Hematology, April 2007; 12(2): 175–178

A comparison of conventional tube test and gel technique in evaluation

of direct antiglobulin test

SUDIPTA S. DAS, RAJENDRA CHAUDHARY, & DHEERAJ KHETAN

Department of Transfusion Medicine, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, India

(Received 22 August 2006; accepted 6 October 2006)

Abstract
In vivo coating of red cells by antibody and/or complement is detected using various sensitive techniques, however most
hospitals even today rely on the conventional tube technique (CTT). We compared the performance of the CTT and recently
introduced gel test (GT) in the evaluation of direct antiglobulin test (DAT).
The CTT and GT were first compared using in-house prepared control cells. The polyspecific DATs were performed
simultaneously by CTTand GTon 170 consecutive blood samples. Positive samples were further tested for monospecific IgG
and C3d by both techniques.
GT demonstrated stronger agglutination scores (60 vs. 43) compared to CTT using control cells. The sensitivity and
specificity of the GT was 98.4 and 95.2%, respectively as compared to CTT for polyspecific DAT. Discordance between the
two test systems was seen in 6/170 patients. Of these, 5 were missed by CTT while GT failed to detect in vivo coating in only 1
case. The agreement between two methods of DAT was 96.4% (k ¼ 0.926) using polyspecific AHG and 95.7% (k ¼ 0.379)
with monospecific anti-IgG. We conclude that GT is a better alternative to CTT for detecting red cell bound antibodies in
various clinical conditions.

Keywords: Immunohematology, autoimmune hemolytic anemia, direct antiglobulin test, gel test, autoantibody

Introduction red cells [3]. All these necessitated the establishment

of a new, precise, easy and sensitive technique, which
The first description of the ABO system and red cell
agglutination by Landsteiner in 1900 and subsequent led to the advent of the gel test (GT) in 1993 [5,6]. The
development of the antiglobulin test by Coombs et al. GT requires no red cell washing and uses gel filtration
in 1945 enabled the immunohematologists to establish media impregnated with AHG reagent to bring about
and improve various serological investigations in agglutination [3]. Furthermore, the technique can be
human blood [1–3]. The direct antiglobulin test used with a panel of monospecific AHG reagents,
(DAT) used to investigate autoimmune hemolytic such as IgG IgM, IgA, C3c or C3d to detect the
anemia (AIHA) is a diagnostic procedure to demon- immunoglobulin classes or IgG subclasses (IgG1,
strate in vivo antibodies and/or complement coating red IgG2, IgG3, IgG4) enabling the characterization of
blood cells. This procedure uses polyspecific antihu- AIHA [4].
man globulin (AHG) reagent containing antibodies to Recently, we at our center introduced gel technol-
IgG and C3d component [4]. Majority of the blood ogy for various immunohematological investigations.
banks in the developing countries perform routine The purpose of this study was to validate the GT DAT
DAT using the conventional tube technique (CTT). and its comparison with the already established CTT
This traditional method is not without problems and in the detection of red cell bound immunoglobullins
demand skilled personnel and meticulous washing of and complements.

Correspondence: R. Chaudhary, Department of Transfusion Medicine, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow
226014, India. Tel: 91 522 2668700. Fax: 91 522 2668017. E-mail: rkcchaud@sgpgi.ac.in

ISSN 1024-5332 print/ISSN 1607-8454 online q 2007 Informa UK Ltd.

DOI: 10.1080/10245330601111862
176 S. S. Das et al.

Material and methods The agglutination scores for each DAT strength were
calculated according to the standard guidelines [7].
Samples
We studied 170 consecutive blood samples sent to our
immunohematology laboratory for DAT after obtain- Statistical analysis
ing consent of the patient and seeking approval of The results of testing samples in the CTTand GT DAT
institutional review board. Blood samples were assays were compared using paired t test. A p value of
collected in K2 EDTA vials and analyzed for ,0.05 was considered significant. The sensitivity and
polyspecific DAT on the day of collection using both specificity were also calculated for GT considering
CTT and GT. Any sample positive with either of the CTT as the standard method. k value was calculated as
techniques was further tested for monospecific DAT a measure of agreement using SPSS, version 9.0.
using anti-IgG and -C3d by both gel and CTT.

Results
CTT
Comparison of GT and CTT using control cells
It was performed following standard method
described by AABB [7]. Briefly, one drop of 2– 5% The GT was able to detect in vitro sensitization of red
suspension of red cells was dispensed into test tubes cells till 1:128 dilutions with total agglutination score
and washed three times with normal saline, final wash of 60, while CTT could detect at 1:32 dilution and
decanted completely. Two drops of polyspecific AHG total score being 43.
reagent (Diaclon Coombs, Diamed, Switzerland)
were then added, mixed well and tube centrifuged at
1000g for 30 s and cells were examined for aggluti- DAT (polyspecific) using GT and CTT
nation. The polyspecific AHG reactive samples were Of the 170 samples evaluated for DAT using CTT and
then similarly tested using monospecific AHG GT, 100 (58.8%) samples were negative and 64
reagents viz anti-IgG and -C3d (Ortho Diagnostics, positive with both the methods (observed
USA). All reactions were graded and recorded. The agreement ¼ 96.4%, k ¼ 0.926). Discrepant results
negative test was further confirmed by absence of were observed in 6/170 samples: 5 were positive by
agglutination on addition of sensitized check cells GT while negative with tube test, whereas only 1
(in house). sample was positive exclusively with the tube test
(Table I). The sensitivity, specificity, positive pre-
GT dictive value (PPV) and negative predictive value
(NPV) of the GT compared to CTT were 98.4, 95.2,
It was performed following manufacturer’s instruc- 92.7 and 99%, respectively, for polyspecific DAT.
tion. Briefly, 50 ml of 1% red cell suspension in low Of these 70 samples that were DAT positive by
ionic strength solution (LISS) was added to each either method, 34 were finally diagnosed as AIHA
microtube of the ID cards (polyspecific AHG, LISS- with evidence of in vivo hemolysis, while the
Coombs card, Diamed, Switzerland) and centrifuged remaining 36 samples were from patients with various
in a dedicated centrifuge device (Diamed, Switzer- autoimmune disorders such as systemic lupus erythe-
land) at 70g for 10 min following the recommended matosus (18), rheumatoid arthritis (5), Hashimoto’s
incubation period of 15 min. Samples reactive with thyroiditis (3) and other (10).
polyspecific AHG were then similarly tested for
monospecificity using gel ID cards (monospecific
AHG, LISS-Coombs card, Diamed, Switzerland). In DAT (monospecific) using GT and CTT
both the techniques, the findings of the agglutination Among the 70 polyspecific DAT positive samples, 4
reactions were graded as 4 þ , 3 þ , 2 þ , 1 þ , weak samples showed discordant results when evaluated
and negative and documented accordingly.
Table I. Comparison of GTand CTT for polyspecific DATs in 170
Comparison of GT and CTT using control cells subjects.

Serial dilutions of cold acid eluate from red cells of the Conventional tube test
(CTT)
55 warm AIHA patient, DAT 4 þ , ranging from 1:1
to 1:256 were prepared. “O” Rh (D) positive red cells Gel test (GT) Positive Negative Total
were incubated with serial dilutions at 378C and
washed with normal saline. The sensitized cells were Positive 64 05 69
Negative 01 100 101
tested in duplicate by tube and gel methods for Total 65 105 170
detection of antibodies coating the red cells for
comparison across range of DAT reaction strengths. Agreement between two methods ¼ 164/170 (96.4%, k ¼ 0.926).
 DAT by get test and conventional test tube method 177

Table II. Concordance between GT and CTT for mono-specific DAT in poly-specific DAT positive samples (n ¼ 70).

GT CTT Anti-IgG Anti-C3d

Positive Positive 66 10
Positive Negative 02 01
Negative Positive 01 00
Negative Negative 1 59
Total agreement 67 69
Percentage of agreement (k) 95.7 (0.379) 98.5 (0.944)

with monospecific sera (3 with anti-IgG and 1 with sensitization. However, Paz et al. 2004 observed that
anti-C3d) (Table II). Table III summarizes the though the GT DAT was more sensitive than CTT,
discordant DAT results. Out of 5 samples negative the clinical significance of a DAT positive only by
by CTT for polyspecific DAT, 2 were from patients GT was doubtful [13]. Others also reported failure of
with AIHA having in vivo hemolysis. Both these CTT as compared to GT in the diagnosis of AIHA
samples were positive by GT. Out of 64 samples that with evidence of hemolysis [4].
were positive by both methods, in 96.8% the strength In the present study (Table II) the CTT failed while
of reaction evaluated by GT was either more or equal the GT succeeded in detecting autoantibodies bound
to that of the CTT ( p , 0.05). Only on two occasions to the red cells in 3 patients using monospecific
the strength of DAT reaction in CTT was more than DAT (anti-IgG or anti-C3d). Only on one occasion
GT (Table IV). was CTT positive while GT negative with both poly
and mono (IgG) DAT; however, this case was later
diagnosed as SLE with a weak DAT reactivity that was
Discussion
not clinically significant. Thus the GT provided more
The GT was introduced more than a decade ago, but information on immunoglobulin and complement
most blood centers rely on the CTT for evaluation of binding on the red cells as compared to CTT. This is
DAT for serological diagnosis of AIHA, HDN and in contrast to the findings of Tissot and Colleagues
DHTR even today [8]. Owing to the simplicity, in 1999 that the tube agglutination assay was more
reproducibility and sensitivity of the GT, many blood sensitive for detection of red cell bound C3d than the
banks are now gradually adopting this technique [9]. GT [14]. This can be explained by the advancement
Most cases of immune mediated hemolytic anemia of gel technology over the years and use of
can be effectively diagnosed by the CTT, but some impregnated AHG in the gel matrix rather than the
patients, despite hemolysis, show a negative tube DAT application of reagents into the micro column during
[10 – 12]. We compared GT with CTT for polyspecific the test as practiced earlier. However, the occurrence
and monospecific DATs. We observed GT to be a of false positive reactions by the GT could not be ruled
good alternative to CTT in terms of sensitivity of out. Thus more studies on a larger sample size
98.4% and specificity of 95.2%. The PPV and NPV including red cells with known monospecific DAT
were 92.7 and 99%, respectively. Our results are in strength are needed to compare the CTT and GT in
accordance with Nathalang et al. 1997 who observed a relation to the detection of red cell bound IgG or C3d.
sensitivity of 100% and specificity of 80% with the GT A close agreement was observed between the two
[4]. In 5 of the 6 patients with discordance between test systems using polyspecific AHG (95.7%) and
two test systems, red cell antibodies could not be monospecific anti-IgG (90.9%) (Table II). Reis et al.
detected by CTT using polyspecific AHG (Table I). described close agreement between CTT and column
Two of these 5 patients were finally diagnosed as agglutination technique using glass beads in 1993 [3].
AIHA with evidence of in vivo hemolysis. Thus, it In our study, two of the 34 patients with AIHA who
appears from our results that the GT DAT is more were anti-IgG positive with the GT were negative with
reflective of clinically significant in vivo red cell CTT. This could be attributed to increased sensitivity

Table III. Details of discordant DAT results of CTT and GT in 6 patients.

CTT GT

S. No. Diagnosis Age (years)/sex Poly AHG Anti-IgG Anti-C3d Poly AHG Anti-IgG Anti-C3d

1 Primary AIHA 25/F Neg Neg Neg 1 þ 1 þ Neg

2 Autoimmune hepatitis 23/M Neg 1 þ Neg 1 þ 2 þ W þ
3 SLE 21/F Neg Neg Neg 1 þ 1 þ Neg
4 NHL 70/M Neg 1 þ W þ 1 þ 1 þ 1 þ
5 Primary AIHA 30/F Neg W þ Neg 1 þ W þ Neg
6 SLE 22/F 1 þ 1 þ Neg Neg Neg Neg
178 S. S. Das et al.

Table IV. Comparison of CTT and GT strength in 64 samples [3] Reis KJ, Chachowski R, Cupido A, Davies D, Jakway J,
positive for polyspecific DAT by both methods. Setcavage TM. Column agglutination technology: The
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Grade of reaction Number of patients n (%) [4] Nathalang O, Chuansunrit A, Prayoonwiwat W, Siripoonya P,
Sriphaisal T. Comprison between conventional tube technique
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