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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 1  |  Issue : 2  |  Page : 46-50

Verification of column agglutination technology with conventional tube technology for naturally occurring antibody titration


Department of Transfusion Medicine, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, Maharashtra, India

Date of Web Publication6-Sep-2016

Correspondence Address:
Nidhi Mehta
Department of Transfusion Medicine, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2455-8893.189846

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  Abstract 


Background: Pre-transfusion compatibility testing is performed in order to prevent transfusion of incompatible donor red cells that might result in an immune mediated hemolytic transfusion reaction, thereby forming a critical element in the process of transfusion to enhance blood safety. Titers are generally determined with a semi-quantitative assay by serial double-fold dilution. Several different techniques have been employed for titration, of which the conventional tube test (CTT) and column gel card test –Column Agglutination Technique (CAT) are the most frequently used. Historically, CTT has been used as the standard technique for immunohaematological studies, such as direct antihuman globulin test for the diagnosis of autoimmune haemolytic anaemia and in screening for specific antibodies in transfusion medicine.
Study Design and Methods: For this study, 51 plasma samples from donors were subjected to antibody titration using the two techniques CAT and CTT. For the purpose of standardization and reproducibility of results, all 51 samples were tested by two technicians and the results obtained were a total of 70 antibody titers. Dilutions were continued to a titer of 1:1024 with the aim of allowing for at least two negative tubes beyond the titer end-point. The statistical analysis of the data has been done in MS-Excel.
Results: Out of the 70 titer samples, 57 samples showed identical titer values in the methods performed by both the technicians. 8 samples showed titer values higher in Technician 2 by one dilution as compared to Technician 1, whereas 5 samples showed titer values lower in Technician 2 when compared to Technician 1 (one dilution low in 4 samples and two dilutions low in 1 sample).
Conlcusion: CAT titers demonstrated up to a twofold difference as compared to CTT titres, as demonstrated in the figures presented. This disparity is not unusual and can be attributed to the lack of standardization which can be reduced by Column Agglutination Technology. CAT uses specific volumes of red blood cells and the reagents are dispensed by pipettes. Also, the procedure is technically simpler due to the elimination of washing steps and results in well-defined end points. Column Agglutination Technique offers not only sensitivity, but also the best titer turnaround time, eliminating almost 45 minutes of incubation period alone. The results of testing by CAT are comparable to CTT but have the advantages of ease of performance, better standardization of cells, most importantly stable results and reproducibility. Implementation of antibody titers by the gel method would clinically benefit the management of ABO incompatible solid-organ transplant patients.

Keywords: Antibodies, antibody titration, column agglutination technique, conventional tube technology, standardization, validation


How to cite this article:
Mehta N, Chakraborty IR, Rane M, Ambre V. Verification of column agglutination technology with conventional tube technology for naturally occurring antibody titration. Glob J Transfus Med 2016;1:46-50

How to cite this URL:
Mehta N, Chakraborty IR, Rane M, Ambre V. Verification of column agglutination technology with conventional tube technology for naturally occurring antibody titration. Glob J Transfus Med [serial online] 2016 [cited 2019 Jan 16];1:46-50. Available from: http://www.gjtmonline.com/text.asp?2016/1/2/46/189846




  Introduction Top


Pretransfusion compatibility testing is performed to prevent the transfusion of incompatible donor red cells that might result in an immunomediated hemolytic transfusion reaction, thereby forming a critical element in the process of transfusion to enhance blood safety. ABO and Rh blood grouping of recipients and donors and cross-matching between donor and recipient in indirect anti-globulin phase to detect clinically significant antibodies are integral steps of the compatibility testing.[1],[2] The titer of an antibody represents the strength of that antibody's response to a specific antigen. Titers are generally determined with a semi-quantitative assay by serial double-fold dilution.[3] Several different techniques have been employed for titration, of which the conventional tube technology (CTT) and the column gel card test (column agglutination technique [CAT]) are the most frequently used. Historically, CTT has been used as the standard technique for immunohematological studies, such as direct antihuman globulin (AHG) test for the diagnosis of autoimmune hemolytic anemia and in screening for specific antibodies in transfusion medicine.[4],[5],[6],[7],[8]

Based on CTT, a number of clinically relevant cutoff values have been set.[9] The traditional CTT method that is used for antibody titration is time- and labor-intensive, prone to technical errors due to complexity in performing the procedure, requires experienced staff to read results, and gives only one chance to read the reaction without disturbing the cell button.[10] Furthermore, this method demonstrates low reproducibility [11] and variable sensitivity,[12] and has been proven to be difficult to standardize between different laboratories.[12],[13],[14] The introduction of newer techniques such as CAT, solid phase red cell adherence assays, and erythrocyte-magnetized technique has tried to overcome these shortcomings, bring about an improvement in the quality of testing and reproducibility of results.[13] CAT is an easy and sensitive technique that requires no red cell washing and uses gel filtration media impregnated with an AHG reagent to induce agglutination.[15],[16] The simplicity factor of CAT is further enhanced with its ability to provide for a stable end point for testing and has encouraged many blood banks and transfusion departments to gradually adopt this technique.[17],[18]

Our study has been formulated on the basis of two hypotheses: (1) Sensitivity and efficiency of CAT is superior compared to conventional tube technique (CTT). (2) The results of CAT are reproducible.


  Materials and Methods Top


Assessment of baseline anti-ABO antibody titers utilizes two methods of titrations:

  • The conventional tube technology CTT (AABB method)
  • The column agglutination technology CAT: BioRad Gel cards.


For this study, 51 plasma samples from donors were subjected to antibody titration using the two techniques CAT and CTT. Out of this, 16 samples were A-positive, 16 samples were B-positive, and 19 samples were O-positive. For the purpose of standardization and reproducibility of results, all 51 samples were tested by two technicians. Since Group O samples contained both anti-A and anti-B titers, the total number of titer samples were seventy as compared to the total number of samples that were tested for CTT and CAT.

Specimen

  • Anticoagulated sample (ethylenediaminetetraacetic acid sample) and clotted sample.


Reagents

  • Donor's red cell suspension 1% LISS
  • Known pooled reagent red cell suspension 2-5%
  • Commercially available AHG antisera
  • NS (Normal Saline)
  • LISS solution.


Master dilution technique for titration studies

A row of test tubes [Figure 1] were labeled according to the serum dilution, usually 1:1 (Neat) through 1:512. Dilutions were continued to a titer of 1:1024 with the aim of allowing for at least two negative tubes beyond the titer end point.
Figure 1: Scheme for serial dilutions for the titration of serum

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Test tube titration of serum for immunoglobulin M and immunoglobulin G

Out of the 200 µl of diluted test serum, 50 µl of test serum is pipetted into a clean set of 10 test tubes, along with 50 µl of 2–5% of red cell suspension in each.[19] The tubes were then kept at room temperature (RT) for 15 min. On completion of 15 min, the test tubes kept at RT are centrifuged at 1000 rpm for 1 min.

The remaining 100 µl of diluted serum is incubated at 37°C for 15 min along with 100 µl of 2–5% red cell suspension. Each tube is then washed thrice with normal saline at 3000 rpm, and the supernatant is removed after the last wash. Two drops of AHG antisera are added to each tube, mixed, and centrifuged at 1000 rpm for 1 min. The cell button was re-suspended by gentle agitation, and agglutination was graded microscopically with the highest dilution showing +1 agglutination.

Gel card titration of serum for immunoglobulin M and immunoglobulin G

Two saline (NaCl) gel cards for immunoglobulin M titer and two AHG immunoglobulin G (IgG) gel cards for IgG titers are selected, and the micro columns are labeled corresponding to the dilution tubes, starting from 1:2,1:4, to 1:1024.

Fifty microliters of 0.8% donor's red cell suspension is dispensed into separate micro columns of the gel cards, followed by the addition of 25 µl of serially diluted serum mixture from the labeled test tubes into the corresponding columns of gel cards.

The saline gel cards were kept at RT for 15 min, whereas the AHG IgG gel cards were kept in the incubator at 37°C for 15 min. On completion of 15 min, both the gel cards were centrifuged. The test results were examined and the reactions were graded macroscopically with the highest dilution showing +1 agglutination.

Statistical analysis

The statistical analysis of the data has been done in MS-excel.


  Results Top


A total of 51 samples were included in the study. Between both methods, titer values ranged from 1:2 to 1:512. The end point of titrations by CTT with their corresponding values by CAT performed by both the technical officers is shown in [Table 1],[Table 2],[Table 3],[Table 4].
Table 1: Antibody titer by CAT compared to CTT at Room Temperature

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Table 2: Antibody titer by CAT compared to CTT at 370C AHG phase

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Table 3: Antibody titer by CAT compared to CTT at Room Temperature

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Table 4: Antibody titer by CAT compared to CTT at 370C AHG phase

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Results by first technician

Analysis of the data from [Table 1] reveals that at RT, 29 samples had identical titer values by both methods. The CAT titers were higher that CTT titer for 41 samples (one dilution higher in 40 samples and two dilutions higher in 1 sample).

Similarly, the data from [Table 2] reveal that at 37°C AHG phase, 17 samples had identical titer values by both methods. The CAT titers were higher than CTT for 51 samples (one dilution higher in 49 samples and two dilutions higher in two samples). However, the CAT titers showed one dilution lower in two samples as compared to CTT.

Results by second technician

Analysis of the data from [Table 3] reveals that at RT, 23 samples had identical titer values by both methods. The CAT titers were higher that CTT titer for 46 samples (one dilution higher in 43 samples and two dilutions higher in 3 sample).

Similarly, the data from [Table 4] reveal that at 37°C AHG phase, 17 samples had identical titer values by both methods. The CAT titers were higher than CTT for 47 samples, with one dilution in all of them. However, the CAT titers showed one dilution lower in two samples as compared to CTT.

The magnitude of difference between the methods performed by the two technicians was stable across the range of titer values and has been shown in [Table 5].
Table 5: Interpersonal validation of 70 CAT titers at 37 0C AHG Phase

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Out of the 70 titer samples, 57 samples showed identical titer values in the methods performed by both the technicians. Eight samples showed titer values higher in technician 2 by one dilution as compared to technician 1, whereas five samples showed titer values lower in technician 2 when compared to technician 1 (one dilution low in four samples and two dilutions low in one sample)


  Discussion Top


Techniques for antibody titration have undergone extensive development, and several methods are routinely employed for ABO blood group antibody titration, with CTT and CAT being most frequently used. CTT is considered the classic test for antibody titration, but is time-consuming because of extensive cell washing required, and is difficult to standardize as compared to CAT which shows simplicity, reproducibility, and sensitivity. This study compared the CTT method with CAT with respect to sensitivity and reproducibility.

Although less than half of samples in AHG phase showed identical results when performed by both technicians, relative to CTT method by CAT was consistently higher by one to two dilutions in majority tested. While CAT titers were slightly higher on average, this difference was not statistically significant. This result was found to be as comparable to the results of the study performed by Adriaansen and Perry.[10] The variance noted in the results can be attributed to the difference in the method of diluting the sample, as well as difference in serologic testing method or both.

Reproducibility has been found to be problematic in this study, in spite of the technical staff belonging to the same laboratory. The end point chosen for tube technique reactivity varies based on the discerning ability of individual. The same problem has been noted in the study by AuBuchon et al.[20] Additional sources of variability in titration using tube technique have been postulated in the study conducted by Cid et al.[21] These could include the reagents utilized, including the type of tubes used and the sources of the gel cards. Although all materials used in this study were licensed and approved by commercial manufacturers, some differences between lots or manufacturer could exist even within the purview of regulatory agency. A control sample of known antibody concentration ideally should be tested repeatedly by both methods to determine the spread of results. The degree of variation in results between the two methods can thereby compared, which can successfully lead to the establishment of standardization system for titration.[21]

CAT titers demonstrated up to a 2-fold difference as compared to CTT test tube, as demonstrated in the figures presented. This disparity is not unusual and can be attributed to the lack of standardization which can be reduced by CAT. CAT uses specific volumes of red blood cells, and the reagents are dispersed by pipettes. In addition, the procedure is technically simpler due to the elimination of washing steps and results in well-defined end points.


  Conclusion Top


CAT offers not only sensitivity, but also the best titer turnaround time, eliminating almost 45 min of incubation period alone. The results of testing by CAT are comparable to CTT, but have the advantages of ease of performance, better standardization of cells, and most importantly, stable results and reproducibility. Implementation of antibody titers by CAT would clinically benefit the management of ABO incompatible solid organ transplant patients.[4]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Downes KA, Shulman IA. Pretransfusion testing. In: Roback JD, editor. Technical Manual. 17th ed. Bethesda, MD: American Association of Blood Banks; 2011. p. 437-62.  Back to cited text no. 1
    
2.
Carlson TH, editor. Standards for Blood Banks and Transfusion Services. 27th ed. Bethesda, MD: American Association of Blood Banks; 2011.  Back to cited text no. 2
    
3.
AuBuchon JP, de Wildt-Eggen J, Dumont LJ. Reducing the variation in performance of antibody titrations. Arch Pathol Lab Med 2008;132:1194-201.  Back to cited text no. 3
    
4.
Shirey RS, Cai W, Montgomery RA, Chhibber V, Ness PM, King KE. Streamlining ABO antibody titrations for monitoring ABO-incompatible kidney transplants. Transfusion 2010;50:631-4.  Back to cited text no. 4
    
5.
Cooling LL, Downs TA, Butch SH, Davenport RD. Anti-A and anti-B titers in pooled group O platelets are comparable to apheresis platelets. Transfusion 2008;48:2106-13.  Back to cited text no. 5
    
6.
Coombs RR, Mourant AE, Race RR. A new test for the detection of weak and incomplete Rh agglutinins. Br J Exp Pathol 1945;26:255-66.  Back to cited text no. 6
    
7.
Coombs RR. Historical note: Past, present and future of the antiglobulin test. Vox Sang 1998;74:67-73.  Back to cited text no. 7
    
8.
Das SS, Chaudhary R, Khetan D. A comparison of conventional tube test and gel technique in evaluation of direct antiglobulin test. Hematology 2007;12:175-8.  Back to cited text no. 8
    
9.
Kamesaki T, Oyamada T, Omine M, Ozawa K, Kajii E. Cut-off value of red-blood-cell-bound IgG for the diagnosis of Coombs-negative autoimmune hemolytic anemia. Am J Hematol 2009;84:98-101.  Back to cited text no. 9
    
10.
Adriaansen MJ, Perry HE. Validation of column agglutination technology for blood group alloantibody titration. N Z J Med Lab Sci 2013;67:92-6.  Back to cited text no. 10
    
11.
Bruce M, Chapman JF, Duguid J, Kelsey P, Knowles S, Murphy M, et al. Addendum for guidelines for blood grouping and red cell antibody testing during pregnancy. BCSH Transfusion Task Force. Transfus Med 1999;9:99.  Back to cited text no. 11
    
12.
AuBuchon JP, de Wildt-Eggen J, Dumont LJ; Biomedical Excellence for Safer Transfusion Collaborative; Transfusion Medicine Resource Committee of the College of American Pathologists. Reducing the variation in performance of antibody titrations. Vox Sang 2008;95:57-65.  Back to cited text no. 12
    
13.
Bajpai M, Kaur R, Gupta E. Automation in immunohematology. Asian J Transfus Sci 2012;6:140-4.  Back to cited text no. 13
[PUBMED]  Medknow Journal  
14.
Rumsey DH, Ciesielski DJ. New protocols in serologic testing: A review of techniques to meet today's challenges. Immunohematology 2000;16:131-7.  Back to cited text no. 14
    
15.
Nathalang O, Chuansumrit A, Prayoonwiwat W, Siripoonya P, Sriphaisal T. Comparison between the conventional tube technique and the gel technique in direct antiglobulin tests. Vox Sang 1997;72:169-71.  Back to cited text no. 15
    
16.
Lapierre Y, Rigal D, Adam J, Josef D, Meyer F, Greber S, et al. The gel test: A new way to detect red cell antigen-antibody reactions. Transfusion 1990;30:109-13.  Back to cited text no. 16
    
17.
Dittmar K, Procter JL, Cipolone K, Njoroge JM, Miller J, Stroncek DF. Comparison of DATs using traditional tube agglutination to gel column and affinity column procedures. Transfusion 2001;41:1258-62.  Back to cited text no. 17
    
18.
Bromilow IM. Gel techniques in blood group serology. Med Lab Sci 1992;49:129-32.  Back to cited text no. 18
    
19.
Roback JD, editor. Technical Manual. 17th ed. Bethesda: American Association of Blood Banks; 2011. p. 907.  Back to cited text no. 19
    
20.
AuBuchon JP, de Wildt-Eggen J, Dumont LJ; Biomedical Excellence for Safer Transfusion Collaborative; Transfusion Medicine Resource Committee of the College of American Pathologists. Reducing the variation in performance of antibody titrations. Arch Pathol Lab Med 2008;132:1194-201.  Back to cited text no. 20
    
21.
Cid J, Nogués N, Montero R, Hurtado M, Briega A, Parra R. Comparison of three microtube column agglutination systems for antibody screening: DG Gel, DiaMed-ID and Ortho BioVue. Transfus Med 2006;16:131-6.  Back to cited text no. 21
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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