Anti-D Alloimmunization After RhD Positive Red Cell Transfusion to Selected RhD Negative Patients

Abstract

Transfusion of RhD positive red cells to RhD negative individuals is not routine transfusion practice for the fear of alloimmunization. Aim of this study was to prospectively evaluate rate of alloimmunization after transfusion of RhD positive red cells in RhD negative individuals and to assess delay in transfusion due to decision making. This was a prospective, observational study conducted from 2014 to 2018. All patients were followed up for a period of three months, at 3, 14, 45 and 90 days with antibody screening. In addition, patients who were immunosuppressed and alloimmunized were followed up at 6 months and one year. During the period of the study, there were a total of 57 RhD negative patients (52 males and five females) who received a mean of 4.42 ± 2.85 transfusions. Alloimmunization was detected in 8 (14.03%) patients at a mean interval of 25.63 ± 16.04 days. Anti-D was detected in seven and one patient developed anti-E alloantibody. Mean number of red cell units transfused in alloimmunized was 1.7 ± 0.26 while it was 5.4 ± 1.82 in non-alloimmunized group. There was no delay in providing units to these patients. The TAT was found to be 68 min. Rate of alloimmunization after transfusion of RhD positive red cells to RhD negative individuals was found to be 12.3%. In life saving conditions, RhD negative patients can be transfused RhD positive red cells without delay in decision making.

Introduction

Transfusion of RhD positive red cells to RhD negative individuals is not a routine transfusion practice because there are concerns about the potential for serious sequelae like alloimmunization. RhD negative mismatched transfusions have the potential for serious sequelae, such as inadvertent alloimmunization of a RhD negative female of childbearing age with RhD positive red cells, and the potential risk of hemolytic disease of fetus and newborn (HDFN). Also of concern is the potential for delayed hemolytic transfusion reaction upon subsequent receipt of RhD positive transfusion(s) in individuals who have been previously alloimmunized.

Regardless of these facts, in life saving haemorrhagic conditions, when there is unavailability of RhD negative units, it is standard practice to transfuse RhD positive red cells to RhD negative individuals [1–5]. Alloimmunization after RhD mismatched transfusions is not obligatory. However, the potential risk due to strong immunogenicity of RhD antigen cannot be ignored. Risk of RhD alloimmunization in RhD negative healthy volunteers transfused with RhD positive red cells has been found to be more than 80 percent., In immunocompetent RhD negative patients, RhD alloimmunization is around 20–30 percent but this incidence is lesser when studied in immunosuppressed patients like patients with bone marrow transplants, solid tumours and persons living with HIV [6–13].

The aim of the present study was to prospectively evaluate the rate of alloimmunization after transfusion of RhD positive red cells in RhD negative individuals and to assess the delay in transfusion due to decision making.

Materials and Methods

Settings and Design

This was a prospective, observational study conducted in the department of transfusion medicine at a tertiary healthcare center in India from the month of December 2014 to December 2018. An algorithm for participation in the study is illustrated in Fig. 1.

Fig. 1.

Algorithm for the study (Group specific transfusion was preferred over group O transfusion)

Inclusion Criteria

• 1.2.1 RhD negative patients admitted in the hospital who required transfusion
• 1.2.2Critical RhD negative red cell inventory defined as < 10 ‘O’Negative units in stock at the time of patient enrolment
• 1.2.3Negative red cell antibody screen performed within 72 h of the required time of transfusion (Patients who did not have an antibody screen report within 72 h of enrolment, were not included)
• 1.2.4Patient’s informed consent for RhD positive red cell transfusion

Exclusion Criteria

• 1.3.1Females < 45 years of age
• 1.3.2Patient’s refusal to RhD positive unit transfusion
• 1.3.3Patient admitted when RhD negative red cell inventory was above critical threshold 1.3.4 Positive antibody screen performed within 72 h of the required time of transfusion
• 1.3.4Patients who received platelet concentrates during the hospital stay (after RhD positive red cell transfusion)
• 1.3.5Patients who could not be followed up after discharge or left against medical advice or patients from other countries who could not be kept under follow-up.
• 1.3.6Patients requiring lifesaving transfusions because consent for lifesaving/emergency transfusion was not necessary [Based on the priority, there are three types of requisition forms: (1) Routine: Transfusion expected after 12 h, (2) Urgent: Time to transfusion around 2 h, (3) Lifesaving: Units arranged on priority, as soon as possible, or at maximum within 10 min of receiving the requisition form. Difference between emergency and urgent is that for emergency, uncrossmatched units are issued whereas for urgent transfusion, units are issued after the compatibility testing has been completed.]

Documentation and Consent

The primary team was informed about the option of RhD positive transfusion. The primary team would discuss the need of RhD positive red cell transfusion with the patient or the next of kin, explaining the benefits and risks. If the patient consented to RhD positive red cell transfusion, this was communicated to the transfusion service with a copy of the consent form and a requisition with special mention of the same signed by the primary team was received in the red cell serology laboratory. All such patients were marked in the hospital information system for traceability. A record of age, gender, diagnosis and indication of transfusion, number of total and D + RBC transfused and all subsequent serological investigations (mentioned below) till discharge and follow up were recorded. A record of group specific and non-group specific units issued to the patient was kept.

Red Cell Transfusion

All red cell concentrates were prepared from 450 mL whole blood units collected in a top and bottom blood bag system with inline leucofilter with greater than 3 log reduction capacity. All units were tested for anti-hepatitis C virus (HCV), anti-human immunodeficiency virus (HIV), hepatitis B surface antigen, malaria, and syphilis using chemiluminescence immunoassay (Vitros 3600 Immunodiagnostic System, Ortho Clinical Diagnostics, USA). All red cell units were phenotyped for Rh and Kell antigens (D, C, E, c, e, K) using solid-phase RBC adherence (Galileo NEO, Immucor, USA). Units were selected from the inventory in first-in, first-out order.

Follow-Up

All patients were followed up for a period of three months, at 3, 14, 45 and 90 days with antibody screening (AS) for alloimmunization to transfused units and direct antiglobulin test (DAT). Samples collected in 3 mL ethylene diamine tetra acetate (EDTA) vacutainers, were tested for: (a) automated ABO group and Rh type (Neo, Immucor, Norcross, USA). (b) DAT, antibody screening and antibody identification were performed manually by column agglutination technique (Ortho-Clinical Diagnostics, USA). In cases where anti-D was detected during the period of observation, titer of anti-D was estimated in coomb’s phase at 37 °C by conventional test tube technique using R₂R₂ red cells from inventory. Laboratory values suggestive of hemolysis including reticulocytes counts, bilirubin, hemoglobin, peripheral smear and lactate dehydrogenase (LDH) were also monitored till 3 months from date of transfusion for which 3 mL sample was collected in EDTA and 3 mL in serum separator tube (SST). For hemoglobin and reticulocyte count, EDTA sample was used and for bilirubin and LDH, blood sample was collected in serum separator tube. The patients were requested to visit the hospital on the requested dates. If they couldn’t turn up due to any reason, they were offered home collection. Two EDTA and a serum separator tube were used every time. In addition, patients who were immunosuppressed and those who developed a positive antibody screen were followed up at 6 months and one year to monitor delayed development of anti-D. Immunosuppressed individuals were identified from the patient records as those taking immune-suppressants during the three-month follow-up period.

Turnaround Time (TAT)

All RhD negative patients transfused with RhD positive units were divided in two groups, those who underwent elective surgeries and gave consent for RhD positive transfusion prior to surgery and those who were provided urgent (not lifesaving) RhD positive transfusion. To assess the importance of implementing and understanding hospital policies, turnaround time for issuing units to these patients from the time the primary team was informed about the choice of transfusing RhD positive units was provided and was compared with the mean TAT for issuing RhD positive units in elective surgeries where the consent was obtained beforehand. For the purpose of calculation, every patient included in the study was considered as a subject and issue of first RhD positive red cell unit to these patients was the event for study. TAT for elective transfusion was calculated from the time a call from anesthetist to initiate the protocol was received in the red cell serology laboratory to the time first RhD positive unit was issued. TAT for urgent (not lifesaving) transfusion was calculated from the time the primary team was informed about the option of RhD positive transfusion to the time first RhD positive unit was issued.

Ethical Committee Approval

The Institutional review board and ethical committee approval was taken. Informed consent was obtained from each patient. None of the patients were charged for follow-up investigations.

Statistical Analysis

Data were entered in an MS excel sheet (Excel (Redmond, WA); numerical values, percentages, mean and standard deviation was calculated. Statistical analysis was performed using SPSS software (Version 25.0.0.0, Chicago, USA). Patients were split in two categories; alloimmunized and non-alloimmunized and variables were compared between these two patient categories. Independent student t test was applied to calculate p-value for comparing TAT in the two groups. P < 0.05 was considered significant.

Results

Demographic Details

This was a prospective analysis of all RhD negative patients who received RhD positive red cell transfusions. A total of 25,447 patients were admitted in the hospital during the study duration out of which requisitions were received for 20,658 patients. 2136 of these 20,658 patients had RhD negative blood group. A total of 48,249 units were requested and a total of 29,855 units were issued for these 20,658 patients. During the period of the study, there were a total of 57 RhD negative patients (52 males and five females) who received a total of 252 RhD positive red cell transfusions with the mean number of red cell unit transfusions being 4.4 ± 2.9 (1–9). ABO non-group specific transfusions were 102 (40.5%). A department-wise distribution of the cases has been illustrated in Fig. 2. Patients details are elaborated in Table 1.

Fig. 2.

Department-wise distribution of a patients and b number of units transfused

Table 1.

Details of patients on the basis of alloimmunisation

		Alloimmunised patients	Non-Alloimmunised patients	Overall
No of patients		8	49	57
Age	> 60	0	9	9
	< 60	8	40	48
Gender	Male	5	47	52
	Female	3	2	5
Diagnosis	Trauma	3	11	14
	Liver Transplant and gastrosurgery	1	17	18
	Obstetrics and Gynaecology	1	4	5
	Cardiac surgeries	1	8	9
	Neurosurgeries (Not included in trauma)	0	2	2
	Hemato-oncology and BMT	2	7	9
Mean number of red cell units transfused		1.7	5.4	3.1
Antibody formation	RhD	7	–	7
	Non RhD	1 (Anti-E)	–	1

Alloimmunization Details

Alloimmunization was detected in 8 (14%) patients at a mean interval of 25.6 ± 16 days from the first RhD positive red cell transfusion considering the day of first transfusion of RhD positive unit as day 0. Anti-D was detected in seven of these eight cases and one patient developed anti-E alloantibody. Therefore, RhD alloimmunization was found to be 12.3%. There were a total of 20 patients who were immunosuppressed at the time of RhD positive transfusion and these patients were followed up at 6 months and one year as well. None of these 20 patients developed anti-D in long term follow up. The mean number of red cell units transfused in alloimmunized patients was 1.7 ± 0.3 while it was 5.4 ± 1.8 in non-alloimmunized group. The median titer of anti-D observed in these seven cases was 8 at the time of detection. Mean haematological parameters to assess hemolysis at different points of time have been listed for alloimmunized and non-alloimmunized patients separately (Table 2).

Table 2.

Mean haematological parameters to assess hemolysis at different points of time

Lab Parameteres	Day 3	Day 14	Day 45	Day 90	6 months	1 year
Alloimmunized
Hemoglobin (g/dl)	8.2	8.4	9.9	8.9	9.8	10.4
S. Bilirubin (mg/dl)	0.9	0.6	1.1	1.3	0.8	1.0
Corrected reticulocyte count (%)	3.5	2.6	3.3	2.3	1.8	1.4
Lactate Dehydrogenase (U/L)	224.2	189.2	231.5	171.2	175.3	198.5
Non- alloimmunized
Hemoglobin (g/dl)	10.8	11.2	10.9	11.8	11.2	12.0
S. Bilirubin (mg/dl)	0.5	0.4	0.5	0.3	0.6	0.8
Corrected reticulocyte count (%)	2.1	1.8	1.7	1.1	1.2	1.2
Lactate Dehydrogenase (U/L)	166.0	165.1	171.5	139.2	159.1	178.3

Follow-Up

During follow-up of these patients, one of these seven became antibody screen negative at one year, rest continued to remain antibody screen positive with a median titer at one year of 4. Patients were monitored for presence of possible clinical signs such as pallor, icterus and tachycardia and laboratory features of hemolysis till three months from the date of transfusion. None of the patients had a hemolytic reaction reported during this period.

Comparison of TAT for Elective and Urgent Transfusions

All 57 patients were divided in two groups on the basis of whether RhD positive red cell transfusion was an elective or an urgent decision. The mean turnaround time for issuing units to patients in both these categories was compared. The purpose of this exercise was to assess whether there was a significant delay in decision making when a patient was offered this option; which in turn is directly influenced by whether the institutional policy of RhD positive transfusion to RhD negative patients is well understood by the clinicians. First group of elective RhD positive transfusion included 18 patients and second group of urgent RhD postitive transfusion included 39 patients. The mean turnaround for elective transfusion group was 97 ± 10.4 min and that for urgent transfusion group was 68.4 ± 16.1 min. On applying student t-test, p-value was found to be 0.0018. Ironically, the TAT for urgent transfusion was found to be significantly less than the TAT for elective transfusion reflecting that contradictory to the usual belief, there was no delay in decision making for transfusing RhD positive to RhD negative recipients.

Discussion

Alloimmunization against red cell antigens is a common complication associated with red cell transfusions and anti-D alloimmunization is observed more frequently than alloimmunization to other RBC antigens. Rh is a complex blood group system and D antigen is second to only ABO in terms of immunogenicity [14, 15]. Prevention of alloimmunization after RhD mismatched transfusion has been less discussed than prevention of alloimmunization in pregnancy, in great part owing to the fact that RhD mismatched transfusions are sporadic events occurring as emergency situations or during inventory shortages. Most of these patients are males or older females in whom the risks of alloimmunization are considered to be minor. However, owing to ethical concerns and practical considerations, a study in which RhD mismatched transfusions are deliberately administered is difficult, and as a result, the establishment of best practice has progressed as an empirical process, in part supported by occasional studies in volunteers, case reports, and uncontrolled studies of patients who received inadvertent mismatched transfusion [8, 11–23].

Prevalence of RhD Antigen

The prevalence of RhD antigen in the Indian population is roughly 95 percent (93.80 to 94.8%) according to various studies [23–26]. Therefore, arranging RhD negative units for patients, especially in cases of hemorrhage and patients with need of frequent blood transfusions can be challenging. Policies and procedures for blood transfusion in such cases are required for management of patients.

Alloimmunization: Incidence and Number of Red Cell Transfusions

In early volunteer studies, 70 to 90 percent of subjects repeatedly given RhD positive antigen exposure became alloimmunized, and it has been reported that a volume as low as 0.1 to 0.5 mL may trigger alloimmunization in normal individuals. Thus, the incidence of alloimmunization has been mostly cited to be more than 80 percent based primarily on studies of healthy volunteers [27]. A more recent retrospective analysis of 78 patients who received RhD mismatched blood transfusions without immunoprophylaxis, reported an incidence of 30% [12]. Interestingly, this analysis suggested a possible inverse correlation between the number of units given and the probability of alloimmunization. Six of the 16 patients developed additional IgG autoantibody and in 3 cases, evidence for prolonged hemolysis was found. In the present study, none of the patients had an autoantibody component and on follow-up no evidence of hemolysis was detected. The authors considered that the discrepancy between the rate of alloimmunization in their study and that seen in earlier volunteer studies could be due to “high zone” tolerance when large volumes of RhD positive RBCs are transfused or due to stress-induced immune suppression. The lack of immunization in RhD negative liver transplant recipients receiving RhD positive transfusion has been demonstrated previously [28, 29]. In the present study, RhD alloimmunization was found in 12.3%. This rate of RhD alloimmunisation is lower than that found in some other studies who transfused RhD positive red cells in RhD negative patients which has been previously discussed that rate of alloimmunization can be different for different races [30, 31]. Gonzalez-porras et al. reported around 21% of their patients (non-immunosupressed) alloimmunised with D antibody while Selleng and colleagues evaluated a total of 85 RhD negative patients admitted in trauma centre who received 2,836 RhD positive red cell concentrates and 22% patients developed documented anti-D alloimmunization [3, 16]. Baldwin et al. reported an incidence of D-alloimmunization of 19 percent in solid tumor patients while in another study of 17 RhD negative liver transplant patients who received from 5 to 41 units of Rh-positive red cells during surgery, no evidence of immunization appeared in any patient [17]. Immunisation to RhD antigen is more in non-immunosuppressed patients while it varies in immunosuppressed patients from no antibody development in some to late development in others [32]. Our patients were mostly from non-immunosuppressed category but 20 patients were followed up for late development of anti-D. None of these patients developed a positive antibody screen in one year of follow-up.

It has been seen that in immunocompetent individuals, the earliest time at which anti-D can be detected in primary immunization ranges from 4 to 10 weeks and the production of anti-D within 2 weeks of a first stimulus has been observed only after the injection of specially treated RhD positive RBCs. [19–234–18] There were only two patients who were alloimmunized at day 14 of follow up. Anti D formation in RhD negative individuals does not seem to be dependent upon number of RhD positive units transfused. The mean number of red cell units transfused in alloimmunized patients in our study was 1.7 while it was 5.4 in non-alloimmunized group. Gonzalez-porras et al. found the mean number of unit transfused in alloimmunized group to be 3.2 while it was higher in non alloimunized group [16]. Various other factors may play a role in alloimmunisation like age, gender and diagnosis. Some of the studies have found patients with hematological malignancies to be more prone to alloimmunisation. 2 out of 8 patients who were allo-immunised in our study were from hemato-oncology.

Alloimmunization to Other Antigens

Many of the studies that evaluated anti-D formation in RhD negative individuals also found development of other non-D antibodies against minor red cell antigen sometime upto an extent of 50% of total antibodies found. Most of these antibodies were against other Rh and Kell antigens [13, 18, 19]. We found only one case where anti-E alloantibody was formed. The probable reason could be because extended Rh and Kell phenotyping of all units are performed and most of the patients are being given Rh and Kell matched units. Also, universal leucodepletion of red cell units also reduces the chances of alloimmunisation. This may also be the reason for low rate of alloimmunisation in our patients despite most of them being immunocompetent.

Delay in Decision Making for Administering RhD Positive Red Cells to RhD negative Recipients

There is concern amongst physicians regarding the considerable, unnecessary delay in decision making when a situation of RhD positive transfusion arises in a RhD negative recipient. Calculation of TAT in elective and urgent procedures in the present study revealed that ironically, the TAT for issuing units in cases who required urgent transfusion was close to an hour and considerably less than TAT for the elective procedure category. This was probably due to the fact that the institutional policies for transfusion in such cases is quite robust and that the physicians work in close co-ordination with the transfusion services.

Conclusion

Rate of alloimmunization after transfusion of RhD positive red cells to RhD negative individuals was found to be 14% for all antigens and 12.3% for D antigen. There was no delay in providing units to such patients. The TAT was found to be 68 min. In life saving conditions, RhD negative patients can be transfused RhD positive red cells. This could be quite useful tool in safeguarding patients’ life as well as saving precious RhD negative units, especially in Indian scenario where inventory of RhD negative units is just 5% of total population. The strengths of this study include prospective study design, inclusion of patients from many specialities; both immunocompetent as well as immunosuppressed individuals, calculation and comparison of TAT as a predictor of complete understanding of hospital transfusion policies, comparison between alloimmunized and non-alloimmunized groups, follow up at 3, 14, 45 and 90 days and long term follow-up at 6 months and 1 year. Limitation of this study was exclusion of patients receiving lifesaving transfusions.

Funding

None.

Declarations

Conflict of interest

The authors declare no conflict of interest.