supplementary Fig. 2).