Introduction

Currently, the immunohematologists are trying to establish as well as improve majority of the serological investigations, after the discovery of ABO system and RBC agglutination by Landsteiner in 1900 and by Coombs et al. in 1945, respectively.^[1]

The principle of the Coomb’s test is demonstration of antibodies or complement coating red cells using Coombs reagent or Antihuman globulin.^[2]

Technically various modifications have been made to bring about added sensitivity including the use of more specific reagents like monospecific AHGs.^[3]

A study done by Lapierre et al. in 1990, showed improved reliability of Gel Card when correlated with Conventional tube results for detecting a variety of clinically significant known antibodies.^[2]

Historically, for immuno-haematological studies, like DCT and screening of antibodies in transfusion medicine, conventional tube method was used as the standard technique.^[4]

But it is time-consuming, in need of an experienced personnel to interpret the results which makes it difficult to automate and involves many cell washing steps.

Gel Card is however an easy and sensitive technique that surpasses the above disadvantages and induces agglutination by the uses gel filtration media impregnated with an antihuman globulin reagent.^[1]

Aim of the Study

To compare the test results of Coomb’s test done by gel card method and by manual method.

Objective

To study the findings of Coomb’s test done by Gel Card method and Conventional tube method and to compare the result of the above two methods.

Materials and Methods

A comparative study was done in the Blood Bank of BVDU (Deemed to be University) Medical College and Hospital, Sangli, India, for a duration of 8 months from March 2022 to October 2022, where in all the samples subjected for Coomb’s test, either DCT or ICT or both were considered in this study. Both Direct and Indirect Coomb’s test performed by Gel Card method and Conventional tube method.

For Gel card method: blood sample, plastic microtube with 6 wells, micro pipette as per volume, LISS diluent, incubator (card warmer), timer, centrifuge machine, card reader.

For Conventional tube method: blood sample, isotonic saline, clean dropper, clean test tubes, already prepared ‘O’ red cell suspension, incubator, Anti-human globulin, centrifuge machine, timer, microscope.

Clotted samples, insufficient quantity, wrong bulb sample and more than 24 hours sample (without refrigeration) were some of the exclusion criteria considered in our study.

Procedure by gel card method

Sample preparation for Direct and Indirect Coomb’s test.

Prepare 0.8% red cell suspension in LISS as follows:

Dispense 1 ml of LISS in a clean labelled test tube.

Add 10 microL packed cells and mix gently.

A. Gel Card Method for DCT:-

Observe the card for appearance of gel and label with patient’s details and remove the foil seal as instructed.

Pipette 50 microL of patient’s red cell suspension (0.8%) into the labelled microtube.

Centrifuge the car in Matrix Cerd Centrifugr for 1 cycle (10 minutes)

Read the reaction.

B. Gel Card Method for ICT:-

Observe the card for appearance of gel and label with patient’s details and remove the foil seal as instructed.

Pipette 50microL of suspension (0.8%) od fresh pooled and washed “O” cells into the labelled microtube. Pipette cell suspension at 45 degrees angle.

Add 25 microL of patient’s serum/plasma into the microtube at 90 degrees angle.

Incubate at 37 degrees for 15 minutes in Matrix Card Warmer.

Centrifuge the card in Matric Card Centrifuge for 1 cycle (10 minutes).

Read the reaction.

Grading	Agglutination	Interpretation
0	No agglutination	Compatible
+ 1	Agglutination of red cells in the lower half of the gel card	Incompatible
+ 2	Agglutination of red cells through the entire length
+ 3	Agglutination of red cells in the upper half of the gel card
+ 4	Agglutination of red cells in the lower half of the gel card
Hemolysis	-	Invalid

Table 1 Grading for gel cardmethod 7

Results

A total of 80 samples taken for this 8 months study, out of which 60 were evaluated for DCT and 40 evaluated for ICT. From the 60 samples for DCT, 42 showed positivity for Gel card method and 30 for Conventional tube method. And from 40 samples for ICT, 24 showed positivity for Gel card method and 18 showed positivity for Conventional tube method.

Sensitivity and specificity and p-values were calculated for the positive results by Gel Card and Conventional tube method.

Discussion

Conducted a comparative study in the blood bank of BVDU (Deemed to be University) Medical College and Hospital, Sangli, India, for 8 months, from March to October of 2022, where in all the samples for Coomb’s test, both Direct and Indirect were implemented by both Gel card and Conventional method simultaneously.

For a better understanding, the principle, indications, advantages and disadvantages will be discussed in the following. As we all know, Coomb’s test is a laboratory investigation done for the detection of either in-vivo (Direct Coomb’s test) or in-vitro (Indirect Coomb’s test) antibodies directed towards RBC’s.^[8] For the detection of antibodies present on the surface of the red cells, Direct Coomb’s test is the diagnostic tool used.^[3]

As mentioned before, the Gel technique has been proven to be more efficacious and simplified technique as well as the interpretation of results along with a better reliability, reproducibility, stability and increased sensitivity.^[3] Initially introduced in Thailand, 1993, and has become popular and used worldwide in several blood banks.^[3] For Gel Card test we use specific microtubes which are being prepared using standard reagents. This method has been widely used for cross matching, the detection of antigen, alloantibody screening/identification.^[3] Since the tube method was the first technique used, some of the blood bank personnels still prefer the Conventional Tube method despite aving many drawbacks like skilled technical expertise especially in the cell washing step (which may lead to false positive results) and also the intervariability complex.^[3]

On the other hand, the benefits of Gel technology have surpassed the Conventional Tube method – quick, safe, elimination of cell washing step, technician friendly, less handling of samples, lesser interpretive errors, lesser protocol errors does not require special skills for performance.^[3], ^[9] It also has a clear cut grading system giving a uniform interpretation by the observers when get for a weeks time due to the stability of the agglutinates. The duration of 1 week gives another advantage in certain medico legal cases. Another most important point to be noted is the high sensitivity towards IgG coated cells, making it a better technique compared to the Conventional tube method.^[3] Despite the above mentioned advantages, there are certain unavoidable disadvantages as well – cost, false positive reactions (macrocytosis, marked leucocytosis and increased ESR), the possibility of missing C3d coated red cells.^[3]

Principle of Gel Card Method: The basic principle of the gel test is, instead of a glass test tube, the serum and cell reaction takes place in a microtube having 6 wells embedded in a plastic card, which allows easy testing, reading as well as handling and disposal. Saphadex gel is used in gel cards which holds agglutinate in semisolid medium, this helps in clear visualization of agglutination than that of the tube method.^[2]

This method introduced by Lappiere et al., was firstly used for the cross matching of blood.^[7] As stated earlier, the tube technique aka conventional technique has been the cornerstone for Coomb’s testing over last 4 decades, but the enhanced sensitivity Gel card technique has made the interpretation more reliable.

Advantages of Gel card method:^[3], ^[10], ^[11], ^[12]

Simple, reliable, rapid, reproducible and sensitive

Greater uniformity amongst repeat test

Less volume of specimen required

Standardized reporting, grading system

No cell washing required

More consistent and reproducible interpretation of results

Higher sensitivity with IgG coated cells

Disadvantage of Gel card method:^[3], ^[10], ^[11], ^[12]

Expensive

Requires special incubator and centrifuge machine

Advantages of conventional method:^[3], ^[10], ^[11], ^[12]

Cheaper

Detection of C3 complement

False positive results:^[3]

Overcentrifugation

Increased ESR, rouleaux formation

Macrocytosis

Leucocytosis

Inappropriate washing, inadequate resuspension of cell button

Hypergammaglobilinemia

False negative results:^[3]

AHG reagent failure

Improper or inadequate or delayed washing

Low serum/cell ratio

Resuspension of cell button too vigorously

Conclusions

Our study showed that gel card is easier to use, more sensitive and less time-consuming with more standardized result and less sample needed for the test. Results of Gel card can be preserved for 3-4 days and this can be interpreted by various observers and compare it with the standardized grading system. Gel card assay appears to be an excellent method for detecting agglutination better than conventional tube method and easy to read weak agglutination and it can also detect ABO incompatibility. The performance of saline tube technique requires more experience and highly accuracy due to its long stages and multiple washing. But one disadvantage of gel card method is that gel cards are costly and require separate incubator and centrifuge.

Source of Funding

None.

Conflict of Interest

None.