Difficulties of immunohematology tests in the case of hematological malignancies

¹Hematology Laboratory, Central Laboratory, Mohammed VI University Hospital, Oujda, Morocco, ²Faculty of Medicine and Pharmacy, Mohammed First University, Oujda, Morocco

^&Corresponding author
Abir Yahyaoui, Hematology Laboratory, Central Laboratory, Mohammed VI University Hospital, Oujda, Morocco

Introduction    

The study of the ABO blood group in transfusion procedures and medical practice has demonstrated the vital role of the ABO system in the immunological safety of transplants, blood transfusions, and the management of fetomaternal incompatibility [1]. The majority of hematological adverse events, which can in some cases be fatal, are linked to genetic variations in ABO antigens between individuals [2]. The distribution of these epitopes is ubiquitous, and their expression is genetically induced and controlled by different regulatory mechanisms, with genetic polymorphism and interference with different pathologies [3,4]. Nevertheless, this expression may be altered or modified in certain pathological profiles, notably in hematological malignancies [5]. Numerous studies have shown that ABO system antigens are susceptible to changes in expression, as is the occurrence of autoimmunization or alloimmunization [6]. Hematological malignancies are recognized as all neoplasia and malignant proliferations developed from hematopoietic cells of the bone marrow and lymphatic system [7]. Over the last decade, the incidence of these pathologies has been rising steadily, and they are responsible for various complications of varying degrees of severity. Indeed, numerous observations have reported the alteration of ABO antigens in subjects with myeloproliferative, myelodysplastic, or lymphoproliferative syndromes, with the appearance of erythrocyte alloimmunization or autoimmunization [8]. To this end, blood transfusion in these patients can be dangerous because of the possible variation in their ABO blood group or the appearance of autoantibodies or alloantibodies during the tumor process [9]. However, few studies have been published on this issue. The aim of our study was therefore to identify the difficulties of immunohematology tests that can be disrupted in the context of hematological malignancies, in particular ABO blood grouping, irregular agglutinin testing (IAT), and direct Coombs test (DCT), and to establish a course of action to validate these results, especially in the transfusion context.

Methods     

Study design and population: this is a retrospective descriptive study conducted in the hematology department of the central laboratory, over 19 months from January 2022 to September 2023.

Study population and inclusion criteria: we included in our study all patients followed for a hematological malignancy who had difficulties during immunohematology tests performed in the hematology department. Whole blood samples, collected in a tube with EDTA (Ethylene Diamine Tetra-Acetic Acid) anticoagulant, were centrifuged at 2,500 rpm for 3 minutes, and the globular pellet was separated from the serum. ABO Rhesus blood grouping is based on the principle of hemagglutination, a gel filtration technique based on antigen-antibody interaction, performed on a gel card, which included the two simultaneous tests, globular and serum, which are compulsory and complementary. The irregular agglutinin test (IAT) and the direct Coombs test (DCT) are based on the same principle, on a Coombs (anti-IgG+C3d) and IAT gel card respectively.

Data collection and study variables: data were collected from the immunohematology laboratory database. The information recorded included demographic data, age, gender, underlying pathology, patient follow-up, disease progression, previously known ABO blood group, the notion of recent transfusion less than 3 months old, and immunohematology test difficulty observed. The results of immunohematology tests were recorded in the department's database. To reduce the risk of bias associated with data collection, this task was performed by the authors.

Statistical analyses: all analyses were carried out using IBM SPSS (Statistical Package for the Social Sciences) version 23.0, used to evaluate all results once they were entered into Microsoft Excel Professional 2019 with the calculation of averages and percentages of all results. Frequency and percentage were used to describe categorical variables, whereas mean, interquartile range, and median were used to express continuous variables.

Ethical considerations: the study was conducted with the approval of the department heads. Data collection was carried out for the anonymity of the patients and the confidentiality of their information, in accordance with ethical considerations.

Results     

Hematological malignancies are becoming increasingly common in our hospital practice. During the study period from January 2022 to September 2023, 4370 blood groupings were performed, resulting in 2.2% of immunohematology test difficulties (2.2% of 4370 is 96 patients), including 14.4% of patients with hematological malignancies (14.4% of 96 is 14 patients) (Table 1). They ranged in age from 30 to 75 years. Females predominated with 8 cases, compared with 6 male patients. The acute myeloid leukemia (AML) was found in the majority of patients, 3 cases of lymphoma with diffuse large-cell B lymphoma (DLBCL) and Hodgkin's lymphomas (HL), 2 cases of multiple myeloma (MM), 1 case of plasma cell leukemia (PCL) and 2 cases of myelodysplastic syndrome (MDS). The search for grouping difficulties in this population, summarized in Table 2, provided 5 cases with reaction defects in globular test induced by an anti-B agglutination deficiency during blood group determination, in 3 patients with diffuse large-cell B lymphoma and Hodgkin's lymphoma and 2 patients with AML (Figure 1 A); 4 cases of mixed-field reaction in the globular test during blood group determination with double population image using anti-A, reported in patients with AML (fFigure 1 B); 4 cases of reaction defect in the plasma test, during blood group determination with an excess reaction, reported in patients with multiple myeloma, plasma cell leukemia and MDS (Figure 1 C) and 1 case of polyagglutinability phenomenon observed in the plasma testing during blood group determination with a positive AB control and a positive direct Coombs test (Figure 1 D) group A.

Rhesus-positive was predominant in 9 patients, group O Rhesus-positive in 4, and group B Rhesus-positive in 1. The direct Coombs test was carried out in 10 patients, returning positive in 6 cases for IgG and c3d complement (Figure 1 E), and the irregular agglutinin testing with the panel of three red cell tests (red blood cells I, II, and III, returned positive in 7 patients (Figure 1 F), revealing the presence of irregular anti-erythrocyte antibodies, with the exception of antibodies directed against A and B antigens. However, the globular test reaction inconsistencies observed in our patients reflect the weakened expression of A and B antigens in the context of hematological malignancies, initially observed in acute leukemia. The changes often correspond to a decrease in A1, A, B or H antigen expression, or even to their total extinction. These changes affect only part of the red blood cells, and a mixed-field reaction is then observed during blood grouping. For this, it is necessary to look for substance H on the red cell surface with anti-H, to look for weak antigen A or B by fixation-elution, and to look for soluble ABH substances in the saliva of secretors. In the case of the mixed-field reaction observed in hematological malignancies, a provisional card with transfusion advice should be issued. Incoherence due to excess reaction to the globular test may be caused by a roller phenomenon, due to the high concentration of abnormal proteins that disrupt the test, as in dysglobulinemia in multiple myeloma, Waldenström's disease or hyperfibrinogenemia. Cold agglutinins are characterized by in vivo sensitization of red blood cells by cold autoantibodies secondary to hematological malignancy. In this case, the red blood cells need to be washed several times in saline at 37°C, and the plasma test requires diluting the plasma by one-half to one-third.

Discussion     

Hematological malignancies can influence blood group antigens, and consequently immunohematology test results. This is why clinical information is essential when validating results, and in the event of reactional ambiguity, more specific tests are used for validation. Observations in Europe and America report a weakening or even loss of certain ABO system antigens responsible for a change in the subject's blood group. The decrease or absence of antigen expression is well-known in acute leukemia, and has been demonstrated in malignant solid tumor cells, which have lost normal expression of blood group antigens; these findings have been associated with more aggressive neoplastic behavior [10]. In our patients with hematological malignancies, the reactional inconsistencies observed in the globular test are due to weakened expression of A and/or B antigens. These changes often correspond to a decrease in A1, A, B, or H antigen expression, without the antibody corresponding to the missing antigen being detected in the patient's plasma. They may be somatic, as the theoretical ABO group observed following these changes is sometimes incompatible with the family's blood group as a whole. Sometimes, they may be acquired, since the group was normal before the onset of leukemia. These changes are not due to chemotherapy either, since they are observed even before the start of treatment [11].

The antigenic deficiency observed corresponds to a deficiency of the corresponding enzyme and not to a deficiency of the precursors. When the changes affect only part of the red blood cells, a mixed-field reaction is observed. Such changes have also been observed in other hemopoietic malignancies, in particular myeloproliferative disorders. Although many arguments suggest a genetic anomaly, their actual significance and the role of these modifications in the malignant process are debatable. However, the alteration of ABH antigens during hematological malignancies is a recurrent, if infrequently reported, finding. A patient with acute myeloid leukemia, who had previously shown normal expression of A antigen, had very low expression of the antigen on his red blood cells, as first reported by Van Loghem et al. Hematological cancers have also been linked to the loss of A, B, or H antigens on the surface of red blood cells [6]. Loss of A, B or H antigens on the surface of red blood cells has also been described in the context of hematological malignancies. In healthy A, B or AB individuals, complete agglutination of red blood cells is observed after incubation with antibodies reactive against their blood group. In patients who have lost ABH antigens, a variable proportion of red blood cells fail to agglutinate, giving a characteristic mixed-field reaction. Mixed-field reactions can also occur in healthy individuals when reactions are associated with rare alleles of the ABO gene such as A3 and B3. The team of Tina et al. analyzed 57 blood samples from patients diagnosed with different types of myeloid malignancies, 38 of whom had AML, 6 subjects with myeloproliferative disorders, and 13 patients with myelodysplastic syndromes [6].

Blood samples from two AML patients were used in the investigation by Rakul et al. The results of cross-matching indicated differences in the blood group, and reverse cross-matching only revealed the presence of anti-A antibodies [12]. The patient's red blood cells were treated with anti-B sera for elution and adsorption experiments, after which the anti-B adsorbed on his cells was eluted. The presence of B antigen on the patient's cells was subsequently confirmed when the elution was tested using group B and group O red blood cells to detect the presence of anti-B antibodies. Group B red blood cells showed a positive reaction, while group O red blood cells showed a negative reaction. Throughout treatment, the patient's red blood cells' responses to anti-B antibodies were stronger. She regained her original blood type at the end of the second consolidation, when the reaction with anti-B antibodies was significant. As a result, during the leukemic phase, the original B blood group antigen was changed, and during remission, the B antigens were expressed again. In a similar vein, in the second instance, the B antigens were expressed after remission after being inhibited throughout the leukemic period [12]. Alterations in the ABO blood group can also develop during multiple myeloma. An instance of a blood group switching from AB positive to O positive has been reported [13].

The weakened state of ABO antigens in hematopoietic disorders can be attributed to two potential processes. A/B transferases' inactivation is the first process, while H-transferase's inactivation is the second [5]. Patients with hematological malignancies are thought to have a variable erythrocyte population that comes from a malignant tumor cell clone [12]. Combining immunophenotyping and cytogenetic analysis has demonstrated that a significant percentage of AML patients contain erythroid lineage cells that are derived from malignant stem cells, which is consistent with this idea [14]. Patients with lymphoid malignancies have occasionally been observed to have lost ABH antigens; this is likely due to pluripotent cells that have a primarily lymphoid character [6]. The preferred method for identifying quantitative and qualitative changes in A, B, and H antigens in patients with hematological malignancies is flow cytometric analysis [15]. The use of this technique in ABO phenotyping has yielded very satisfactory and reliable results [16]. Several studies have confirmed its effectiveness in determining the frequency of change of ABH antigens in hematological malignancies, making it possible to determine the total or partial loss of these antigens and correctly identify the cellular phenotype [17].

For the majority of MDS patients, transfusion assistance continues to be the mainstay of care in myelodysplastic syndromes. Red cell transfusions are necessary for up to 90% of patients during their illness, and 30-45% of patients develop a dependency on them [18]. Patients with MDS are more susceptible to transfusion-related complications, which can result in considerable morbidity and mortality. These consequences include iron overload and immediate or delayed hemolysis. Anti-erythrocyte autoantibodies may also develop as a result of alloimmunization [19]. Approximately 28% of patients with autoantibodies also exhibited alloantibodies, most of which were produced by transfusion and detected concurrently [20]. However, as alloimmunization develops, some MDS patients need further red cell transfusions. Data on alloimmunization in these patients receiving continuous transfusions, despite the high prevalence of MDS, primarily represent the experience of single sites with small patient populations and brief follow-up periods [8]. Regarding the limitations of our study, the small number of patients may limit the applicability of our results to a larger population; resource limits and the potential for more specialized examinations may have restricted the scope for additional specialized investigations.

Conclusion     

Determining the difficulties and particularities of ABO Rhesus grouping, as well as direct Coombs tests and irregular agglutinin testing in patients being followed for hematological malignancies, who are particularly susceptible and vulnerable to various complications, will allow these tests to be better managed and particular consideration given when performing them, taking into account the particularities and possible changes in this population. Larger-scale studies with greater sampling and more extensive investigations are needed to better understand the difficulties of immunohematology testing during hematological malignancies, and the possible changes in blood group that require constant research. It must therefore be correctly performed and monitored throughout the course of the malignant disease, to avoid fatal transfusion reactions.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

All authors have read and approved the final version of this manuscript.

Acknowledgments     

We would like to thank all the staff of the biological hematology department of our laboratory for their valuable contributions.

Tables and figure     

Table 1: the diverse diagnoses of hematological malignancies that were identified in the population under study

Table 2: the difficulties of immunohematology tests observed in our patients, divided according to their hematological malignancy

Figure 1: the various difficulties encountered when carrying out immunohematology tests in the population studied; (A) globular test reaction defect induced by an anti-B agglutination deficiency during blood group determination; (B) mixed field reaction in the globular test during blood group determination with double population image using anti-A; (C) reaction defect in the globular test, during blood group determination with an excess reaction with anti-B; (D) polyagglutinability phenomenon observed in the serum testing during blood group determination. (E) Positive direct Coombs test (DCT) for IgG and c3d complement; (F) positive irregular agglutinin test (IAT) with the panel of three red cell tests (red blood cells I, II and III

References