記住我
ORIGINAL ARTICLE
Year : 2021 | Volume
: 46
| Issue : 2 | Page : 105-110
Comparative study between different alloadsorption techniques in warm autoimmune hemolytic anemia
Noha A.M Emam1, Soha Ezzelarab1, Noha B Hassan1, Heba N El Saeyed1, Nermeen A Nabih2, Gehan M Hamed MD 1
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Internal Medicine, Clinical Hematology and Bone Marrow Transplantation, Faculty of Medicine, Ain Shams University, Cairo, Egypt
Correspondence Address:
Gehan M Hamed
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, 67 El Nasr Street, Sheraton Heliopolis, Cairo, 11799
Egypt
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejh.ejh_52_20
Background Autoantibodies in patients with autoimmune hemolytic anemia (AIHA) are encountered with difficulties in ABO grouping and cross-matching; moreover, they may mask the presence of alloantibodies, leading to hemolytic transfusion reaction. Therefore, an efficient and time-saving method is required to detect alloantibodies underlying autoantibodies for safe transfusion in AIHA.
Aim To compare the efficiency of different alloadsorption techniques in detection of alloantibodies after complete removal of autoantibodies.
Patients and methods A total of 70 patients with warm AIHA were enrolled in this study; antibody screening was done by using screening cells (I+II+III cells). Allogenic adsorption was performed using conventional, polyethylene glycol (PEG), and low ionic strength solution (LISS)/papain methods followed by rescreening to ensure the adsorption of the autoantibodies, and whenever screening test revealed the presence of alloantibodies, antibody identification was done. Antibody identification and cross-matching with phenotyped red cells were done by the plasma obtained with this method. The best adsorption method was chosen depending on its ability to preserve the alloantibody.
Results A significant difference was found among the three alloadsorption methods regarding number of alloadsorptions and time for complete autoantibodies removal. PEG showed the lowest mean number of alloadsorptions (2.6±1.2) followed by LISS/papain (3.1±1.5) compared with the conventional method (3.7±1.4) (P<0.001). Regarding the time, PEG was the most rapid method (38.6±17.6 min) followed by LISS/papain (45.9±22.8 min) and then the conventional method (110.1±14.4 min) (P<0.001). However, no significant difference was found between PEG and LISS/papain (P=0.014). Alloantibodies were detected in 35% of cases, with predominance of anti-Rh system (61%), mainly anti-c and anti-E. Alloantibodies belonging to Rh and Kidd were best identified in adsorbed plasma by LISS/papain in comparison with PEG and the conventional techniques.
Conclusion Among the evaluated methods, LISS/papain displayed the highest diagnostic accuracy, sensitivity, and specificity of alloantibody detection. Compared with the conventional method, LISS/papain and PEG minimized the time and number of alloadsorptions, enhancing turnaround time and reducing the labor of pretransfusion testing in AIHA.
Keywords: alloadsorptionimmune hemolytic anemia, low ionic strength solution/papain, polyethylene glycol
Throughout the history of hemotherapy, various challenges and concerns have been encountered in its practical application [1]. Pretransfusion testing is necessary to ensure ABO and Rh type compatibility between donor and patient blood, as well as to disclose the presence of red blood cell (RBC) alloantibodies potentially causing transfusion reaction [2].
Autoimmune hemolytic anemia (AIHA) is an uncommon disorder characterized by hemolysis mediated by autoantibodies directed against self-RBC, with the incidence of one to three per 100 000/year and mortality rate of ∼11% [3],[4]. Transfusing AIHA patient is a challenge to the immunohematologist, as it is encountered with difficulties in ABO grouping and cross-matching requiring specialized serological tests such as alloadsorption or autoadsorption. At times, it may be almost impossible to find a fully matched unit to transfuse these patients [5].
In patients with AIHA, more than one-third to one-half of the patients have underlying alloantibody(s) [6]. The problem is that autoantibodies mask underlying alloantibodies, and failure to recognize alloantibody(s) may cause hemolytic transfusion reactions, which may be at times even life-threatening and also limit the availability of subsequent safe transfusion(s) [7].
Adsorption techniques such as auto and alloadsorption using reagents such as polyethylene glycol (PEG) or low ionic strength solution (LISS) are widely applied to detect such alloantibodies, although autoadsorption using patient’s own red cells is cheap and safe, but it is not suitable for use in recently transfused or severely anemic patients [8],[9],[10]. The use of alloadsorption with enhancement additive such as PEG or LISS/papain participates in solving their detection by decreasing the number of steps and processing time, and also minimizes the delay of urgent transfusion to patient with warm AIHA [11],[12],[13]. The aim of this study is to evaluate and compare the efficiency of different autoantibodies adsorption techniques using PEG and LISS/papain-treated/untreated RBCs to facilitate detection and identification of alloantibodies.
Patients and methodsThis study was conducted on 70 patients with warm AIHA who were admitted to Ain Shams University Hospitals from July 2018 to July 2019. An informed consent was obtained from each patient before enrollment. The study was approved by the Ethical Committee of Ain Shams University.
Diagnosis of AIHA was based on combination of clinical and laboratory signs of RBC hemolysis (reticulocytosis, polychromasia, microspherocytes with or without nucleated RBCs in peripheral blood smear, indirect hyperbilirubinemia, and elevated lactate dehydrogenase), together with the detection of autoantibodies and/or complement deposition on RBC as mostly evidenced by a positive direct antiglobulin test or direct Coombs test [14],[15].
Sampling and measurements
Blood samples were collected under complete aseptic conditions on EDTA (1.2 mg/ml) for complete blood picture, peripheral blood smears, reticulocyte count, ABO/Rh grouping, and Coombs test. Serum obtained from clotted samples by centrifugation for 15 min at 1000g was used for biochemical analysis. Further 8–10 ml of blood was collected on citrate tubes for RBC phenotyping and then plasma was separated by centrifugation at 4000g for 5 min for antibody screening and adsorption.
All studied patients were subjected to detailed history and clinical examination, laying stress on pallor, fatigue, jaundice, history of blood transfusion, previous operations, pregnancy/abortion, and presence of organomegaly or lymphadenopathy. Laboratory workup for hemolytic anemia included complete blood picture using Coulter LH 750 (Beckman Coulter Inc., Fullerton, California, USA), peripheral blood smear stained with brilliant cresyl blue to estimate reticulocyte count, and biomarkers of hemolysis (lactate dehydrogenase and bilirubin) using Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany).
Detection of autoantibodies
Patients’ samples were confirmed to contain warm autoantibodies by routine screening procedure using direct Coombs test by gel card method (Diamed system LISS/Coombs) and antibody screening by using screening cells (I+II+III cells) provided by Diagnostic Grifols SA (Parets Del Valles, Barcelona, Spain). Positive reaction of auto control together with the screening panels (pan-positivity) suggested the presence of autoantibodies with or without alloantibodies. Allogenic adsorption followed by rescreening was done to ensure the adsorption of the autoantibodies. Whenever screening test revealed alloantibodies antibody, identification using 11 panel cells (Diagnostic Grifols panels) was done.
Alloadsorption methods
Selection of three blood bags of group O to prepare a set of adsorbing cells with known phenotypes; R1, R1, R2R2 and rr, at least one of the adsorbing cells should be negative for K, E, C, c, Jka, Jkb, S, s, Fya and Fyb. They were freshly prepared with one month expiry and washed to remove any remaining antibodies (anti-A and anti-B).
All 70 patients’ samples were subjected to the three alloadsorption techniques: (a) the conventional adsorption procedure is to mix equal volumes of the patient’s plasma and adsorbing RBC and then incubate at 37°C for 30 min. (b) PEG adsorption method: PEG 20% was prepared by adding 20 g PEG (4000 MW which is locally supplied) in 100 ml of phosphate buffered saline, pH 7.3. Equal volumes of the patient’s plasma, adsorbing RBCs, and PEG solution were mixed and then incubated at 37°C for 15 min. (c) LISS-papain alloadsorption: LISS and papain (Palerm, ref:69015) both are supplied by DIAGAST company (251 Avenue Eugène Avinée, Loos, Nord-Pas-De-Calais, France). Preparation of 1% papain by adding 30 μl of papin to 3 ml of LISS and then added to adsorbing RBCs for 5 min (papinization of RBCs) followed by adding patient’s plasma and incubation at 37°C for 15 min.
Two steps of adsorption were done, and the supernatant of the adsorbed plasma was tested against screening cells to identify the specificity of antibodies that remain after adsorption. When screening cells show no reaction (negative), adsorption was ceased by this method, and the case was defined to have only autoantibody with no underlying alloantibodies with completion of adsorbing steps by the remaining two methods to record their results. On the contrary, whenever one or two of screening cells were positive, it means presence of alloantibody, and the suitable adsorption method was chosen depending on its ability to preserve the alloantibody (with enhanced reactivity). Antibody identification and cross-matching with phenotyped red cells were done by the plasma obtained with this method.
Statistical analysis
Data were analyzed using statistical program for Social Science SPSS (SPSS Inc., Chicago, IL, USA), version 23. Quantitative data were described in the form of number and percentage, mean±SD when the data were parametric and median with interquartile range when the data were nonparametric. Qualitative data were described as frequency and percentage. Student t, Mann–Whitney U, χ2 tests were used for intergroup comparison. Comparison between more than two independent groups with quantitative data was done by one-way analysis of variance when the data were parametric, and Kruskal–Wallis test was done when the data were nonparametric. A P value less than 0.05 was considered significant.
ResultsClinical and laboratory data of studied patients are listed in [Table 1].
Table 1 Clinical and laboratory data of studied patients, as well as comparison between alloantibodies positive and negative autoimmune hemolytic anemia patientsAutoantibodies were detected in all studied patients by Coombs test, whereas alloantibodies were detected in 25 (35%) patients together with the existing autoantibodies. Anti-Rh (mainly anti-C and anti-E) was the predominant alloantibody that was detected in 17 (61%) of studied patients [five (20%) were anti-E, six (24%) were anti-C, three (12%) were anti-e, two (8%) were anti-c, and one (4%) was anti-D). Anti-MNs alloantibodies were detected in five (18%) patients [three (12%) was anti-S and two (8%) was anti-M], anti-Kidd alloantibodies were detected in four (14%) patients [two (8%) were anti-JK-a and two (8%) were anti-JK-b], and the remaining two (7%) patients had anti-Kell alloantibody.
Among the 25 patients with alloantibodies, higher incidence was detected in females [15 (60%)] than in males [10 (40%)], in patients with history of blood transfusion [23 (92%)] than patients without previous transfusion [two (8%)], as well as in patients with secondary AIHA [23 (92%)] mainly in systemic lupus erythromatosus (SLE) eight (32%) than in idiopathic AIHA [two (8%)].
Comparison between patients with and without alloantibodies revealed no significant difference (P>0.05) regarding their age, sex, cause of AIHA, blood group, or phenotype ([Table 1]).
Technical comparison between different adsorption methods regarding number of and time of alloadsorptions
The total number of alloadsorptions and time for complete autoantibody removal were 257 alloadsorptions and 7710 min, respectively, by the conventional method; 214 alloadsorptions and 3210 min, respectively, by LISS-papain method; meanwhile using PEG method required only 180 alloadsorptions and 2700 min, respectively.
As shown in [Table 1], a significant difference was found among the three alloadsorption methods regarding number of alloadsorptions and time for complete autoantibodies removal. PEG showed the lowest mean number of alloadsorptions (2.6±1.2) followed by LISS-papain (3.1±1.5) compared with the conventional method (3.7±1.4) (P<0.001). Similar results were obtained regarding time of alloadsorption, where PEG showed the shortest mean alloadsorption time (38.6±17.6 min), followed by LISS-papain (45.9±22.8 min) and then the conventional method (110.1±43.3 min) (P<0.001). However, no significant difference was found between PEG and LISS/papain regarding time and number of alloadsorptions (P=0.014).
Efficiency of adsorption methods used in detecting alloantibodies
For confirmation of the results, antibody identification was carried out for the adsorbed plasma of the three methods. Samples were considered true positive when the positive test in the antibody screening was associated with the identification of the antibody specificity. On the contrary, samples were considered false positive when the positive results in the antibody screening were not followed by subsequent specificity antibody identification (unspecified).
The present study revealed better alloantibody preservation and identification after complete adsorption of autoantibodies using LISS-papain method, where alloantibodies were detected in 25 samples out of the 70 tested samples in contrast to PEG method in which alloantibodies were detected in 21 samples, whereas four alloantibodies were lost (one anti-C, two anti-JK-a, and one anti-S). Moreover, 23 alloantibodies were detected using the conventional method, whereas two alloantibodies (one anti-C and1 anti-JK-a) were lost ([Table 2]).
Table 2 Result distribution of alloantibodies detection, diagnostic accuracy, sensitivity and specificity, among the three alloadsorption methodsThe accuracy, sensitivity, specificity, and negative predictive value and positive predictive value were calculated for each method and are illustrated in [Table 2]. LISS/papain method showed the highest diagnostic accuracy, sensitivity, negative predictive value, and positive predictive value in detection and identification of alloantibodies after autoantibody adsorption.
DiscussionThe safe transfusion of patients with warm AIHA requires an efficient and time-saving assay to detect alloantibodies underlying autoantibodies [10]. Various adsorption techniques are available to remove serum autoantibodies and subsequently detect the underlying alloantibody in previously transfused patients with AIHA [16]. To establish a rapid, effective, and low-cost alloadsorption technique in the reference laboratory of the central blood bank in Ain Shams University Hospitals, we evaluated different adsorption techniques using PEG, LISS-papain treated, and untreated RBCs for detection and identification of alloantibodies.
In the present study, the overall incidence of red cell alloimmunization constitutes 35%. This was in agreement with previous studies that reported alloimmunization rates ranged from 17.5 to 38%, with an average rate of 32% [15],[17]. Among the 25 patients with alloimmunization, we detected higher incidence in females (60%) than males (40%), in blood transfused patients 23 (92%) than without transfusion (8%), as well as in secondary AIHA (92%) mainly SLE (32%) than idiopathic AIHA. However, no significant difference was found between patients with and without alloantibodies regarding their age, sex, type of AIHA, blood transfusion, blood group, or phenotype. Karafin et al. [18] reported that alloimmunization was more common in females than males, in RhD-negative patients, and in certain diseases (SCD, SLE, and myelodysplastic syndrome).
The alloantibodies’ specificity in this study was mainly anti-Rh (anti-D, anti-C, anti-E, anti-c, and anti-e) in 60% of cases, whereas 18% were anti-MNS antibodies followed by 14% anti-Kidd and 7% anti-Kell alloantibodies. Similar results were reported by Das and Chaudhary [16], as they detected alloantibodies in seven (30.4%) patients with predominance of anti-Rh allo-antibodies. Another study done by El Dewi and Metwally [19] revealed that 23% of patients with warm AIHA had concomitant alloantibodies with 45% anti-Rh, 17% anti-Kell, 15% anti-MNS, 8% anti-Kidd, and 4% anti-Duffy alloantibodies.
Since the assay turnaround time to determine ABO blood type and test for RBC alloantibodies is important in the hospital transfusion service, different methods were used for detection of alloantibodies in the present study: the conventional method, PEG, and LISS/papain. The mean number of alloadsorptions and time necessary for complete autoantibody removal were estimated to choose the suitable method for autoantibody removal and optimum detection of coexisting alloantibodies. According to our results, PEG was the most rapid with less alloadsorption steps followed by LISS/papain method, with no significant difference. The conventional method showed significant longest time and higher number of adsorption steps. Chiaroni et al. [10] had to perform a mean of 2.88 alloadsorptions for autoantibody removal on 50 sera using LISS-papain, which required only a mean of 57.6 min to remove all autoantibodies.
Das and Chaudhary [16] found that the mean number of alloadsorption for complete autoantibody removal using PEG was 1.43, which was lower than the mean number of 3.9 using the LISS-papain method. However, compared with our results, they reported longer mean time for autoantibody removal by PEG and LISS-papain alloadsorption (93.6 and 177.7 min, respectively).
On the contrary, alloantibodies showed a higher reactivity with LISS/papain method in comparison with PEG technique in which alloantibodies were lost in four cases (one anti-C, two anti–JK-a, and one anti-S). This was in contrast to what had been stated that papain increases Rh antigen antibody reaction with subsequent decline in their detection [20]; this can be explained by using Rh antigen-negative adsorbing RBCs, thus preventing the RBCs sensitization with these antibodies. Regarding anti-Kidd and anti-MNS antibodies, they were saved in adsorbed plasma by LISS/papain method more than PEG method, which showed less reactivity. This can be explained by the potentiating effect of LISS and the proteolytic activity of papain enzyme that breaks down MNS antigens on adsorbing RBCs making the corresponding antibodies free in plasma and easily identified. Moreover, PEG may cause enhancing of adsorption of these antibodies and subsequently missed in detection in adsorbed serum, especially if they were in low titer. In 2013, Lambert [21] recorded in his study in the Irish Blood Transfusion Service that addition of LISS to prepapainized RBCs with incubation of 10 min only to alloadsorption process was more efficient and was helpful in detection of anti-C and anti-JK-a. However, in our study, preservation of alloantibody could be owing to addition of papain and LISS to first alloadsorption only with 15 min incubation. Das and Chaudhary [16] also documented the weaker strength of the indirect antiglobulin test reactions with the PEG method than that with the LISS/papain method but both methods could detect underlying alloantibodies. This finding has been documented by other authors and has been attributed to PEG-induced immunoglobulin precipitation, resulting in a weaker strength of reaction [22].Regarding sensitivity and specificity for the three methods, according to our results, LISS/papain was the most sensitive method (100%) compared with 92 and 84% for the conventional and PEG methods, respectively. The specificities by the PEG and the LISS/papain were equal (95.5%) and by the conventional method was 93%.
ConclusionIn conclusion, LISS/papain method successfully adsorbed the autoantibodies and preserved alloantibodies with 100% sensitivity. LISS/papain and PEG method were rapid and less laborious compared with the conventional alloadsoption technique. Regarding pretransfusion testing, always sensitivity is the most crucial element, as it is reflected on the patient’s safety. So, we recommend the usage of LISS/papain as an enhancement media in allogenic adsorption.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
留言 (0)