Abstract
Background
Alloimmunization to red cell antigens during antenatal period is a serious complication. Antibody titration of maternal blood sample corroborates with severity of disease and helps in planning further management. Conventional Tube Testing (CTT) method is widely accepted for antibody titration in antenatal cases but it can be replaced by Gel Microcolumn Agglutination (GMA) as it offers many advantages. Critical titer levels have been recommended by CTT method, but corresponding levels by GMA are yet to be established due to lack of adequate data.
Methods
Total of 1020 Rh negative antenatal females were evaluated over a period of four years and those found to be positive on antibody screening were further investigated. Antibody titration by CTT and GMA was carried out and the titers were correlated with the outcome of pregnancy.
Results
Out of the 1020 cases screened for antibodies, 112 (10.98%) were detected to be positive by GMA while 40 (3.92%) cases were detected by CTT. Titration was performed by both methods and results were statistically correlated. A moderate correlation was observed where one or more than one antibodies were involved (Pearson’s Coefficient 0.756), while a strong correlation (0.893) was observed between the two methods when single antibody (Anti-D) was involved. Titer values obtained by GMA were also higher than CTT.
Conclusion
Gel microcolumn agglutination (GMA) was found to be more sensitive and precise method for titration. A titer of 512 by GMA (IgG only) mostly corresponded to the values of 16 by CTT and had good correlation with clinical outcome.
Keywords: Red blood cell alloimmunization, Gel microcolumn agglutination, Conventional tube testing, Hemolytic disease of fetus and newborn, Rh-negative pregnancy
Introduction
Alloimmunization to red cell antigens can complicate antenatal cases (ANCs) and may lead to serious medical conditions like hemolytic disease of the fetus and newborn (HDFN). Maternal IgG type antibodies associated with ABO, Rh, or Kell blood group system(s) cross placental barrier in immune-mediated HDFN, causing hemolysis of fetal red blood cells.1 While morbidity and mortality associated with HDFN has significantly reduced in the developed countries, it still remains a critical problem in many developing countries1 where it is most frequently associated with Rh “D”-negative ANCs. Serologic testing in combination with clinical tests during pregnancy can help to determine the level of antibody(ies) present in maternal circulation, their potential to cause HDFN, and also the severity of red blood cell destruction during the period of gestation.2
Antibody titration method is a semi-quantitative technique used to estimate the concentration and strength of alloantibody(ies) in maternal serum sample.3 Historically, antibody titers have been found to corroborate with severity of HDFN, and this has helped the obstetricians in formulating their treatment protocols. Consistent and precise titers are important to monitor the cases for fetal anemia by nonserologic methods like Doppler ultrasound or potentially invasive measures, such as amniocentesis.
The methods of qualitative and quantitative analysis in immunohematology have evolved, with the availability of better technology and automation to ensure that results obtained from different laboratories are precise and comparable in determining antibody(ies) titration.4 Many centers and laboratories have already graduated to gel microcolumn agglutination (GMA) from conventional tube testing (CTT) for titrating IgM as well as IgG type anti-A1 and anti-B antibodies.5 However, we at our center are yet to validate GMA for titration of other clinically significant red cell alloantibodies(ies).
The determination of anti-D antibody titer(s) faces various challenges, due to the complex structure of the Rh-D antigen as well as due to the associated factors affecting the titration process. As per the current Association for the Advancement of Blood and Biotherapies (AABB) guidelines, titration using normal saline antihuman globulin still remains the only recommended method for titration of antibodies in ANCs,6 and validation is required to be carried out at the center or the laboratory employing a different technology(ies)6 like GMA. In the literature, there is dearth of studies on the value of “critical titer” for alloantibody(ies) in ANCs and the precise method for their titration by GMA. The study was conducted to correlate GMA with the CTT in the performance of anti-D antibody titration in Rh(D)-negative ANCs.
Materials and methods
The study was conducted at a tertiary care hospital in Western India over a period of four years after obtaining approval and ethical clearance of the institutional ethical committee. Written informed consent was obtained from all the participants and the participation was completely voluntary, with the option to withdraw from the study anytime.
A study population of 1020 Rh-negative ANCs were enrolled for the study and evaluated from February 2018 to March 2022. Their blood samples were collected in 3 ml K3 EDTA (Krupa Labequi, India) tube as well as 5 ml plain tube (Becton, Dickinson and company, UK). It was ensured that there were no labeling errors, and the sample volume was adequate for performing test by both the techniques. The samples were stored at 2–6 °C and analyzed within 24 h of collection for antibody screening followed by antibody identification and titration for anti-D antibodies by CTT as well as GMA method for samples found positive on antibody screening.
Antibody screening
For CTT method, a 3% cell suspension of O group red cells in normal saline (NS) was used. One drop of this red cell suspension was added to two drops of test serum in tube followed by incubation at 37 °C for 1 h. The cells were washed with NS thrice and polyspecific antihuman globulin (Arkray Healthcare, IVD, Surat, India) was added followed by centrifugation of the tube. Positive and negative controls were run along with the test samples, and the reactions were recorded.
For GMA testing, a commercial 3 cell screening panel (ID-Diacell I-II-III, Diamed, Cressier, Switzerland) was used. 50 μL of 0.8% suspension of cells in low ionic strength solution were taken in gel cards with 25 μL of test serum. A 15 min incubation at 37 °C was provided to the cards followed by centrifugation (ID-Centrifuge 12 S-II, Diamed, GmbH, CH-1785, Cressier, Switzerland), and the reactions were recorded.
Titration
The reactions by both the methods for antibody screening were observed and the positive samples were subjected to antibody(ies) identification by using 11 cell antibody identification panel (ID-Diapanel, Diamed GmbH, Switzerland) and further subjected to titration by CTT and GMA (IgG only, Biorad, Diamed GmbH, Switzerland).
Serial dilutions of test serum were made. The dilutions were tested for both the tests (CTT and GMA) in parallel. For CTT method, one drop of 3% R2R2 red cell suspension for anti-D antibodies was added to the tubes containing test serum in serial dilution followed by incubation at 37 °C for 1 h. The cells were washed with NS thrice, and antihuman globulin was added followed by centrifugation of the tubes and the reactions were recorded.
For GMA testing, 50 μL of 0.8% suspension of R2R2 red cells (for anti-D antibodies) in low ionic strength solution was added to 25 μL of serial dilutions of the test serum in GMA (IgG only, Biorad, Diamed GmbH, Switzerland) and incubated at 37 °C for 15 min as per the manufacturer's instructions followed by centrifugation. The highest dilution with 1+ agglutination was noted as the titer for the respective test serum.
The titers were further correlated with the clinical context, recent history of Anti-D administration, intervention like Intra uterine transfusion (if performed) and final outcome of the pregnancy.
Statistical analysis was carried out with the help of department of biostatistics, using the SPPS software (IBM Corp., version 26.0). Box and Whisker plot was used to check for presence of any outliers in the titer values and Karl Pearson coefficient of correlation was used to establish any correlation between the titer values by the two methods.
Results
Out of the 1020 ANCs tested, 112 (10.98%) were ICT positive by GMA of which 40 cases also tested positive by CTT (Fig. 1). Out of 72 cases positive only by GMA, 66 had a history of anti-D administration in the last 3 months (6 cases were lost to follow up). The antibody(ies) identified by GMA was anti-D in 108 cases and a combination of anti D + anti C in 4 cases. On the other hand, the antibody(ies) identified by tube method was anti-D in 36 cases and a combination of anti D + anti C in 4 cases (Fig. 1). Presence of anti-G antibody was ruled out using differential adsorption and elution method as described by Das, et al.7
Fig. 1.
Distribution of alloantibodies detected by conventional tube testing (CTT) and gel microcolumn agglutination (GMA) technique.
A total of 122 different samples were obtained from 40 ANCs on which titration was conducted and only the first reported titers by both the methods for each ANC were included for an accurate clinical correlation with the outcome of the pregnancy(ies).
The anti-D antibody titer values obtained by both the methods were compared. The GMA titer values were higher than the CTT (Table 1). Also, with increasing CTT titer values, the GMA titer values were also found to be increasing (Fig. 2). The differences were threefold in 2 cases, fourfold in 6 cases, fivefold in 24 cases, sixfold in 4 cases, and sevenfold in 4 cases (Table 2). As the titers obtained by the GMA span a very large range, Box and Whisker plot was employed (Fig. 3) to check for presence of any outliers. Four titer values were identified as outliers and these samples had a combination of Anti-D and Anti-C antibodies. The Karl Pearson coefficient of correlation was evaluated to be 0.756 with a p value <0.001, when all these samples were included, which suggested moderate correlation, but for cases involving single antibody (only anti-D), it was evaluated to be 0.893 with a p value <0.001, which suggested a strong correlation (Table 3).
Table 1.
Anti-D antibody titer values by conventional tube testing (CTT) and gel microcolumn agglutination (GMA) method.
| S. No. | Titer by CTT | Titer by GMA | Antibody(ies) detected |
|---|---|---|---|
| 1 | 32 | 512 | Anti-D |
| 2 | 16 | 512 | Anti-D |
| 3 | 32 | 2048 | Anti-D |
| 4 | 32 | 1024 | Anti-D |
| 5 | 64 | 1024 | Anti-D |
| 6 | 16 | 512 | Anti-D |
| 7 | 16 | 512 | Anti-D |
| 8 | 32 | 1024 | Anti-D |
| 9 | 32 | 1024 | Anti-D |
| 10 | 64 | 2048 | Anti-D |
| 11 | 128 | 16,384 | Anti-C + Anti-D |
| 12 | 64 | 2048 | Anti-D |
| 13 | 16 | 512 | Anti-D |
| 14 | 32 | 2048 | Anti-D |
| 15 | 32 | 1024 | Anti-D |
| 16 | 64 | 8192 | Anti-C + Anti-D |
| 17 | 128 | 4096 | Anti-D |
| 18 | 4 | 64 | Anti-D |
| 19 | 32 | 1024 | Anti-D |
| 20 | 2 | 16 | Anti-D |
| 21 | 16 | 512 | Anti-D |
| 22 | 32 | 1024 | Anti-D |
| 23 | 16 | 512 | Anti-D |
| 24 | 128 | 16,384 | Anti-C + Anti-D |
| 25 | 32 | 512 | Anti-D |
| 26 | 32 | 1024 | Anti-D |
| 27 | 32 | 2048 | Anti-D |
| 28 | 16 | 512 | Anti-D |
| 29 | 64 | 2048 | Anti-D |
| 30 | 32 | 2048 | Anti-D |
| 31 | 64 | 8192 | Anti-C + Anti-D |
| 32 | 16 | 512 | Anti-D |
| 33 | 4 | 64 | Anti-D |
| 34 | 32 | 1024 | Anti-D |
| 35 | 64 | 1024 | Anti-D |
| 36 | 128 | 4096 | Anti-D |
| 37 | 2 | 16 | Anti-D |
| 38 | 32 | 1024 | Anti-D |
| 39 | 32 | 1024 | Anti-D |
| 40 | 64 | 2048 | Anti-D |
Fig. 2.
Scatter plot chart showing anti-D antibody titer values by conventional tube testing (CTT) and gel microcolumn agglutination (GMA) method.
Table 2.
Comparison of Anti-D antibody titers on conventional tube testing (CTT) and gel microcolumn agglutination (GMA) method.
| GMA titers | CTT titers | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Neg | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | |
| Neg | – | – | – | – | – | – | – | – | – |
| 1 | – | – | – | – | – | – | – | – | – |
| 2 | – | – | – | – | – | – | – | – | – |
| 4 | – | – | – | – | – | – | – | – | – |
| 8 | – | – | – | – | – | – | – | – | – |
| 16 | – | – | 2 | – | – | – | – | – | – |
| 32 | – | – | – | – | – | – | – | – | – |
| 64 | – | – | – | 2 | – | – | – | – | – |
| 128 | – | – | – | – | – | – | – | – | – |
| 256 | – | – | – | – | – | – | – | – | – |
| 512 | – | – | – | – | – | 8 | 2 | – | – |
| 1024 | – | – | – | – | – | – | 10 | 2 | – |
| 2048 | – | – | – | – | – | – | 4 | 4 | – |
| 4096 | – | – | – | – | – | – | – | – | 2 |
| 8192 | – | – | – | – | – | – | – | 2 | – |
| 16,384 | – | – | – | – | – | – | – | – | 2 |
Fig. 3.
Box and Whisker plot showing anti-D antibody titer values by conventional tube testing (CTT) and gel microcolumn agglutination (GMA) method.
Table 3.
Coefficient of correlation (r) of titer values by conventional tube testing (CTT) and gel microcolumn agglutination (GMA) method.
| Titer by CTT and GMA for all samples having antibodies | Pearson Correlation (r) | 0.756 | 0.756 |
| Sig. (2-tailed) | <0.001 | <0.001 | |
| N | 40 | 40 | |
| Titer by CTT and GMA for samples having only Anti-D antibody | Pearson Correlation (r) | 0.893 | 0.893 |
| Sig. (2-tailed) | <0.001 | <0.001 | |
| N | 36 | 36 |
The anti-D antibody titers were further analyzed in terms of the clinical outcome of the pregnancy for a better correlation of the titer values by these two methods. Fifteen out of the 40 ANCs (positive by CTT) had undergone intrauterine transfusion (IUT) resulting in a viable delivery, 8 cases were reported to have IUFD with the remaining cases being managed successfully without any active intervention. The titer values for all the ANCs requiring IUT or which had resulted in intrauterine fetal demise (IUFD), was noted to be more than 16 by CTT and more than 512 by GMA. On analyzing the follow-up samples of these cases, titer values by GMA were noticed to be elevated much earlier compared to that by CTT and the results of the analysis corroborated with the clinical outcomes.
Discussion
The reported studies regarding titration by GMA in ANCs8,9,10,11 had different outcomes, depending upon the reagents and methodology used. There have been other studies3 which tried to correlate titration of IgG antibodies in other (nonantenatal) cases by GMA and CTT. Multicentric studies correlating titration by CTT and GMA for IgG antibodies in ABO incompatible renal transplants12 have also been published. To the best of our knowledge, this is the first study to employ use of IgG only cards for titration of antibodies in ANCs, which is highly specific for detection of clinically significant (IgG) antibodies. The results were much more accurate and precise with this method. In renal transplant recipients, use of IgG only type of cards has been recommended for titration of IgG antibodies of ABO system, as the polyspecific Coombs card may cause aberrant readings sometimes.13
While GMA had detected 112 cases, CTT could detect only 40 cases. There were 72 cases that were positive by GMA only with the anti-D antibody titer remaining below 16 in all of these cases. The reaction in GMA only positive samples mostly remained below 3+ grade and most commonly observed grades were 2+ grade and 1+. All these low-titer cases had history of prophylactic anti-D administration as low anti-D titers are known to be observed till 12 weeks after administration. The increased sensitivity of the GMA system is a well-established fact, and similar results were observed in most of the previous studies.8,9,10,11
The antibody(ies) identified by GMA and CTT method in our study were mainly anti-D and anti-C antibodies. Other clinically significant antibodies related to Rh and Kell blood group system were not detected. A possible reason could be the sample size of our study and prevalence of these red cell antigens in the population, such as the antigen frequency of antigen e in the Indian population is 98%14 which explains the low possibility of individuals developing anti-e antibodies. On the other hand, the antigen frequencies of K and E antigens are 3.5% and 20%, respectively.14 Therefore, the possibility of a pregnant female getting exposed to these antigens and developing corresponding antibodies (anti-K, anti-E) would be low. Our findings related to anti-C and anti-c were similar to results observed in other previous studies.8,15
One of the major limitations of this study is the relatively smaller number of cases analyzed (as compared to previous studies), which is expected with decreasing incidence of alloimmunization in ANCs. This study only compared the anti-D antibody titer values by GMA with CTT at a single center. Future studies may employ additional platforms like solid phase red cell adherence and erythrocyte-magnetized technique for a more elaborate evaluation of the titer values.
Conclusion
The GMA titer values were higher than that obtained by CTT in our study and a GMA (IgG only) titer of 512 mostly corresponded to CTT titer value of 16 with good correlation with the clinical outcome of the cases at our center. Therefore, the GMA testing technique can be reliably employed for detection and titration of red cell alloantibodies in Rh-negative ANC. Periodic screening for alloantibodies in ANCs would help in early detection of HDFN and initiating appropriate treatment.16 If the titers are to be reported by the GMA, the higher values should be discussed with the treating physician and should be apprised about the differences in the values obtained by these methods. The prospect of reporting GMA titer along with CTT can be employed as an initial approach to create awareness about the difference. It will also provide a better overview about the advantages of using a more sensitive and precise method like GMA, which also offers scope for automation.
On the basis of these observations, we conclude that GMA is a better technique than CTT for detection as well as titration of antibodies because of higher sensitivity and technical ease. Therefore, titration by GMA should be considered for ANCs though, further studies and more data would be helpful in establishing a critical titer by GMA.
Disclosure of competing interest
The authors have none to declare.
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