Implementing type and screen method replacing conventional antiglobulin crossmatch procedure for compatibility testing in elective protocol in a tertiary care hospital

Abstract

INTRODUCTION:

Pretransfusion testing is an essential and important investigation to provide safe and fully compatible packed red blood cell (PRBC) unit to the recipient to prevent the fatal hemolytic transfusion reaction. Type and screen (T and S) procedure is one of the effective approaches in pretransfusion testing in which antibody screen (AS) negative recipients are transfused with an immediate saline phase compatible PRBC unit. In order to explore the feasibility of implementing T and S protocol in our institute, this study was carried out. The main aim of our study was to compare the T and S protocol in AS-negative recipients with anti-human globulin (AHG) phase crossmatch in patients requiring PRBC under elective protocol.

MATERIALS AND METHODS:

Our study was a cross-sectional analytical study carried out in all the elective blood samples with a history of transfusion or pregnancy. In our study, 849 elective blood samples were subjected to antibody screening by reagent 3 red cell screening panel and AS-negative blood samples were subjected to immediate saline phase crossmatch, and compatible units were issued to the patients. The same samples were subjected to AHG phase crossmatch for validation.

RESULTS:

Among 14,705 elective requests for PRBC 849 patients had a history of transfusion or pregnancy. Of 849 patients, no case was found to be positive for antibody screening. Totally around 1028 PRBC units were saline crossmatched and totally 677 PRBC units were issued to the patients under T and S protocol. Not even a single case where AHG crossmatch was incompatible when antibody screening was negative. There was a perfect agreement between T and S method and AHG phase crossmatch. Crossmatch transfusion ratio was reduced from 2.1–1.5 with the implementation of T and S protocol.

CONCLUSION:

This study validated and showed that there was a perfect agreement between T and S protocol and AHG phase crossmatch. All blood requests under elective protocol, the T and S method can be implemented for better utilization of technical workforce and better inventory management.

Introduction

Blood component transfusion forms an important therapeutic intervention for many diseases, surgeries, etc. Pretransfusion testing is an important mandatory investigation before issuing blood components for transfusion. Among various blood component transfusions, packed red blood cell (PRBC) is most frequently transfused component. Pretransfusion testing for PRBC is performed in view of avoiding immune-mediated hemolytic transfusion reaction (HTR) due to incompatible donor RBCs. Complete pretransfusion testing protocol for PRBC transfusion includes ABO grouping, Rh typing, auto- and alloantibody screening, antibody identification, and compatibility testing.[1] Major cross-match or compatibility testing is done commonly in two phases. First phase is done at room temperature (20°C–25°C) - Immediate spin (IS) Saline phase. It is done to rule out IgM antibodies, for example, ABO mismatch which has potential to cause Hemolytic Transfusion Reactions due the IgM anti-A and anti-B antibodies. The duration of this phase is about 10–15 min. At 37°C, antihuman globulin (AHG) or coombs phase is to detect nonagglutinating incomplete IgG antibodies.[2] This procedure takes about 30–60 min or more. Antibody screen (AS) was established as a part of pretransfusion testing in the early 1960s along with AHG phase crossmatch. It is mostly deployed for the non-ABO antibodies of IgG type which are found to be clinically significant.[3] However, several workers then questioned the importance of performing the AHG phase crossmatch when AS screening was negative. This resulted in the introduction of type and screen (T and S) approach, wherein any patient with AS negative could be transfused with red cell unit after a compatible immediate-spin crossmatch which will save precious time and also detect clinically significant alloantibodies.[4] In our center, we have three different protocols for issuing blood components. They are emergency, urgent, and elective protocols. In emergency protocol, which is usually uncommon, mainly for massive transfusions O negative blood units are issued without patient’s samples work up and later complete pretransfusion testing is done. In urgent protocol, PRBCs are issued after patient’s blood grouping with Rh typing and crossmatch in immediate saline phase at room temperature. In elective protocol, PRBCs are issued with patient’s blood grouping with Rh typing, AS by the Indirect Coombs Test (ICT), followed by IS crossmatch and AHG crossmatch. For ICT-pooled, “O” cells from 5 local donors were taken and the antigen representation of the donor cells was unknown. This takes a long time due to the lengthy incubation and the safety profile of blood is also questionable.

Currently, each elective protocol crossmatch at most of the blood centers takes longer time and might not detect clinically significant low-frequency antibodies as it involves one-time AS with local pooled donor cells followed by IS saline crossmatch at room temperature and AHG phase crossmatch at 37°C with each unit of PRBC. Mostly, incomplete alloantibodies are of IgG which plays a major role in causing delayed hemolytic transfusion reaction without being significantly seen in pretransfusion testing. These can be missed during routine ICT AS. Ultimately, Indian laboratories preferably perform AHG phase crossmatch to rule out these clinically significant IgG antibodies. If AS is performed by commercial (reagent) 3 red cell panel which has representation of almost all significant antigens, routine AHG phase crossmatch can be replaced as it takes a long time due to incubation and duplication. This is mostly needed in tertiary care hospitals with heavy workload and which caters to a variety of patients requiring multiple transfusions. Most of the studies demonstrated the absence of delayed hemolytic transfusion reactions in patients receiving blood transfusion using T and S approach. Even though the studies inferred the incompatibility of some of the transfused red cell units by the subsequent AHG crossmatch, the evidence of hemolysis was not found in any of the patients either clinically or serologically.[5] Hence, pretransfusion testing can be done excluding the AHG crossmatch without putting patients at risk.[4] Some studies also demonstrated the safety of TS approach in patients requiring multiple and massive transfusions.[6] The aim of the present study was to compare T and S procedure by commercial 3 red cell panel in AS-negative recipients with AHG phase crossmatch for patients requiring PRBC under elective protocol. This study is the first of its kind in our institute and also in south India. Most centers in India including our blood bank use pooled donor O cells for antibody screening in patient’s serum. In this study, we used commercial (reagent) 3 red cell panels for AS which represent almost all clinically significant antigens followed by immediate saline phase room temperature crossmatch with each unit.

Materials and Methods

Study design

This was cross-sectional analytical study.

Study participants

Inclusion criteria

All blood requests and blood samples which were received as per elective protocol for red blood cell transfusion. Patients with a history of previous transfusion and pregnancy were included in our study.

Exclusion criteria

Any patient’s blood request and sample with positive AS. Any patient sample which showed incompatibility in immediate saline phase crossmatch at room temperature after negative T and S.

Study period

The duration of the study was from November 2017 to August 2019.

Sampling

Sampling population

All the elective blood samples that have reached the blood bank for processing were taken.

Sample size calculation

In a study done by Chaudhary and Agarwal[7] (Lucknow), the specificity of IS cross matching to predict compatibility was 99.25% With expected specificity of 99.25% and expected prevalence of compatibility as 98.0%, the sample size is calculated as 823 with 95% confidence interval, 5% absolute precision, We have processed totally 849 samples at the end of the study period.

Sampling technique

Consecutive sampling.

Ethical clearance was obtained from the Institutional Ethical Committee, project no: Being JIP/IEC/2017/0421.

Study Protocol

All the samples received for the patients requiring RBC transfusion under elective protocol were processed according to Figure 1.

Figure 1.

Study protocol

Compatible units (AS negative, IS compatible) work up

Were worked up for validation and comparison: Same samples and units were subjected to AHG phase crossmatch at 37°C simultaneously [Figure 2]

Figure 2.

Compatible unit's further work

Tube technique or column agglutination technology (CAT) was used to perform patient and donor blood grouping, Rh typing, IS and AHG crossmatch. In all the samples, antibodies were screened using a commercial 3-cell reagent, and AHG crossmatch was done for compatibility testing. The actual AHG crossmatch was carried out routinely by the blood bank technologists and the antibody screening by 3 red cell panel was carried out by the investigator and this blinding was carried out without knowing the result of the compatibility test. After completing all the tests, the results of the two tests were compared. Crossmatch transfusion ratio (C/T ratio) is one of the effective and important indicators to determine the effectiveness of T and S policy. It was determined by

Statistical tests used for data analysis

All statistical analyses were done using SPSS version 21. Categorical variables such as compatibility and blood group were expressed as proportions. Continuous variables such as age were expressed as mean due to its normal distribution. The results of T and S procedure and AHG crossmatch were compared to determine the level of agreement between two tests by Kappa statistics.

Results

During the study, totally 105,500 requests came for different blood components. Out of 105,500 requests, the highest requests were received for PRBC which comprises 40,193 (38.1%) followed by 33,108 (31.3%) requests were received for FFP, 30,225 (28.6%) requests were received for RDP and 1974 (1.9%) requests were received for cryoprecipitate. Among these requests, 14,705 were received for elective protocol. The gender distribution of total blood requests under elective protocol was almost equal. Among 14,705 elective requests, the number of requests with a history of transfusion or pregnancy was 849 [Table 1]. These 849 patients’ requests were evaluated for T and S protocol and conventional AHG phase crossmatch. Among 849 patients with the previous history of blood transfusions, majority of patients were transfused more than 3 months which comprised 79% (N = 671) and 21% (N = 178) of patients were transfused < 3 months. The indications for transfusions were broadly classified. “Anemia” - in which PRBC is ordered in elective protocol and transfused as top-up to meet surgical fitness or as a therapeutic measure. “Surgery” - Preoperatively, crossmatched or ordered in anticipation of blood loss (MSBOS) and these units may be transfused or may not be.

Table 1.

Data regarding study samples crossmatched and transfused

Character		n
Total number of patients who underwent antibody screen (study sample)		849
Patients in whom the antibody screen was negative		849
Patients in whom the antibody screen was positive		0
Number of PRBC units saline crossmatched in patients with negative antibody screen and further validated with AHG phase crossmatch		1028
Number of PRBC units where the AHG crossmatch was incompatible when antibody screen was negative		0
Number of PRBC units issued and transfused to the study population		677
C/T ratio for the study samples with type and screen protocol		1.5
Number of units crossmatched during the study period with routine AHG phase crossmatch		30,184
Number of units issued and transfused under routine AHG phase crossmatch during the study		14,984
C/T ratio for total blood samples processed during the study with routine AHG phase crossmatch		2.1

PRBC=Packed red blood cell, AHG=Anti-human globulin, C/T: Crossmatch transfusion ratio

“Blood loss-” it may be acute blood loss intra- or post-operative (preoperative blood units were not cross-matched and kept) or in chronic blood loss as in UGI bleed. In our study population, majority of blood requests were indicated for surgery (N = 628) as impending blood loss (MSBOS), followed by therapeutic transfusions for acute or chronic blood loss (N = 119) and then top-up (anemia) for surgery and other intervention procedures (N = 102).

Totally 849 patients with the history of previous transfusion or pregnancy were typed and screened by commercial 3-cell panel and validated by AHG phase crossmatch for compatibility testing. Of 849 patients, none of these cases was found to be positive for T and S as well as showed incompatibility for AHG crossmatch [Table 2]. In our study, totally 1028 PRBCs were ordered and all the units were saline cross-matched, type and screened and further validated by AHG phase crossmatching. In these units, 677 PRBC units were issued to the patients with different indications. The C/T ratio was found to be 1.5, during the same period, totally 30,184 PRBC units were cross-matched under AHG phase and 14,984 PRBC units were issued to the patient under routine AHG phase crossmatch protocol with C/T ratio 2.1. Thus, C/T ratio was 2.1 in total elective samples and 1.5 with study samples. Among 849 study samples, totally 169 (20%) patients had repeat transfusions. Among 169 patients with repeat transfusions, 152 (90%) patients were transfused within 3 days and 17 (10%) patients were transfused between 3 and 7 days.

Table 2.

Comparison of type and screen with anti-human globulin phase crossmatch

Antibody screening		AHG crossmatch compatibility
		Compatible		Incompatible
Negative		849		0
Positive		0		0

AHG=Anti-human globulin

In our study, by Kappa statistics, there is a perfect agreement (Kappa score = 1; P < 0.001) between T and S method and AHG phase crossmatch. All the study samples (n = 849) were negative for T and S and similarly, for all the study samples, no incompatibility was found during further validation with AHG crossmatch. This concurrence result suggests that T and S method is as safe as that of the AHG phase for elective protocol with a significant reduction of CT ratio.

Discussion

Our study was a cross-sectional analytical study done on patient samples under elective protocol with a history of transfusion or pregnancy. The main purpose of our study was to compare T and S procedure with conventional AHG phase crossmatch in the elective protocol. During the study from November 2017 to August 2019, totally 40,193 PRBC requests were received. Among them, totally 14,705 PRBC requests were processed under elective protocol. Among these 14,705 PRBC request, we selected 849 patient samples under elective with previous history of transfusion or pregnancy as RBC alloimmunization is one of the most common risks associated with RBC transfusion and pregnancy particularly Rh-negative mothers. Most of the blood requests to our blood bank come under urgent protocol (more than 50%) and under elective protocol (36%), majority of requests came mainly from surgery, obstetrics and gynecology, and orthopedics departments which is comparable with Arora et al.[8] which also had a high number of requests from medicine and allied departments. Our results were not comparable with Thakral et al.’s[9] study which may have different protocols.

In broad, the incidence of alloimmunization in multi-transfused adult population in whom pretransfusion antibody screening has been done ranges from 2% to 30%.[9,10,11,12] In most of the studies compared, all studies had alloantibody and antibody positivity rates varied from 0.15% to 3.4% as they have not excluded ICT-positive samples. In our study, we have excluded all the samples in which ICT was positive with in house Pooled “O” cells as per methodology, but the positivity rate during our study period was 2% of all ICT samples received and most commonly implicated antibody was anti-D mostly in pregnant in women. Some of the studies [Table 3] had a similar result when compared to our study (which had 100% concordance). In our study, though we excluded all ICT-positive samples and we did not find any AS positivity with commercial red cell panel, and all the samples were compatible in both immediate saline and AHG phase cross matching which was in concordance with studies of Pathak et al.[13] and Agrawal.[14] However, in the studies conducted by Tiwari et al.[4] and Chaudhary and Agarwal[7] there was a single unit in which AHG phase crossmatch was incompatible in AS-negative recipients. In our study, the 3 red cell screening panel almost covered all significant antigens and was able to detect all the clinically significant antibodies. However, this panel did not include Mi III phenotype of the Miltenberger subsystem (or GPMur) and in Chaudhary and Agarwal et al.’s[7] study the antibody for this antigen was implicated for discordant result for T and S versus AHG crossmatch. As the other Indian studies have also not found any discordant result, hence this “Mi III” and “‘In” phenotype may be low-frequency antigen. This has to be further validated by multi-centric phenotyping for these antigens in large population across India to be included in red cell panels.

Table 3.

Comparison of type and screen and anti-human globulin phase crossmatch among similar studies

Various studies		Concordance between posttransfusion AHG phase crossmatch and immediate saline crossmatch in antibody screen negative recipients (%)
Pathak et al.[13] (n=45,373)		100
Agarwal[14] (n=354)		100
Tiwari et al.[4] (n=32,650)		99
Heddle et al.[5] (n=9128)		98
Our study (n=849)		100

AHG=Anti-human globulin

The effective indicators to measure the efficacy of implementing T and S policy is C/T ratio. The C/T ratio corresponds to real blood usage. A C/T ratio >2.5 is an indicator of poor blood utilization, and hospitals should aim to keep a C/T ratio of 2 or less.[15] Our C/T ratio is almost as par with standard available guidelines which says it should not be more than 2, even if it improved (to 1.5) after the implementation of T and S. All other studies shown in [Table 4] also had C/T ratio as par with standard available guidelines. In our study, when C/T ratio is compared with different departments (elective protocol), obstetrics and gynecology (2.6) show the highest C/T ratio and Zewdie et al.’s[19] study which was conducted in Ethiopia also has the highest C/T for obstetrics and gynecology (31.0) and this study has very high C/T ratio describing their ineffective or poor blood utilization.

Table 4.

Comparison of crossmatch transfusion ratio among various studies

Various studies		Decrease in C/T ratio after implementation of type and screen protocol
Aggarwal et al.[16] (n=24,724)		1.94-1.04
Chow[17] (n=1024)		2.9-1.3
Alavi-Moghaddam et al.[18] (n=472)		1.41-1.3
Our study		2.1-1.5

C/T ratio=Crossmatch transfusion ratio

Conclusion

In our study, T and S protocol was completely validated by AHG crossmatch and we did not find any incompatibility. Thus, our study depicts 100% safety value for T and S protocol among the elective patients with the history of transfusion or pregnancy. The results of our study may provide a baseline data for implementing T and S procedure not only for elective protocol but also can be extended with further studies (after implementing the same protocol and validation) for urgent protocol in our institute which has the highest number of requests.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.