Detection of Alloantibodies in Multiple Transfused Recipients using Gel Technology

 

Dr. Suresh Kumar Sutrakar¹*, Dr. Uday Raj Singh², Prabha Verma³

¹Assistant Professor, Department of Pathology, S.S. Medical College and Asso. S.G.M. Hospital, Rewa (M.P.), India-486001

²Associate Professor and Head Department of Pathology, S.S. Medical College and Asso. S.G.M. Hospital, Rewa (M.P.)

³Lecturer and Head Department of Biochemistry, M.L.B. Medical College Jhansi (U.P.), India-284001

 

ABSTRACT:

Allogeneic blood transfusion is a form of temporary transplantation. This procedure introduces a multitude of foreign antigens and living cells into the recipient that will persist for a variable time. A recipient who is immunocompetent often mounts an immune response to the donor antigens, resulting in a variety of clinical consequences depending on the blood cells and specific antigens involved. Delayed hemolytic transfusion reactions usually occur in patients who have been previously sensitize to an antigen through transfusion or pregnancy. They can result in symptomatic or asymptomatic hemolysis several days after a subsequent transfusion due to an anamnestic recall of the antibody.

Approximately 0.1-2% of patients who receive transfusions develops anti-RBC antibodies. In patients who are transfused regularly (e.g. patients with sickle cell disease and thalassemia), the frequency of alloimmunization is much higher, affecting 10-38%. A hemolytic transfusion reaction occurs following transfusion of an incompatible blood component. Traditionally, red cell antibody screening and identification are done by agglutination methods in the tube. This study has been undertaken on the multiple transfused recipients selected randomly in the Blood Bank, N.S.C.B.medical College Jabalpur (Madhya Pradesh), for detection of alloantibodies and autoantibodies using gel technology as methodology. The study constitutes various clinically diagnosed patients of thalassemia, sickle cell anemia, acute leukemia, aplastic anemia and severe anemia. The alloantibodies detected were 13/64 (20.3%) and autoantibodies 02/64 (3.1%) by micro typing gel system, auto control was performed for all indirect antiglobulin test (IAT) and direct antiglobulin test (DAT) positive samples. The (P value-0.073) and (P value-0.30) > (P value-0.050)   had been calculated for rate of alloimmunization in relation to age and number of transfusions found to insignificant whereas (P value-0.023) < (P value-0.050) was calculated for rate of alloimmunization in clinically diagnosed patient found to be significant. All alloantibodies found to be of Rh and Duffy blood group systems.    

 

 

 

INTRODUCTION:

Delayed hemolytic transfusion reactions, acute hemolytic transfusion reaction and febrile transfusion reactions usually occur in patients who have been previously sensitize to an antigen through transfusion or pregnancy. They can result in symptomatic or asymptomatic hemolysis several days after a subsequent transfusion due to an anamnestic recall of the antibody.

 

Approximately 0.1-2% of patients who receive transfusions develops anti-RBC antibodies. In patients who are transfused regularly (e.g. patients with sickle cell disease), the frequency of alloimmunization is much higher, affecting 10-38%. 24-week-old male infant with interstitial pneumonia and hepatitis because of CMV developed an RBC auto antibody and two RBC alloantibodies^.1 3 episodes of delayed hemolytic transfusion reaction due to multiple red cell antibodies.² DHTR/H syndrome occurs in pediatric SCD patients, typically 1 week post transfusion.³ the risk of immunological reactions (haemolytic transfusion reactions, alloimmunization and immunosuppression) are predominant.⁴

The rate of alloimmunization was detected by micro typing gel system, the direct antiglobulin test (DAT),  indirect antiglobulin test (IAT) and autocontrol tests performed at 37°C on all 64 samples, out of which 13/64(20.3%) and 02/64(3.1%) were detected alloantibodies and autoantibodies respectively. The rate of alloimmunization, 20.3% which was reported by various authors was range from 0.02%-20.8%.^{5, 6} The results correlate with previous studies.^7-9 The gel test, as developed by Lapierre, is an innovative approach to blood group serology. This technology addresses the issue of standardization and incorporates sensitivity, specificity, and efficiency. The gel test was released in Europe in 1988 and was available for use in 1995.¹⁰ The gel micro typing system utilizes a sephadex gel to capture agglutinates in a semi-solid medium. This enhances visualisation of agglutination as compared to the traditional tube techniques. In the latter, the agglutinate, particularly in weak reactions, mixes with the free cells at the bottom of the tube, making visualization difficult. The capacity of the gel test to separate RBCs from their surrounding fluid permits an antiglobulin test to be performed without washing. At the beginning of centrifugation, the RBCs are pulled away from the suspension medium (unbound serum globulin) and enter the gel first. The surrounding medium remains above the gel and the characteristics of the gel prevent the medium from interfering with the antiglobulin reaction. Sensitized RBCs agglutinate as they were expose to the antiglobulin reagent in the gel and are trapped. Unsensitized RBCs are not agglutinated and pass through the gel to pellet at the bottom of the microtube.

 

MATERIAL AND METHODS:

The present study was conducted in the Blood bank, Department of Pathology, N.S.C.B. Medical College and Hospital, Jabalpur from April 2005 to October 2006. The study comprised of 64 multitransfused patients of various clinically diagnosed groups.  The Comparison of DATs using traditional tube agglutination to gel column and affinity column procedures and found that Gel micro column DATs were more sensitive than tube agglutination and affinity micro column DATs.¹¹ 125 sickle cell anemia (SCA) patients using tube indirect antiglobulin test (PEG, LISS or enzyme) and gel centrifugation test (LISS or enzyme). The alloimmunization rate was 20.8% and the gel test detected a higher number of alloantibodies than the tube test (26 v 21, p = 0.02). ¹²

 

The serum is use as a sample material; the serum must be well cleared, by centrifugation at 1500g for 10 minutes, before use to avoid fibrin residues, which may interfere with the reaction pattern. Where samples are not for immediate testing they should be stored at 2-8°C after separation for a maximum of 48hours, thereafter at –35°C. The basic pieces of equipment and materials required for the gel test are the dedicated incubator, centrifuge, and RBC diluents. Accessories are also available to make the testing run smoothly.These include specially designed workstations, automatic pipettes, and disposable tips. The micro tubes of the ID-Card “Liss/Coombs” contain plolyspecific AHG, was used for DAT, IAT and Autocontrol tests.

1.        Direct Antiglobulin test (DAT)

 

Sample:

Red Cell Concentrate is prepare by taking 10µl patient’s whole blood in test tube containing 1.0 ml of LISS (Low Ionic Saline Solution).

 

Test Procedure:

1      Allow all reagents to reach room temperature before use.

2      Identify the ID-micro typing card with the patient and donor unit name and number. Remove the aluminium foil.

3      Prepare a 0.8% suspension of test red cells: -

10µl red cell concentrate + 1.0ml ID-Diluent 2

Or

20µl whole blood   + 1.0ml ID-Diluent 2

Mix well.

4      Add 50µl of test red cell suspension to the appropriate microtube.

5      Centrifuge the microtyping card for 10 minutes in the ID-Centrifuge.

6      Interpret the results.

 

Interpretation:

Positive reactions, grade 1-4, in the micro tube indicates a positive DAT.

Negative reactions in the micro tube indicate a negative DAT.

 

Autocotrol:

Sample:

Red Cell Concentrate/Whole Blood - from Patient

Serum or Plasma of patient

 

Test Procedure:

1      Allow all reagents to reach room temperature before use.

2      Identify the ID-microtyping card with the patient and donor unit name and number. Remove the aluminium foil.

3      Prepare a 0.8% suspension of test red cells: -

10µl red cell concentrate + 1.0ml ID-Diluent 2

Or

20µl whole blood    + 1.0ml ID-Diluent 2

Mix well.

4      Add 50µl of test red cell suspension to the appropriate microtube.

5      Add 25µl of test serum or plasma to each microtube

6      Centrifuge the microtyping card for 10 minutes in the ID-Centrifuge.

7      Interpret the results as shown in diagram no.4

 

Interpretation:

Positive reactions, grade 1-4, in the micro tube indicates a positive Auto control.

Negative reactions in the micro tube indicate a negative Auto control.

2.       Indirect Antiglobulin Test (IAT):

The IAT (antibody screen) is perform by incubating patient serum with reagent screening red cells for approximately 20 minutes and then observing for agglutination. If the antibody screen is positive, additional testing is required to determine the specificity of the antibody. 

Preparation of Screening Panels: 

 

We have screened randomly selected “O” blood group voluntary donors for minor antigen typing by using commercial antisera (DiaMed Company) for following antigens; D, C, E, e, c, K, k, Fya, Fyb, M and N. The blood sample are washed toughly using normal saline thrice and 10% red cell suspension is prepared using LISS in a test tube, 10 µl of 10% RBC suspension and 10 µl of Antisera were added on slide and agglutination were noted against the respected antisera’s. Out of screened donors 3 panels were selected which are of R1R1, R2R2 and rr  phenotype covering almost all above mentioned antigens, these panels were used as reagent screening red cells for antibodies detection against representative antigens of various blood group systems as shown in table-1.

 

Sample:

Serum is collect from patient whole blood by centrifuging the blood collected in a test tube at 2000 Rpm for 5 minutes.

 

Test Procedure:

·         Allow all reagents to reach room temperature before use.

·         Identify the ID-micro typing card with the patient name and number. Remove the aluminium foil.

·         Add 50µl of Antibody Screen Cells (O Cells) to microtubes “1”, “2” and “3” respectively.

·         Add 25µl of test serum or plasma to each microtube.

·         Incubate the micro typing card for 15 minutes at 37°C in the ID-Incubator. Centrifuge the micro typing card for 10 minutes in the ID-Centrifuge.

·         Interpret the results.

 

Interpretation:                                                                                                    

Positive reactions, grade 1-4, in the micro tube indicates the presence of an antibody.

Negative reactions in the micro tubes indicate the absence of an antibody.

 

 

Table No.1.

 

RESULT:

Table No.2-Alloimmunization in different age groups

Age Group in years	Total		Positive cases
	No. of cases	Percent	DAT	%	IAT	%	AUTO CONTROL	%
<9 10-19 20-29 30-39 >40	26 15 10 5 8	40.6 23.4 15.6 7.8 12.5	4 0 4 0 2	15.4 0 40 0 25	4 1 0 0 0	15.4 6.7 0 0 0	1 0 1 0 0	3.8 0 10 0 0

Table no.1showing, the (P value-0.073) > (P value-0.050) states no significant correlation between age and alloimmunization.

 

Table No.3-Alloimmunization in clinically diagnosed patients

Clinical Diagnosis	Total		Positive cases
	No. of cases	Percent	DAT	%	IAT	%	AUTO CONTROL	%
Thalassemia Sickle cell Anemia Acute Leukemia A plastic Anemia Severe Anemia	17 13 9 4 21	26.6 20.3 14.1 6.2 32.8	4 0 2 1 3	23.5 0 22.2 25 14.3	4 1 0 0 0	23.5 7.7 0 0 0	1 0 0 1 0	5.9 0 0 25 0

Table no.3 showing, the (P value-0.023) < (P value-0.050) was calculated for rate of alloimmunization in clinically diagnosed patient found to be significant

 

Table No.4-Alloimmunization in accordance to number of transfusions

Table no.4 showing, the (P value-0.30) > (P value-0.050) states there is no significant correlation between number of transfusions and alloimmunization.

Table no. 5-Alloantibodies in different blood group systems

Table no. 5-Alloantibodies in different blood group systems

Antigen	Blood Group System	No. of Alloantibodies
E	Rh	6
Kell (Kl)	Kell	0
D	Rh	0
c	Rh	1
Fy(a)	Duffy	6
C	Rh	0
e	Rh	0
Fy(b)	Duffy	0
M	MNS	0
N	MNS	0

 

The table no.5 shows most frequent alloantibodies are Anti-E (46.1%), Anti-Fya (46.1%), and Anti-c (7.6%), most of  alloantibodies are of Rh  system and Duffy blood group systems. 

 

DISCUSSION:

The rate of alloimmunization in our study was 20.3% which correlate with previous studies done by various authors^.6,12 few authors studies shows lower alloimmunization could be due immunogenecity of blood group antigens which varies from 0.06-1.0.^13-14 and RBC intrinsic immunogenicity depends on the distribution of DRB1* restriction molecules.¹⁵   In our study in females had alloantibodies of 6.4% twice of males having 3.2% Which correlate to previous studies^.14,16 and in both sexes’ autoantibodies were 3.1%. The (P value-0.073) > (P value-0.050) states no significant correlation between age and alloimmunization.

 

In present study a total of 17 cases of thalassemia was included, the alloantibodies detected was 17.6% which correlate to previous studies.^17-20, but few authors reports low number of alloantibodies^14,21 could be due to antibody formation is less when transfusion is start in the first year of life. The induction of immunological tolerance by starting

repeated transfusions at this time was believed to account for the low rate of alloimmunization. ²²

 

And 5.8% autoantibodies were also detected. A total of 13 cases of sickle cell anemia was included in study, present study shows alloantibodies of 7.7% which correlate with previous study. ²³ but most of previous studies ^18,24,25,26 show high number of alloantibodies this could because of heterogeneity of blood group antigens in different race and population, lack of better-matched blood for the recipients and use of post storage leukodepleted blood for transfusion. The (P value-0.023) < (P value-0.050) was calculated for rate of alloimmunization in clinically diagnosed patient found to be significant. In our study the alloantibodies found maximum of 33.3% in patients who received blood transfusions of 21-30 units, it correlate to previous author’s studies. ^27-29 The (P value-0.30) > (P value-0.050) states there is no significant correlation between number of transfusions and alloimmunization.

 

In the present study the almost all the alloantibodies were of Rh system and Duffy system which correlate to previous studies.^{14, 21, 30, 31}

CONCLUSION:

The present dissertation emphasizes on the compulsory screening of all the multitransfused recipients prior to transfusion to prevent alloimmunization. The study shows that the prevalence of alloantibodies was more common in thalassemia and sickle cell anemia; emphasizing that in these patients, the antigen typing and extended cross matching should be performed for Rh and Duffy blood group systems.

 

ABBREVIATIONS:

AHG       -       Anti-human globulin

AGT       -       Antiglobulin test

DAT      -       Direct antiglobulin test

IAT         -       Indirect antiglobulin test

No.          -       Number

AIHA    -       Autoimmune hemolytic anemia

RBC        -       Red blood corpuscles

WBC      -       White blood corpuscles

°C            -       Degree centigrade

DHTR     -       Delayed hemolytic transfusion reaction

SCD        -       Sickle cell anemia

LISS        -       Low ionic saline solution

 

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Received on 01.09.2014          Modified on 10.09.2014

Accepted on 16.09.2014          © RJPT All right reserved