Hematological and immunological manifestations in systemic lupus erythematosus Tunisian patients

¹Laboratory of Hematology, Military Hospital, Tunis, Tunisia, ²Department of Internal Medicine, Military Hospital, Tunis, Tunisia, ³Laboratory of Immunology, Military Hospital, Tunis, Tunisia, ⁴Molecular Biology Unit (UR17DN06), Laboratory of Hematology, Military Hospital, Tunis, Tunisia

^&Corresponding author
Najiba Fekih-Mrissa, Molecular Biology Unit (UR17DN06), Laboratory of Hematology, Military Hospital, Tunis, Tunisia

Introduction    

Systemic lupus erythematosus (SLE) is an autoimmune, chronic, multi-systemic disease that is characterized by various clinical manifestations. Hematological manifestations in SLE are commonplace and diverse. The principal indications are anemia, leukopenia, thrombocytopenia (also known as thrombopenia), antiphospholipid syndrome (APS), circulating immune complexes, elevated autoantibody production, and significant tissue damage [1]. These symptoms may be brought on by iatrogenic injury, another comorbid condition, or the disease itself. Different populations have differing rates of them [2]. Both the 1997 update of the 1982 American College of Rheumatology (ACR) [3], and the 2012 Systemic Lupus International Collaborating Clinics (SLICC) [4], consider these impairments. The current objective here is to evaluate the more commonly occurring characteristics and patterns of hematological and immunological manifestations among SLE Tunisian patients, as studies are scarce regarding this cohort.

Methods     

Study design: a descriptive, retrospective, cross-sectional, quantitative, and qualitative study of 99 adult SLE patients was used in this investigation.

Setting: this research was conducted in 2020 at the Department of Internal Medicine of the Military Hospital of Tunisia. The study was conducted during 19 years from 2000 to 2019. Data were collected from the case notes of patients.

Participants: a total of 99 consenting SLE patients (77 women, 22 men) were included in the study. Diagnoses were established according to the criteria of the American College of Rheumatology (ACR) [3].

Inclusion criteria: all medical records of patients who were diagnosed with SLE based on the American College of Rheumatology's (ACR) 1997 criteria, as well as those of patients who presented with one or more symptoms, signs, and/or pathologies that would have led to a lupus diagnosis based on the same criteria.

Exclusion criteria: incomplete files or files that did not include the ACR criteria necessary for an SLE diagnosis were excluded. Patients presenting a set of signs and symptoms satisfying criteria for several systemic diseases were also excluded.

Variables: a study questionnaire (socio-demographic and clinical data) designed for this study was administered to each patient. All patients underwent standard laboratory testing at diagnosis that adhered to our laboratory protocols. Leucopenia was defined as a white blood cell (WBC) count of less than 4x10⁹/L, lymphopenia as a lymphocyte count of less than 1.0x10⁹/L, and anemia as a hemoglobin level of less than 12.0 g/dL. Platelet counts below 100x10⁹/L were considered to be indicative of thrombocytopenia in lupus patients. Patients were considered to suffer hematological comorbidities with lupus only if they presented with at least one hematological manifestation that was linked to disease activity at the time of admission, and that they additionally met the following criteria: thrombocytopenia (platelets less than100x10⁹/L), hemolytic anemia (hemoglobin of less than 12 g/dL with the presence of elevated reticulocytes), leucopenia (leukocytes less than 4x10⁹/L), and/or neutropenia (neutrophils less than 1x10⁹/L) [5]. Immunological variables included in the analysis, which were all established either before or during hospitalization, include antinuclear antibodies (AAN, indirect immunofluorescence), anti-dsDNA antibodies (anti-dsDNA) (indirect immunofluorescence on Crithidia luciliae), anti-SSA, anti-SSB, anti-Sm, anti-RNP, anti-SCL70, anti-JO1, anti-centromeres (line immunoblot assay), serum complement C3 and C4 (nephelometry), anti-cardiolipin antibodies (aCL), anti-β₂ glycoprotein 1 (ELISA), lupus anticoagulant (chronometric method), anti-CCP (ELISA), and ANCA (indirect immunofluorescence on ethanol/formol-fixed neutrophils).

Statistical analysis: descriptive statistics included both qualitative and quantitative variables expressed in percentages and standard deviations. The Statistical Package for the Social Sciences v.16 (IBM, [Armonk, NY, USA]) was used for all association analyses between immunological and hematological manifestations. The Chi-square test for qualitative variables was used to assess differences. Probability values that were deemed statistically significant were for those at p<0.05. Furthermore, the 95% confidence intervals (CI) and odds ratio (OR) were computed.

Ethical consideration: the local Ethics Committee gave its approval to the study. The research complies with the Declaration of Helsinki's tenets. Additionally, anonymity and confidentiality were preserved.

Results     

Descriptive data of the study participants: the study included 99 SLE patients in total (77 women and 22 men). Biographical data at the initiation of the study recorded an average age of 38.46 ± 13.98 years and an average age at onset of 35.3 years ± 14.1 (Table 1). Eighty-six of 99 SLE patients (86.9%) presented with hematological pathologies.

Hematological manifestations: hematological abnormalities were documented in 86.9% of patients. The cell line most frequently affected were lymphocytes (lymphopenia 47 cases 47.5%), followed by leucocytes (leucopenia 41 (41.4%) cases), erythrocytes (hemolytic anemia 20 (20.2%) cases), and platelets (thrombocytopenia14 (14.1%) cases) (Table 2). The mean values of leukocytes, hemoglobin, and thrombocytes upon admission were: 5.2x10⁹/L (1.1-14.5x10⁹/L),10.3 g/L (4.2-16.1 g/dL), and 215.3x10⁹/L (5-570x10⁹/L) respectively (Table 1). There were 41 cases (41.4%) in which more than one cell line was involved Twelve cases (12.1%) were diagnosed with Evans syndrome and three cases (3.03%) with pancytopenia.

Positive immunological tests: Table 3 provides a summary of the immunological test results. The vast majority of patients (95%) had positive ANA, followed by those with anti-dsDNA antibodies (64.3%). Unexpectedly frequently, anti-Sm antibodies, anti-cardiolipin, and anti-RNP were positive in 33.4%,16.3%, and 13.5% of cases respectively. Anti-SSB antibodies were positive in 7.1% of patients and were seen more commonly than lupus anticoagulant (1%). Anti-SCL70 antibodies were seen in 4.1% of cases while both anti-centromeres and anti-CCP were present in 2.1%. Anti-JO1, anti-β₂ glycoprotein1, and ANCA were observed in 1%. Low levels of complement C3 and C4 were observed in 42.8% of subjects.

Association between hematological abnormalities and immunological laboratory analysis: the associations between different hematological abnormalities and various immunology laboratory analyses in SLE patients are given in Table 4. Table 5 lists the associations that the univariate analysis determined to be significant (p<0.05). Patients with thrombopenia presented with low C4, although it did not rise to statistical significance (p=0.8), while the presence of anti-dsDNA autoantibodies was statistically associated with lymphopenia.

Discussion     

In our present study, the hematological and immunological abnormalities in 99 SLE research subjects residing in Tunisia are reported. It should be noted that, while this study was conducted at one hospital (a monocentric study), the cases represent the wider Tunisian population as this location serves the country´s military and family members from all regions. Among the white cell abnormalities, lymphopenia was the most commonly occurring (47.5%), followed by leukopenia (41.4%). Thrombocytopenia, a comparatively less common occurrence in SLE, was present in 14.1% of our patients. In the present investigation, hematological abnormalities were found in 86.9% of cases. While this result is consistent with many other studies, it is significantly higher than the percentage found in the European study (12.8%) (Table 6) [6].

Leukopenia: leukopenia is defined as a WBC of less than 4x10⁹/L on two or more occasions following the ACR and SLICC criteria for classifying SLE [4,7]. The actual pathogenic mechanism of the disease, along with several additional elements (e.g. immunosuppressive medications), may be a factor in the low white blood cell count in some patients. Although 50-60% of SLE patients have leucopenia, only 17% have a WBC below 1x10⁹/L [8]. In our present study, we found leucopenia in 41.4% of our patients. This result is similar to studies conducted in Tunisia (2002), Egypt, and China (Table 6) [9-11].

Lymphopenia: lymphopenia is defined as a lymphocyte count below 1.5x10⁹/L on at least two occasions, according to the 1997 revision of the ACR criteria [12]. Similar to the SLE classification criteria, lymphopenia (less than 1x10⁹/L at least once) is also included in the SLICC classification criteria [4]. Lymphoma is now acknowledged as one of the most prevalent hematological findings in SLE [13]. The prevalence of low lymphocyte counts in SLE ranges from 20 to 93% [14], and they are often seen in patients who have active or severe disease [15]. Furthermore, regardless of treatment, lymphocyte levels may change throughout the disease [14]. Immunosuppressive medications and glucocorticoids may confound results and cause lymphopenia in severe illness. Although it happens separately from neutropenia, lymphopenia may also be a factor in the leucopenia these patients experience.

Lymphopenia represents the most commonly occurring hematological abnormality in our patients (47.5%). This is in concordance with most studies (Table 6). The main cause of variations in the frequencies of hematological abnormalities, across studies, is typically due to the differences in the frequencies of lymphopenia and leucopenia [1,16,17]. The pathogenesis of lymphopenia in SLE remains largely unclear. However, titers of IgG antibodies to lymphocytes have been demonstrated to correlate inversely with complement and lymphocyte levels. This suggests that autoantibodies to lymphocytes are a heterogeneous group and may contribute to the pathogenesis [18].

Another study of SLE patients found that IgG anti-lymphocyte antibodies were independently linked to lymphopenia with sensitivity (42.3%) and specificity (96.7%) [19]. The decrease in both the quantity and function of lymphocytes has long been attributed to anti-lymphocyte antibodies. Rivero et al. studied 158 SLE patients with active disease, examined from the time of diagnosis, and found that 75% suffered from lymphopenia. Later, another 18% experienced lymphopenia following the reactivation of the disease [14]. According to some authors, there is a correlation between disease activity and lymphopenia. Mirzayan et al., in their prospective study to characterize prognostic parameters for SLE disease, concluded that lymphopenia may be a predictor of flares [20]. Clinically silent lymphopenia can also be linked to active SLE and/or an elevated risk of infection. The use of immunosuppressive treatments complicates and confounds the data regarding the elevated risk of infection.

Neutropenia: the standard definition of neutropenia is an absolute neutrophil count below 1x10⁹/L. It is unclear the etiology of neutropenia in SLE. There may be a role for both humoral and cellular immune mechanisms. Increased peripheral granulocyte destruction, increased margination and/or increased splenic pool shifting, and decreased marrow production are three possible explanations for neutropenia in SLE [21]. Both primary and secondary neutropenia can occur in autoimmune diseases. The precise target of the autoantibodies is unknown, and it frequently happens that these cases coexist with hemolytic anemia or thrombocytopenia [22].

Anemia: anemia is characterized by a hemoglobin level below 12g/dL in women and 13.5 g/dL in men [23]. Throughout the disease, it affects over 50% of patients thereby making it the most prevalent hematological abnormality observed in SLE [24,25]. Anemia in SLE can have a variety of etiologies, including immune and non-immune factors [13,26-29]. Iron deficiency anemia (IDA), autoimmune hemolytic anemia (AIHA), anemia of chronic disease (ACD), and drug-induced myelotoxicity are some of the more prevalent causes of anemia in SLE [13,26,27]. Less common causes of anemia include aplastic anemia [30,31], myelofibrosis, pernicious anemia, sideroblastic anemia, and pure red cell aplasia [32]. Approximately 20% of our patients were diagnosed with hemolytic anemia. This result is marginally higher (e.g., Egypt 18.8%) [11] to higher (e.g., Turkey 6.5%) [33]. than other populations studied. However, this result is substantially less than the frequencies found in two previous Tunisian studies (2002: 71%, 2013: 73.4%) [9,34].

Thrombocytopenia: a platelet count of less than 100x10⁹/L without any other discernible cause is considered thrombocytopenia, per the ACR and SLICC classification criteria for SLE [3,4,12]. Although it is frequently mild, thrombocytopenia is a common symptom in SLE patients. Numerous factors can lead to true thrombocytopenia, including decreased bone marrow platelet production, splenic sequestration of platelets or accelerated peripheral circulation platelet destruction, immune-mediated peripheral circulation platelet destruction by antiplatelet antibodies [13,26,35], medications, infections, and bone marrow suppression [27,36]. Immune thrombocytopenia is a common clinical manifestation of SLE, affecting 7-30% of patients [37-39]. In our present study, we found thrombocytopenia in 14.1% of our patients.

Immunological abnormalities: the ANA frequency (95.9%) was commensurate to that found in other studies (range: 87%-100%, (Table 6)). Anti-dsDNA antibodies were found in 64.3% of our patients. This result is comparable to the China and Moroccan series [10,40], but frequencies were found significantly higher in other series (e.g., UAE: 85.3%, Iran: 83%, (Table 6)) [41,42]. The United States appears to be an outlier as only 27% tested positive in a 2002 study [43]. Many studies failed to report any complement C3/4 results; however, similar to Saudi Arabian and Iranian studies [42,44], low C3 and C4 were both found in 42.8% of patients. A Spanish study although reported a significantly higher frequency of 76.3% of cases with low C3 [45] (Table 6). Similar to many studies, we found that 38% of our patients tested positive for anti-SSA antibodies; however, in other studies conducted in Tunisia, the authors found a significantly higher frequency of anti-SSA antibodies (2002: 64%, 2013: 56.4%).

Association between hematological and immunologic abnormalities: there is a variable association of hematological abnormalities with different organ system involvement and immunologic features reported in the literature. This variability is possibly due to differences in disease patterns between various ethnic groups and populations. The association of hematological abnormalities with immunologic features has not been well-studied. In our study, no autoantibody considered was statistically associated with the ensemble of tested hematological abnormalities in SLE patients. However, when hematological abnormalities were considered individually, there was a statistically significant correlation between anti-dsDNA antibodies and lymphopenia (p=0.043) (Table 5).

Vilá et al. also determined there was such an association (p<.0001) [46]. No such association was noted, however, between anti-Sm and this disease whereas Yavuz et al. observed that anti-Sm was significantly associated with lymphopenia [47]. Although this study failed to show a statistical association between thrombocytopenia and low complement C4 (p=0.8, (Table 5)), further research with a larger sample size may better estimate the effect size. A positive result by Ziakas et al. found that patients with low C3 or CH50 were more likely to be thrombocytopenic [35]. The size limitation of our series, variations in test kits, and the cut-off levels in each population could explain variations in frequencies between our findings and those of other studies. This study derives its strength from the broad extent of hematological parameters studied and the association between hematological and immunology abnormalities in SLE patients. The study was limited due to the availability of the number of patients admitted to a single hospital.

Conclusion     

The pathophysiology encompassing the wide range of hematological disorders that affect SLE patients is still unclear. They may be a side effect of SLE treatments, a comorbid condition, or a manifestation of SLE itself. Studies of any population may contain a collection of constellations of hematological disorders thereby making statistical inferences challenging. Both the ACR and SLICC criteria for classifying SLE include thrombocytopenia, leucopenia, and autoimmune hemolytic anemia (AIHA). Although hematological abnormalities are common in SLE, their frequency also varies in different populations due to significant genetic and environmental influences. The etiology of these hematological abnormalities needs to be better defined through additional research.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

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

Acknowledgments     

We would like to thank Dr. Christian Winchell for his precious help in correcting this manuscript. We are especially grateful to all the study participants.

Tables     

Table 1: summary of the reported patients with systemic lupus erythematosus

Table 2: hematological findings at diagnosis for 99 systemic lupus erythematosus patients

Table 3: autoantibody profile of this systemic lupus erythematosus cohort

Table 4: immunological analysis associated with Hematological abnormalities

Table 5: analysis of the variables potentially associated with hematological abnormalities

Table 6: comparison of the studied systemic lupus erythematosus patients to other countries

References