Rhesus factor is a stronger predictor for the risk of Sars-CoV-2 and mortality than ABO blood types

Objectives: In this study, we aimed to evaluate the relationship between ABO blood groups and Rhesus factor (Rf) and severe acute respiratory syndrome coronavirus-2 (Sars-CoV-2), as well as the risk of infection susceptibility and death according to pre-existing comorbidities. Methods: This retrospective study included patients medical record between March 2020 and March 2021. A total 470 patients were included in the study. The subjects were categorized according to diagnose of Sars-CoV-2. Also, we evaluated the subject according to severity of Sars-CoV-2 infection. The logistic and multivariate regression analysis were performed to predict possible e ﬀ ect of ABO and Rf types as well as comorbidities on indicators of Sars-CoV-2 severity including Intensive care unit (ICU) hospitalization, intubation, and mortality. Results: The distribution of ABO blood type and Rf were not statistically di ﬀ erent cases with and without Sars-CoV-2. Blood type B and A were the most groups in intubation and mortality among patients with Sars-CoV-2. However, ABO blood types had no signi ﬁ cant e ﬀ ect on risk of Sars-CoV-2 and mortality while, Rf had a signi ﬁ cantly e ﬀ ect on it. Additionally, Rf had a statistically signi ﬁ cant e ﬀ ect on all severity indicators of Sars-CoV-2 but ABO had not. Conclusions: While Rf was signi ﬁ cantly associated with risk of Sars-CoV-2 and had a strong e ﬀ ect on ICU admission, intubation, and mortality, ABO groups were not associated with risk of disease. Intubation and mortality rates were higher in patients with blood group B (OR: 2.93 p:0.390 95 % CI [0.253 – 33.9], OR: 0.217 p:0.211 95 % CI [0.020 – 2.37]) and Rh factor + (OR: 1.63 p:0.027 95 % CI [0.046 – 0.828]).


Introduction
Severe Acute Respiratory Syndrome Coronavirus-2 (Sars-CoV-2) or Coronavirus disease-2019 (COVID-19) was identified in Wuhan, China on December 31, 2019, and soon spread worldwide to become a global pandemic [1,2].According to World Health Organization (WHO), from the first day the pandemic was declared (March 11, 2020) to the end of February 2023, more than 750 million people have been affected [3].The Sars-CoV-2 pandemic is not over yet and continue affecting people.Although a significant majority of the world's population has now been vaccinated, the pandemic has so far caused numerous health issues and high rates of mortality.In Turkey, the first Sars-CoV-2 case was declared on March 11, 2020.Similar to other countries, Turkey has also suffered from serious health concerns and deaths due to the pandemic [4].
The blood types are determined depending on whether specific antigen on erythrocytes surface or not.This surface antigen has been suggested to play a role in the development of several diseases.In addition to this, blood groups are divided into two categories according to the presence or absence of the Rhesus (Rh) factor protein [5,6].ABO blood types have been investigated to illuminate possible mechanisms of susceptibility or protection for Sars-CoV-2 infection.
One of the early studies about ABO and Sars-CoV-2 has suggested that blood type A was more susceptible to infection whereas blood type O was less associated with the risk of Sars-CoV-2.After this study, reports from Europe supported that blood type A was prone to severe symptoms of viral infection but, blood type O had a lower risk of Sars-CoV-2 [7,8].However, available data on the relationship between ABO groups and Sars-CoV-2 infection have so far been controversial [6,[9][10][11].
Within this scope, the purpose of the present study is to investigate the possible relationship between ABO blood types as well as Rh factor and Sars-CoV-2 infection by using patients' medical data.A second aim of this study is to evaluate possible associations between Sars-CoV-2 and clinical outcomes, the severity of infection, and mortality.

Study design
This retrospective study was conducted with the medical records of patients who were admitted to Taksim Training and Research Hospital (Istanbul, Turkey) between March 2020 and March 2021.A total of 470 patients with an age range of 18-99 were categorized into two main groups as patients who tested positive for Sars-CoV-2 and were hospitalized due to coronavirus infection (n=363) and patients who tested negative for Sars-CoV-2 and were hospitalized for other health issues (n=107) such as traffic accidents, bone fracture, anemia, and neurologic diseases.The CONSORT diagram was summarized patient selection in Figure 1.A priori power analysis was performed to determine the sample size for detecting a difference between the groups at an effect size of f=0.40 in laboratory measurements based on the statistical methods (α=0.05,power=0.80).The diagnosis of Sars-CoV-2 was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and computed tomography (CT).Patients whose negative test results were not also confirmed by their CT scans were excluded from the study.
Clinical data including duration of hospital stay, intensive care unit (ICU) hospitalization, intubation, case fatality (CF) percentage, lung involvement, presence of comorbidities such as diabetes mellitus (DM), hypertension (HT), coroner artery disease (CAD), chronic obstructive pulmonary disease (COPD), cerebrovascular disease (CVD), and asthma were recorded and evaluated.

Laboratory data
The diagnosis of Sars-CoV-2 was confirmed by RT-PCR via the analysis of the viral genome at the Cemil Tascioglu City Hospital (Istanbul, Turkey), using nasopharyngeal swab samples.Alanine aminotransferase (ALT), glucose, creatinine, and C-reactive protein (CRP) was measured at the Roche cobas c501 clinical chemistry autoanalyzer (Roche Diagnostics, Mannheim, Germany).D-Dimer tests were analyzed using a Stago Compact Max3 analyzer with a commercial kit (Stago Diagnostics).The whole blood count analysis was conducted with a Mindray BC8000 autoanalyzer.Data concerning ABO and Rh factors of the patients were obtained from the database of the Taksim Training and Research Hospital.

Statistical analyses
All statistical analyses were conducted with SPSS software version 23.0 (Armonk, USA).The normality analysis of variables was performed with the Kolmogorov-Smirnov test.Normally distributed variables were presented as mean + standard deviation (SD), non-normally distributed variables as median (inter-quartile range), and categorical variables as percentages.The ANOVA test and independent samples t-test were used to compare normally distributed variables, whereas Kruskal-Wallis and Mann Whitney-U tests were performed for non-parametric variables.The Pearson Chi-square and the Fisher's Exact tests were conducted for the analysis of categorical variables.Logistic regression was performed to predict the potential risk of susceptibility and mortality for ABO blood type and Rhesus factor, and multivariate linear regression analyses were used to predict the possible effect of demographic and clinical characteristics, such as comorbid diseases, on the severity indicators of Sars-CoV-2 infection, including ICU hospitalization, intubation, and case fatality.All comparisons were two-tailed and a p value of <0.05 was defined as showing statistical significance.

Results
The demographic and clinical characteristics of subjects with and without Sars-CoV-2 are summarized in Table 1.There were not any significant differences with respect to gender, the distribution of blood type, and rhesus factor (p>0.05).On the other hand, ICU hospitalization, the number of intubated patients, and case fatality (CF) were higher in the subjects with Sars-CoV-2 (p<0.0001 for all).
The comparison of demographic and clinical characteristics according to ABO blood type and rhesus factor in patients with Sars-CoV-2 are shown in Tables 2 and 3.There were no differences between age and gender in terms of the ABO and Rh groups.Additionally, the groups had no differences concerning the presence of DM, HT, CAD, CVD, and asthma (p>0.05).The number of intubated patients and CF were statistically different in groups in terms of ABO blood type (p=0.045 and p=0.021, respectively).Furthermore, there were significant statistical differences in ICU hospitalization, intubated patients, and CF in terms of the rhesus factor (p=0.032, p=0.014, and p=0.012; respectively).
We also assessed the subjects diagnosed with Sars-CoV-2 in terms of severity of infection as shown in Table 4. Mean age, percentage of comorbidities such as DM, HT, CAD, COPD, and as expected CF rate were significantly higher in the severe Sars-CoV-2 group than the subjects with mild/moderate Sars-CoV-2 (p<0.0001,p=0.002, p=0.003, p=0.001, p<0.0001; respectively).However, the presence of asthma in the mild/moderate group was higher than the subjects with severe Sars-CoV-2 (p<0.0001).
In addition, we performed logistic regression analysis to predict ABO blood types to susceptibility and mortality.were not predictive for susceptibility of Sars-CoV-2 and mortality respectively.On the other hand, the Rh factor was significantly predictive for both susceptibility and mortality (Odds ratio: 1.63 p:0.027 95 % CI [0.046-0.828],Odds ratio: 2.16 p:0.035 95 % CI [0.015-0.861])respectively.
Furthermore, we performed multivariate linear regression analysis to assess possible effects of age, gender, and likely comorbidities on ICU hospitalization, intubation, and CF as summarized in Table 5. Age, gender, and ABO blood type were not found to affect ICU hospitalization, intubation, and mortality in all cases.However, age had an effect on indicators of severity of infection in cases with Sars-CoV-2, while gender and ABO blood type were not observed to have an effect on indicators in the Sars-CoV-2 group.Rh factor was more predictive on ICU hospitalization, intubation, and CF in all cases and in cases with Sars-CoV-2.

Discussion
The present study exhibited the association between patients with and without Sars-CoV-2 and ABO blood type as well as Rh factor from a single center medical record.There were not any differences in the distribution of ABO blood types among patients with and without Sars-CoV-2.Our results showed that intubation and CF were statistically different between ABO blood types.The number of patients with B blood type were the highest with respect to intubation, followed by A blood type as the second group, and lastly O blood type.Similarly, the CF was significantly different between ABO blood types.In addition, ICU hospitalization, intubation, and CF were significantly higher in patients with Rh+ than the patients with Rh−.According to multivariate and logistic regression analyses, ABO blood types did not have an effect on indicators of Sars-CoV-2 infection severity, but Rh factors had a significant effect on all severity indicators of infection in all cases and cases with Sars-CoV-2.
The association between blood types and bacterial or viral diseases has recently been in the scope of researchers, and the potential relationship between blood types and several diseases have been investigated.For instance, some studies have evaluated the possible relationship between blood types and severe acute respiratory syndrome (SARS) or influenza infection [12,13].After the Sars-CoV-2 pandemic, it has been suggested that ABO blood types are related to susceptibility, severity, and mortality of Sars-CoV-2 infections and that the possible relationship results from that Anti-A and Anti-B of ABO blood type could provide a protection from Coronavirus spike protein binding cell receptors.Because of this finding, non-O blood types were thought to increase the risk of infection severity [14].
There are controversial results in the literature with respect to blood type and Sars-CoV-2 infection.A nationbased study carried out in Sweden has shown that people with blood types A, AB, and B are at increased risk of Sars-CoV-2 infection [15].Another study suggested that blood types B and AB were more predictive of infection risk, and blood type B was significantly associated with mortality.In the same report, a relationship between Rh and Sars-CoV-2 infection was not observed [16].Koc et al. have reported that the rate of mechanical ventilation and hospitalization in ICU was higher in patients with A and AB blood groups than other blood types.On the other hand, it was shown that the blood group O was more related to the risk of infection and worse prognosis than other blood types.However, researchers did not find any relationship between mortality and blood type [17].A meta-analysis consisting of 23 studies have concluded that majority of reports indicated the blood type A to be more susceptible for Sars-CoV-2 infection, but there were some reports showing that the B blood type was the most vulnerable for infection.Furthermore, A and AB blood types were more related to severity of Sars-CoV-2 and mortality whereas, the O blood type had some protective properties against worse outcomes of Sars-CoV-2 or mortality [18].In parallel to some reports [19], our results revealed no statistically significant relationship between ABO groups and Sars-CoV-2 infection meanwhile, the most frequent blood types were B and A with respect to ICU hospitalization and CF in the present study.Moreover, none of the ABO blood types were associated with the mortality of Sars-CoV-2.Additionally, the ABO blood type had no effect on severity indicators of Sars-CoV-2 including ICU hospitalization, intubation, and mortality.Another interesting relationship with susceptibility of Sars-CoV-2 is Rh factor.The blood type is determined as positive or negative based on presence of Rh factor protein.Like ABO blood types, even though a significant number of studies have investigated the Rh factors in Sars-CoV-2, the results are controversial.We showed that the Rh factor was significantly predictive of susceptibility to Sars-CoV-2 and mortality.In addition, multivariate regression analysis revealed that the Rh factor had a significant effect on indicators of Sars-CoV-2 severity including ICU hospitalization, intubated patients, and CF in both all cases and cases with Sars-CoV-2.Furthermore, there was a statistically significant difference between mild and severe cases, the number of Rh+ patients being higher in severe cases than mild cases.Recently, it was reported that patients with blood types B and AB as well as Rh+ were more likely to test positive for Sars-CoV-2 [11].Another study from India also supported these findings, indicating that Rh+ individuals were more susceptible to Sars-CoV-2 infection [20], whereas being Rh-was associated with a lower risk of infection in another reports [21].However, in a study conducted in Turkey, the Rh factor was shown to be related to hospitalization in ICU while it was not statistically associated with mortality [22].On the contrary, some studies suggested that the prevalence of Sars-CoV-2, rate of intubation, and mortality were significantly higher in Rh-patients [23].There are some reports that suggest no relationship between the Rh factor and Sars-CoV-2 [24].
We assessed the effect of comorbidities on ICU hospitalization, intubation, and CF in the present study.In our study, Although, HT was the most frequently observed  [26].In parallel with our results, the COPD had a higher prevalence in patients with severe symptoms and worse outcome of infection [27].It was reported that the patients with COPD had a four folds higher risk for severe clinical manifestations of Sars-CoV-2 compared to patients without COPD [28].Additionally, the mortality rate in subjects with Sars-CoV-2 was higher than in cases with non-Sars-CoV-2 [29,30].
The present study had some restrictions.Firstly, we had a relatively small sample size.A larger population data could be more useful to understand the possible association between blood types and Rh factor and Sars-CoV-2.Secondly, the data on vaccination and detailed demographic characteristics of subject could be another limited part of our study.

Conclusions
We evaluated the relationship between ABO blood types, Rh factors and Sars-CoV-2.The distribution of ABO groups and Rh factors were similar between the patients with and without Sars-CoV-2.Meanwhile, intubation and mortality rates were higher in patients with B blood type with Rh+.The most of patients with Rh-were blood type O with a lower rate of ICP hospitalization, intubation, and CFR.However, logistic regression analysis indicated that Rh factor was more predictive risk of infection and death but ABO blood types were not.Additionally, multivariate linear regression analysis proved that ABO blood types did not have an effect on ICU hospitalization, intubation, and mortality whereas, Rh factors had significantly higher predictive power on severity indicators in both all cases and cases with Sars-CoV-2.

Table  :
Demographic and clinical characteristics of all subjects with and without Sars-CoV-.

Table  :
Demographic and clinical characteristics of subjects with Sars-CoV- according to blood types.

Table  :
The comparison of blood type and rhesus factor according to severity of Sars-CoV-.

Table  :
Demographic and clinical characteristics of subjects with Sars-CoV- according to Rhesus factor (Rh).

Table  :
Multivariate Linear Regression analysis to predict likely comorbidities and demographic characteristics on indicators of the severity of Sars-CoV-.DM, diabetes mellitus; HT, hypertension; CAD, coroner artery disease; COPD, chronic obstructive pulmonary disease; CVD, cerebrovascular disease; ICU, intensive care unit; CF, case fatality.Adjusted R square for ICU hospitalization: . for intubation: ., and for CFR: . in all cases.Adjusted R square for ICU hospitalization: ., for Intubation: ., and for CFR . in cases with Sars-CoV-.Bold values indicate the statistically important ones (<.).