Removal of Antibodies from Direct Antiglobulin Test Positive Red Blood Cells: A Comparison of Three Elution Methods

Vijayalakshmi Kuttatha, Biju Georgeb, Suji Sudhakarc

a. Department of Transfusion Medicine, Regional Cancer Centre, Thiruvananthapuram, Kerala, India; b. Department of Community Medicine, Medical College, Calicut, Kerala, India; c. Department of Medical Laboratory Technology, Kannur University, Kannur, Kerala, India*

Corresponding Author: Dr. Vijayalakshmi Kuttath, Department of Transfusion Medicine, Regional Cancer Centre, Thiruvananthapuram, Kerala, India. Email:
Published on 26th June, 2015


Irregular antibodies of the immunoglobulin G type or complement components may be seen in several hematological conditions either freely in the serum or attached on the red cell surface. The red cell coated antibodies test positive in direct antiglobulin test (DAT) test and may be auto or alloantibody in nature. Elution of these antibodies from the red cell is essential before it can be adequately characterized. Furthermore, the intact red cell that remains after elution can be used for antigenic phenotyping. From the several eluting reagents available, the method of choice should be one that uses a minimum of the sample, gives quick and maximum elute, with no damage to the red cell membrane. Three eluting agents, namely heat elution, cold glycine hydro chloric acid (HCl), and glycine (HCl) ethylenediaminetetraacetic acid (EDTA) were compared for their efficacy in removing antibodies from DAT positive red blood cells. Elution was performed on in vitro sensitized DAT positive cells. All 51 samples were tested with all three eluting agents. Total negativity in DAT or decrease in strength of DAT was looked for.

A total of 51 in vitro sensitized samples with Grade 2 agglutination were studied. Glycine EDTA gave negative results 38 samples (74.5%), but there was lysis in 12 samples (23.5%), heat elution method gave a negative DAT reaction in only 24 (47.1%) samples, but done of the samples were lysed by heat elution glycine HCl, failed to give a negative DAT reaction, also there were lysis in 4 (7.8%) samples. Among the three reagents studied, glycine EDTA has the best ability to elute coated antibodies completely, but cell lysis is a problem with this reagent. Heat elution is better over other reagents when intact red cells are to be retrieved for antigen phenotyping.

Key words: Elution, Direct Antiglobulin Test, Immunoglobulin G, Complement

Background and Rationale

Issue of blood to patients with irregular antibodies is one of the major challenges to a blood bank professional. Irregular antibodies can be seen freely in the serum or attached to the red cell surface. The red cells can be coated with immunoglobulin G (IgG) or complement or both. A positive direct antiglobulin test (DAT) signifies in vivo coating of red cells.

Elution is the technique to remove adherent antibody protein from the red cell surface. Characterization of antibody from elute is an important part of the investigation of autoimmune hemolytic anemia, hemolytic disease of newborn (HDN), and when multiple anti bodies are present in a recipient. Intact red blood cells (RBCs), post elution can be used for antigenic phenotyping to select antigen negative units for cross match.1,2

The recovery of antibody from a cell sensitized with complement alone is not likely. When the cell is coated with IgG antibody alone or in combination with complement, we can expect a successful elute. Hence, cells testing positive in the DAT reaction with monospecific anti-IgG alone or polyspecific anti IgG-C3d can be eluted.2

Although several techniques to detach antigen antibody complexes are described, such as heat (56°C), freeze thawing, alterations in pH, and organic solvents, each method suffers from one of three faults; methods may be laborious, elute obtained may be weak, or the method may end up in cell lysis.

A quick, easy method that gives maximum elution of antibodies, with minimal damage to cell membranes should be the method of choice.3-5

Aims and Objectives

The objectives of this study are

  1. Comparison of three eluting agents namely heat (56°C), glycine ethylenediaminetetraacetic acid (EDTA), and glycine hydro chloric acid (HCl)

  2. Selection of the best technique for detecting and recovering bound IgG from DAT positive RBCs.


The study was performed as a three-month study at the Department of Transfusion Medicine; Regional Cancer Centre, Thiruvananthapuram, Kerala, India.

The study protocol was approved by the Ethics Committee of the Institute.

Study design: Experimental

Study subjects: In vitro sensitized and DAT positive RBC

Sample size: 51 samples

Inclusion criteria: Grade two agglutination in DAT

Exclusion criteria: Negative, microscopic, and Grades 1, 3, 4 agglutination in DAT

IgG coated red cells were prepared with pooled washed O Rh positive cells incubated with one in four diluted monospecific anti IgG (Tulip)

  1. Heat elution:6

    Equal volumes of 6 times washed DAT positive cells were mixed with 6% bowine albumin, heated with intermittent agitation in a water bath at 56°C for 10 min, centrifuged an removed the elute,

  2. Glycine HCl elution (Cold acid elution):6

    1. Glycine HCl (0.1 M, pH 3.0.), prepared by dissolving 3.75 g glycine and 2.922 g of sodium chloride in 500 ml distilled water. Adjusted pH to 3.0 with 12 N HCl, stored at 4°C.

    2. Phosphate butter (0.8M, pH 8.2) prepared by dissolving 109.6 g Na2HPO4 and 3.8g KH2 PO4 in 600 ml distilled water. Adjusted pH with IN NaOH or IN HCl. Dilute to a final volume of IL with distilled water, stored at 4°C.


DAT positive cells were first chilled in an ice bath for 5 min, added 1 ml of chilled saline and 2 ml of chilled glycine HCl to 1 ml of washed RBC mixed and incubated the test tube in ice bath for 1 min. Spin at 1000 g for 2-3 min, supernatant transferred out, we may add 0.1 ml of Phosphate buffer pH 8.2 to each 1 ml of elute, mix spin again for 2-3 min (addition of phosphate buffer restores neutrality of the acid elute, this avoids hemolysis of reagent red cells used to characterize the elute).

  • 3. Glycine HCl/EDTA solution6

    1. Disodium EDTA was prepared (10% w/v) using Na2EDTA 2H2O, 10 g in 100 ml distilled water.

    2. Glycine HCl (0.1 m at pH 1.5): Glycine 3.75 g NaCl 2.922 g distilled water to 500 ml, adjust pH to 1.2 with 12 N HCl stored at 4°C

    3. Tris Base (M) SIGMA chemicals 12.1 g distilled water to 100 ml

Procedure: Mix 4 ml glycine HCl and 1 ml EDTA and add 1 ml DAT positive cells, incubate 1-2 min at room temperature, light spin for 2-3 min transfer out supernatant, adjust pH to 7.5 with 1M Tris base, mix and centrifuge, and transfer out supernatant.

The residual red cells are retested for DAT agglutination was graded. Reduction in agglutination or negativity of DAT was looked for, lysis was monitored. On performing DAT after elution, a score of 0 was assigned to samples showing negative agglutination, 1 for weak agglutination and 2 for Plus 1 agglutination.


Statistical analysis of data was done using SPSS software version 13. P≤0.05 was taken as significant.


The study was approved by the Institutional Review Board.



Three eluting agents namely heat (56°C), glycine HCl (cold), and glycine HCl-EDTA were applied on 51 in vitro sensitized DAT positive RBCs, to compare the efficacy of eluting agents. On analyzing the outcome glycine HCl-EDTA elution give negativity in DCT in 38 (74.5%) samples, compared to 24 (47.1%) for heat elution and nil (no negativity) for glycine HCl elution.

Decrease in strength or weakening (plus 1 or microscopic agglutination) of DAT was also variable for the three reagents. Only 1 (2%) samples of glycine EDTA gave a microscopic reaction, whereas 22% (43.1%) of the 51 heat eluted samples gave microscopic agglutinations. Regarding glycine HCl, even though none of the samples gave negative DCT, there was 7 (13.7%) samples showing grade1agglutination, and 40 (78.4%) samples showing microscopic agglutination (Tables 1-3).

Table 1: Heat


Table 2: Glycine EDTA


Table 3: Glycine HCl


Another significant finding was the lysis of red cells obtained, glycine EDTA, though superior in producing DCT negativity shared cell lysis in 12 (23.5%) samples.

Glycine HCl showed lysis in 4 (7.8%) of samples, heat elution preserved the maximum integrity of cell membrane that it did not cause lysis of any of the samples (Table 4).

Table 4: Descriptive statistics (Friedman ANOVA)


Statistical comparison of the decrease in agglutination produced by three reagents was done using Friedman ANOVA. To study the reagent wise difference, Wilcoxon signed rank test was used. Those 16 samples which showed lysis of red cells in either of the three methods was excluded from analysis, leaving 35 samples for analysis.

Mean of scores was calculated glycine EDTA had the lowest score, thus statistically implying to be the better reagent, heat was the second best eluting agent, glycine HCl the least. A significant correlation P≤0.05 was obtained with Friedman ANOVA for all three reagents, glycine EDTA having the highest statistical significance.


There are a number of uses for elution procedure including removing an auto antibody from red cell surface, for auto adsorption of an elute to separate auto and alloantibodies in case of multiple irregular antibodies, accurate phenotyping of red cell antigens, and characterization of an irregular antibody.

Elution procedures are useful only in cases where a cell has attached IgG alone or IgG and complement. It fails to give anelute when cells are sensitized with complement alone.

In our study, we have done in vitro sensitization of RBC with mono specific IgG, so that a successful elute is obtained.

Elution removes antibody from red cell membrane either by a dissociation of antigen antibody complex or by disrupting the red cell antigen. Many techniques of elution are available such as Landsteiner heat method, Lui freeze-thaw method, ultrasonic vibration, Acid elution, cold acid, organic solvents as ether, xylene, chloroform, chlorquine, glycine EDTA. However, no one technique serves the purpose fully. The eluting agent must be chosen to serve our purpose, whether to characterize the antibody in elute or antigenic phenotyping or RBC, in which case intact red cell membrane after elution is a must. An ideal eluting agent must be quick easy procedure giving maximum elution from DAT positive cell with minimal RBC membrane damage.

In our study comparing the efficacy of the three eluting agents, glycine EDTA was clearly superior in producing negativity in DCT, but the main drawback was cell lysis. Heat elution, even though negativity was less than glycine EDTA, was better in preserving the integrity of red cell membrane, giving no lysis.

In comparison to the above two reagents, glycine HCl was inferior to glycine EDTA and heat elution in terms of decrease in DCT positivity and preservation of cell membrane integrity.


From our study, we conclude that choice of an eluting agent should relate to purpose, that is if maximum elution of antibody is our need, we may choose glycine EDTA whereas heat elution should be chosen if we intend to phenotype the antigens on red cell surface.


IgG: Immunoglobulin-G

DAT: Direct antiglobulin test

DCT: Direct coombs test

AHG: Anti human globulin test

HCl: Hydro chloric acid

EDTA: Ethylenediaminetetraacetic acid

Na2HPO4: Disodium hydrogen phosphate

NaOH: Sodium hydroxide

HDN: Hemolytic disease of newborn

End Note

Author Information

  1. Vijayalakshmi Kuttath, Associate Professor and Head of Department, Department of Transfusion Medicine, Regional Cancer Centre, Thiruvananthapuram, Kerala, India.

  2. Biju George, Assistant Professor, Department of Community Medicine, Medical College, Calicut, Kerala, India.

  3. Suji Sudhakar, Department of Medical laboratory technology, Kannur, Kerala, India.

Conflict of Interest



We thankfully acknowledge the help of Dr. Biju George, Assistant Professor, Department Of Community Medicine, Government Medical College Calicut and Dr. Preethi Sarah George, Assistant Professor in Biostatistics, Division of Epidemiology and Biostatistics, Regional Cancer Centre, Thiruvananthapuram for their valuable help and advice during statistical analysis of data.


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