|Year : 2014 | Volume
| Issue : 3 | Page : 96-100
Prevalence of red cell allo-antibodies among muti-transfused autoimmune hemolytic anemia Egyptian patients
Ibrahim Youssef Abdel-Messih, Hanan Mohamed Mahmoud, Deena M. M. Habashy
Department of Clinical Pathology, Ain Shams University, Cairo, Egypt
|Date of Web Publication||30-Sep-2014|
Hanan Mohamed Mahmoud
Department of Clinical Pathology, Ain Shams University, Eldemerdash Hospital, Ramsis Street, Cairo
Source of Support: None, Conflict of Interest: None
Background: The aim is to detect the prevalence and specificity of red cell allo-antibodies underlying pan reactive auto-antibodies in Egyptian AIHA patients Patients and Methods: Sixty multi-transfused Egyptian AIHA patients received 715 units of blood were studied during the period from February 2013 to November 2013. Initial antibody screening and identification (using pre-warming technique in cases of cold auto-antibodies), direct antiglobulin, auto-control and immediate spin, allogenic adsorption for patients` plasma then detection of possible underlying allo-antibodies were done. Results: Five patients with cold auto-antibodies were identified with absence of underlying allo-antibodies. From the 55 patients with warm auto-antibodies, 23 (41.8%) had allo-antibodies with a total number of 40 underlying allo-antibodies indicating that Egyptians carry the highest percentage of allo-antibodies compared to other studies. Conclusion: Red cell allo-immunization has a very high frequency among multi-transfused Egyptian AIHA patients.
Keywords: Autoimmune hemolytic anemia, multi-transfusion, red cell alloimmunization
|How to cite this article:|
Abdel-Messih IY, Mahmoud HM, Habashy DM. Prevalence of red cell allo-antibodies among muti-transfused autoimmune hemolytic anemia Egyptian patients. J Appl Hematol 2014;5:96-100
|How to cite this URL:|
Abdel-Messih IY, Mahmoud HM, Habashy DM. Prevalence of red cell allo-antibodies among muti-transfused autoimmune hemolytic anemia Egyptian patients. J Appl Hematol [serial online] 2014 [cited 2019 Jun 16];5:96-100. Available from: http://www.jahjournal.org/text.asp?2014/5/3/96/141996
| Introduction|| |
The diagnosis of autoimmune hemolytic anemia (AIHA) is a challenge for both the immunohematology laboratory and the clinician as the laboratory investigation can be troublesome and often requires extensive time-consuming serological testing. 
When autoimmune hemolytic anemia patients need a blood transfusion, detection of allo-antibodies can be difficult because auto-antibodies can mask the presence of clinically significant allo-antibodies. 
The presence of undetected allo-antibodies may be the cause of increased hemolysis following transfusion, which may be falsely attributed to an increase in the severity of autoimmune hemolytic anemia. 
About 32% of patients with auto-antibodies to red blood cells (RBC) are associated with allo-antibodies.  Patients exposed to foreign red cells by prior transfusion or pregnancy may produce IgG antibodies to antigens of certain systems, primarily Rh (C, c, D, E, e), Kell, Duffy, Kidd, and Ss, etc., but many less common possibilities exist. These red cell antibodies are clinically significant as they can react at 37°C causing hemolysis. 
This study was aimed to detect the prevalence and specificity of red cell allo-antibodies underlying pan reactive auto-antibodies in multi-transfused Egyptian AIHA patients.
| Patients and methods|| |
Patients and Controls
Blood samples were available from 60 multi-transfused Egyptian patients with AIHA referred to the National Blood Transfusion Center (NBTC) to receive blood transfusion (26 males and 34 females) aged between 30 and 65 years (mean: 49.3 ± 10.1 years) and from 20 age- and sex-matched healthy blood donors control subjects during the period from February 2013 to November 2013.
Thirty-four patients had idiopathic AIHA and 26 had secondary AIHA to other diseases including other systemic autoimmune disease (such as systemic lupus erythematosus and rheumatoid arthritis) or malignancy (lymphoma and chronic lymphocytic leukemia). None of the patients has been immunized before the blood transfusion, patients with allo-antibodies detected before the first transfusion were not eligible for this study. History of blood transfusion was obtained from the patients' records; all patients had transfusion more than once. The study design was approved by the Research Ethics Committee at Ain Shams University, Faculty of Medicine and conducted according to the Helsinki declaration of 1975. All samples were withdrawn after signing a written consent by the patient and the healthy donor groups.
A volume of 3 ml of peripheral blood was drawn into ethylene diamine tetra acetic acid (EDTA) anticoagulant tube. Samples were then centrifuged at 3,500 g for 3 min to obtain plasma for reverse ABO grouping, antibody screening and identification, auto control, immediate spin and prewarming technique. In cases with detected warm auto-antibodies, another 7 ml peripheral blood was drawn into EDTA anticoagulant tube.
Blood grouping (ABO/Rh) was performed, according to standard serologic method using column agglutination technique (Ortho-Clinical Diagnostics, Inc., Rochester, NY ABO-Rh/reverse grouping cassettes) following manufacturer's instructions.
Antibody identification techniques
Antibody screening test (indirect antiglobulin (AHg) test) was performed according to standard serologic methods using antihuman globulin, anti-IgG,-C3d poly-specific-ortho-BioVue system poly cassettes, , results were interpreted according to earlier research. 
Antibody identification antibody identification panels (reagent RBC-resolve® panel A, and reagent RBC-resolve® panel b-ortho-clinical diagnostics), each formed of 11 vials of different phenotype RBC, were used and performed by the same technique used in the screening test (column agglutination technique). These panels fulfilled the specifications regulated by the British Guidelines,  direct antiglobulin test (DAT) was performed if cells are positive and its positivity confirms the presence of auto-antibodies.
Autocontrol/direct antiglobulin test
These were performed according to standard serologic methods using column agglutination technique for direct and indirect anti-globulin test using anti-human globulin, anti-IgG,-C3d; poly-specific as well as mono-specific ortho-BioVue system cassettes. Test results were expressed as either positive or negative based on the presence or absence of reactivity. Positive auto-control and DAT indicate presence of auto-antibodies.
A volume of 100 μl of patients' plasma was added to 50 μl of reagent red cell suspension of antibody screening cells being tested; tubes were mixed and centrifuged for 1 min at 1000 rpm. The red cells were gently re-suspended and examined for agglutination that indicated the presence of cold reacting antibody as testing was performed at room temperature.
Adding 50 μL of 3% reagent red cell suspension to tubes one containing red cells and the other containing a small volume of the patient's plasma placed in the water bath at 37°C then transfer 100 μL of prewarmed plasma to each tube and mix. Incubate at 37°C for 45 min. Fill each tube with prewarmed saline. Centrifuge and wash three times with 37°C saline. Add 100 μL AHg to each tube and mix. After centrifugation for 1 min at 1000 rpm we observe for reaction. Grade and record the results. 
Allo-adsorption was done using three red cell specimens expressing: R1R1, R2R2 and rr red cells phenotypes, and lacking the antigens: K, Fya, Fyb, JKa, JKb, Lea, Leb, M, N and S, these red cell specimens were washed separately with saline, serial adsorptions were performed by mixing 1 ml of the patients' plasma to 1 ml of each of the three washed packed cells in three separate test tubes, incubating them at 37°C for 45 min, then adding the harvested three supernatant plasma produced from centrifugation, to a new set of the washed three red cell specimens and continue as previously described. The three samples of adsorbed plasma were tested individually against antibody detection panels. Antibody identification was performed as mentioned before. Positive antibody screening results using adsorbed plasma indicated that the patient had allo-antibodies in addition to auto-antibodies that were masked by the pan-agglutination reactivity of the auto-antibodies; in this case identification of the co-existing allo-antibodies was done through antibody identification test.
Data were collected, verified, revised, edited on PC and then analyzed using SPSS V17 statistical software package, SPSS Inc., Chicago, USA under Windows Vista operating system Microsoft Corporation, USA, using Student's t-test, Chi-squared test or fisher exact test. P < 0.05 was used as the level of significance.
| Results|| |
All patients were positive for DAT and auto-control. Healthy blood donors exhibited a negative DAT. Allo-antibodies against RBC Ag were not detectable in their plasma. Cold auto-antibodies were detected in 5/60 (8.3%) patients with absence of underlying allo-antibodies as indicated by their negative antibody screening using prewarming technique, patients with warm auto-antibodies were 55/60 (91.7%), 23/55 (41.8%) of which were positive for allo-adsorption. With a total number of 40 allo-antibodies in the 23 patients, anti-E allo-antibody was found to be of the highest incidence being present in 9/40 (22.5%) of antibodies, followed by anti-K antibody being detected in 6/40 (15%) of antibodies, anti-S 5/40 (12.5%) and anti-Jk a 4/40 (10%) of detected antibodies. Anti-D, anti-C and anti-C followed each by an incidence of 7.5% (3/40) of antibodies, which is found in female patients who were known to have been pregnant previously. Anti-M was detected in 2/40 (5%) of antibodies. The least prevalent allo-antibodies were anti-e, anti-Fy a , anti-Fy b , anti-Jk b and anti-Le a being detected in just one case each (2.5%) [Table 1] 11 (47.8%) were positive only for one allo-antibody, 7 (30.4%) of patients had 2 different allo-antibodies and 5 (21.8%) patients were positive for 3 allo-antibodies [Table 2].
|Table 1: Specificity and prevalence of alloantibodies in alloimmunized studied patients|
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Association of allo-immunization with Clinical and Laboratory Data
Patients received a total of 715 units with a mean of 13 units, a statistically high significant association was revealed between the development of allo-immunization and the number of transfused units specially those who received nonleucodepleted blood and in patients who underwent splenectomy (P = 0.007, 0.006, 0.046 respectively).
| Discussion|| |
It has been previously described that transfusion therapy in patients with classical AIHA is not associated with a high risk of alloimmunization or with a tendency to aggravate the hemolysis. 
It is well-known that autoimmunity is frequently associated with allo-antibodies,  not only could we confirmed this relationship, but we demonstrated that Egyptians carry the highest percentage of allo-antibodies compared to other studies ,,,,,, [Table 3].
In this study, none of the five patients with cold auto-immune antibodies were found to have allo-antibodies.
Eldeeb et al.  found that the patients who had an older age had a higher alloimmunization rate, while transfusion at an early age (<3.5 years old) may offer some immune tolerance and protection against alloimmunization in chronically transfused patients, and alloimmunization will be a less significant problem. Another factor that could possibly contribute to the higher prevalence of alloimmunization in AIHA is that the dysfunctioning immune system, either hyper or hyposensitive can result in an enhanced or reduced antibody production respectively. 
Regarding the prevalence of allo-antibodies detected in the present study, with the majority of detected antibodies against antigens of the Rh-Kell system, anti-E (22.5%) and anti-Kell (15%) antibodies were found to be the most frequent. The finding that these two antibodies account for the predominance of cases of alloimmunization agrees with the data reported by many workers. , On the other hand, a study conducted in United Kingdom  did not encounter anti-Kell among the most frequent antibodies. This may be explained by the antigenic discrepancy between different populations with different ethnic and genetic backgrounds.
To evaluate the repeated transfusion as a risk of allo-antibody development in this study the number of units of blood transfused, and the transfusion episodes were significantly associated with the RBC alloimmunization rate.  On the other hand, another study  observed a tendency to form antibodies in the first transfusion episode and that the frequently transfused patients were not necessarily the ones that formed antibodies.
The results of studying the role of leukodepletion in red cell alloimmunization showed a significantly lower rate of allo-antibody formation in patients who received leukodepleted blood transfusions as reported before.  In spite that Blumberg et al.  suggested that the non leuco-filtered RBCs often contain large number of platelets which are rich in CD40 L, and this co-stimulatory molecule is well-known to activate B cells and is crucial for class switching from IgM to IgG during the immune response to antigen, and that leukodepletion of allogenic RBC transfusion reduces stimulus associated with such transfusions as an explanation, but still this suggestion needs further studies to confirm it.
Another contributing factor revealed by this study is a significant association between alloimmunization and splenectomy. Patients who had a splenectomy had a higher alloimmunization rate than those who didn't, as absence of spleen may further enhance the immune response to the infused foreign antigens, which are not effectively filtered. 
In conclusion, red cell alloimmunization is highly frequent among multi-transfused Egyptian AIHA patients and is significantly associated with the frequency of blood transfusion, use of nonleucodepleted blood and in patients who underwent splenectomy. As alloimmunization is shown to be a risk in multi-transfused patients, we advise undertaking extended cross-match when such patients get their first ever transfusion
| Acknowledgment|| |
The technical and material support provided by the NBTC for accomplishing this work is greatly appreciated.
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[Table 1], [Table 2], [Table 3]