Matrix Gel System PDF

ISO 13485:2016
Performance Evaluations

MATRIX GEL SYSTEM
Gel card system for Blood Banking Applications
Performance Evaluations ISO 13485:2016
INDEX
S. No. Name of the Publication Pg Nos
1. Journal of Forensic Science & Criminology Vol 6 Issue 1, 2018 1- 8

2. International Journal of Current Research, Vol 9 Issue 07, July 2017 52800- 53803
3. Indian Journal of Basic and Applied Medical Research Vol 2, Issue 4, Sep 2016 28-131
4. Vox Sanguinis, 2015, International Society of Blood transfusion 109 (Suppl)1-379 495
5. Vox Sanguinis, 2015, International Society of Blood transfusion 109 (Suppl)1-379 689
6. Indian Society for Blood transfusion and Immunohematology, Transcon 2015 066
7. Asian Journal of Transfusion Medicine, Vol 12, Issue1, January- June 2018 51-56
8. Journal of Evolution of Medical and Dental Science, Vol.4, Issue 73, Sep 2015 12659-12667

MATRIX GEL SYSTEM
EXTERNAL EVALUATIONS
INDEX
S. No. Name of the Evaluating Body
9. Institute of Transfusion Medicine and Immunohematology, German Red Cross Center, Frankfurt
Evaluation of Matrix AHG (Coombs) Test Card for antibody detection (screening)and identification.
10. Institute of Transfusion Medicine and Immunohematology, German Red Cross Center, Frankfurt
Evaluation of MatrixAHG (Coombs) Test Card for autocontrols.

MATRIX GEL SYSTEM
Journal of Forensic Science & Criminology
Volume 6 | Issue 1
ISSN: 2348-9804
Research Article Open Access
The Efficiency and Specificity of Matrix Gel Method from the Forensic Point of
View, in Determination of ABO Blood Grouping and Rhesus Factor
Harel VS*, Pawar SG, Mahajan KD, Palaskar SG, More BP and Kulkarni KV
Directorate of Forensic Science Laboratories Mumbai, Maharashtra, India
*
Corresponding author: Harel VS, Directorate of Forensic Science Laboratories Mumbai, Maharashtra,
India, Tel: 8369095511, E-mail: [email protected]
Citation: Harel VS, Pawar SG, Mahajan KD, Palaskar SG, More BP, et al. (2018) The Efficiency and
Specificity of Matrix Gel Method from the Forensic Point of View, in Determination of ABO Blood
Grouping and Rhesus Factor. J Forensic Sci Criminol 6(1): 107
Abstract
In today’s crime investigation world determination of ABO blood grouping is still a very vital and effective precess in the field of forensic
crime scenes. This investigation involves the identification of blood group, on the clothes (Accuse, Victim, injured, Complainer) collected
from the scene of crime, and its cross comparision with the blood sample send by medical officer. Hence for this purpose total of 200
cases were included in the study having the samples of blood of accuse, victim, injured, complainer consisting of male and female... The
determination of ABO/Rh factor was performed by conventional tube method and matrix gel card. The comparison of both techniques
shows a very comparative result. As the red blood cells are sensitized with antibody will get agglutinate in the presence of anti human
reagent in the matrix gel card and this will be trapped in the gel column this helps for easy analysis of blood group. However spin tube
method is an operator-dependent assay, and is more susceptible to handling errors, the results are not more objective. The matrix gel
card method requires Small sample volumes, and gives standardized performance with technical ease, and is with ready automation, and
increased biosafety; all these factors have made this technology advantageous. In both techniques the reaction strength for ABO grouping
and Rh factor is mainly govern by agglutination reaction intensity between red blood cells and anti-human reagent.
Keywords: Forensic serology; Blood group; Conventional tube technique; Haematology; Matrix gel card
Introduction
Blood grouping technique is widely used in forensic laboratories for investigation of biological fluids collected from crime scenes.
In 1900, Sir Karl Landsteiner discovered the blood grouping technique known as the “ABO” system for which he was awarded
with Nobel Prize in 1930 [1]. Edmond Locard a pioneer of forensic science proposed that every criminal carries some trace of
evidence with him or her from the scene of crime by which he or she can be linked with the crime. The strongest evidence from
the crime scene available is blood or blood stains because the source of blood and their stains help in solving the crime of violence,
sexual offences, vehicular accident cases or murder. In cases of natural disasters the prime identification of body part is ABO blood
grouping then later comes DNA matching. This is the most commonly followed techniques in today’s forensic laboratory analysis
[2,3].
The identification of biological fluids and the comparison of individual characteristics of biological evidence with known standards
of its class, the biological samples can be identified. It is vital scientific evidence as it forms an important link in chain of evidence
or supports circumstantial evidence. A careful investigation of blood group from the blood samples and blood stain is therefore
important. Investigation of blood and blood stain gives very insight information about accused or victim which is useful in the
court of law with a varying degree of reliability [4].
The presence of ABO blood group and Rhesus factor is applied to inherited antigens detected on red blood cells by specific
antibodies [5,6]. Once the blood group and the Rh typing are established it remains unchanged throughout life [7,8]. The antigenic
determinants on the surfaces of red blood cells are the A, B, and O blood group proteins, which are for convenience called A, B, and
O antigens. Matrix gel is technique of blood group cross matching. The matrix gel card technique is introduced by lapierre, using
the sephadex gel containing within micro-tube. (Lapierre1988; letich et.al. 1993). [9-11].
The aim of this study is to determine ABO blood group from the blood samples collected as exhibits from the crime scene and the
methods used are spin tube method and matrix gel System. The comparative study for these two method was carried out by testing
total 200 samples of blood in the form of clothes or different types of exhibits which having blood strains.
Annex Publishers | www.annexpublishers.com Volume 6 | Issue 1

Journal of Forensic Science & Criminology 2
This study is carried out for the comparative study between spin tube method and matrix gel card method for blood group
identification on the basis of efficacy sensitivity and specificity [12-14].
Material and Method

Chemicals and reagents
1. The Matrix AHG (Coombs) test card:- Matrix forward and reverse grouping card with auto control has six micro-tubes prefilled
with auto control a gel in a suitable buffer. The first three micro-tubes contains Monoclonal Anti-A, Anti-B and Anti-D. The forth,
five and six number Micro-tubes has neutral gel. The Forth tube is used as negative control. The fifth and sixth are used for reverse
grouping.
2. Red Blood cells:- The Fresh blood samples of “A, B and O” blood group were kindly procided by blood bank of J.J. group of
Hospitals, Mumbai, Maharashtra, India.Matrix card reader ( Matrix Auto Max-80)
3. Matrix Diluent- LISS:- for three preparation of fresh cells.
4. Gel card centrifuge (Imperial Biotech LLP, Tulip gel card centrifuge)
5. Micropipette
Methodology
In the conventional spin tube method the ABO forward grouping is determined in the presence of the blood group antigens A, B
and O by testing the RBC’s with known antisera, specifically Anti-A, and Anti-B. On the other hand, the ABO reverse grouping
method results into the presence of the expected ABO blood group antibodies by testing the serum or plasma with known A1 and
B red blood cells. In case of matrix gel test the ABO forward grouping were performed in the Anti-A, Anti-B, micro-tubes, which
contain the specific antibody incorporated into the gel.
Conventional method of ABO grouping

ABO blood group can be determined from the fresh blood samples by detection of antigens on RBC’S (forward method) or
detection of antibodies in serum (reverse method). The blood samples are received in sterile bulb by forensic laboratories from
the medical officers. The testing sample is taken into the tube and saline 0.2%, was added to the sample. This mixture was then
centrifuged at about 3000 rpm for five minutes. The supernatant was then discarded and the suspended cells were rewashed with
saline for two times. Resuspend the button of cells in fresh normal saline. After this the cell suspension was prepared in 2% saline.
Forward method
In the tile with two cavities added one drop of anti-A serum and anti-B serum in the above marked cavities A and B respectively.
Added one drop of about 2% cell suspension in each cavity and mixed the contents thoroughly. The tile was roated for 5 minutes
and results were examined macroscopically as well as microscopically for agglutination.
Reverse Method
Removed the serum carefully. Marked the cavities of tile as A and B. Added one drop of serum in each cavity. Added one drop of
2% cell suspension of A and B cells in the respective cavities and rotated the tile for 5 minutes results are examined macroscopically
as well as microscopically for agglutination.
Matrix gel card Method

In this method ABO reverse grouping was performed in the buffer gel micro-tubes. Formation of agglutination indicates the
presence of an antigen-antibody reaction, while lack of agglutination indicates the absence of an antigen-antibody reaction.
Agglutinated red blood cells are trapped in the gel at various levels within the micro-tube depending on the size of the agglutinates.
Free non agglutinated red cells pass through the gel and form a button of red blood cells on the bottom of the micro-tube. The
control micro-tube must be negative for the results to be valid. In the gel test, the reagent red blood cells are combined with sample
serum/plasma in the upper reaction chamber of the micro-tube of card. Following an incubation period to enhance antigen/
antibody interaction, the sensitized red blood cells react with the antibodies incorporated in the gel of the micro-tube during a
centrifugation step. Agglutination indicates the presence of an antigen/antibody reaction while lack of agglutination indicates the
absence of an antigen/antibody reaction. Agglutinated red blood cells become trapped in the gel at various levels within the micro-
tube, depending on the size of the agglutinates. Free non agglutinated red blood cells pass through the gel and form a button of red
blood cells on the bottom of the micro-tube.
Sample preparation for forward grouping

Prepared 5% of blood cells suspension in matrix diluent LISS, by taking 0.5 mL LISS + 50 µL of whole blood or 25 µL of packed
blood cells. Mixed gently and used for forward grouping.

3 Journal of Forensic Science & Criminology
Sample preparation for reverse grouping

Prepared 0.8% blood of cells suspension in matrix diluent LISS as follows: - Washed fresh A, B cells with 0.9% saline till the
supernantant is clear. Add 1 mL of LISS in Each labelled test tubes as A and B. Used for reverse grouping.
Procedure
Pipetted 10 µL of 5% blood cell suspension in the microtubes 1 to 4 (A-B- -D- Control). Pipetted 50 µL of 0.8% known ‘A’ blood
cell suspension in the microtube 5. Pipetted 50 µL of 0.8% Known ‘B’ blood cell suspension in the microtube 6. Pipetted 50 µL of
serum of A and B in the microtubes 5 and 6. Allowed the card to incubate for 10 min.at room temperature. The card was removed
and the results were recorded.
Agglunation in the forwarding grouping and either haemolysis or Agglunation in the reverse grouping were interpreted as a
positive reaction. The results of ABO and Rh type of samples were recorded [15-17].
Results
The reaction strength may be recorded by grading of the Agglutination Reaction Intensities:-
The Red blood cells possesing the corresponding antigen will agglutinate in the presence of specific antibody, and will trapped
in the gel column, and gets settle at the bottom of the microtube. The control microtube (Ctrl) must be negative to validate the
forwarding results [8-10].
Positive Reaction:- A clear line on the surface of the gel column is formed by the agglutinated blood cells or sometimes it dispersed
in the gel column [8-10].
Negative Reaction:- Non Agglutinated red blood cells settle at the bottom of micotube forming a compact button [8-10].
Strength of reaction Comments

G++++ (G4+):- Agglutinated red blood cells form a line at the top of the gel microtube.
G+++ (G3+):- Most agglutinated red blood cells remain in the upper half of the gel microtube.
G++ (G2+):- Agglutinated red blood cells are observed throughout the length of the microtube. A small button of red blood cells
may also be visible at the bottom of the gel microtube.
G+ (G1+):- Most agglutinated red blood cells remain in the lower half of the microtube. A button of cells may also be visible at the
bottom of the gel microtube.
G± Most agglutinated red blood cells are in the lower third part of the gel microtube.
Negative: - Negative all the red blood cells pass through and form a compact button at the bottom of the gel microtube.
Mixed field agglutination

Agglutinated red blood cells form a line at the top of the gel and non-agglutinated red blood cells form a compact button at the
bottom of the gel microtube.
H:- Hemolysis of red blood cells
Out of 200 blood samples (which include Accuse, Victim, Suspect, injured, Deceased, Vitness etc.) cases, 5% (10 out of 200) shows
“inconclusive or invalid” results.
Blood Group Type A B AB O

Samples with correct results 45 80 36 29
Table 1: Number of samples Showing blood group are as follows
Results for:- “A “Blood Group:-
Figure 1: G (4+): Rh; G (3+): A-cells, B-Serum; Negative (-): B-cells, Ctrl, A-serum

Figure 1 shows, G (3+) agglutination reaction in forward A-cells and G (3+) agglutination reaction in reverse grouping B-serum
and shows negative reaction for B-cells and A-serum. Hence the blood group is ‘An’ Rh+.
Figure 2: G (4+): A-cells, Rh, B-Serum; Negative (-): B-cells, Ctrl, A-serum
Figure 2 shows, G (4+) agglutination reaction in forward A-cells and G (4+) agglutination reaction in reverse grouping B-serum,
and shows negative, reaction for B-cells and A-Serum. Hence the blood group is ‘An’ Rh+”
Figure 3: G (4+): A-cells, Rh; G (2+): B-Serum; Negative (-): B-cells, Ctrl, A-serum
Figure 3 shows, G (4+) agglutination reaction in forward A-cells and G (2+) agglutination reaction in reverse grouping B-serum,
and shows negative, reaction for B-cells and A-Serum. Hence the blood group is ‘An’ Rh+”
Results for:- “B “Blood Group:-
Figure 4: G (4+): B-cells, Rh, A-Serum; Negative (-): A-cells, Ctrl, B-serum
Figure 4 shows, G (4+) agglutination reaction in forward B-cells and G (4+) agglutination reaction in reverse grouping A-serum,
and shows negative, reaction for A-cells and B-Serum. Hence the blood group is ‘B’ Rh+.
Figure 5: G (4+): B cells, Rh; G (2+): A-Serum; Negative (-): A-cells, Ctrl, B-serum
Figure 5 shows, G (4+) agglutination reaction in forward B-cells and G (2+) agglutination reaction in reverse grouping A-serum,

Figure 6: G (4+): B-cells, Rh; G (1+): A-Serum; Negative (-): A-cells, Ctrl, B-serum
Figure 6 shows, G(4+) agglutination reaction in forward B-cells and G (1+) agglutination reaction in reverse grouping A-serum,
Results for:- “AB “Blood Group:-
Figure 7: G (4+): A-cells, B-cells, Rh; Negative (-): A-serum, B-Serum, Ctrl
Figure 7 shows, G (4+) agglutination reaction in forward A-cells and B-cells and negative agglutination reaction in reverse grouping
for A-serum and B-serum. Hence the blood group is ‘AB’ Rh+.
Results for:- “O “Blood Group:-
Figure 8: G (4+): Rh, A-serum; G (3+): B-Serum; Negative (-): A-cells, B-cells, Ctrl
Figure 8 shows, Negative agglutination reaction in forward A-cells and B-cells and G (4+) agglutination reaction in reverse grouping
for A-serum and G (3+) agglutination reaction for B-serum. Hence the blood group is ‘O’ Rh+.
Figure 9: G (4+): Rh; G (2+): A-serum, B-Serum; Negative (-): A-cells, B-cells, Ctrl
Figure 9 shows, Negative agglutination reaction in forward A-cells and B-cells and G (2+) agglutination reaction in reverse grouping
for A-serum and B-serum. Hence the blood group is ‘O’ Rh+.

Figure 10: G (4+): Rh, A-serum; G (2+): B-Serum; Negative (-): A-cells, B-cells, Ctrl
Figure 10 shows, Negative agglutination reaction in forward A-cells and B-cells and G (4+) agglutination reaction in reverse
grouping for A-serum and G (2+) agglutination reaction for B-serum. Hence the blood group is ‘O’ Rh+.
grouping for A-serum and B-serum. Hence the blood group is ‘O’ Rh+.
grouping for A-serum and B-serum. Hence the blood group is ‘O’ Rh+.
Figure 13: G (4+): Rh; G(2+): B-Serum; G(1+): A-serum; Negative(-): A-cells, B-cells, Ctrl
grouping for A-serum and G (1+) agglutination reaction for B-serum. Hence the blood group is ‘O’ Rh+.

Results for:- “Inconclusive “Blood Group:-
Figure 14: G (4+): A-cells, B-cells, Rh Ctrl; G (3+): A-serum, B-Serum
Figure 14 shows, G (4+) agglutination reaction in forward A-cells and B-cells and G (3+) agglutination reaction in reverse grouping
for A-serum and B-serum. Hence the blood group is cannot be concluded.
Figure 15: G (4+): Rh, A-serum, B-Serum; G (3+): Ctrl; G (2+): B-Cells; Negative(-): A-Cells
Figure 15 shows, G (2+) agglutination reaction in forward B-cells and negative agglutination reaction A-cells and G (4+)
agglutination reaction in reverse grouping for A-serum and B-serum. Hence the blood group is cannot be concluded.
Figure 16: G (4+): B-Cells, Rh; G (3+): A Serum; Negative (-): A-Cells, Ctrl, B-serum
Figure 16 shows, G (4+) agglutination reaction in forward B-cells and negative agglutination reaction A-cells and G (3+)
agglutination reaction in reverse grouping for A-serum and negative agglutination reaction for B-serum. Hence the blood group
is cannot be concluded
Figure 17: G (4+): A-Cells, B-Cells, Rh, Ctrl, A-serum, B-Serum

Figure 17 shows, G (4+) agglutination reaction in forward A-cells and B-cells and G (4+) agglutination reaction in reverse grouping
for A-serum and B-serum. Hence the blood group is cannot be concluded

Discussion
This study presents number of advantages of blood grop cross matching by using Matrix gel card method over routine spin tube
method. The hemagglutination in positive wells was strong, so that they were easily seen with the naked eye.
The ideal condition for the agglutination reaction for blood group type “A” is Figure 2, for blood group type “B” is Figure 4. For
blood group type “AB” is Figure 7, for blood group type “O” is Figure 12. It was observed that Figure 1 shows G (3+), reaction and
Figure 3 shows G (2+) reaction, but when these samples were preocessed, the correct reaction for reverse (Serum) grouping was
some times observed difficult to conclude. Similarly the results of samples in Figure 5, Figure 6, Figure 14 shows G (2+), G (1+)...
etc reaction and but when these samples were preocessed with routine spin tube method, the very poor agglutination reaction was
observed.
Figure 15, Figure 16, Figure 17, and Figure 18 shows the inconclusive or invalid results by both routine spin tube method, and
Matrix gel card method.
In our study it was observed that 5% (10 samples out of 200) samples which showed invalid/ Haemolysed / Inconclusive results by
our routine spin tube method, was giving correct agglutination by gel card technique. Which is coated in the studies by Kaur R,
Rumsey DH, and Malyska H. et.al?
It wass observed that Matrix gel card test is better than Spin saline tube method because of its simplicity, stability of results, better
handling, long time recorded, dispensation of controls with comparable sensitivity and specificity which is follow with this study
(Col et al., 2008).
As the Matrix gel test uses an increased serum to cell ratio and there is no need of wash phase, thus reducing possibility of elution
of weakly bound antibodies hence the false positive screens of results were reduced using the matrix gel test system. (Bromilow et
al, 1992).
Matrix gel card test results remain stable within the gel, allowing rereading and it can be photocopied. The disposal of plastic cards
is also very easy, which increases standardisation of laboratory techniques and introduces more objective reading of agglutination
reaction. (Bromilow et al, 1992).
The blood group antigens for ABO and Rh factor were detected upto 12 Months. The number of non-specific anti bodies and false
positive sereens of results were reduced using matrix gel system.
Conclusion
From above observations it was concluded that the Matrix gel card technique is more suitable and less time consuming. The results
are more stable can be recorded after long time.The test can be carried out with very small sample. In conclusion Matrix gel card
test has been shown to be very efficient in forensic fields to get the blood group from the blood samples of Accuse, Victim, injured,
Complainer Consisting of Male and Female.
Acknowledgment
Author thanks to Director General (Legal & Technical) Home Dept. Govt. of Maharashtra and Forensic Science Lab Laboratory,
Mumbai, for the facilities to do this analysis.
References
1. Lapierre Y (1998) Proceedings of XX Congress of the International Society of Blood Transfusion Society. British Blood Transfusion Society; Manchester, UK.
2. Lapierre Y, Rigal D, Adam J, Josef D, Meyer F, et al. (1990) “The gel test: A new way to detect red cells antigen - antibody eactions.” Transfusion. 30: 109-13.
3. Leitch K, Forrest A, Mitchell R (1993) “A preliminary trial of the gel test for blood group serology.” Br J Biomed Sci 50: 64-6.
4. Mollison PL (1993) Blood Transfusion in Clinical Medicine (9th Edn) Blackwell Scientific Publications, USA.
5. Novaretti MCZ, Jeus ES (1994) Evaluation of a gel test system for the detection of transplacental haemorrhage. Transfusion 34: S110.
6. Kaur R, Kakkar N, Dhanoa J (2003) “Use of gel-based DiaMed-ID microtyping system for cross matching enhances sensitivity.” Indian J Pathol Microbiol 46:
617-20.
7. Rumsey DH, Ciesielski DJ (2000) “New protocols in serological testing: A review of techniques to meet today’s challenges. “Immunohematology 16: 131-7.
8. Malyska H, Weiland D (1994) “The gel test.” Lab Med 25: 81-5.
9. Novaretti MC, Silveira EJ, Filho EC, Dorlhiac-Llacer PE, Chamone DA (2000) “Comparison of tube and gel technique for antibody identification.” Immunohe-
matology 16: 138-41.
10. Bromilow IM, Adams KE, Hope J, Eggington JA, Duguid JK (1991) “Evaluation of the ID gel test for antibody screening and identification.” Transfus Med 1:
159-61.
11. Bromilow IM (1992) Gel Techniques in blood group serology. Med Lab Sci. 49: 129-32.
12. Cate JC 4th, Reilly N (1999) “Evaluation and implementation of the gel test for indirect antiglobulin testing in a community hospital laboratory.” Arch Pathol
Lab Med 121: 693-7.

13. Swarup D, Dhot PS, Kotwal J, Verma AK (2008) Comparative Study of Blood Cross Matching Using Conventional Tube and Gel Method. Med J Armed Forces
India 64: 129-30.
14. Kaur R, Kakkar N, Dhanoa J (2003) Use of gel based DiaMed-ID microtyping system for cross matching enhanced sensitivity. Indian J Pathol Microbiol 46:
617-20.
15. Coombs RRA, Mourant AE, Race RR (1945) A new test for the detection of weak and ìncomplete’ Rhagglutinins. Br J Exp Pathol 26: 255-66.
16. Coombs RR, Mourant AE, Race RR (1946) In-vivo isosensitisation of red cells in babies with haemolytic disease. Lancet 1: 264-6.
17. Jaiprakash M, Gupta PK, Kumar H (2006) Role of gel based technique for coomb’s test. Indian J Pathol Microbiol 49: 370-2.
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INTERNATIONAL JOURNAL
OF CURRENT RESEARCH
International Journal of Current Research
Vol. 9, Issue, 07, pp.53800-53803, July, 2017
ISSN: 0975-833X
RESEARCH ARTICLE
EVALUATION OF METHODOLOGY AND COMPARATIVE STUDY BETWEEN SPIN SALINE TUBE AND
MATRIX GEL CARD TECHNIQUES FOR BLOOD COMPATIBILITY
1,*Dr. Nouratan Singh, 2Neeraj Singh, 2Reeba Rachel Joseph,
2Anil Kumar Gautam and 3Dr. Neeraj Tandan
1, 2Department of Pathology and Blood Bank (Transfusion Medicine), UPUMS, Saifai, Etawah, India
3 Executive Director, SARC, Meerut, Uttar Pradesh, India
ARTICLE INFO ABSTRACT
Article History: Introduction: A study on Evaluation of methodology and comparative study between Spin saline tube
Received 20th April, 2017 using without AHG, with AHG and Gel card technique for blood cross crossmatching on the basis of
Received in revised form efficacy, sensitivity and specificity was undertaken on approximately 500 samples processed in Blood
16th May, 2017 Bank of U.P. University of Medical Sciences, Hospital, Saifai, Etawah, India.
Accepted 19th June, 2017 Material and Methods: Most commonly
ommonly Spin saline tube method are used widely in blood banks. A
Published online 26th July, 2017 new technique of cross matching is introduced as AHG gel card.
car . In thi
this study we used Matrix gel card
method based on indirect coombs test (ICT) for cross match and tube method including Spin saline
Key words: tube method with AHG and without
wit AHG.
Spin saline tube method,
Result: five hundred samples are taken for the study and out of this 490 samples are compatible using
AHG, LISS, Spin saline tube method without coombs reagent, 10 sample shows incompatibility, whereas in Spin
Matrix Gel card technique. saline method by using coombs reagent shows 99.2% 99.2% compatibility, 06 samples show false positive
and 04 samples show true positive of previously result. As per findings specificity and sensitivity is
100% of gel card and tube test using AHG, whereas Spin saline tube test specificity is 98.8 %.
Spin saline
saline tube method at room temperature, shows 98% compatibility due to 06 06- sample false
positive and 04 sample true positive, whereas Spin saline tube with coombs reagent at 370C, shows
99.2% compatibility due to 496 sample were found compatible and 04 sample true positive. In matrix
gel card also shows 99.2% compatibility.
Conclusion: The usage of Matrix Gel card in Blood Bank for cross match is easy to performed with
recorded test result and more sensitive and specific then Spin saline tube method whereas ind indirect
coombs tube test is also sensitive and specific but more time consuming as compare to Gel card but
cannot recorded result and more time consuming than Spin saline and gel card method.
Copyright©2017, Nouratan Singh et al. This is an open access article distributed under the Creative Commons Attribution
ribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
Citation: Dr. Nouratan Singh, Neeraj Singh, Reeba Rachel Joseph, Anil Kumar Gautam and Dr. Neeraj Tandan Tandan, 2017. “Evaluation of methodology
compatibility International Journal of Current Research
and comparative study between Spin saline tube and matrix gel card techniques for blood compatibility”, Research, 9, (07),
53800-53803.
INTRODUCTION It also used for various test such as ABO and Rh typing,
identification of alloantibodies, indirect and direct coombs test
This study based on the recorded data analysis, was done in (ICT & DCT) (Mollison,, 1993; Lapierre and Rigal, 1990). This
blood bank of U.P. University of Medical Sciences, Hospital, study is also carried out for evaluation of methodology and
Saifai, Etawah (U.P.) with the presence and supervision of comparative study between Spin saline tube using without
Pathologist. Matrix Gel Card a newly introduced technique for AHG, with AHG and matrix gel card technique used for blood
blood cross matching. The Spin saline tube method is used crossmatching on the basis of efficacy, sensitivity an and
previously for blood cross match which is mainly Spin saline specificity.
tube method (Spin saline tube method at RT) and indirect
coombs tube method. Matrix gel card technique is introduced Aims and objectives of study
by Lapierre, which was based on controlled centrifugation of
red blood cells in sephadex gel contained within microtube gel Evaluation of methodology and comparative study between
techniques (Lapierre, 1988; Letichet al., 1993). Spin saline tube using without AHG, with AHG and matrix gel
card technique for blood crosscross- matching on the basis of
*Corresponding autour: Dr. Nouratan Singh, efficacy, sensitivity and specificity.
Department of Pathology and Blood Bank (Transfusion Medicine),
UPUMS, Saifai, Etawah, India.
53801 Dr. Nouratan Singh et al. Evaluation of methodology and comparative study between spin saline tube and matrix gel card techni
techniques
for blood compatibility
MATERIALS AND METHODS RESULTS

The study based on data analysis total 500 sample randomly Total 500 random blood sample cross matched by using Spin
selected from day wise round the clock duty wise stored saline tube method with and without using AHG and Matrix
sample of requisition for cross matching blood in blood bank Gel Card. Result are observed in Spin saline tube method
of UPUMS, Hospital, Saifai, Etawah. Most commonly Spin without AHG, 500 sample shows 98% compatibility but in 06
saline tube method are useded widely in blood banks. A new sample (1.2%) shows false positive (FP) and four sample
technique of cross matching is introduced as AHG gel card.
car In shows true positive (TP), if we Add AHG (IAT) calculated
this study we used Matrix gel card method based on ICT for after compare the result of Spin salin
saline tube method with AHG
cross matching and tube method including Spin saline tube and Matrix Gel card method which shows 496 (99.2%) sample
method and indirect coombs test tube method used.
u Sample compatible and 04 sample (0.8%) True Positive (TP) found in
comes from ward with issuing form, the collection of blood observation, incompatibility of 06 samples (FP) disappear after
from healthy donors who have >45 kg of weight with negative incubation with AHG reagent at 370C.
all serology of HIV, HbsAg, HCV, VDRL and Malaria. In this
study first we follow the steps: Blood grouping of patient
blood and donor blood from pilot tube by the help of antisera
A, B, D. After matched of blood group we proceed to perform
cross matching of blood of both donor and patient’s blood by
three methods.
Spin saline tube method without AHG and with AHG reagent,
other third method is Matrix Gel card method which is recently
introduced in blood bank. Centrifuge the both blood samples
and extract the serum and red cells from patient and donors
samples,
les, prepared cell suspension of the donor’s red cells and
patient’s red cells. The method which is apply in Spin saline
tube method, marking tubes as major and minor with marker,
in major tube we mixing of patient’s serum and red cells of
donor, in minor tube serum of donor and red cell from patient
sample. After that we add the AHG reagent in spin saline tube
method and kept it incubate for one hour at 370C and then Fig. 1. Compatibility
mpatibility shows by Matrix Gel card Technique
centrifuge the both tubes, see the result if clumping or
agglutination or hemolysis presentt in both test tubes, blood bag
is incompatible for patient. If clumping or agglutination not
present blood is compatible for patient (Coombs
Coombs, 1945 &
1946). In Gel Card technique we used Matrix Gel Card
incorporated with AHG reagent (each plastic card containing
cont
six microtubes), incubator (cartridge warmer), Card centrifuge
for centrifuge of Gel Card, Diluent-22 LISS, test tubes and
micropipette. Firstly we prepared a 0.8% red cell suspension
by adding 10µl of packed red cells of donor in 1ml Diluent-2
Diluent
LISS in to clean test tube by micro pipette, after that we take a
Matrix gel card, open the foil of one microtube gently and
write the patient details, ID number at below part of microtube,
add 50µl of 0.8% donor red cell suspension, after this add 25µl
patientt serum in same microtube by proper way. Incubate the
Matrix gel card in card incubator (cartridge warmer) for 15
minutes at 370C. After incubation centrifuge the Matrix gel
card in card centrifuge machine for 10 minutes at preset/ 950
rpm, at the end of centrifuge
entrifuge read the result. If gel card shows
RBCs are settled bottom of particular microtube means No
agglutination (Negative result) that means Donors blood is Fig. 2. Results plotted in graph
compatible to the recipient and suitable for transfusion to
patient. If RBCs are trapped or floated ated between upper and In table:1, five hundred samples are taken for the study and out
bottom of tube that means something is wrong and result are of this 490 samples are compatible using Spin saline tube
called Positive result and incompatible for recipient. Positive method without coombs reagent, 10 sample shows
result shows grading in to 1+ to 4+. (1+ means nearne to bottom incompatibility, whereas in Spin saline method by using
of micro tube and 4+ means top of micro tube). In case of 4+ coombs reagent shows 99.2% compatibility, 06 sample shows
reaction, indicated if a solid band of red blood cells (RBCs) on false positive and 04 sample shows true positive of previously
top of the gel card’s microtube, 3+ reaction displays if result. Sensitivity and specificity is 100% of gel card and
agglutination of RBCs in the upper half, 2+ reaction is indirect coombs tubee test using AHG, whereas Spin saline tube
indicated by RBCs agglutinate dispersed throughout the test specificity is 98.8 %. Spin saline tube method at room
microtube,, while a 1+ reaction shows if RBCs aggregate in temperature, shows 98% compatibility due to 06 06- sample false
mainly lower half part of the microtube with dotted structure in
column.
53802 International Journal of Current Research, Vol. 9, Issue, 07, pp.53800-53803, July, 2017
Table 1. Result observation
Method Used Samples Compatible Incompatible

TN FP TP FN
1. Spin saline tube method without AHG (RT) 500 490 06* 04 00
2. Spin saline tube method with AHG (370C) 500 496 00 04 00
3. Matrix Gel Card (370C) 500 496 00 04 00
*Result obtained only if AHG not used with Spin saline tube method otherwise it shows compatible result
with AHG.
positive and 04 sample true positive, whereas Spin saline tube CONCLUSION
with coombs reagent at 370C, shows 99.2% compatibility due
to 496 sample were found compatible and 04 sample true Matrix Gel card is more sensitive and more specific than Spin
positive. In matrix gel card also shows 99.2% compatibility. saline tube methods and also less time consuming but more
costly than Spin saline tube methods. Matrix Gel Card
DISCUSSION technique is more stable and fully recordable for a long period.
We can shoot the picture or scan of result and share or stored
Matrix gel card technique recently introduced for blood cross- for further investigations. As per result, time consuming,
matching and ABO & Rh Blood Grouping system in India and recording, handling, less exposure, we concluded and advice
other country. The matrix gel card test performed in various for use of gel card in various blood banking services as
institutions and hospitals for blood cross match, matrix gel routinely test performed in cross matching for blood
card have six microtube embedded in a plastic card (Malyska, transfusion because of high sensitivity and specificity then
1994). The advantages of matrix gel card as easy reading of Spin saline tube methods. Matrix gel card method is better than
microtube, easily recording for a long time, handling and Spin saline tube method because of its simplicity, stability of
disposal (Malyska, 1994). In this study 0.8% sample out of 500 results, better handling, long time recorded, dispensation of
sample shows incompatibility (agglutination) by gel card controls with comparable sensitivity and specificity which is
method and also spin saline tube method using AHG. Whereas follow with this study. The result shows that gel test is more
Spin saline tube method without AHG shows 98% sensitive than tube test for identifying clinically potentially
compatibility which is not correct because 06 sample shows significant of antibodies. Matrix gel card test also less time
False Positive if we subjected to AHG. The specificity and consuming than tube method with AHG reagent but cost
sensitivity is 100% of both gel card and Spin saline tube effective method. We recommended that the usage of Matrix
method with AHG, whereas specificity of Spin saline tube Gel card for routine blood cross-matching, blood grouping
without AHG is 99.2%. Matrix gel card method is better than (forward and reverse) in all blood bank.
Spin saline tube method because of its simplicity, stability of
results, better handling, long time recorded, dispensation of Acknowledgement
controls with comparable sensitivity and specificity which is
follow with this study (Colet al., 2008). Matrix gel test at least We are highly thankful especially to all faculty, technical and
assensitive as an LISS AHG tube test with a better balance of supportive staff of department of transfusion medicine (Blood
both sensitivity and specificity in blood cross-matching Bank), UPUMS, Saifai, Etawah.
(Rumsey and Ciesielski, 2000). The number of non-specific
antibodies and false-positive screens of results were reduced
REFERENCES
using the matrix gel test system. In antibody titers performed
using the gel system were more sensitive than without AHG
Bromilow, I.M. et al. 1991. Evaluation of the ID gel test for
tube method (Bromilow, 1992). The matrix gel system was
antibody screening and identification. Transfusion
easy to use and provide reproducible and reliable results. The
Medicine,1: 159-61.
results of my study obtained with tube AHG same as matrix
Bromilow, I.M. et al. 1992. Gel Techniques in blood group
gel card method. The result shows that gel test is more
serology. Med Lab Science,49: 129-32.
sensitive than tube test for identifying clinically potentially
Cate John, C., Reilly, N. 1999. Evaluation and implementation
significant of antibodies (Bromilow et al., 191). The testing
of the gel test for indirect anti-globulin testing in a
efficiency improved by the using of the matrix gel test into
community hospital laboratory. Arch of Pathology and Lab
routine use (Novaretti et al., 2000). It’s proved that matrix gel
Med., 121: 693-7.
cardsystem also easy to use and its finding suggest more
Col D. Swarup, Brig P.S. Dhot, Lt Col J. Kotwal, Lt Col A.K.
sensitive than the Spin saline tube agglutination technique
Verma, 2008. Comparative Study of Blood Cross Matching
without AHG (Cate John and Reilly, 1999). Matrix gel card
Using Conventional Tube and Gel Method. Air force
system is better than Spin saline test tube method and simple to
journal India, 129-130.
perform and less exposure of blood bank personal to blood
Coombs, R.R.A et al. 1945. A new test for the detection of
specially area with HIV, HbsAg and HCV infections
weak and ìncomplete' Rh agglutinins. British Journal of
(Nathalang et al., 1993). It also concluded that matrix gel card
Experimental Pathology,26: 255-266.
test is better alternative to the Spin saline tube test for blood
Coombs, R.R.A et al. 1946. In-vivo isosensitisation of red
crossmatching as well as coombs tests (Direct and Indirect)
cells in babies with haemolytic disease.Lancet, i: 264-266.
(Jai prakash et al., 2006). In some study author concluded that
Jai Prakash, M. et al. 2006. Role of gel based technique for
Spin saline tube method show false negative result but in my
coomb’s test. Indian J pathology Microbiol.,49(3):370-2.
study Spin saline tube method provided six false positive,
which is different from previous studies.
53803 Dr. Nouratan Singh et al. Evaluation of methodology and comparative study between spin saline tube and matrix gel card techniques
for blood compatibility
Kaur, R., Kakar, et al. 2003. Use of gel based DiaMed -ID Mollison, P.L. 1993. Blood Transfusion in Clinical Medicine.
microtyping system for cross matching enhanced 9th Edition. Blackwell Scientific Publications.
sensitivity. Indian J Pathology Microbiol.,46: 617-20. Nathalang, O., Kuvanont, S., et al. 1993. A preliminary study
Lapierre, Y. 1988. The gel test: A new approach for detection of the gel test for cross matching in Thailand. J Med Tech
of red cell antibodies/ antigen in a solid phase. Proceedings Associat Thai., 21:101–106.
of XX Congress of the International Society of Blood Novaretti, M.C.Z., Jens, E.S., et al. 2000. Comparison of tube
Transfusion Society. Manchester: British Blood and gel technique for antibody identification.
Transfusion Society, 145. Immunohaematology, 16: 138-41.
Lapierre, Y., Rigal, D. 1990. The gel test: A new way to detect Novaretti, M.C.Z., Jeus, E.S. 1994. Evaluation of a gel test
red cells antigen - antibody reactions; Transfusion, 30: 109- system for the detection of transplacental haemorrhage.
13. Transfusion, 34 (Supp): S 110.
Letich, K., Forrest, A., Mitchell, R. 1993. A preliminary trial Rumsey, D.H., Ciesielski, D.J. 2000. New protocols in
of the gel test for blood group serology. Br J Biomed serological testing: A review of techniques to meet today’s
Sciences,50-1. challenges. Immunohaematology, 16: 131-7.
Malyska, H., Weiland, D. 1994. The gel test. Lab Med., 25: 81-
5.
*******
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Indian Journal of Basic and Applied Medical Research; September 2016: Vol.-5, Issue- 4, P. 128-131
Original article:
A comparative study of blood cross match using newly introduced gel
technique and conventional tube Method
*Dr. Santosh Kumar Gond1 , Dr.S.K.Mishra 2 , Dr. Ashutosh Garg3,Dr.Priyanka Mishra4
1- P.G. Resident ,Department Of Pathology,S.S.Medical college ,Rewa
2- Associate Professor,Department of pathology,S.S.Medical college ,Rewa
3- Demostrator ,Department of Microbiology,S.S.Medical college ,Rewa
4- Post Graduate Student ,S.S.Medical college Rewa * Corresponding author
Abstract:
Introduction: A comparative study of blood cross match between gel card technique and conventional tube method was
undertaken on approx 1000 sample conducted in Sanjay Gandhi Memorial Hospital ,Rewa associated with Shyam shah
medical college Rewa .
Material & Methods: most commonly conventional tube method are used widly. Now new technique of cross matching is
introduced . In this study Matrix gel card[16] method based on indirect coombs test for cross match and conventional tube
method including saline tube method and indirect coombs tube method used.
Observation and result: one thousand sample is taken for the study and out of this 996 sample is compatible using indirect
coombs gel card and indirect coombs tube test and 04 sample shows incompatibility of both test ,whereas in saline method
without using coombs reagent shows 100% compatibility, if we use coombs indirect test, 04 sample shows false positive
and 04 sample shows false negative of previously 100% compatible result. Sensitivity and specificity is 100% of gel card
and indirect coombs tube test using AHG, whereas saline tube test specificity is 99.6% .And positive predictive value 100%
for Gel card and indirect coombs test[5][6] using AHG and 99.6%for saline tube method if AHG not used.
Conclusion : Gel card used in blood cross match is easy to performed and recorded test and more sensitive and specific then
convention saline method whereas indirect coombs tube test is sensitive and specific as Gel card but cannot record and more
time consuming than saline and gel card method.
Keywords: Gel technique ; conventional tube methods
Introduction: (IAT&DAT)Present study is carried out for com-

the current study was done in blood bank of Sanjay parison between gel card and conventional tube
Gandhi memorial hospital ,Rewa, (M.P) , newly test for sensitivity and specificity , time and cost
introduced a technique called Matrix Gel Card efficieny .
technique for blood cross matching. Previously Aims and objectives:
conventional tube methods are used for blood cross Comparative study between conventional cross
match which is mainly saline tube method(Spin match and matrix gel card technique on the basis
tube method) and indirect coombs tube method . of sensitivity and specificity.
gel technique is introduced by Lapierre, which Material and methods:
based on controlled centrifugation of red cells 1000 randomly sample selected and collected
though sephadex gel contained within microtu- from donors attending blood bank of Sanjay
be.gel techniques also used for various test like Gandhi memorial Hospital ,Rewa .Donors are
ABO and Rh typing, ide-ntification of all antibo- healthy and >47 kg of weight with negative
dies , indirect and direct antiglobulin test serology of HIV,HCV, HbsAg, VDLR and
128
www.ijbamr.com P ISSN: 2250-284X , E ISSN : 2250-2858
Malaria. In present study first we done the blood clean and ready for conduc-ting the test Gel card
grouping by using Antisera A,B,D of patient blood technique: first prepare a 0.8% red cell suspe-nsion
and donors blood bag. After matching of blood by adding 1ml diluents-2 in to clear test tube then
group we proceeds to cross matching of the donor add by micro pipette 10µl of packed red cells of
and recipient blood by using two methods first is donor to it. after this take a Matrix gel card open
Conventional tube method with AHG (IAT) and the foil of one micro tube gently and write the pati-
without it. Second method is Matrix Gel Card ent id no. below particular micro tube then add
method which is newly introduced in our blood 50µl of 0.8 donor red cell suspension to it after this
bank. Method which is apply in Conventio-nal tube add 25µl patient serum to it. Incubate the gel card
method first marking the patient and donor test in Matrix gel card incubator for 15 minutes at
tube with marker ,centrifuge the both blood sample 370C. After incubation centrifuge the card in Mat-
and extract the serum of patient and donor red rix gel card centrifuge machine for 10 min-utes and
cells, mixing of serum of patient and red cells of then read the result.If gel card result shows RBCs
donor in clean test tube and after this we add the are settled bottom of particular micro tube means
Anti Human globulin (AHG, Coombs Reage- No agglutination (Negative result) that means
0
nt[5][6]) and incubate in 37 C and then see the Donors blood is compatible to the recipient and
result if clumping present in test tube blood bag is suitable for trans-fusion. If RBCs are trapped betw-
incompatible. if not present blood is compa-tible een upper and bottom of tube that means somet-
for patient. Second method is Matrix Gel Card hing is wrong and result are called Positive result,
method in this method special machine used for incompatible for recipient. Positive result are grade
centrifuge of Gel Card and also incubator for Gel in to +4 to +1.(+4 means top of micro tube and +1
card ,LISS, test tubes and micropipette first we near to bottom of micro tube).
Observation and result:
Method Used Sample Compatible Incompatible
Size TN FP TP FN
01 Conventional tube method without AHG 1000 992 04* 00 04**
02 Conventional tube method with AHG(IAT) 1000 996 0 04 00
03 Matrix Gel Card 1000 996 0 04 00
*,** Result obtained only if AHG used with Conventional method otherwise it shows 100% compatible result.
Table no. 01 shows 1000 random blood sample is false positive(FP) and 04 sample shows False Neg-
cross matched by using conventional tube method gtive(FN) if we Add AHG (IAT) this is calculated
with and without using AHG(IAT) and Matrix Gel after compare the result of conventional tube meth-
Card . Result are observed in conventional tube od with AHG and Matrix Gel card method which
method without AH-G ,1000 sample shows 100% shows 996(99.6) sample compatible and 04 (0.4%)
compatibility but in the table 04 sample shows True Positive(TP)
129
Indian Journal of Basic and Applied Medical Research; September 2016: Vol.-5,
Vol. Issue- 4, P. 128
128-131
992
1000 996
996
500
4
4 0
4 0
0 0
SalineTube without AHG 0
4
SalineTube with AHG 0
Matrix Gel card False positive

True positive
False Negative
True Negative
True Negative False Negative True positive False positive
Graph No.01
Observation and result plotted in graph
Discussion: which is agreement with this stu-dy.
dy.Rumsey DH
In India and other country the gel test performed proposed that the gel test at least as
et al[18]proposed
in various institutions and hospitals for blood sensitive as an LISS IAT tube test with a better
cross match it is first introduced by Lapierre et balance of sensitivity and specificity.
specificity.Bromillow et
al.[11] he gives the idea of six micro tube embe-
embe al[2][3] proposed that The number of non
non-specific
dded in plastic card. Microt-ubes filled with antibodies and false-positive
positive screens were reduced
specif-ic
ic gel medium which allows to testing using thee gel test system. Antibody tit
titers perform-
,easy reading , recording, handling and disposal . ed using the gel system were more sensitive than
In our present study 0.4% sample shows incomp-
incomp with our tube IAT method. The gel system was
atibility (agglutination)
ion) by gel card method and easy to use and gave reliable, reproducible results.
also convent-ional
ional tube method using IAT(AHG). My study agree-ment
ment with result but my result
Where as conventional tube method (Spin tube) obtained with tube IAT same as gel card method
without IAT shows 100% compatibility which is .Noveretti MCZ et al.[14] result shows that gel
not correct because 04 sample shows False test is more sensitive than tube test for identifying
Negative and 04 sample shows False Positive if we potentially clinically significant antibo
antibo-dies. Cat
subjected to IAT.The specificity and sensitivity is et al[4] testing efficiency was improved following
100% of both gel card and conventional tube introduction of the gel test into routine use.
method with IAT(AHG) ,where as specificity of Kaur et al[8] study shows that DiaMed gel card
conventional tube (Spin tube) without IAT is system easy to use and his finding suggest it
99.6%.Swarup et al[15] concluded that gel card proved to be more sensitive than the conventional
method is better than
an conventional spin tube tube agglutination technique. Nathlang et al
method because of its simplicity, stability of [17]study
study proposed that the gel test equal or better
results, dispensation of controls, absence of wash than conventional testt tube method and simple to
phase with comparable sensitivity and specificity performed and less exposure of blood bank person
130
www.ijbamr.com P ISSN: 2250-284X
2250 , E ISSN : 2250-2858
to blood specially area with HIV infection is Conclusion

prevalent. My study agree with above both prior Gel card is more sensitive and more specific than
study and its result. Jai prakash et al [7]conc- conventional tube methods and also less time
luded that gel test is better alternative to the con- consuming but more costly than conventional
ventional tube test for both DAT and IAT.Over all, tube methods. As per result we concluded and
prior study which is mention above are correlate advice for use of gel card in vari-ous blood ban-
with my present study with maximum findings. king services as routine test in cross matching for
prior blood transfusion because of high sensit-ivity
and specificity than conventional tube methods.
References:
1. Blundell et al. Observations on transfusion of blood by Dr.Blundell with a description of his gravitator. Lancet.1828; ii: 321-
324.
2. Bromilow IM et al. Gel Techniques in blood group serology. Med Lab Sci 1992; 49: 129-32.
3. Bromilow IM et al. Evaluation of the ID gel test for antibody screening and identification. Transfusion Medicine 1991; 1:
159-61.
4. Cate John C, Reilly N. Evaluation and implementation of the gel test for indirect antiglobulin testing in a community
hospital laboratory.Arch of Pathol and Lab Med 1999; 121: 693-7.
5. Coombs, R.R.A et al. A new test for the detection of weak and ìncomplete' Rh agglutinins. British Journal of Experimental
Pathology. 1945; 26: 255-266.
6. Coombs, R.R.A et al. In-vivo isosensitisation of red cells in babies with haemolytic disease.Lancet.1946; i: 264-266.
7. Jai prakash M et Al. Role of gel based technique for coomb’s test. Indian J pathol Microbiol.2006;49(3):370-2.
8. Kaur R,Kakar et al. Use of gel based DiaMed -ID microtyping system for cross matching enhanced sensitivity.indian J
Pathol Microbiol.2003,46:617-20.
9. Landsteiner, K. On agglutination of normal human blood [Translation of article originally published in 1901: UÈ ber 766
Historical Review q 2000 Blackwell Science Ltd, British Journal of Haematology.1961; 110: 758-767 .
10. Landsteiner, K.S. & Wiener, A.S.An agglutinable factor in human blood recognized by immune sera for rhesus blood.
Proceedings the Society for Experimental Biology and Medicine.1940; 43: 223.
11. Lapierre Y. The gel test: A new approach for detection of red cell antibodies/ antigen in a solid phase. Proceedings of XX
Congress of the International Society of Blood TransfusionSociety. Manchester: British Blood Transfusion
Society;1988:145.
12. Lapierre Y, Rigal D. The gel test: A new way to detect red cells antigen - antibody reactions; Transfusion. 1990; 30: 109-13.
13. Novaretti MCZ, Jeus ES. Evaluation of a gel test system for the detection of transplacental haemorrhage. Transfusion 1994;
34 (Suppl): S 110.
14. Novaretti MCZ, Jens ES, et al. Comparison of tube and gel technique for antibody identification. Immunohaematology
2000; 16: 138-41
15. Col D Swarup, Brig PS Dhot, Lt Col J Kotwal, Lt Col AK Verma. Comparative Study of Blood Cross Matching Using
Conventional Tube and Gel Method. Air force journal india.2008:129-130.
16. Tulip group web page: http://www.tulipgroup.com/Common_New/instrumentation.htm#
17. Nathalang O, Kuvanont S, Suwanasit T, Yensuang K, Sriphaisal T, Krutvacho T. A preliminary study of the gel test for
cross matching in Thailand. J Med Tech Assoc Thai 1993;21:101–106.
18. Rumsey DH, Ciesielski DJ. New protocols in serological testing:A review of techniques to meet today’s challenges.
Immunohaematology 2000; 16: 131-7.
131
Vox Sanguinis © 2015 International Society of Blood Transfusion
Vox Sanguinis (2015) 109 (Suppl.), 1-379, P-495
DETECTION AND IDENTIFICATION OF RED CELL ALLOANTIBODIES IN

MULTIPLY TRANSFUSED THALASSEMIA MAJOR PATIENTS
Jain RJ1, Jain P1, Choudhary NC2 and Mahadik VK1
1
C.R.G. Hospital and R. D. Gardi Medical College, Ujjain, India 2Fortis Hospital, Gurgaon, India
BACKGROUND:
Lifelong red blood transfusion remains the main treatment for â-thalassemia major patients. Transfusion therapy could be
complicated with the development of anti RBC antibodies (alloantibodies and/or autoantibodies). Some alloantibodies
are haemolytic and may cause haemolytic transfusion reactions and limit the availability of further safe transfusion.
Alloimmunization to red cells antigens is one of the most important immunological transfusion reaction and causes
delayed type of transfusion reaction.
AIM AND OBJECTIVE:

(i) To provide frequency and distribution pattern of various types of irregular red cell alloantibodies in patients with
thalassemia major. (ii) To determine the mean red cell transfusion requirement and mean transfusion duration.
METHOD:
A prospective study was conducted from January 2014 to December 2014 at Transfusion Medicine and Blood Bank Dept.
Seventy eight diagnosed thalassemia major patients were included in this study and samples collected and investigated
for the development of alloantibody to red cell antigens by using Matrix Gel System (Tulip Diagnostics). Five to seven ml
of blood was collected in plain tube serum was separated. Separated serum was taken in two aliquots, labelled properly
and stored in two different boxes at -30°C in deep freezer, till the antibody screening and identification was performed.
Tests for antibody screening and identification were performed on preserved sample to investigate prevalence of red cell
alloimmunization by standardized laboratory techniques by the same person. Antibody screening was carried out on
serum employing commercial three-cell panel (Matrix Gel System, Tulip Diagnostics) using standardized blood bank
techniques. If patients were found to have irregular red cell alloantibody then the antibody identification was performed
using commercial 11 cell panel cells (Matrix Gel System, Tulip Diagnostics).
RESULTS:
A total of 78 patients were included in the study. Forty eight patients were males and thirty females. Mean age was 8.2
years. Irregular red cell antibodies were found in 6 patients (7.69%). Mean age of patients who developed red cell
alloantibodies was 12.48 years. Three patients developed single antibodies (50%) (2 patients anti-K and 1 patient anti-C),
while other three developed multiple antibodies (50%) (anti-D and anti-E, anti-D and anti-C, anti-E and anti-K).
CONCLUSIONS:
Red cell alloimmunisation should be kept in mind in the patients receiving multiple transfusions. In present study,
alloimmunisation rate was 7.69%. Mean transfusion duration in these patients was 21.80 days, probably due to the
presence of alloantibody. We also suggest that red cells alloimmunisaton should not be overlooked in patients receiving
regular blood transfusion. RBC alloantibody detection on regular interval and antibody negative blood transfusion is
strongly recommended in transfusion depended thalassemia patients.
Vox Sanguinis © 2015 International Society of Blood Transfusion
Vox Sanguinis (2015) 109 (Suppl.), 1-379, P-689
DETERMINATION OF THE MEAN RED CELL TRANSFUSION REQUIREMENT

COMPARED ON THE BASIS OF IRON OVERLOAD AND TYPE OF CHELATION
THERAPY AND DEVELOPMENT OF ALLOANTIBODIES IN MULTIPLY TRANSFUSED
THALASSEMIA MAJOR PATIENTS
Jain PJ1, Jain RJ1, Choudhary NC2 and Mahadik VK1
1
C.R.G. Hospital and R. D. Gardi Medical College, Ujjain, India 2Fortis Hospital, Gurgaon, India
BACKGROUND:
Transfusion-dependant thalassemia patients, in the absence of chelation therapy, develop progressive accumulation of
iron, which is responsible for tissue damage, and eventually, death. The factors which influence the iron burden are of
chelation therapy and mean red cell transfusion requirement. Increasing red cell transfusion requirement, iron deposit
and development of alloantibodies complicate transfusion therapy in thalassemia patients.
AIM AND OBJECTIVE:

(i) To investigate the patient for the red cell transfusion requirement on the basis of iron overload and type of chelation
therapy. (ii) To determine rate of development of red cell allo0antibodies in thalassemia major patients.
METHOD:
A prospective study was conducted from February 2013 to December 2014. Ninety eight patients were included in this
study and 3 consecutive samples collected after every 6 months and investigation for red cell requirement, compared on
the basis of iron overload and type of chelation therapy. Iron overload was measured by serum ferritin levels.
RESULTS AND OBSERVATIONS:

In present study, mean red cell transfusion requirement was 206.20ml/kg/year (SD = 28.62) majority of the children in this
study i.e. 40 (41.67%) were undergoing hypertransfusion therapy and transfused red cells were in the range of 208-248
ml/kg annually. It was observed that the requirement of red cell transfusion increases with the age of the patient. Out of 96
patients, 86 (89.58%) of thalassemia children were on chelation therapy. Maximum number of patients 37 (38.54%) were
on oral chelation therapy and after this 35 (36.45%) of patients on combined chelation therapy. (Desferroxamine & oral
chelation). Only 14 (14.58%) were on parenteral (desferroxamine) chelation therapy out of 96 patients, 10 (10.41%)
patients were not taking any chelation therapy. In present study the difference of mean red cell transfusion requirement
among all the chelation therapy groups when compared with each other were found highly significant (P<0.01). The mean
red cell transfusion requirement were minimum in combination therapy group (combination of two iron chelators such as
parenteral desferroxamine plus oral deferiprone) followed by parenteral desferroxamine chelation therapy group, oral
chelation therapy group and maximum in patients those started chelation therapy but discontinued.
Irregular red cell alloantibodies were found in 8 patients (8.16%). Five patients developed single antibodies, while other
three patients developed multiple antibodies. (Matrix Gel, Tulip Group).
CONCLUSION:
Red cell transfusion requirement and chelation therapy should be kept in mind in the patients receiving multiple
transfusions. In present study the difference of mean red cell transfusion requirement among all the chelation therapy
groups when compared with each other were found highly significant (P<0.01). The mean red cell transfusion
requirement was minimum in combination therapy group (combination of two iron chelators such as parenteral
desferroxamine plus oral deferiprone) and maximum in patients who started chelation therapy but discontinued it and this
difference was found highly significant (P<0.01). Combination of two iron chelators (such as parenteral desferroxamine
plus oral deferiprone) have been shown to produce addictive and synergistic effects, may produce enhanced iron
excretion, minimize side effect, decrease mean red cell transfusion requirement and improve compliance is strongly
recommended in transfusion dependant thalassemia patients.
Issue 1 January-June 2018
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Original Article
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An approach to incompatible
cross‑matched red cells: Our
experience in a major regional
blood transfusion center at Kolkata,
Eastern India
Website:
www.ajts.org
DOI:
10.4103/ajts.AJTS_157_16 Prasun Bhattacharya, Eeshita Samanta, Nowroz Afroza, Archana Naik,
Rathindranath Biswas
Abstract:
INTRODUCTION: With the increased utilization of immunohematology (IH) analyzers in the
transfusion medicine, type, and screen policy is the method of choice. Still, the importance of routine
crossmatching could not be overruled. Here, we tried to understand the clinical conditions and safety
of red cell transfusion and their outcomes.
MATERIALS AND METHODS: This prospective study was conducted by IH laboratory,
Medical College Kolkata, Blood Bank from October 1, 2015 to March 31, 2016. A set of 3cc
ethylenediaminetetraacetic acid and clotted blood samples of the patients were received according to
sample acceptance criteria. Blood grouping by conventional tube technique followed by crossmatching
was performed by column agglutination technology (CAT) in polyspecific (IgG + C3d) gel media.
Any positive result was rechecked in duplicate with additional two group‑specific donor units. The
persistent incompatibility was further evaluated using direct anti‑human globulin test, auto control,
antibody screening, and antibody identification by CAT.
RESULTS: On the evaluation of 14,387 sets of patients’ sample, only 100 were found to be
incompatible (0.69%). Incompatibility rate is higher in females (59%). Eighty‑five of these patients
were repeatedly transfused. Only 38% of incompatible crossmatch were positive on indirect
anti‑human globulin test/antibody screening. Antibody could be identified in 16 of them. Seventeen
of 100 incompatible samples (17%) presented with panagglutination, were managed with Rh, Kell
phenotype/best‑matched red cell units. In these 16 patients, 23 alloantibodies were identified; allo
anti‑E was the most common.
CONCLUSION: This study showed antibody against the Rh system as the most common cause of
incompatibility.
Keywords:
Department of
Immunohaematology and Antibody screening, antibody identification, column agglutination test, conventional tube
Blood Transfusion, technique, direct anti‑human globulin test, immunohematology analyzer, incompatible crossmatch,
Kolkata Medical College, indirect anti‑human globulin test, panagglutination, polyspecific (IgG + C3d) gel media
Kolkata,
West Bengal, India
Address for Introduction therapeutic support and minimal red cell

correspondence: destruction. With the increasing utilization
O
Dr. Prasun Bhattacharya, ne of the essential goals in
Department of
of automated immunohematology (IH)
Immunohaematology
crossmatching of red cells is that analyzers, the routine cross‑matching is
and Blood Transfusion, the transfused blood must be compatible predominantly replaced by ABO and Rh
Kolkata Medical College, with the patient to provide maximum type and antibody screen or type and
88 College Street,
Kolkata ‑ 700 073, This is an open access article distributed under the terms of the
West Bengal, India. Creative Commons Attribution-NonCommercial-ShareAlike 3.0 How to cite this article: Bhattacharya P, Samanta E,
E‑mail: pbhattach@gmail. License, which allows others to remix, tweak, and build upon Afroza N, Naik A, Biswas R. An approach to incompatible
com the work non-commercially, as long as the author is credited cross-matched red cells: Our experience in a major
and the new creations are licensed under the identical terms. regional blood transfusion center at Kolkata, Eastern
Submission: 22‑12‑2016 India. Asian J Transfus Sci 2018;12:51-6.
Accepted: 29‑05‑2017 For reprints contact: [email protected]
© 2018 Asian Journal of Transfusion Science | Published by Wolters Kluwer - Medknow 51

Bhattacharya, et al.: An approach to incompatible cross‑matched red cells
screen (T/S) policy. In the Eastern part of India, major observed in the blood samples, they were referred to the
cross‑matching between the recipient’s serum and blood transfusion officer (BTO) / resident doctor on duty
donor red cells by anti‑human globulin is the most for further decision making. Blood sample(s) showing
common practice in most of the blood banks. These visible evidence of gross deterioration/hemolysis
tests are carried out either by the conventional tube were excluded from the study. If the patient had a
techniques (CTT) or by the semi‑automated column previous history of blood transfusion, the transfusion
agglutination technology (CAT). This is due to the records related to blood group and any other relevant
constraints related to trained workforce and availability information were verified.[2]
of regular supply of reagents and other logistics.
Preparation of the blood sample and blood
It appears once the recipient’s ABO and Rh blood type is grouping
known, a transfusion of compatible blood can be given. The EDTA and clotted vial were centrifuged at 3000 ×g
However, in practice, donor red blood cells (RBCs) may for 3 min to separate red cells and serum/plasma.[3] ABO
still be incompatible as it contains other minor antigens and Rh typing was done by CTT using commercially
against which the recipient is alloimmunized/sensitized. available monoclonal antibodies (Tulip Diagnostics Pvt.
Therefore, a cross‑match is done to ensure that the donor Ltd., India). Reverse grouping was performed by CTT
RBCs actually do match against the recipient’s serum. using freshly prepared in‑house reagent pooled A, B,
There are times when even after an exhaustive workup, and O cells.[4] Tests were validated by a negative saline
a unit of compatible red cells becomes unavailable for control. Any discrepancy in blood grouping results
the patient. The commonly observed clinical conditions was resolved according to their type and classification,
and the insights obtained on how safe to transfuse as per departmental standard operating procedures
the best unit of blood available was reviewed here and recorded. The concerned treating facility was also
along with their outcomes. The clinical and serologic intimated of their significance.
evaluation, which allows for the transfusion of the most
compatible (or “least incompatible”) blood, requires a Cross‑matching of patient’s sample
joint effort between the clinician and the transfusion Once the blood group of the recipient’s sample was
medicine physician.[1] determined, a major cross‑match using group‑specific
donor red cell units (1% donor red cell suspension in
Materials and Methods low ionic strength saline solution) was done by CAT in
polyspecific (IgG + C3d) gel media (Matrix gel system,
A prospective analysis was conducted in all the Tulip Diagnostics Pvt. Ltd., India). The tests were
incompatible cross‑matched blood samples at the IH performed according to the manufacturer’s instructions.
laboratory of Kolkata Medical College Hospital blood
bank since October 1, 2015–March 31, 2016 (6 months). A positive and negative control were run daily in parallel
This blood bank is one of the major regional blood with the tests to validate the test results.[5]
transfusion centers in the state of West Bengal (WB),
Eastern India, with an average annual blood collection Evaluation of an incompatible cross‑matched
over 30,000 units. The center supplies an overall annual sample
average of 50,000 units of blood components to the In case of any incompatible major cross‑match results,
patients who were admitted in the hospital itself as well a repeat cross‑match with the same donor unit was
as patients referred from other hospitals located within performed along with two additional group specific
or outside the city of Kolkata. Red cell concentrates donor units. This repeatation was done to rule out any
constitute a major volume of the supplied blood possibility of technical errors (contamination, direct
components to the extent of 60% approximately. This anti‑human globulin test [DAT] positive donor unit,
study had been approved by the Institutional Ethics mis‑grouping, etc.) as well as clerical/transcriptional
Committee. errors. If the incompatibility persisted on repetitions in
any of these 3 units, a further evaluation of the recipient’s
Sample acceptability criteria sample was done in the departmental IH laboratory. The
Any request for blood component(s) was accompanied IH laboratory could not routinely perform T/S policy so
by a duly filled and authorized blood requisition form as such an alternative protocol is chosen.
designed by the WB State Blood Transfusion Council along
with properly labeled 3 cc ethylenediaminetetraacetic An initial workup of the recipient’s sample was done
acid (EDTA) and 3 cc clotted blood samples. The by DAT, auto‑control and antibody screening using
samples should be freshly collected mentioning the commercially available cells or in‑house prepared
name of the patient with patient identity number and screening cells.[3] Any reaction with a strength of 2+ or
phlebotomist’s signature. If any nonconformity were above was considered to be strong and below these were
52 Asian Journal of Transfusion Science - Volume 12, Issue 1, January-June 2018
weakly reactive. Antibody identification was done in monitored with a posttransfusion 24 h increase in
antibody screen positive samples, using commercially hemoglobin (Hb) and clinical improvement of signs
available reagent 11 cell panel (Ortho‑clinical Diagnostics and symptoms. A fresh set of blood samples (EDTA and
Inc., USA) by CAT in Ortho BioVue system on clotted) were required for each and every transfusion
polyspecific AHG (IgG + C3d) cassettes. The workflow requisition, irrespective of the time interval between
of evaluation of an incompatible cross‑match was shown two consecutive transfusions.
in Figure 1. However, a detail of clinical history of the
patient along with the history of medications and relevant Results
history suggestive of alloimmunization/sensitization is
recorded by the transfusion medicine resident doctor, Demographic distribution, clinical history,
wherever was possible.
and history of alloimmunization in the study
Selection and issue of appropriate donor red cell population
A total of 14,387 sets of patient’s samples were accepted
unit
at the blood bank during 6 months period. Only
Wherever any alloantibody(s) were being detected
100 (0.69%) of these 14,387 were found to be cross‑match
corresponding antigen(s) negative compatible or
incompatible and subjected to evaluation and selection
best‑matched unit was issued after consultation with
the treating clinician.[1] In situations where no specific of appropriate donor units [Figure 2]. The cross‑match
alloantibody could be pointed, group‑specific, extended incompatibility was much higher in the females (59%)
Rh and Kell phenotype matched (where the patients than the males (41%) [Figure 3]. An overall distribution
were transfusion free for more than 3 months) red of incompatibility ranges from 1 to 68 years of age, with
cell units were provided as emergency lifesaving a maximum incidence of 39% (n = 39) in the 11–20 years
resort. In situations where patients received recent age group. A minimum incidence of 5% (n = 5) was
transfusions (within 3 months) best‑matched units observed in the persons above 50 years of age [Figure 4].
(less strength than autocontrol) were provided.[6,7]
On an overall 14,387 red cell demands, majority were for
Each of these transfusions was under the supervision anemia (n = 8925), surgical procedures (n = 3455), and
of the treating clinician or the transfusion medicine obstetric cases (n = 1005). The rest of the 1002 belonged
resident. In the event of any adverse outcome, the to other category which was requested for miscelleneous
transfusion was stopped immediately and the blood reasons, namely, acute hemorrhage, trauma, dialysis,
bank resident doctor was informed for further etc., [Figure 5]. The majority population of anemic
proceedings. Every successful transfusion events were patients were suffering from thalassemia (n = 4115, 46%),
Patient’s sample + Request form
Acceptable Unacceptable
ABO & Rh typing Request for fresh

sample
Blood group Referred to BTO
Blood group
discrepancy in acute life
confirmed
saving
conditions
Past record verification & or resolution
as per departmental SOPs
Cross match with group

specific donor units
Compatible Incompatible
Repeat cross match with same donor

unit along with 2 more donor units
Detail IH work-up + Information

Incompatible to the treating clinician for
further proceeding and follow up
Figure 1: Evaluation of an incompatible cross-match sample
Asian Journal of Transfusion Science - Volume 12, Issue 1, January-June 2018 53

hematological malignancies (n = 1865, 21%), autoimmune causative antibody could be identified in 16 of them,

hemolytic anemia (AIHA) (n = 88, 1%), and other causes with an overall antibody identification rate of 42.10%
of anemia (n = 2857, 32%) [Figure 5a and b]. on IAT/antibody screen positive samples. In the rest 22
of these 38 patients, the specific antibody identification
Out of these 100 patients, 85% (n = 85) were repeatedly could not be done with the available logistics. On
transfused. Thalassemia, hematological malignancies and the other hand, 17 of the 100 samples presented with
autoimmune anemia (primary/secondary) constitute an DAT positive and panagglutination, where only blood
overall 78% (n = 78) of the total burden of cross‑match group specific, best‑matched or extended Rh and Kell
incompatible samples. Comparative details of the total phenotype‑matched red cells were transfused [Figure 8a
study population versus the incompatibility results are and b]. The complete analysis of the rest 45 patients could
shown in Figure 6. not be done as either they were lost to follow‑up or the
patient’s blood sample was not received again.
Laboratory workup and immunohematology
analysis of blood samples Out of the 16 patients where antibody detection could
DAT was positive in 53% (n = 53) incompatible samples be done, 6 of them were multiple antibodies and 10 were
and 44 of these were strongly positive (more than 2 in single. An association of c, E antibodies was observed
strength/ grade) [Figure 7]. Of these 44 strongly positive in 5 out of 6 patients with multiple alloantibodies. The
DAT samples, 21 weakened their strength on auto control. other patient with multiple alloantibody was E, S, and N.
A total of 38 (38%) of the incompatible cross‑match

blood samples were positive on indirect anti‑human
globulin test (IAT)/antibody screen on CAT. The
41
Male 41
100
Female 59
59
Total Sample
Incompatible
Cross Match
14387
Figure 3: Gender distribution of the incompatible patients
10000 8925
9000
8000
Figure 2: Total sample vs incompatible cross match 7000
6000
5000
4000 3455
3000
45 2000 1005 1002
1000
39 0
40 Anaemia Surgical Obstetrics Others
a
35
30
25 Thalassemia
19 20 2857(32%)
20 Hemato Oncological
4115(46%)
15 AIHA
10 88(1%)
10 7 Others
5 1865(21%)
5
0
00-10 11-20 21-30 31-40 41-50 above 50 b
Figure 5: (a) Overall disease distribution in the study population (b) Further
Figure 4: Overall age distribution of 100 incompatible patients distribution of disease under Anemic population

Table 1: Antibody profile in incompatible cross‑match

70%
Anemic Population patients
60% 58%
Incompatible patients Antibody profile in Type specificity of antibody
50% 46% patients (n=16)
Patients with multiple Anti (c + E) (n=5)
40%
32% antibodies (n=6) Anti (E + S + N) (n=1)
30% Patients with single Anti E (n=4)
21% 22%
20%
antibody (n=10) Anti c (n=3)
14%
Anti D (n=1)
10% 6%
1% Anti Kell (n=1)
0% Anti JKb (n=1)
Thalassemia Hemato-Oncology AIHA Others
Figure 6: Comparative disease distribution among Anemic population and phenotype matched red cells transfusion along with
incompatible patients
steroid/rituximab therapy, recovered uneventfully
with an appropriate rise in Hb level and became DAT
negative after 3 months. An overall transfusion reaction
was observed in 2 of these 17 patients (11.7%). There
9 was no event of death due to adverse outcome.
Discussion
Strong (> 2+) Incompatibility in cross‑matching during pretransfusion
testing is not uncommon. There is hardly any evidence‑based
Weak (< 1+)
study on frequency of incompatible cross‑matched red cells
and how to approach these cases for better transfusion
practice from the eastern part of India till now.
44
In our study, we rechecked all the ABO and Rh (D)
group specific incompatible cases with the same donor
unit (along with two other separate donor units) to exclude
Figure 7: Distribution of DAT +ve samples clerical error, as clerical error is the most common cause
of incompatibility as shown by Stainsby et al. in UK.[8] The
incidence of persistent incompatible cases were 0.69%,
whereas the study by Bhatt et al. in Western India showed
an overall incidence of incompatibility were 0.21%.[9]
38
45 16 In the present study, majority of incompatible crossmatches
22 were found in females (59%) which is comparable to the
study conducted by Bhatt et al. in western part of India.[9]
17
Incompatibility was most prevalent in the age group of 11–
20 years (39%) and they were mostly thalassemic patient.
Ab-Screening/IAT +ve DAT +ve and Pan
A total of 58% of incompatible patients were thalassemics.
Lost follow-up Agglutination Ab - identified Ab Unidentified The other important causes of incompatibility were
a b
AIHA (14%) and hematological malignancy (6%). This is
Figure 8: (a) Overall serological status of incompatible samples (n = 100) in contrast to the study conducted by Bhatt et al. where
(b) Results of further evaluation of IAT positive samples (n = 38)
peak incidence seen in AIHA (40%).[9] The present study
had shown repeated red cell transfusion was the major
The specificity of the alloantibody detected in 16 patients factor associated with incompatible cases (85%).
is given in Table 1. A total of 23 alloantibodies were
identified in 16 patients. Majority of these antibodies On analysis of these incompatible blood samples, only
identified were of the Rh system (19/23 [82.60%]) with 38 cases were found to be IAT/antibody screening
anti‑E being the most common antibody (10/23 [43.47%]). positive. Among these 38 IAT/antibody screening positive
cases alloantibody against red cell antigens was detected
It was also observed that 6 of these 17 patients (initially in 16 of them (42.1%), panagglutination (agglutination
showing DAT positivity and panagglutination) who with all reagent cells) with DAT positivity was found in
came for further follow‑up after receiving best match/ 17 patients (44.73%). A single alloantibody was detected
Asian Journal of Transfusion Science - Volume 12, Issue 1, January-June 2018 55
in 10 patients (62.5%), and the rest 6 patients were Dr. Biswajit Halder, Dr. Sukanya Banerjee and
having multiple alloantibodies (37.5%). A total of seven Dr. Krishna Basu Choudhuri.
different types of alloantibodies were observed in these
16 patients [Table 1] having both single and multiple Financial support and sponsorship
antibodies. Most of the alloantibody detected belonged to Nil.
the Rh system (82.6%, 19 out of 23), of which anti‑E (43.47%)
was the most common followed by, Anti-c (34.78%), and Conflicts of interest
anti‑D (4.34%). This result is comparable to the study as There are no conflicts of interest.
observed by Goldfinger and Lu.[10]
References
Conclusion
1. Petz LD. “Least incompatible” units for transfusion in autoimmune
This study showed the antibodies against Rh system hemolytic anemia: Should we eliminate this meaningless term? A
commentary for clinicians and transfusion medicine professionals.
antigens were the most common cause of incompatibility
Transfusion 2003;43:1503‑7.
in multi‑transfused patients. A significant number of 2. Saran RK. Transfusion Medicine Technical Manual. 2nd ed.
incompatible cross‑match were found due to AIHA, New Delhi: Directorate General of Health and Family Welfare
presented with positive DAT and panagglutination Government of India; 2003. p. 117‑8.
in antibody screening panel and were managed by 3. Datta SS, Mukherjee S, Talukder B, Bhattacharya P, Mukherjee K.
best‑matched red cells. The treating clinicians were Frequency of red cell alloimmunization in thalassemia
patient: A report from Eastern India. Adv Hematol 2015;1‑6.
informed about the type of AIHA (warm/cold/mixed) Doi:10.1155/2015/61093.
to start the definitive treatment. 4. Fung MK. Technical Manual. 18th ed. Bethesda, Maryland, USA:
AABB; 2014. [Methods 2‑2].
A significant number of these AIHA patients were 5. Fung MK, Technical Manual. 18th ed. Bethesda, Maryland, USA:
followed up for 3 months, and on follow‑up, they showed AABB; 2014. p. 376.
clinical improvement following steroid/rituximab along 6. Plapp FV, Beck ML. Transfusion support in the management of
with transfusion therapy. immune haemolytic disorders. Clin Haematol 1984;13:167‑83.
7. Ness PM, Shirey RS, Thoman SK, Buck SA. The differentiation of
delayed serologic and delayed hemolytic transfusion reactions:
Since the majority of alloantibodies are detected against Incidence, long‑term serologic findings, and clinical significance.
the Rh system (82.6%), extended Rh phenotyping Transfusion 1990;30:688‑93.
of the donor red cells and the recipients at the onset 8. Stainsby D, Russell J, Cohen H, Lilleyman J. Reducing adverse
of initial transfusion may prevent the development of events in blood transfusion. Br J Haematol 2005;131:8‑12.
alloantibodies in the multitransfused patients. 9. Bhatt JK, Patel TR, GajjarMD, Solanki MV, Bhatnagar NM,
Shah SD. Evaluation of incompatible crossmatch at blood bank
of a tertiary care teaching hospital in Western India. Pathol Lab
Acknowledgment Med 2016;7(1). www.openventio.org
The authors would like to acknowledge the continuous 10. Goldfinger D, Lu Q. The incompatible crossmatch. 2013. Available
support and patronage from Prof. Krishnendu Mukherjee, from: www.uptodate.com [Last accessed on 2016 May 30].

DOI: 10.14260/jemds/2015/1825
ORIGINAL ARTICLE
A STUDY OF IRREGULAR ANTIBODIES IN 200 MULTI-TRANSFUSED
PATIENTS
Rakesh P. Pimpaldara1, Arpit C. Patel2, Jitendra Patel3, Snehal Patel4, A. N. Pandya5,
Sangita Wadhwani6
HOW TO CITE THIS ARTICLE:

Rakesh P. Pimpaldara, Arpit C. Patel, Jitendra Patel, Snehal Patel, A. N. Pandya, Sangita Wadhwani. “A Study of
Irregular Antibodies in 200 Multi-Transfused Patients”. Journal of Evolution of Medical and Dental Sciences
2015; Vol. 4, Issue 73, September 10; Page: 12659-12667, DOI: 10.14260/jemds/2015/1825
ABSTRACT: BACKGROUND: Alloimmunization is one of the major concern in the management of

patients who required repeated blood transfusion as a lifesaving treatment. The knowledge of
incidence of such alloantibodies is essential for selecting appropriate red blood cells for transfusion.
AIMS: This study was carried out to get the frequency and type of unexpected red cell antibodies in
the multi-transfused patient at a tertiary level government hospital in South Gujarat. MATERIALS
AND METHODS: This prospective study was carried out in 200 patients who required multiple
blood transfusions. The antibody screening was done with 3 & 11 commercial cell screening &
identification panel by column agglutination technique (Matrix Gel System & Matrix Erygen AS-ID,
Tulip Diagnostics, India) at saline & anti-human globulin phase. RESULTS: The overall prevalence of
alloimmunization was 7.0%. The majority of these had a single alloantibody (11 cases, 84.62%)
whereas the remaining 2 cases (15.38%) had multiple antibodies. The anti-c and anti-D antibodies
comprised the most common alloantibody (27% each both) followed by, anti-N (20%), anti-C (13%),
anti-e & anti-M (7%) antibodies. Gender & number of blood units were found to be risk factors of
alloimmunization in transfused patients. In our study we found females (79%) are more prone to
alloimmunization. Those who were transfused more than 2 units have higher frequency of
alloimmunization. The highest incidence of alloimmunization was observed in obstetrics and sickle
cell patients. CONCLUSIONS: The majority of alloantibodies detected in the current study were
clinically significant and of mainly belonging to Rh blood group system. Thus pre-transfusion
antibody screening on patients’ samples prior to cross-match needs to be initiated in India and we
can at-least provide corresponding Rh antigen negative blood to ensure safe transfusion practice
KEYWORDS: Red Cell, Red cell antigen, Alloimmunization, alloantibodies, Indirect Antiglobulin Test.
MESHTERMS: Erythrocyte, Isoantibodies, Coombs test.
INTRODUCTION: Alloimmunization is one of the major concern in the management of patients who
required repeated blood transfusion as a lifesaving treatment. In the patients affected with
haemoglobinopathies, haematologic diseases, various types of cancers, recipients of organ
transplantation, and patients with renal failure, the prevalence of alloimmunization has been
reported to be up to 60 per cent.[1] Alloimmunization further complicates the transfusion therapy due
to difficulty in getting compatible blood & delayed haemolytic transfusion reaction.[2] The knowledge
of such alloantibodies is essential for selecting appropriate red blood cells for transfusion. This study
was carried out to get the frequency and type of unexpected red cell antibodies in the multi-
transfused patient at a tertiary level government hospital in South Gujarat.
J of Evolution of Med and Dent Sci/ eISSN- 2278-4802, pISSN- 2278-4748/ Vol. 4/ Issue 73/ Sept 10, 2015 Page 12659
DOI: 10.14260/jemds/2015/1825
ORIGINAL ARTICLE
MATERIALS AND METHODS: The study was performed between the years 2012 to 2014 at blood
bank attached to department of Immunohematology & blood transfusion of tertiary level Government
Medical College and Hospital of South Gujarat after obtaining ethical committee clearance from the
institute and assessed for the presence of alloantibodies. Antibody screening was carried out in 200
multi-transfused patients prior to compatibility testing. A detailed clinical and transfusion history
was taken using a set performa which included the name, identification number, age, sex, diagnosis,
blood group, transfusions done till date of request, transfusions during the present study period,
result of serological testing like direct antiglobulin test and auto control, antibody screen tests in the
study period with results and antibody identification results.
The blood requisition for these patients were received along with samples (plain & EDTA) for
antibody screen testing and compatibility testing as a protocol. ABO and Rhesus blood grouping tests
were done by forward and reverse grouping in all patients so as to confirm the blood group.
Subsequent antibody screening was performed on all samples using a commercially available three
cell panel (Matrix gel system & Matrix Erygen AS; Tulip Diagnostics, India) by the column
agglutination method, using saline, antiglobulin & enzyme phase.
Antibody screening was done for antigens of blood groups which include Rh, Kell, Kidd, Duffy,
Lewis, P and MNS antigens along with an auto-control. Antibody screen positive samples were
further analysed for the specificity of the alloantibody with an eleven cell identification panel (Matrix
gel system & Matrix Erygen ID, Tulip Diagnostics, India). Later on compatible blood at anti-human
globulin phase was issued for transfusion whenever required. An auto control using the patient's own
cell and serum was tested in parallel with each screen to exclude presence of autoantibodies. The
criteria for antibody screening and identification were based on the standard recommendations and
Manufacturer Company.[3,4]
STATISTICAL ANALYSIS: The patient with positive screen was assessed based on gender, age, and
history of transfusion, clinical diagnosis and alloantibody specificity. The two sided chi square t test &
odds ratio were performed to determine the difference in antibody rate by gender and no of
transfusions. P<0.05 was considered significant. The analyses and data management were performed
using Epi Info software version.
RESULTS: A total of 200 patients (114 male & 86 female) were included in the present study.
Different diagnosis of 200 patients was: 36(18%) of thalassemia, 30(15%) of sickle cell disease,
29(14%) with surgical illness, 23(12%) of other anaemia, 22(11%) with renal disease, 16(08%) of
leukaemia, 14(07%) with GIT diseases, 10(05%) with obstetrics condition and 20(10%) with other
diseases (Figure 1). Age of the patients included in the study ranged from 2 to 85 years with a mean
age of 28.21±16.78 years. Among the alloimmunized cases, the age range was 18 to 35 years with a
mean age of 26.61±5.3 years. Among total number of cases, 14(07%) patients were positive for
different type of irregular antibodies while remaining 116(93%) patients were negative for
alloimmunization. 13 patients were included in study as one patient was having auto antibody.
Among the total 13 number of patients with alloantibodies, three male patients had positive
results for antibody which is 2.70% of total male patients and 23.07% of total positive cases while ten
female patients were positive for alloantibody which is 13.33% of total female patients and 76.92%
of total positive cases.
DOI: 10.14260/jemds/2015/1825
ORIGINAL ARTICLE
Female patients were significantly more positive for irregular antibodies (Chi-square test 2
tailed P value is 0.012). The odds ratio for male and female positivity was 5.4 indicating that female
were 5 times more prone to develop alloantibodies in comparison to male patients. Among the
positive cases, blood group distribution is shown in Figure 2. Out of 200 patients, 94(47%) patients
had received ≤2 units of blood transfusion, among which 01(01.06%) had developed irregular
antibodies while 106(53%) patients had received >2 units of blood transfusion, among which
12(11.32%) had developed irregular antibodies which showed significant difference between these
two groups (chi-square test 2 tailed P value is 0.008). Among the 13 patients with alloantibodies, 11
patients (84.62%) had a single alloantibody, whereas two patients (15.38%) had multiple
alloantibodies.
Among the total 13 patients with alloantibody/alloantibodies, four (31%) patients were
having anti-c antibodies, Three (23%) patients were having anti-N antibodies, two (15%) patients
were having Anti-D, two (15%) patients were found positive for both anti-D & C, one (08%) each
patient was having Anti-e and anti-M (Figure 3). The adsorption & elusion study to find out
possibility of Anti G antibody in two patients who were found positive screen for both anti D and anti
C was not done. Among the positive cases, four (31%) cases were that of sickle cell anaemia, four
(31%) cases of obstetrics, two (15%) cases of anaemia, and one each case of P. vivex (8%), hemolytic
anaemia (7%) and Bernard soulier syndrome (8%).
DISCUSSION: It is a routine practice to perform pre transfusion compatibility testing before blood
transfusion to prevent immune mediate haemolytic transfusion reactions. The steps of pre-
transfusion testing involve reviewing the acceptability of blood sample, checking the ABO group and
Rh D type, antibody screening test, determining the specificity of antibodies detected unexpectedly,
choosing donor RBC units suitable for recipients, and carrying out cross-match.[3] As blood is
routinely matched with respect to major blood group antigens i.e. ABO and Rh D antigen, there is a
high probability that the donor will have minor blood group antigens not present in the recipients
which will result in alloimmunization. Factors for immunization are complex and involve at least
three main contributing elements. This includes RBC antigenic difference between the blood donor
and the recipient, the recipient's immune status and immuno-modulatory effect of the allogenic blood
transfusions on the recipient's immune system.[5]
The development of red cell antibodies (Allo as well as autoantibodies) occurs in a variable
number of multiple transfused patients. In such circumstances, transfusion therapy may become
significantly complicated. Effects of alloimmunization may include difficulty in finding compatible
RBC units because of the presence of clinically significant RBC antibodies, transfusion reactions, or
platelet refractoriness.[6] Present study is an effort to characterize blood group alloantibody
formation in the patient population.
Few studies of multiple transfused patients in India had revealed rate of alloimmunization
ranging from 3 to 13% as mentioned in Table 1. The rate of present study was 07% which is similar
to the studies conducted by J Shukla et al (9.87%), Pradhan et al (08%) and Gupta et al (9.48).[7,9] The
studies done by Pahuja et al (3.7%) and Dhawan et al (5.64%) had lower rate while study of V
Sangole et al had higher rate of 13.04 %.[10,12]
Females have been observed to be more prone to development of alloimmunization than
males probably due to the fact that females, especially in developing countries, are anaemic and
pregnancy is an important risk factor for alloimmunization.[13]
DOI: 10.14260/jemds/2015/1825
ORIGINAL ARTICLE
In present study, 11(79%) out of 14 alloimmunized patients were women, with significant
association in chi-squares test (P value <0.05). This finding of the present study is in agreement with
the study done by Alick et al while studies done by Bhaskar S et al & Makroo et al did not show such
association with gender.[13,15] In the present study, female were five times more prone to develop
alloantibodies in comparison to male patients. Clinical diagnosis of the study group may lead to a
vulnerable immune status which may predispose to altered or increased immune response to various
antigens. In our study, significant number of sickle cell anaemia patients developed alloantibodies.
Out of 30 sickle cell cases, alloimmunization was found in four (13.33%) cases, which is comparable
to study by Elliott et al (30%) and Murao et al (9.9%).[16,17]
Though antigen typing before transfusion of people with sickle cell disease and providing
antigen negative units is now widely employed by sickle cell centers, the alloimmunization rate
remains quite high in contemporary sickle cell populations and may be due in large part to
transfusions received at institutions not providing extended matching. Two out of 21 patients of
chronic anemia developed alloantibodies (9.52%), which is comparable to study by Elliott et al
(05%).[16] Out of 5 patients of obstetrics, 4 (80%) patients developed alloantibodies. (Figure 3).
The specificity of most alloantibodies detected in the present study was against Rh system
(85%) due to their high immunogenicity, which is similar to previous reports of Thakral et al (61%),
Hmida et al (59%) and Dhawan et al (52%).[11,18,19] Anti c was detected in four patients, Anti-D in four,
Anti-N in three, anti-C in two and Anti-M, Anti e in one patient each. Anti-c and anti-D (27%) were the
most common antibodies in our study, which is comparable to Thakral et al (38.8%).[18] Hence, the
transfusion of blood matched for Rh could prevent alloimmunization resulting in a significant
difference in the alloimmunization rates, but the potential to form RBC alloantibodies to unmatched
antigens will exist.[20] In our study, majority of the patients with anti-D (either singly or in
combination) were multiparous females who might have formed anti-D due to previous pregnancies
or transfusions. (Figure 3)
In the present study, there was an absence of anti-Kell antibody in all subjects which was
similar to the findings of the study done by Thakral et al while other studies found anti Kell anti-
bodies.[11,13,15,18] This could be due to differences in blood group antigen frequencies in different
populations. According to the study on blood donors of the South Gujarat, Kell antigen positivity was
found to be 6%.[21] Thus, the lower frequency of Kell antigen in donated blood might be the reason
behind the lesser risk of alloimmunization from transfusion of a Kell antigen positive unit and the
result was absence of anti Kell antibody in present study.
In the present study we detected single antibody in 84.62% of cases and multiple antibody in
15.38% of cases, which is comparable to study by Alick et al who found single antibody in 78.6% and
multiple antibody in 21.4%.[14] Similar results was also found by Dhawan et al (22%cases had dual
allo antibodies).[11] Since pre-transfusion antibody screening in patients' samples is not a routine
practice in India, these patients might have received antigen mismatched blood leading to formation
of multiple alloantibodies.
The risk of developing alloimmunization was very clearly associated with the number of
transfusions received. In our study 11.32% patients of patient group who received more than 2 units
of blood transfusion were developed alloantibodies and it is significant statistically (P<0.05). This
finding is supported by some of the earlier studies done by Alick et al, Dhawan et al, Vishinski E et al
& Jensen LS et al who have found a strong correlation between the numbers of blood units transfused
and alloantibody formation.[11,14,20,22]
DOI: 10.14260/jemds/2015/1825
ORIGINAL ARTICLE
CONCLUSION: By considering the results of present and other reference Indian studies, blood banks
of India should go for universal type and screen policy for finding the prevalence of alloantibodies in
general patient and donor population. The indigenous development of local cell panels would be a
better option to ensure adequate supplies of reagent red cells and introduction of type and screen
policy for all the patients. Patients with identified alloantibodies can be flagged in a database and the
information can be shared between institutions and shared with the patient in the form of report
issuing to the concern person as well as patient education if possible. To avoid the effects of
alloimmunization, after antibody screen and identification, corresponding antigen negative blood
should be given to the patient.
The other approach to avoid alloimmunization in regularly transfused patients like sickle cell
disease & thalassemia is to allot a group of 10-15 donors to such single patient. Whenever the
transfusion required, donor will be selected from this group. In this way we can minimize
alloimmunization as well as better safety in terms of transfusion transmitted infections also.
STUDY LIMITATION: The adsorption & elusion study to find out possibility of Anti G antibody in two
patients who were found positive screen for both anti D and anti C was not done. The limitations of
this study was follow up data was not available due tovarious reasons & phenotyping of each & every
donor was not possible so only cross match compatible blood were issued.
Tables:
Sl. No Studies No. of Cases % of Positive Cases
1 J Shukla et al 81 9.87
2 Pradhan et al 100 8
3 Pahuja et al 211 3.79
4 Gupta et al 116 9.48
5 Dhawan et al 319 5.64
6 V Sangole et al 46 13.04
7 Present study 200 7
Table 1: Incidence of Alloimmunization in
Multi-Transfused Patients in various Studies
Studies Most common Antibody %

Satyam arora et al anti kell 35
J Shukla et al anti C & anti E 50
R Gupta et al anti E 36.4
B Shenoy et al anti C & anti kell 43
R Makroo et al anti E 37
Thakral et al anti c 39
Present study anti c and anti D 27
Table 2: Major Antibody type in various Studies
DOI: 10.14260/jemds/2015/1825
ORIGINAL ARTICLE
Fig. 1: Diagnosis of Patients (n = 200)
Fig. 2: Blood Group Distribution of Positive Patient
DOI: 10.14260/jemds/2015/1825
ORIGINAL ARTICLE
Fig. 3: Antibody specify & Diagnosis of Positive Patients
REFERENCES:
1. Khademi Reyhaneh, Gharehbaghian Ahmad, Karimi Gharib, Vafaiyan Vida, Khademi Raheleh &
Tabrizi Namini Mehdi. Frequency & specificity of RBC alloantibodies in patients due for surgery
in Iran. Indian J Med Res 138, August 2013, pg. 252-256.
2. Domen R.E., Ramirez G: Red cell alloimmunisation in chronic renal failure patients undergoing
hemodialysis. Nephron 48:284-285, 1998.
3. SaranR K, editor. Transfusion Medicine Technical Manual. 2nd edition. Directorate General of
Health services, Ministry of health & welfare, Government of India, New Delhi; 2003. f.
4. Kathy D Blaney, Paula R Howard. Basic & applied concepts of blood banking and transfusion
practices. 3rd edition. Mosby, elsevier inc.; 2009. p.
5. Lasky L. C. Ross P. R., Polesky H. F.: Incidence of antibody formation and positive direct
antiglobulin tests in a multi-transfused hemodialysis population: Transfusion 24:198-200,
1984.
6. Brecher E. Technical Manual. Chap 18, 19. 12th edition. United States: American Association of
Blood Banks; 2009. p. 389-91,407.
7. Shukla JS, Chaudhary RK. Red cell alloimmunization in multi-transfused chronic renal failure
patients undergoing hemodialysis. Indian J PatholMicrobiol 1999; 42:299-302.
8. Pradhan V, Badakere S, Vasantha K, Korgaonkar S, Panjwani S, Jajoo N. Antibodies to red cells in
beta thalassemia major patients receiving multiple transfusions: A short report. Indian J
Hematol Blood Transfus. 2001; 19:100–1.
9. Gupta R., Singh D. K., Singh B., Rusia U. Alloimmunization to red cells in thalassemics: emerging
problem and future strategies. Transfusion and Apheresis Science. 2011; 45(2):167–170.
10. Pahuja S., Pujani M., Gupta S. K., Chandra J., Jain M. Alloimmunization and red cell
autoimmunization in multitransfused thalassemics of Indian origin. Hematology. 2010;
15(3):174–177.
DOI: 10.14260/jemds/2015/1825
ORIGINAL ARTICLE
11. Dhawan HK, Kumawat V, Marwaha N, Sharma RR, Sachdev S, Bansal S, Marwaha RK, and Arora
S. Alloimmunization and autoimmunization in transfusion dependent thalassemia major
patients: Study on 319 patients. Asian J Transfus Sci. 2014 Jul-Dec; 8(2): 84–88.
12. Sangole VM, Chaudhari DR. Alloimmunisation of red blood cells in multitransfused patients. Int
J Health Sci Res. 2013; 3(5):39-41.
13. Makroo RN, Vimarsh Riana, Rosamma NL, Rashmi S.; Detection of alloimmunization to ensure
safer transfusion practice; Asian Journal of Transfusion Science, Vol. 7, No. 2, July-December,
2013, pp. 135-139.
14. M Alick, S Nathan. Frequency and Distribution of RBC Alloantibodies among Transfused
Patients at Ndola Central Hospital, Zambia: International Journal of Science and Research.
2014; 3 (5): 1.
15. BhaskarShenoy, Murali Mohan Voona, Shivaram C, Nijaguna, Shivananda. Red Cell
AlloimmunizationIn Multi Transfused Patients with Beta Thalassemia Major-A Study from
South India. Int J Med Pharm Sci, June 2013; 03 (10): 31-40.
16. Elliott PV, Earles A, Johnson RA, Hoag MS, Williams A, Lubin B. Allo-immunization in Sickle Cell
Anemia and Transfusion of Racially Unmatched Blood: N Engl J Med 1990; 322:1617-1621.
17. Murao M, Viana MB. Risk factors for alloimmunization by patients with sickle cell disease. Braz
J Biol Med Res 2005; 38:675-82.
18. Thakral B, Saluja K, Sharma RR, Marwaha N. Red cell alloimmunization in a transfused patient
population: a study from a tertiary care hospital in north India. Haematology. 2008; 13(5): 313-
8.
19. Hmida S, Mojaat N, Maamar M, Bejaoui M, Mediouni M, Boukef K. Red cell alloantibodies in
patients with haemoglobinopathies. Nouv Rev FrHematol. 1994 Oct; 36(5):363-6.
20. Vishinski E, Earles A, Johnson R, Hoag M, Williams A, Lubin B. Alloimmunization in sickle cell
anaemia and transfusion of racially unmatched blooding Engle. J Med 1990; 322:1617-21.
21. Kahar MA, Patel RD. Phenotype frequencies of blood group systems (Rh, Kell, Kidd, Duffy, MNS,
P, Lewis, and Lutheran) in blood donors of south Gujarat, India. Asian J Transfus Sci. 2014 Jan;
8(1):51-5.
22. Jensen LS, Kissmeyer-Nielsen P, Wolff B, Qvist N. Randomised comparison of leucocyte -
depleted versus buffy-coat-poor blood transfusion and complications after colorectal surgery.
Lancet 1996; 348:841-5.
DOI: 10.14260/jemds/2015/1825
ORIGINAL ARTICLE
4. 3rd Year Resident, Department of
AUTHORS:
Immunohematology and Blood Transfusion
1. Rakesh P. Pimpaldara
Department, Government Medical College,
2. Arpit C. Patel
Surat.
3. Jitendra Patel
5. Professor and HOD, Department of
4. Snehal Patel
Immunohematology and Blood Transfusion
5. A. N. Pandya
Department, Government Medical College,
6. Sangita Wadhwani
Surat.
6. Blood Transfusion Officer, Blood Bank, New
PARTICULARS OF CONTRIBUTORS:
Civil Hospital, Surat.
1. 3rd Year Resident, Department of
Pathology, Government Medical College, NAME ADDRESS EMAIL ID OF THE
Surat. CORRESPONDING AUTHOR:
2. 3rd Year Resident, Department of Dr. Jitendra Patel,
Immunohematology and Blood Blood Bank, 2nd Floor,
Transfusion Department, Government New Civil Hospital,
Medical College, Surat. Majura Gate,
3. Assistant Professor, Department of Surat-395001, Gujarat.
Immunohematology and Blood E-mail: [email protected]
Transfusion Department, Government
Medical College, Surat. Date of Submission: 25/08/2015.
Date of Peer Review: 26/08/2015.
FINANCIAL OR OTHER Date of Acceptance: 05/09/2015.
COMPETING INTERESTS: None Date of Publishing: 08/09/2015.
EXTERNAL EVALUATIONS

MATRIX GEL SYSTEM
Performance evaluation of MatrixTM AHG (Coombs) Test Card for antibody
detection (screening) and identification.
EVALUATING CENTER:
This study was conducted at the Immunohematology Laboratory of the German Red Cross Blood Center in Frankfurt
(Institute of Transfusion Medicine and Immunohematology), under the supervision of Dr. med. C. Geisen (MD). (Data on
file Tulip Diagnostics Pvt. Ltd.).
SUMMARY
In this evaluation study 551 samples for antibody screening were tested in parallel with MatrixTM AHG (Coombs) Test card
and DiaMed ID LISS/Coombs card using a Tecan pipetting machine. Out of 551 samples tested 42 samples showed
positive results for antibody screening and were followed up by antibody identification.
Overall, semi-automated system using automated pipetting system and card reading with respective Saxo card reader
MatrixTM Gel System showed a 4.7% lower sensitivity and a 1.6% lower specificity with respect to antibody screening. In
contrast to antibody identification MatrixTM Gel System showed a 2% higher sensitivity. Antibodies which, in one case
each, were detected in the antibody identification only with MatrixTM Gel Cards included anti-Jk(a), E and K; antibodies
which, in one case each, were detected only by DiaMed ID Cards included anti-c and anti-Cw.
INTRODUCTION:
The purpose of this study was to evaluate the performance of MatrixTM AHG (Coombs) Test Card manufactured by Tulip
Diagnostics Pvt. Ltd, against DiaMed ID LISS/Coombs test cards manufactured by DiaMed. Both the gel card consists of
6 microtubes prefilled with polyspecific anti-human globulin.
The performance evaluation of MatrixTM AHG (Coombs) Test Card was carried out under routine conditions for antibody
detection and antibody identification. All tests were performed in parallel with both the test systems in the
Immunohematology Laboratory of the German Red Cross Blood Center in Frankfurt (Institute of Transfusion Medicine
and Immunohematology). The study was performed in two parts. First part of the study was performed from November
18, 2008 to December 16, 2008. Second part was performed from February 9, 2009 to February 27, 2009.
MATERIALS AND METHODS:

Blood samples used were the residual patient's blood samples which were sent for routine diagnostic testing. In first part,
404 fresh (not frozen) samples were tested: 371 random routine samples (unknown allo-antibody status) and 33
additional samples with known allo-antibody were used. In second part, 158 fresh (not frozen) random routine samples
were tested. Antibody detection was performed with ID-DiaCell I-II-III in both MatrixTM AHG (Coombs) Test Card and
DiaMed-ID LISS/Coombs Cards. Invitroscreen I-II-III test cells were used in combination with MatrixTM AHG (Coombs)
Test Card and ID test cells were used with ID-Cards under routine testing procedure.
RESULTS OF STUDY IN PART 1:

Of the originally 404 samples, sufficient plasma was available from 398 samples. Out of 398 samples 39 samples were
positive for antibody detection. On performing identification with 11 cell panel 46 Coombs reactive antibodies were
detected. Data is summarized in below tables:
Table 1.1: Concordance of results of the antibody detection test on 398 routine samples tested with MatrixTM Gel System
and DiaMed ID.
DiaMed ID
Antibody Detection n=398
+ -
TM
Matrix Gel System + 38 3
- 4 353
Table 1.2: Antibody detection results for the DiaMed ID-Gel Card system:
Coombs reactive antibodies

Present (39) Not Present (359)
DiaMed ID Gel Card System + 38 4
(True positive) (False positive)
- 1 355
(False negative) (True negative)
TM
Table 1.3: Antibody detection results for Matrix Gel System.

MatrixTM Gel System + 36 5
- 3 354

Of the originally 158 samples sufficient plasma was available in from 153 samples. Out of 153 samples 3 samples were
positive for antibody detection. On performing identification with 11 cell panel 4 Coombs reactive antibodies were
detected. Data is summarized in below tables:
Table 2.1: Concordance of results of the antibody detection test on 153 routine samples tested with MatrixTM Gel System
and DiaMed ID.
DiaMed ID
+ -
TM
- 2 139
Table 2.2: Antibody detection results for the DiaMed ID-Gel Card system

DiaMed ID Gel Card System + 3 2
- 0 148
Table 2.3: Antibody detection results for MatrixTM Gel System.

- 0 141
CALCULATED SENSITIVITY AND SPECIFICITY OF ANTIBODY DETECTION TEST IN PART 1 & 2:

The calculated sensitivity of the antibody detection test using DiaMed ID system and MatrixTM Gel System (for part 1 & 2)
were 96.7% and 92.9% respectively. The specificities of DiaMed ID system and MatrixTM Gel System (for part 1 & 2) for
Coombs reactive antibodies were 98.8% and 97.2% respectively.
RESULTS OF ANTIBODY IDENTIFICATION IN STUDY PART 1:

In 39 samples 46 Coombs reactive antibodies were detected. Result data is summarized in below tables:
Table 3.1: Summary of results of antibody identification in MatrixTM Gel System and DiaMed ID.
DiaMed ID
Confirmed antibody by identification panel n=46
+ -
TM
Matrix Gel System + 41 3**
- 2* 0
* 2 antibodies with the specificity Cw, c (one each) identified only by the DiaMed ID system.
** 3 Antibodies with specificity E / Jk(a) / K (one each) identified only by MatrixTM Gel System.
Table 3.2: Spectrum of 46 identified antibodies in 39 samples
Antibody specificity Total antibodies Detected by DiaMed ID System Detected by MatrixTM Gel System
c 2 2 1
C 2 2 2
Cw 4 4 3
E 11 10 11
D 9 9 9
Jk(a) 3 2 3
K 5 4 5
Le(b) 1 1 1
Lu(a) 3 3 3
M 1 1 1
S 1 1 1
Auto-e 2 2 2
Pan agglutination 2 2 2
Total 46 43 44
RESULTS OF ANTIBODY IDENTIFICATION IN STUDY PART 2:
In 3 samples 4 Coombs reactive antibodies were detected. Result data is summarized in below tables:
Table 4.1: Summary of results of antibody identification in MatrixTM Gel System and DiaMed ID.
DiaMed ID
Confirmed antibody by identification panel n=4
+ -
TM
- 0 0
Table 4.2: Spectrum of 46 identified antibodies in 39 samples
Antibody specificity Total antibodies Detected by DiaMed ID System Detected by MatrixTM Gel System
C 1 1 1
E 2 2 2
D 1 1 1
Total 4 4 4
CALCULATED SENSITIVITY OF ANTIBODY IDENTIFICATION IN PART 1 & 2:

Antibody identification with 11 cell ID test panel in DiaMed ID gel system and Matrix Gel System in study part 1 and 2
achieved a sensitivity of 94% and 96% respectively.
NOTE
Data on file: Tulip Diagnostics (P) Ltd.
Performance evaluation of MatrixTM AHG (Coombs) Test Card for auto-controls.
EVALUATING CENTER:
This study was conducted at the Immunohematology Laboratory of the German Red Cross Blood Center in Frankfurt
(Institute of Transfusion Medicine and Immunohematology), under the supervision of Dr. med. C. Geisen (MD). (Data on
file Tulip Diagnostics Pvt. Ltd.).
SUMMARY
In this evaluation study 544 samples for auto-control were tested in parallel with MatrixTM AHG (Coombs) Test card and
DiaMed ID LISS/Coombs card using a Tecan pipetting machine. Out of 544 samples 146 samples showed positive auto-
control and were further investigated by monospecific DAT.
Overall, semi-automated system using automated pipetting system and card reading with respective Saxo card reader
MatrixTM Gel System showed a 3.1% lower sensitivity and a 1.7% lower specificity in auto-controls.
INTRODUCTION:
The purpose of this study was to evaluate the performance of MatrixTM AHG (Coombs) Test Card manufactured by Tulip
Diagnostics Pvt. Ltd, against DiaMed ID LISS/Coombs test cards manufactured by DiaMed. Both the gel card consists of
6 microtubes prefilled with polyspecific anti-human globulin.
The performance evaluation of MatrixTM AHG (Coombs) Test Card was carried out under routine conditions for auto
controls of patient's blood samples. All tests were performed in parallel with both the test systems in the
Immunohematology Laboratory of the German Red Cross Blood Center in Frankfurt (Institute of Transfusion Medicine
and Immunohematology). The study was performed in two parts. First part of the study was performed from November
18, 2008 to December 16, 2008. Second part was performed from February 9, 2009 to February 27, 2009.
The auto-control was performed routinely with both MatrixTM AHG (Coombs) Test Card and ID LISS/Coombs Test card. A
positive result with either or both was followed by re-testing the sample with monospecific DAT (IgG and C3d) cards of ID
and ScanGel Direct Coombs (DAT) cards.
RESULTS:
Whenever divergent results were obtained in auto-control with DiaMed ID LISS/Coombs Test Cards and MatrixTM AHG
(Coombs) Test Cards, analysis was repeated in duplicate within each system. Auto-controls were scored positive when at
least two of the three tests were positive; for all positive samples, monospecific DAT was performed. Of the originally
tested 404 random patient's samples of the first part of the study, 386 were subjected to further analysis. Analysis of 18
samples could not be completed due insufficient sample. In second part of the study, all 158 samples were tested. Data is
summarized in below tables:

Table 1.1: Results of the auto-controls of 386 routine samples with MatrixTM AHG (Coombs) Test Cards and DiaMed ID
LISS/Coombs Test Card:
DiaMed ID
Auto-control n=386
+ -
TM
84 monospecific DAT positive 2 monospecific DAT positive
9 monospecific DAT negative 1 monospecific DAT negative
- 7 139
6 monospecific DAT positive
1 monospecific DAT negative
Table 1.2: Results of the auto-controls of 386 routine samples with DiaMed ID LISS/Coombs Test Card:
92 294
Auto-control n=386
Confirmed by monospecific Coombs -
DiaMed ID + 90 10
(true positive) (false positive)
- 2 284
(false negative) (true negative)
Table 1.3: Results of the auto-controls of 386 routine samples with MatrixTM AHG (Coombs) Test Cards:
92 294
Auto-control n=386
- 6 279

Table 2.1: Results of the auto-controls of 158 routine samples with MatrixTM AHG (Coombs) Test Cards and DiaMed ID
LISS/Coombs Test Card:
DiaMed ID
Auto-control n=158
+ -
TM
33 monospecific DAT positive 1 monospecific DAT positive
6 monospecific DAT negative 2 monospecific DAT negative
- 1 115
1 monospecific DAT positive
Table 2.2: Results of the auto-controls of 158 routine samples with DiaMed ID LISS/Coombs Test Card:
35 124
Auto-control n=158
DiaMed ID + 34 6
- 1 117
Table 2.3: Results of the auto-controls of 158 routine samples with MatrixTM AHG (Coombs) Test Cards:
34 124
Auto-control n=158
- 1 115
CALCULATED SENSITIVITY AND SPECIFICITY OF ANTIBODY DETECTION TEST IN PART 1 & 2:

The calculated sensitivity of the auto-control test using DiaMed ID system and MatrixTM Gel System (for part 1 & 2) were
97.6% and 94.5% respectively. The specificities of DiaMed ID system and MatrixTM Gel System (for part 1 & 2) for auto-
control were 96.2% and 94.5% respectively.
NOTE
Data on file: Tulip Diagnostics (P) Ltd.
TULIP DIAGNOSTICS (P) LTD

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1. Journal of Forensic Science & Criminology Vol 6 Issue 1, 2018 1- 8

Annex Publishers | www.annexpublishers.com Volume 6 | Issue 1

Material and Method

Conventional method of ABO grouping

Matrix gel card Method

Sample preparation for forward grouping

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Sample preparation for reverse grouping

Strength of reaction Comments

Mixed field agglutination

Blood Group Type A B AB O

Results for:- “A “Blood Group:-

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Results for:- “Inconclusive “Blood Group:-

Figure 14: G (4+): A-cells, B-cells, Rh Ctrl; G (3+): A-serum, B-Serum

Figure 17: G (4+): A-Cells, B-Cells, Rh, Ctrl, A-serum, B-Serum

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Evaluation of methodology and comparative

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ARTICLE INFO ABSTRACT

MATERIALS AND METHODS RESULTS

Table 1. Result observation

Method Used Samples Compatible Incompatible

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Introduction: (IAT&DAT)Present study is carried out for com-

Matrix Gel card False positive

True Negative False Negative True positive False positive

to blood specially area with HIV infection is Conclusion

DETECTION AND IDENTIFICATION OF RED CELL ALLOANTIBODIES IN

AIM AND OBJECTIVE:

DETERMINATION OF THE MEAN RED CELL TRANSFUSION REQUIREMENT

AIM AND OBJECTIVE:

RESULTS AND OBSERVATIONS:

Access this article online

Address for Introduction therapeutic support and minimal red cell

© 2018 Asian Journal of Transfusion Science | Published by Wolters Kluwer - Medknow 51

Patient’s sample + Request form

ABO & Rh typing Request for fresh

Cross match with group

Repeat cross match with same donor

Detail IH work-up + Information

Figure 1: Evaluation of an incompatible cross-match sample

Asian Journal of Transfusion Science - Volume 12, Issue 1, January-June 2018 53

hematological malignancies (n = 1865, 21%), autoimmune causative antibody could be identified in 16 of them,

A total of 38 (38%) of the incompatible cross‑match

54 Asian Journal of Transfusion Science - Volume 12, Issue 1, January-June 2018

Table 1: Antibody profile in incompatible cross‑match

56 Asian Journal of Transfusion Science - Volume 12, Issue 1, January-June 2018

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