Evaluation of Erythrocytes Magnetized Technology for Measurement of ABO Isoagglutinin Titers

Introduction

ABO antibodies are naturally occurring antibodies. Individuals normally produce antibodies directed against the A and/or B antigen(s) absent from their red blood cells (RBC). The ABO antibodies are predominantly immunoglobulin M (IgM), activate complement, and react at room temperature or colder. However, there may be small quantities of immunoglobulin G (IgG) type of ABO antibodies present. The predominant immunoglobulin class of antibodies in group O serum is IgG.^[1] These antibodies are of clinical significance due to their ability to cause hemolytic transfusion reactions, hemolytic disease of the newborn, acute rejection in solid organ transplantation, and delay in engraftment of erythrocytes and megakaryocytes in ABO-incompatible stem cell transplantation.^[2-5] Therefore, ABO isoagglutinin titer measurements are useful for monitoring engraftment and predicting prognosis in ABO incompatible stem cell transplantation, identifying subgroups of ABO blood type, and monitoring transfusion reactions in ABO-mismatched transfusions.^[6,7] Even for management purpose, an isoagglutinin titer is especially useful for the administration of treatment like plasmapheresis, splenectomy, and immunosuppressives like rituximab. It has been shown that the ABO barrier in renal transplantation can be circumvented by pretreatment of the recipient with a combination of immunosuppression and apheresis to reduce the ABO antibody concentrations permitting successful engraftment of an ABO-incompatible kidney transplant.^[8,9]

Performing an antibody titration can help determine antibody concentration levels. Serial twofold dilutions of serum that contains the required antibody are prepared and tested against an RBC suspension having the target antigen. The titer is considered to be the reciprocal of the greatest dilution in which agglutination is observed. The strength of reactivity is also noted by assigning it a score. When new serum samples from the same patients are tested for titer, the initial titer specimen is always tested in parallel to nullify the effect of subjective variation among technologists and the relative strength of the target antigen on the red cells being used. A change in titer level of two or more tubes (fourfold increase) is considered to be significant.

There are many methods for measuring ABO isoagglutinin titers such as conventional tube hemagglutination technique (TT), microcolumn agglutination technique (CT), microtiter plate method using solid phase red cell adherence (SPRCA) assay, and flow cytometric analysis. Hemagglutination at room temperature is used for measuring the IgM titer. Indirect antiglobulin test (IAT) is used to measure IgG antibodies. Dithiothreitol (DTT) is used to specifically differentiate IgG antibodies.^[10,11]

TT for ABO antibody titers is limited by the subjective variation of the agglutination reading. It is time consuming and labor-intensive, prone to technical errors, requires experienced staff to read results, and the reaction can be read only once without disturbing the cell button. Furthermore, TT demonstrates low reproducibility and variable sensitivity and has been proven to be difficult to standardize between different laboratories. The CT is considered to be more effective in standardizing the titer readings as it eliminates the interreader variability and also produces more stable and reproducible result. Its other advantages are that it is less time-consuming and uses lower volumes of serum and RBCs, and can also be used as part of an automated system. It is being used despite the need for expensive reagents.

Another technique is SPRCA assays (fully automated systems available by IMMUCOR, United States). In this one of the components of the antigen antibody reaction is immobilized on a solid medium usually the microtiter plates which reacts with free antigen or antibody in the sample. The end point of the reaction is denoted by use of RBCs that may be used as indicator cells or may be a part of the antigen–antibody reaction. Erythrocytes magnetized technology (EMT; DIAGAST, France) is another technology using microtiter plates and magnetized erythrocytes. These RBC are reacted with patient's serum on a microplate with magnets underneath and the results are read after magnetization and magnetized shaking. Other advanced and sensitive techniques like flow cytometry (FCM) have been claimed to detect both IgG and IgM ABO antibodies more accurately by the use of iso-type-specific Ab.

A variation in the measurement of the ABO antibody titer has been seen among different laboratories due to the absence of a standard method of titration.^[12] Because of the many limitations for the TT, automated instruments using microcolumn agglutination, SPRCA assay, and EMT are being commonly used these days.^[13] The advanced methods have improved the quality of testing but none of them has been found to be better than the other. Using automated techniques have improved the quality of results by reducing manual or clerical errors, decreasing subjectivity, reproducibility in testing, increasing throughput in laboratories with high workload, and keeping records of patient results in form of images that can be retrieved later.

Not many studies have been done for the evaluation of isoagglutinin titers by EMT. Therefore, in this study, we evaluated automated ABO isoagglutinin titer measurements by EMT by QWALYS-3 (DIAGAST, Loos Cedex, France). It is used for ABO blood typing, antibody screening, identification, and cross matching.^[14] Here, we have compared the EMT-based ABO isoagglutinin titer measurements with the conventional TT in Indian scenario.

Materials and Methods

Results

The distribution of subjects into various BGs, their mean age, and male to female ratio is given in ►Table 1.

Frequency and isoagglutination titers of IgG and IgM by both TT and EMT for anti-B in BG A, anti-A in BG B, and anti-A and anti-B in BG O were plotted and are shown in ►Fig. 1A–D and 2A–D, respectively. The distribution of the measured IgG and IgM ABO isoagglutinin titers for each BG by the tube hemagglutination technique and EMT is shown in ►Figs. 3A and B. The median titers of IgG, anti-B for BG A and anti-A for BG B, were same by both methods: 64 (4–512) by TT and 64 (2–2,048) by EMT for anti-A in BG B; 64 (2–256) by TT and 64 (2–512) by EMT for anti-B in BG A. IgG titer was higher for both anti-A and anti-B by EMT 256 (64–2,048), 256 (32–2,048), respectively, than TT method 128 (64–1,024), 128 (16–2,048), respectively.

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Fig. 1

Isoagglutination titers on x-axis and frequency on y-axis of immunoglobulin G (IgG; A–D) by both methods, conventional tube technique (TT) and erythrocytes magnetized technology (EMT), for anti-B in blood group (BG) A, anti-A in BG B, and anti-A and B in BG O.

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Fig. 2

Isoagglutination titers on x-axis and frequency on y-axis of immunoglobulin M (IgM; A–D) by both methods, conventional tube technique (TT) and erythrocytes magnetized technology (EMT), each for anti-B in blood group (BG) A, anti-A in BG B, and anti-A and B in BG O.

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Fig. 3

Box plot showing frequency distribution, minimum, first quartile, median, third quartile and maximum immunoglobulin G (IgG; A) and immunoglobulin M (IgM; B) isoagglutinin titers of anti-B of blood group (BG) A, anti-A of BG B, anti-A of BG O, and anti-B of BG O obtained by the conventional tube technique (TT) and erythrocytes magnetized technology (EMT).

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For IgM, the median titer for anti-B in BG A and BG O was higher by EMT than TT: 32 (0–512) by EMT and 16 (1:256) by TT in BG A; 64 (8–512) by EMT and 48 (8–256) by TT in BG O. It was equal for anti-A in BG B by both methods 16 (1:2,048) by EMT and 16 (0:512) by TT method. For anti-A IgM titer in BG O was lower by EMT than TT method 32 (4:128) by EMT and 48 (8:256) by TT.

The agreements between the two methods are shown in ►Table 2. One twenty one (121/134, 90.3%) cases of IgG titer showed an agreement by both methods (within ± one titer difference). All the cases for IgG titers gave results within two titer differences by both methods. One hundred seven cases (107/116, 92.2%) and one hundred and fourteen cases (114/116, 98.2%) for IgM titer were within one and two titer differences, respectively, by both the methods. Fifty cases (50/134, 37.3%) in case of IgG and forty-one (41/116, 35.3%) in case of IgM titer were giving same results by both TT and EMT. For IgG, 53.7% cases gave higher titers, while only 9% gave lower titers by EMT as compared with TT. For IgM, 36.2% cases gave higher and 28.5% cases lower titers by EMT as compared with that by TT.

Discussion

The main findings of this study showed that the median titers and their range obtained by EMT were higher or equal to that obtained by the TT for all IgM and IgG ABO-antibodies in all BGs except anti-A IgM in BG O that was lower by EMT than TT method: 32 (4:128) by EMT and 48 (8:256) by TT. Around one-third of the cases showed similar results for IgG and IgM titer of ABO antibodies in all BGs. However, for IgG titer ABO antibodies by EMT around 50% samples gave higher titers than TT. In case of IgM ABO antibody titers, around one-third samples gave higher and one-third gave lower titer when EMT was compared with TT.

The significant interlaboratory variations in TT and availability of methods with higher sensitivity for ABO Ab titer measurement have led to increased use of techniques like EMT, CT, and FCM recently.^[13] Many clinicians, however, are unaware of these differences that arise due to use of different methods and take into consideration only the target titer obtained. There are many possible factors that may cause these differences to be produced, most importantly the interference of IgG and IgM measurement with each other. The anti-A and anti-B of BG B and A, respectively, are mostly IgM and small amounts of IgG. These antibodies are mostly IgG in persons with BG O. In a study comparing isoagglutinin titers by TT, EMT and microcolumn agglutination showed that EMT-based automated instrument showed higher titration level than the conventional tube hemagglutination technique. The median titers obtained by EMT in their study for IgG anti-B in BG A, anti-A in BG B, anti-A and anti-B in BG O were 16 (2–64), 16 (4–256), 2,048 (8–8,192), and 256 (16–8,192), respectively. The median titers obtained by EMT for IgM anti-B in BG A, anti-A in BG B, anti-A, and anti-B in BG O were 16 (2–64), 16 (4–256), 32 (2–256), and 16 (2–128), respectively. The titers for all antibodies were highest as measured by EMT except median titers of IgG, anti-A in BG B and anti-B in BG A, that were highest by microcolumn agglutination method.^[15] The agreements between two methods in their study were taken as the percentage of cases within one titer difference. The percentage of agreement for the conventional tube hemagglutination technique and EMT for IgM was 80% for anti-B of group A, 70% for anti-A of group B, 88% for anti-B of group O, and 60% for anti-A of group O, respectively. For IgG, the percentage of cases in agreement with the conventional tube hemagglutination technique and EMT for IgG was 83% for anti-B of BG A, 35% for anti-A of BG B, 0% for anti-B of BG O, and 3% for anti-A of BG O, respectively. Even on comparison of card method with TT and EMT, agreement results were comparable in case of IgM but low in IgG.^[17] Taking all BGs together, our study gave more than 90% results of IgG as well as IgM within one titer difference. Another study comparing five methods for anti-ABO titration on 50 BG O healthy donors, the median titers of IgG anti-A and anti-B antibodies by SPRCA were 64 (8–2,048) and 64 (4–512), respectively. These values were lower than ours in BG O individuals.^[18]

The testing for isoagglutinin titers depends on the temperature differences. IgM can be measured at room temperature and IgG after an incubation at 37°C that causes complement activation. But both IgM and IgG may react at room temperature and activate complements at 37°C leading to interferences in assay methods. When plasma is not treated with DTT or heated at 63°C for 10 minutes to inactivate IgM molecules, indirect agglutination titers may be a mix of IgM and IgG antibodies, reacting at 37°C. However, both IgG and IgM antibodies are likely to be active in humoral rejection, and cold-reacting (30°C) antibodies are usually not of clinical significance.^[19] In a study by Nayak et al, reduction in titers by DTT treatment in nearly 50% samples tested for both anti-A and anti-B titers as compared with the gel card titers read without the use of DTT indicating that there is a good amount of IgM type of anti-A and anti-B antibodies in the serum samples, which should be avoided before reporting the titers of candidates of ABO-incompatible transplants.^[18] For the conventional TT tests in our study, we have not used DTT. We used heat inactivation as a method as well as monospecific anti-IgG antibodies to remove the nonspecific IgM antibodies. Other studies have also used heat inactivation as a reliable method for the estimation of IgG isoagglutinins.^[16,19]

Few studies have also compared the performances of other methods for anti-A and anti-B titers. There are some studies comparing TT with gel method, one of which stated that gel is more sensitive,^[20,21] column agglutination results being approximately two and half fold higher (one more dilution) than that of conventional TT method.^[20]

Another study comparing the TT, gel card, and FCM for isoagglutinin titers showed that there were significant differences in the titers obtained by these methods and each method showed a different detection capacity for each ABO antibody depending on the BG tested. They showed that in BG O, the mean titer in gel IAT was significantly higher than that of tube IAT for anti-A. FCM with anti-IgM showed the highest titer compared with tube or gel method in all of the BGs.^[10] Another study comparing five different methods for anti ABO titration concluded that due to poor agreements between the results obtained by different methods, application of consistent and uniform method for titration throughout treatment of patient was recommended.^[18]

Since most of the methods other than TT are or can be automated, they reduce the risk of manual errors, using standardized protocols with higher precision and accuracy, and minimize manual work providing an increased throughput. Another advantage of using EMT above TT for titration is that pictorial result files are converted to quantitative results that can be compared with follow-up pictures of the patient results, thus helping to decide the course of management for the patient.

It has been established that the titers of IgG are clinically more significant.^[22] The titer of isoagglutinins helps in managing patients as well as determining their prognosis. The tolerable amount of isoagglutinin titer varies among hospitals or clinicians. Risk of antibody-mediated hyperacute rejection, successful transplant by reducing anti-A and anti-B levels by giving immunosuppressant, or procedures like immunoadsorption/plasma exchange can only be determined if an adequate and consistent method for measurement is used. Also, an increase in titer levels of more than two is considered to be significant while patient is on follow-up. Therefore, the standardization of test methods and target isoagglutinin titer can contribute to increasing success rates in cases of transplantation.

In conclusion, the EMT-based automated instrument, QWALYS-3, when compared with conventional test tube method does not give similar results and may vary by one titer dilution in majority (around 90%) of the cases. Therefore, the use of consistent method along with clinical correlation of the isoagglutinin titer is a must for patient management.