|Year : 2022 | Volume
| Issue : 1 | Page : 54-59
Comparative Evaluation of heat-inactivated ABO isoagglutinin titers with solid-phase red cell adhesion titers
Shweta Ranjan, Prashant Pandey, Divya Setya, Supriya Kumari
Department of Transfusion Medicine, Histocompatibility and Molecular Biology, JAYPEE Hospital, Noida, Uttar Pradesh, India
|Date of Submission||11-Jun-2021|
|Date of Decision||09-Dec-2021|
|Date of Acceptance||07-Feb-2022|
|Date of Web Publication||29-Apr-2022|
Dr. Prashant Pandey
Department of Transfusion Medicine, Histocompatibility and Molecular Biology, JAYPEE Hospital, Noida, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background and Objectives: Measurement of actual concentration of IgG requires methods like heat inactivation (HI) of serum. This study was aimed at comparing of heat-treated ABO titers performed by conventional test tube technique (CTT) and column agglutination technique (CAT) with hemagglutination (HA)/solid-phase red cell adherence (SPRCA). Methods: This was a prospective, observational study conducted between October 2019 and March 2020. All consecutive A, B, and O group donors who gave consent for participation in the project were included. All samples were tested by CTT and CAT before and after HI (post DTT treatment performed by CTT [pCTT] and post DTT treatment performed by CAT [pCAT]) and with HA/SPRCA. Results: A total of 300 donors were included: 100 each with A, B, and O blood groups. For group O, IgG titers were higher than IgM titers among group O, whereas that among group A and group B, IgM titer was found to be higher than IgG titer by both HA/SPRCA and pCTT. The titer values among all the three blood groups were highest when the test was performed by pCAT, though the results of pCTT and SPRCA, pCTT, and pCAT were significant in group O donors. For groups A and B, results of pCTT and pCAT, pCAT, and HA/SPRCA were found to be statistically significant. Conclusion: Results obtained by HA/SPRCA were closer to pCTT, with respect to time consumption, expertise required, and lack of inter-observer variation. Titers obtained by pCAT were higher in comparison to pCTT obviously due to its relative higher sensitivity. Hence, these methods cannot be used interchangeably.
Keywords: Column agglutination technology, comparison of conventional tube technique, hemagglutination/solid-phase red cell adherence, heat inactivation, isoagglutinin titer values
|How to cite this article:|
Ranjan S, Pandey P, Setya D, Kumari S. Comparative Evaluation of heat-inactivated ABO isoagglutinin titers with solid-phase red cell adhesion titers. Glob J Transfus Med 2022;7:54-9
|How to cite this URL:|
Ranjan S, Pandey P, Setya D, Kumari S. Comparative Evaluation of heat-inactivated ABO isoagglutinin titers with solid-phase red cell adhesion titers. Glob J Transfus Med [serial online] 2022 [cited 2022 Jun 26];7:54-9. Available from: https://www.gjtmonline.com/text.asp?2022/7/1/54/344336
| Introduction|| |
The two most important immunoglobulin types in transfusion medicine are IgG and IgM. Anti-A and anti-B of blood groups A and B are predominantly IgM type while those of blood group O are predominantly of IgG type. Determination and monitoring of ABO isohemagglutinin titers play an important role in the outcome of solid organ or hematopoietic stem cell transplant. It is the IgG-type antibodies which are clinically significant and play a pivotal role in the transplant. Measurement their titer is of extreme importance as to regulate immune reactions with respect to transfusion or transplantation.,,,,,,,
The concentration of IgG antibodies may be masked by IgM antibodies that lead to overestimation of IgG titers and may result in increased overall expenses in terms of using immunosuppressants, therapeutic plasma exchange, and the hospital stay. In order to determine the actual concentration of IgG antibodies, interference of the IgM antibodies needs to be eliminated. Several methods have been described in the literature to inactivate IgM, including heat inactivation (HI) at 63°C a use of sulfhydryl reagents such as 2-mercaptoethanol (ME) or dithiothreitol (DTT)., Of these, the HI method has several advantages over DTT including less time consuming and not diluting the sample and requires no additional chemical agents so is inexpensive. Heat may denature the heat-sensitive proteins, but the IgG immunoglobulin is relatively more stable at certain temperatures.
Measurement of ABO antibodies is routinely performed by titration, a semi-quantitative method, using conventional test tube technique (CTT). However, there are a few limitations of CTT such as intensive labor oriented and more time consumption, low reproducibility, and inter-observer and inter-laboratory variability. Other semi- and fully automated methods for titration like column agglutination technology (CAT) or solid-phase red cell adherence (SPRCA)/hemagglutination (HA) are now used by laboratories.,,, Many studies have compared CAT with CTT and have concluded that CAT has more advantages over CTT such as high reproducibility and objectivity as well as stable well-defined end points of agglutination reactions.,,,, Fully automated methods such as SPRCA are also being widely used to increase the efficiency and reproducibility of testing.,, While automation has the clear advantage of objectivity, ease of use, and reproducibility with well-defined end points of agglutination reaction, it requires well-neat standardization to be used profitably as it is tagged with significant variation among methods employed and the laboratories involved.,,,,, The aim of the present study was to compare the results of titers obtained by HA/SPRCA and by CTT and CAT (the latter using of heat-treated plasma).
| Materials and Methods|| |
Settings and design
This was a prospective, observational study conducted in the department of transfusion medicine at a tertiary health-care center through a 6-month period between October 2019 and March 2020. Heat-treated serum from each donor was titrated for anti-A and anti-B by CTT and CAT in parallel to the untreated samples by HA/SPRCA.
The study sample included in this study came from all consecutive A, B, and O blood group donors recruited at our center as per the standard protocol laid down., After performing the routine testing of the blood groups, transfusion-transmitted infections (TTI), etc., the antibody titration was performed either on the same day or the next day, with a maximum delay of 1 day of the blood collection. The samples were stored at 4°C throughout. The donors who did not give consent to participate in the study or those found reactive for TTI or those with a positive direct antiglobulin test (DAT) or the or positive antibody screen test were excluded.
Statement of ethics
All donors who gave informed consent to participate in the study were included in this study. The identity of the donors was concealed and only donors' blood unit numbers were used data analysis. No extra blood sample was collected from individual donor for this study and the pilot blood samples meant for routine blood bank testing were used. The study was approved by the institutional review board (IRB) and the institutional ethics committee.
Heat inactivation of plasma
HI of plasma was performed by the method described in the Laboratory Manual published by the American Society for Histocompatibility and Immunogenetics (ASHI). Desired amount of plasma was taken in a test tube and was placed in preheated 63°C heat block for exactly 13 min, followed by centrifugation. The supernatant was removed into a clean prelabeled tube on the very day of titration by CTT and CAT.
Methods of titration
Conventional test tube technique
Titration was performed as per the method described in the technical manual published by the American Association of Blood Banks (AABB). The titer end point was the reciprocal of the highest dilution yielding 1+ agglutination with naked eye. The reactions were recorded for IgM and IgG on a case reporting form.
Column agglutination technique
The Neutral Ortho BioVue System cassettes (Ortho Clinical Diagnostics, Raritan, New Jersey, USA) were used for IgM antibody titer, while for IgG, Anti-IgG Monospecific Ortho BioVue System cassettes (Ortho Clinical Diagnostics, Raritan, New Jersey, USA) were used. Dilutions of test sample were prepared as was used for CTT and the test protocol was followed as per the manufacturer's instructions. The reactions were read and recorded on the case reporting form. The end point as the titer value was considered the highest dilution yielding visible agglutination with naked eye.
Hemagglutination/solid-phase red cell adherence
Antibody titration was carried out using automated device on NeoImmunohematology analyzer. IgM titers were performed by HA technique and IgG titers were performed by solid-phase red cell adherence (SPRCA) technique as per manufacturer's instructions. Agglutination reactions were read by automated cameras. The titer end point was the reciprocal of the highest dilution yielding agglutination.
Data were entered in an MS Excel sheet; numerical values, percentages, mean, and standard deviation was calculated. Statistical analysis was performed using SPSS software (version 184.108.40.206, Chicago, USA). Median IgM and IgG titers were calculated for anti-A and anti-B obtained by post DTT treatment performed by CTT (pCTT), post DTT treatment performed by CAT (pCAT), and HA/SPRCA. Correlation between the methods was tested using Spearman's rho. The strength of the correlation was calculated using the following guide for the absolute value of rs:
- 0.0–0.19 – very weak
- 0.20–0.39 – weak
- 0.40–0.59 – moderate
- 0.60–0.79 – strong
- 0.80–1.0 – very strong.
Nonparametric Wilcoxon signed-rank paired test was used to test for significance comparing IgM and IgG results between pCTT and pCAT, pCTT and HA/SPRCA, pCAT and HA/SPRCA for a given sample. For this purpose, a total of 10 samples (every 10th sample) were included.
| Results|| |
A total of 303 blood donors were included in the study after getting their consent to participate. The three of the donors were seropositive reactive for TTI. The remaining 300 donors were tested negative for DAT, and antibody screen test was divided into three categories in equal number of 100 each as per their blood groups A, B, and O. For group O, the mean age of participants was 31.91 ± 7.8 years with 5% of them being females. For group A, the mean age of participants was 31.25 ± 6.12 years with 6% of females. Likewise, for group B, the mean age of participants was 32.59 ± 7.62 years and 10% (10 of 100) were females.
[Figure 1] illustrates the distribution of anti-A and anti-B, IgG titers of A, B, and O blood groups performed by pCTT and HA/SPRCA with the help of a box and whisker plot. Group O titers were higher than group A/B titers. Titer results for group O individuals were higher when performed by HA/SPRCA as compared to pCTT both for IgG. IgG titer results were similar when performed by SPRCA and pCTT for group A and B individuals.
|Figure 1: Box and whisker plot to illustrate distribution of anti-A and anti-B titers, performed by hemagglutination/solid-phase red cell adherence and conventional test tube technique (post heat inactivation): (a) Anti-A IgG titers, (b) Anti-B IgG titers|
Click here to view
[Figure 2] shows a comparison between median IgG and IgM titers for anti-A and anti-B performed by pCTT, pCAT, and HA/SPRCA of A, B, and O blood groups. For group O individuals, pCTT and pCAT results showed that IgG results were higher than IgM titers. However, when performed by HA/SPRCA, while anti-A IgG titers were higher than IgM titers, anti-B IgG titers were similar to IgM titers. For group A and B individuals, median IgM titers were found to higher than IgG titers when performed by HA/SPRCA and pCAT. However, when performed by pCTT, while anti-A IgG titers were similar to IgM titers, anti-B IgG titers were higher than IgM titers.
|Figure 2: Comparison of median IgG and IgM titers for anti-A and anti-B by post DTT treatment performed by conventional test tube technique, post DTT treatment performed by column agglutination technique, and hemagglutination/solid-phase red cell adherence: (a) Anti-A IgG, (b) Anti-A IgM, (c) Anti-B IgG, (d) Anti-B IgM|
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[Figure 3] illustrates the trend of IgG results obtained for every 10th sample by all three methods: pCTT, pCAT, and HA/SPRCA. A comparison between pCTT and pCAT, pCTT and HA/SPRCA, and pCAT and HA/SPRCA was performed using Wilcoxon signed-rank paired test for significance. When comparing anti-A and anti-B IgG results from group O individuals, results of pCTT and SPRCA, pCTT, and pCAT were found to be significant, whereas results of pCAT and HA/SPRCA were not found to be significant. When comparing anti-A and anti-B IgG results from group A and B individuals, results of pCTT and HA/SPRCA were not found to be significant, while results of pCTT and pCAT and those of pCAT and HA/SPRCA were found to be significant.
|Figure 3: Comparison of IgG titers performed by post DTT treatment performed by conventional test tube technique, post DTT treatment performed by column agglutination technique, and hemagglutination/solid-phase red cell adherence for 10 samples. Wilcoxon signed-rank test was used for calculating significance using P < 0.05. S indicates significant and NS indicates not significant. (a) Anti-A (blood group B), (b) Anti-A (blood group O), (c) Anti-B (blood group A), (d) Anti-B (blood group O)|
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[Table 1] lists the Spearman's rho (rs) for correlation between pCTT and HA/SPRCA as well as between pCAT and HA/SPRCA. The statistical analysis was performed for IgG titers for both anti-A and anti-B antibodies of group O and non-O individually. For blood groups A and B, correlation for IgG titers between pCTT and HA/SPRCA, pCAT, and HA/SPRCA was found to be weak. For blood group O, correlation for IgG titers between pCTT and HA/SPRCA, pCAT, and HA/SPRCA was found to be moderate.
|Table 1: The correlation of ABO immunoglobulin G isohemagglutinin titer results between the three methods for blood groups A, B, and O|
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| Discussion|| |
Quantification of ABO isohemagglutinins is performed by preparing serial dilutions of plasma. CTT is the age-old method that has been used for ABO antibody titration. Automated ABO titration methods (SPRCA/HA) have the advantages of high throughput, less time consumption, minimal training of existing staff, and the ability to quantify both IgM and IgG antibodies individually, but their antibody titration end points and clinical utility have not been defined.
While HA measures level of only IgM titres, SPRCA does it specifically for IgG titres. Hence, IgM inactivation is not needed as is required for CTT and CAT. ABO isohemagglutinin titration by CTT and CAT after the treatment of plasma by heat is a time-consuming procedure. In the present study, we therefore compared the results on ABO titration by CTT and CAT (after HI) and HA/SPRCA.
Heating of plasma in order to inactivate IgM antibodies was first described in 1981 by Steinberg and Cook. Studies performed previously for pretransplant work-up of solid organ transplant recipients have concluded that the presence of IgM antibodies can cause false-positive complement-dependent cytotoxicity (CDC) crossmatch and subsequent use of HI technique can eliminate this false positivity by decreasing interference of IgM antibodies., However, the effect of HI on the ABO isohemagglutinin titers has not been studied in detail when compared to study of the effect of DTT or 2-ME on ABO titers. Hasekura and Ishimori studied the effects of heat on IgG, IgM, and IgA titers in 300 females with ABO incompatible fetus or newborns. The present study was performed for 300 healthy whole blood donors which included both males and females.
In the present study, IgG titers for anti-A and anti-B in group O individuals were higher than IgM titers. When comparing both IgM and IgG titers, titers performed by pCAT were found to be more than titers obtained by CTT. It has been reported that O blood group individuals possessed more IgG ABO isohemagglutinins as compared to A or B blood groups.
Historically, HI of sera has been performed at 56°C for 30 min to destroy the complement., Hasekura and Ishimori heated sera at 70°C for 10 min and observed a significant decline titer values for both IgM and IgG anti-A or anti-B. Riley et al. heated plasma at 63°C for 10 min to ameliorate the effect of IgM in false-positive CDC crossmatch. In the present study, HI was performed on serum heated at 63°C for 13 min as per ASHI laboratory manual. Hasekura and Ishimori expected more difference in titers with the use of heat, but there was not much difference observed by him.
CTT has been the conventional method for all immunohematology investigations including ABO antibody titration, and it is being replaced at various centers with other semi- and fully automated techniques which reduce the inter-observer variation and require much less expertise. However, these techniques are expensive and despite various advantages are not available to transfusion services in resource constraint settings. In the present study, the median IgM and IgG titers for both anti-A and anti-B belonging to A, B, and O blood groups were higher when performed by CAT in comparison to CTT. Park et al. compared only IgG titers of CTT with CAT and found that for blood group O, the titers were more in CAT than CTT. Median anti-A and anti-B titers by CTT for were found to be 32 in their study, and in the present study, it was also 32. Nayak et al. compared results of 50 samples and concluded that there was poor agreement between IgG titers performed by CAT and CTT. In the present study, for groups A and B, correlation for IgM titers was found to be strong between HA/SPRCA and pCTT and pCAT while correlation for IgG titers was found to be weak. For group O individuals, correlation for IgM titers was weak, whereas for IgG titers, it was found to be moderate. Lally et al. compared titer results obtained by automated, solid-phase, and agglutination-based antibody titer platform versus manual gel testing on 54 patient samples. Of these 54 patient samples obtained for the study, 17 belonged to group O individuals. They found that in group O individuals, for anti-A and anti-B both, results obtained by CAT and HA/SPRCA were found to be significant. In the present study, when comparing anti-A and anti-B IgG results from group O individuals, results of pCTT and SPRCA, pCTT, and pCAT were found to be significant, whereas results of pCAT and HA/SPRCA were not found to be significant. When comparing anti-A and anti-B IgG results from group A and B individuals, results of pCTT and HA/SPRCA were not found to be significant, while results of pCTT and pCAT and those of pCAT and HA/SPRCA were found to be significant.
HI helps in reducing the interference of IgM antibodies and its use is a simple, low-cost method requiring simple equipment like a heat block or water bath which is available in most laboratories for IgM inactivation. However, it is slightly time consuming when compared to HA/SPRCA which does not require any treatment of plasma for IgM inactivation. Strengths of this study include a robust sample size. This is the first study which assesses the effect of heat on anti-A and anti-B titers in 300 individuals, 100 each for groups A, B, and O with the use of two different methods: CAT and CTT. Limitations of this study include inability to assess clinical impact of titration performed before and after HI of plasma.
| Conclusion|| |
IgG isoantibody titers were found to be higher than IgM titers in group O whereas IgM titers were found to be higher in group A and B individuals. Results obtained by pCAT were higher than results obtained by pCTT obviously due to higher sensitivity of CAT. Results obtained by HA/SPRCA were closer to those obtained by pCTT, with HA/SPRCA having the advantage of less time consumption, automation requiring less expertise and better precision no inter-observer variation in comparison to pCAT. To discern the most suitable method, a clinical impact of these results needs to be studied.
The authors would like to acknowledge all the personnel and study participants.
Financial support and sponsorship
Conflicts of interest
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