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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 6  |  Issue : 2  |  Page : 150-155

The evaluation of M-TRAP technology in detection of ABO groups and its subgroups and its efficacy as a point-of-care test for blood grouping


1 Department of Pathology, UN Mehta Institute of Cardiology and Research Centre, Ahmedabad, Gujarat, India
2 Department of IHBT, BJ Medical College, Civil Hospital, Ahmedabad, Gujarat, India

Date of Submission07-Jan-2021
Date of Acceptance25-Sep-2021
Date of Web Publication30-Nov-2021

Correspondence Address:
Dr. Sangita D Shah
Department of IHBT, BJ Medical College, Civil Hospital, Ahmedabad, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/gjtm.gjtm_4_21

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  Abstract 


Background and Objectives: Accurate ABO blood grouping including detection of subgroups is a core component in transfusion medicine to prevent adverse transfusion reactions. This article highlights the importance of using M-TRAP technology in a pad-based platform as a point-of-care test for offsite blood donation camps, pre release of blood bags from blood center and at patient's bedside for rapid evaluation of blood groups as compared to conventional methods. Methods: ABD-PAD works on principal of membrane trapping technology used for the evaluation of blood group and their subgroup as a point of care test. Result: Total 1,03,987 blood group test were performed, of which 71,885 of patients' and 32,102 of donors' by EMT (Model – QWALYS 3, Manufacturer – Diagast, France). Among them, 4516 blood group discrepancies were detected. Out of 4516 blood group discrepancies, 17 (00.38%) group discrepancies were due to ABO subgroups. Among them, 13 (76.47%) were subgroup A and 4 (23.53%) were subgroup B. These subgroups were detected by M-TRAP technology. Conclusion: The use of M-TRAP technology (ABD PAD) is a reliable method for manual blood grouping and is useful to detect blood group at patient's bedside, outside blood donation camp, and at the time of issue of blood from blood center.

Keywords: ABD pad, ABO grouping, ABO subgroups, M-TRAP technology


How to cite this article:
Patel TR, Shah SD, Bhatnagar N, Shah MC, Tripathi S, Soni S, Mathew AM, Ahuja T. The evaluation of M-TRAP technology in detection of ABO groups and its subgroups and its efficacy as a point-of-care test for blood grouping. Glob J Transfus Med 2021;6:150-5

How to cite this URL:
Patel TR, Shah SD, Bhatnagar N, Shah MC, Tripathi S, Soni S, Mathew AM, Ahuja T. The evaluation of M-TRAP technology in detection of ABO groups and its subgroups and its efficacy as a point-of-care test for blood grouping. Glob J Transfus Med [serial online] 2021 [cited 2022 Aug 10];6:150-5. Available from: https://www.gjtmonline.com/text.asp?2021/6/2/150/331610




  Introduction Top


The ABO blood group system was discovered by Karl Landsteiner at the beginning of the 20th century and is the most important system for clinical transfusion medicine. Depending upon the person's ABO blood group, naturally occurring immunoglobulin M (IgM) anti-A and/or anti-B may be present in serum, constituting a major barrier against ABO-incompatible blood transfusion and organ transplantation.[1] Identification of subgroups of A, e.g., A2, A3, Aend, Ax, Am, Ay, and Ael,[2] and subgroups of B, e.g., B3, Bx, and Bel,[3] is important because these donors may be mistyped as group O individuals. Wrongly grouped as O, weak subgroups of A or B red cells (if transfused to O group individuals) can show decreased survival. This is due to naturally occurring anti-A and anti-B antibodies in the latter.[4] All the agglutinins of the ABO system fix complement and are capable of causing intravascular hemolysis of incompatible red cells. For these reasons, an error in ABO grouping of a patient or donor could turn out to be fatal during blood transfusion process.

Transfusion safety depends on a series of strictly inter-related processes among which pretransfusion tests have a predominant role.[5],[6] In recent years, some of the new technologies that integrate the classical techniques in immunohematology have become valid for improving the safety of transfusions.[7]

Predonation blood grouping of the donor is the preliminary result which is repeated again during pretransfusion testing from the donor unit and at the time of issuing the blood for transfusion to the patient.[8],[9] The current practices of ABO and RhD blood group testing include conventional test tube technique (TTT), column agglutination technique (CAT), solid-phase technique, erythrocyte-magnetized technique (EMT), slide/tile method, and membrane trapping technology (M-TRAP technology). Of all these six techniques, the latter two techniques are more appropriate for offsite testing of predonation ABO and RhD blood grouping of the donor and pretransfusion ABO and Rh “D” blood grouping of recipients. Besides the test reagents, other methods require additional equipment such as centrifuge, incubator, and reader for the testing procedure. Slide/tile method and M-TRAP technology (ABD–PAD) are the only portable, quick, and simple method. M-Trap is feasible and appropriate for offsite blood donation drive, bedside blood group confirmation and repeat grouping at the time of issue of blood. However, only cell grouping can be performed by these methods, and it should be reconfirmed by other techniques which include both cell and serum grouping.

In this study, we evaluated a new manual pretransfusion testing technique, the ABD–PAD working with M-TRAP technology by comparing its results to those of the standard manual process and other technologies. The purpose of validation is to test the competence of a new M-TRAP technology in detection of subgroups.[10],[11]

Aims and objectives

  1. Check the accuracy of M-TRAP technology
  2. Comparison of M-TRAP technology with other techniques
  3. Ability of M-TRAP technology in detection of subgroups
  4. Advantage of using M-TRAP technology for offsite blood donation drive, bedside blood group confirmation and at the time of issue of blood from blood center.



  Materials and Methods Top


During the study period from November 2019 to October 2020, Department of Immunohaematology and Blood Transfusion affiliated with B. J. Medical College, Ahmedabad, Gujarat, and tertiary care civil hospital has performed total 1,03,987 blood groups, among them 71,885 of patients' and 32,102 of donors' by erythrocyte-magnetized technology (EMT) (QWALYS 3, Diagast, France). Among them, 4516 ABO blood group discrepancies were detected. Among them, more number of discrepancies (3097) were detected in newborns <6 months' age group, having problem in reverse group because of the absence of naturally occurring ABO antibodies. The rest of the discrepancies were due to autoantibodies, autoimmune diseases, infections, Bombay blood group, old age, and subgroups. All the discrepant samples were again checked by gold standard TTT, CAT, and M-TRAP technology. Total 17 subgroups were detected and confirmed by adsorption and elution method.

Erythrocyte-magnetized technology (Model - QWALYS 3, manufacturer - Diagast, France)

The principle of this technology is based on the use of magnetic particles. During the analyzing process, these particles are fixed on the surface of red blood cells by adsorption or by binding on the GPA glycoprotein. When they are exposed to a magnetic field in the microplate, these cells migrate to the bottom of each well. Thereby, it aggravates the antigen–antibody reaction. The centrifugation step is completely eliminated during the analytical process.

Test tube technique

The blood grouping by test tube method is based on the principle of agglutination and it is a gold standard technique. The results were interpreted macroscopically and microscopically and graded as 0, weak, 1+, 2+, 3+, or 4+. The ABO types were recorded according to the results of forward and reverse typing.

Column agglutination technique (ABO Diaclon, Bio-Rad)

In this method, there are 6 microtubes in one gel card. Each microtube consists of reaction chamber that narrows to become a column containing Sephadex gel suspended in a buffer solution. The gel is premixed with the antisera. It acts as a sieve. Large agglutinates remain on or near the top of the gel interface. Smaller agglutinates pass through the gel, depending on the size. Unagglutinated cells pass to the base of the microtube. The results were interpreted macroscopically and graded as 0, weak, 1+, 2+, 3+, or 4+.

Adsorption and elution technique

Polyclonal antisera of human origin from group B, group A, and group O individuals were used for adsorption to determine ABO subgroups. The technique was performed as described in AABB Technical Manual 20th Edition. Heat elution using 6% bovine serum albumin was done at 56°C for 10 min, and the elute was tested against three pooled reagent cells (A and B). Agglutination was noted on immediate spin in few test tubes, whereas tubes not showing agglutination in the above step were incubated at 37°C for 60–90 min. In these tubes, agglutination was observed after adding anti-human globulin reagent.

M-TRAP technology

In this technology, the monoclonal anti-A, anti-B, and anti-D antibodies are covalently adhere on the porous membrane. In addition to red cells to these antibodies, there will be initiation of the immunobinding reactions. Only the red blood cells having the corresponding antigen are fixed into the membrane revealing immediate reaction, symbolized by the red color of the red cells bound to the antibodies. Otherwise, the red cells will not be retained, which indicates a negative reaction with no color appearing. Whenever subgroup is present, it shows light color [Figure 1].
Figure 1: Sample number 32923 shows light stain with anti-A in reaction well, suggesting subgroup of A. (a) ABD PAD. (b) Buffer

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Procedure

  1. Activation – One drop of buffer is dispensed to hydrate the antisera
  2. Blood dispensing – The blood sample is added to the well
  3. Revelation – The well is filled with the buffer revealing the reaction if antigen is present.



  Results and Observation Top


From November 2019 to October 2020, Department of Immunohaematology and Blood Transfusion affiliated with B. J. Medical College, Ahmedabad, Gujarat, and tertiary care civil hospital has performed total 1,03,987 blood groups, among them 71,885 of patients' and 32,102 of donors' by erythrocyte-magnetized technology (EMT) (QWALYS 3, Diagast, France). Among them, 4516 blood group discrepancies were detected. Out of 4516 blood group discrepancies, 17 (00.38%) group discrepancies were due to ABO subgroups.

Among them, 13 (76.47%) were subgroup A and 4 (23.53%) were subgroup B. As compared to the subgroup of A, their counterparts in the B blood group were less.

The EMT and CAT techniques are almost comparable except one difference that detection of IgM antibodies is not possible in EMT. Although TTT is a gold standard method, it has many limitations.

We cannot compare M-TRAP technology with these three methods as reverse grouping is not possible in M-TRAP technology. Only purpose of comparison is to detect the accuracy of M-TRAP technology in forward grouping. However, M-TRAP technology is very appropriate for offsite use as it is based on portable PAD.

There are many advantages of M-TRAP technology over slide/tile method.


  Discussion Top


ABD PAD based on M-TRAP technology was received in our department in November 2019. As a routine protocol of kit verification, we started comparing the results of ABD PAD (M-TRAP technology) with other methods. Initially, we tested 200 samples for blood grouping by EMT, CAT, TTT, and M-TRAP technology. The result of M-TRAP technology was comparable with other standard methods. Among those 200 samples, forward/reverse discrepancy was detected in two samples, by standard (EMT, TTT, and CAT) methods. M-TRAP technology was showing light color in the reaction well suggesting possibility of subgroup. It was later on confirmed by adsorption and elution technique which indicated the possibility of using M-TRAP technology in the detection of subgroups. This study was undertaken to evaluate the performance of the M-TRAP technology in detection of subgroup in the blood center of tertiary care hospital.

During the said period our department performed a total 1,03,987 blood groups, among them 71,885 were patients and 32,102 were donors. Among them, 4516 group discrepancies were detected. The results were described as the reaction grades in EMT. The results obtained with the EMT system and the manual method were interpreted as discrepant according to the following criteria. (1) Forward typing results showing a ≤2+ reaction grade (weak red cell reaction) and a mixed field reaction were regarded as discrepant. (2) Reverse typing results showing unexpected reaction were regarded as discrepant because serum reactions are usually weaker than cell reactions.[12],[13].(3) Results showing extra cell or serum reactions (including any reaction grade) were regarded as discrepant results.

All the discrepant samples in EMT were run in other three methods, CAT, TTT, and M-TRAP technology. The results were almost similar in CAT, TTT, and EMT. The result of M-TRAP technology was comparable to the above methods in forward grouping in most of the samples. In 17 samples, M-TRAP technology was showing the possibility of subgroups. From total 4516 discrepant results, 3097 (68.57%) results showing absent serum reaction were neonatal patients <6 months' age group due to lack of antibody development in this age group. 1038 (22.98%) group discrepancies were due to autoantibodies and 346 (7.66%) due to autoimmune diseases. Infections, Bombay blood group, and old age were accounted for 5 (00.11%), 3 (00.07%), and 10 (00.23%) discrepant results, respectively. All of them were resolved by different methods. Seventeen (00.38%) group discrepancies were due to subgroup [Table 1].
Table 1: Total discrepancy from total 4516 discrepancies

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In most of the discrepancies due to subgroups, forward grouping showed Grade I, Grade II, or absent reaction with the respective antisera and reverse grouping showed reactions in B-cell (in case of subgroup A) and reactions in A-cell (in case of subgroup B). In regular A or B group, there were always Grade III or IV reactions in forward grouping. This type of discrepant result was suggestive of subgroup. All these subgroups were clearly reported positive by M-TRAP technology. We have confirmed them by adsorption and elution technique.

From total 103,987 samples tested for grouping, we found subgroups in 17 samples. Among them, 13 (76.47%) were subgroup A and 4 (23.53%) were subgroup B. As compared to the weak variants of A, their counterparts in the B blood group were less [Table 2]. The frequency of subgroups is 0.000163 in our study. The frequency of subgroup A is 0.000125 and the frequency of subgroup B is 0.000038 in our study [Table 3]. Another study done in North India also showed more frequency of subgroup A than subgroup B.[4] The phenotypic frequencies of these weak subgroups differ between ethnic regions. Weak B subgroups were found at a higher frequency in our population than those seen in French donors.[14]
Table 2: Subgroup detected

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Table 3: Frequency of subgroups in our study from total 103,987 blood groups performed

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The ABO and RhD are the two most significant blood group systems in transfusion medicine[15] discovered a century ago, but the occurrence of its weaker variants due to heterogeneity of the A and B alleles still poses an enigma for immunohematologists because of two characteristics of the ABO system. First, unlike other blood group systems, antibodies of the ABO system are present in the serum of almost every person who does not have the corresponding antigen. Second, all the agglutinins of the ABO system fix complement and are capable of causing intravascular hemolysis of incompatible red cells. For these reasons, an error in ABO grouping of a patient or donor could turn out to be fatal during blood transfusion process. Hence, these blood groups are being tested for all healthy blood donors as well as all patients prior to blood transfusion to ensure that the patients are given the right blood for transfusion.[16]

Identification of the subgroups is important because these donors may be mistyped as group O individuals. Wrongly grouped as O, weak subgroups of A or B red cells (if transfused to O group individuals) can show decreased survival. This is due to naturally occurring anti-A and anti-B antibodies in the latter. Similarly, since Ax individuals almost always have anti-A1 antibodies in their serum, they can lead to fatal transfusion reactions on transfusing their whole blood or plasma to group A individuals.[17]

During our journey of this study, we also compared the different methods of blood grouping. From [Table 4], we can interpret that the EMT and CAT techniques are almost comparable except one difference that detection of IgM antibodies is not possible in EMT. Although TTT is a gold standard method, there are many limitations such as elution of low-affinity antibodies during washing, variability in the red cell concentrations, improper cell-to-serum ratio, lack of consistency in reporting the results due to interobserver variability, multiple washing steps, and larger volume of sample required.[18],[19]
Table 4: Comparison of different methods

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We cannot compare M-TRAP technology with these three techniques as reverse grouping is not possible in M-TRAP technology. The only purpose of comparison is to detect the accuracy of M-TRAP technology in forward grouping. M-TRAP technology is a very good technique with a sensitivity and specificity of 100%. Few advantages of this technology over previous methods are:

  1. It is a good and user-friendly technique to use in outdoor blood donation camp
  2. As it detects weaker antigens, it is very useful in detection of weaker expression of subgroups of blood group A and B
  3. The method is also very simple with only three steps
  4. Very small sample volume is required
  5. Uniformity of results in repeat testing is observed
  6. Results are clear and easily readable macroscopically, so there is no chance of subjective error
  7. Results are conserved after use
  8. Accurate results can be obtained even up to 40°C atmospheric temperature and 90% humidity.


M-TRAP technology is very appropriate for use in outdoor campsites because it is a portable PAD. There is no need of electric power or any other equipment. Accurate results can be obtained even up to 40°C and 90% humidity. The most important factor is that it detects weaker expression of blood groups/subgroups of blood group A and B. Provisional donor card which mentions the subgroup of the donor (if present) can be issued which can be confirmed at the blood center by further evaluation. We can also explain to the donor about his/her special type of blood group, thereby preventing major transfusion reactions.

To prevent major transfusion reactions, prerelease check of blood group and bedside blood group confirmation also play a major role.[20] Because grouping at the time of issue is the final check and in some situations like sample discrepancy, bedside blood grouping is very important where M-TRAP technology can be incorporated.

Slide/tile method is the commonly used portable and simple method that is feasible and appropriate for offsite donation drive, bedside blood group confirmation, and blood group at the time of issue. However, this method is less sensitive than M-TRAP technology and drying up of the reaction mixture can cause aggregation of cells giving false-positive results as well as weaker results are difficult to interpret. Moreover, this slide/tile method, though feasible, has its own limitations like the requirement of the testing reagents to be brought to offsite where it may not be kept in the optimal storage temperature of 2°C–6°C, especially in the tropical countries like India. Furthermore, the slide/tiles that have already been used for ABO and RhD testing will be contaminated with donors' blood and these will pose a risk of contamination to the operator as well as the environment [Table 5].
Table 5: Comparison of membrane trapping technology (ABDPAD) with slide/tile method (for blood grouping at bedside, outdoor camp, and at the time of issue)

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Moreover, this test kit is stable at room temperature and the procedure is simple and user-friendly. Besides, it does not need any additional reagents and can be kept or stored at 2°C–30°C. This method is easy to use and does not require any special equipment and technical expertise. The results can be read in 30 s. Interestingly, M-TRAP technology results compare with conventional agglutination method and the end point of M-TRAP technology results are stable and can be read subjectively by the operator. Especially at outdoor blood donation camp, bedside blood grouping, and at time of issue. It has a low contamination risk to the operator and environment. Thus, this test kit is very appropriate for field use.

Limitations of the study

This study has its limitations as we were not able to confirm the subgroups by molecular methods.


  Conclusion Top


The use of M-TRAP technology (ABD PAD) is a reliable method for manual blood grouping. It can be used in a very simple way. The results are easy to read and interpret and the test is fast and safe.

It is very useful to detect blood group at patient's bedside, outside blood donation camp, and at the time of issue of blood from blood center.

It detects subgroup accurately, so we can detect subgroups of donors at the campsite itself and can inform personally about their group. We can give a special blood group card clarifying their donor as well as recipient status.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Mujahid A, Dickert FL. Blood group typing: From classical strategies to the application of synthetic antibodies generated by molecular imprinting. Sensors (Basel) 2015;16:E51.  Back to cited text no. 8
    
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Garretta M, Muller A, Salmon C. Fréquéncé réelle dés phénotypés B faible. Rév Fr Transfus Immunohématol 1978;21:193-200.  Back to cited text no. 13
    
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Bhagwat SN, Sharma JH, Jose J, Modi CJ. Comparison between conventional and automated techniques for blood grouping and crossmatching: Experience from a tertiary care centre. J Lab Physicians 2015;7:96-102.  Back to cited text no. 15
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