The interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia.

Life-threatening thrombotic events at unusual sites have been reported after vector-based vaccinations against severe acute respiratory syndrome coronavirus 2. This phenomenon is now termed vaccine-induced immune thrombotic thrombocytopenia (VITT). The pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT) and is associated with platelet-activating antibodies (Abs) against platelet factor 4 (PF4). Therefore, current guidelines suggest nonheparin anticoagulants to treat VITT patients. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux, and argatroban with VITT-Ab/PF4 complexes using an ex vivo model for thrombus formation as well as in vitro assays to analyze Ab binding and platelet activation. We found that immunoglobulin Gs (IgGs) from VITT patients induce increased adherent platelets/thrombus formation in comparison with IgGs from healthy controls. In this ex vivo flow-based model, the procoagulant activity of VITT IgGs was effectively inhibited with danaparoid and argatroban but also by heparin. Interestingly, heparin and danaparoid not only inhibited IgG binding to PF4 but were also able to effectively dissociate the preformed PF4/IgG complexes. Fondaparinux reduced the in vitro generation of procoagulant platelets and thrombus formation; however, it did not affect platelet aggregation. In contrast, argatroban showed no effect on procoagulant platelets and aggregation but significantly inhibited VITT-mediated thrombus formation. Taken together, our data indicate that negatively charged anticoagulants can disrupt VITT-Ab/PF4 interactions, which might serve as an approach to reduce Ab-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulants in VITT patients could be made.

In vitro Hemostatic Functions of Cold-Stored Platelets.

Background: Transfusion of platelets is a life-saving medical strategy used worldwide to treat patients with thrombocytopenia as well as platelet function disorders. Summary: Until the end of 1960s, platelets were stored in the cold because of their superior hemostatic functionality. Cold storage of platelets was then abandoned due to better posttransfusion recovery and survival of room temperature (RT)-stored platelets, demonstrated by radioactive labeling studies. Based on these findings, RT became the standard condition to store platelets for clinical applications. Evidence shows that RT storage increases the risk of septic transfusion reactions associated with bacterial contamination. Therefore, the storage time is currently limited to 4-7 days, according to the national guidelines, causing a constant challenge to cover the clinical request. Despite the enormous efforts made to optimize storage conditions of platelets, the quality and efficacy of platelets still decrease during the short storage time at RT. In this context, during the last years, cold storage has seen a renaissance due to the better hemostatic functionality, reduced risk of bacterial contamination, and potentially longer storage time. Key Messages: In this review, we will focus on the impact of cold storage on the in vitro platelet functions as promising alternative storage temperature for future medical applications.

Antibody Binding and Angiotensin-Converting Enzyme 2 Binding Inhibition Is Significantly Reduced for Both the BA.1 and BA.2 Omicron Variants.

BACKGROUND: The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated individuals showed a substantial reduction. METHODS: We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals. RESULTS: While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to Omicron only improved after administration of a third dose. CONCLUSIONS: Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies.

Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies (Ab) reactive to complexes of platelet factor 4 and heparin. Platelets (PLT) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations in this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient Ab induce a new PLT population that is characterized by increased P-selectin expression and phosphatidylserine (PS) externalization. Formation of this procoagulant PLT subpopulation was dependent on engagement of PLT Fc-γ-RIIA by HIT Ab and resulted in a significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLT propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLT intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of P-selectin and PS was dissected. While inhibition of P-selectin did not affect thrombus formation, the specific blockade of PS prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLT are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients.

Cerebral venous sinus thrombosis and thrombocytopenia due to heparin-independent anti-PF4 antibodies after adenovirus infection.

Not available.

Treatment of drug-induced immune thrombocytopenias.

Several therapeutic agents can cause thrombocytopenia by either immune-mediated or non-immune-mediated mechanisms. Non-immune-mediated thrombocytopenia is due to direct toxicity of drug molecules to platelets or megakaryocytes. Immune-mediated thrombocytopenia, on the other hand, involves the formation of antibodies that react to platelet-specific glycoprotein complexes, as in classic drug-induced immune thrombocytopenia (DITP), or to platelet factor 4, as in heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT). Clinical signs include a rapid drop in platelet count, bleeding or thrombosis. Since the patient's condition can deteriorate rapidly, prompt diagnosis and management are critical. However, the necessary diagnostic tests are only available in specialized laboratories. Therefore, the most demanding step in treatment is to identify the agent responsible for thrombocytopenia, which often proves difficult because many patients are taking multiple medications and have comorbidities that can themselves also cause thrombocytopenia. While DITP is commonly associated with an increased risk of bleeding, HIT and VITT have a high mortality rate due to the high incidence of thromboembolic complications. A structured approach to drug-associated thrombocytopenia/thrombosis can lead to successful treatment and a lower mortality rate. In addition to describing the treatment of DITP, HIT, VITT, and vaccine-associated immune thrombocytopenia, this review also provides the pathophysiological and clinical information necessary for correct patient management.

Antibody-mediated procoagulant platelets in SARS-CoV-2-vaccination associated immune thrombotic thrombocytopenia.

The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. To prevent severe infection, mass COVID-19 vaccination campaigns with several vaccine types are currently underway. We report pathological and immunological findings in 8 patients who developed vaccine-induced immune thrombotic thrombocytopenia (VITT) after administration of SARS-CoV-2 vaccine ChAdOx1 nCoV-19. We analyzed patient material using enzyme immune assays, flow cytometry and heparin-induced platelet aggregation assay and performed autopsies on two fatal cases. Eight patients (5 female, 3 male) with a median age of 41.5 years (range, 24 to 53) were referred to us with suspected thrombotic complications 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission. Patients had a median platelet count of 46.5 x109/L (range, 8 to 92). Three had a fatal outcome and 5 were successfully treated. Autopsies showed arterial and venous thromboses in various organs and the occlusion of glomerular capillaries by hyaline thrombi. Sera from VITT patients contain high titer antibodies against platelet factor 4 (PF4) (OD 2.59±0.64). PF4 antibodies in VITT patients induced significant increase in procoagulant markers (P-selectin and phosphatidylserine externalization) compared to healthy volunteers and healthy vaccinated volunteers. The generation of procoagulant platelets was PF4 and heparin dependent. We demonstrate the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.

The relationship between intoxication severity and blood interleukin 6, interleukin 10 and CRP levels in carbon monoxide-poisoned patients.

Carbon monoxide (CO) is one of the most common causes of death due to intoxications. No biochemical marker is available to evaluate the severity of CO intoxication. We measured high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6) and interleukin-10 (IL-10) levels in patients with different degrees of CO poisoning. We prospectively included 40 CO-poisoned patients admitted to emergency services. Blood samples were collected from the patients at admission (0 hour) and after treatment (six hours). While all patients received normobaric oxygen (NBO2) therapy, patients with severe CO poisoning received additional sessions of hyperbaric oxygen (HBO2) therapy. Blood samples were also collected from a group of healthy volunteers (n=40). Serum IL-6 and IL-10 levels were measured with the ELISA method while hs-CRP was quantified by turbidimetric analysis. At admission, IL-6 levels were significantly higher in the patient group compared to the control group (P=0.001), but IL-10 and hs-CRP levels were not significantly different between the groups. Compared to admission levels, IL-6 levels were higher at six hours (P=0.014). The patients were grouped according to treatment type (NBO2, HBO2) and history of syncope, but no significant differences were detected in patient subgroups regarding IL-6, IL-10 and hs-CRP levels. A weak positive correlation was found between COHb and lactate levels in patients (P=0.013; r=0.390).This study shows that IL-6 level increases in CO-poisoned patients, but it is not correlated with the severity of the intoxication.

The effect of hyperbaric oxygen therapy on rhabdomyolysis-induced myoglobinuric acute renal failure in rats.

Myoglobinuric acute renal failure (MARF) may develop after severe muscle injury. Heme oxygenase-1 (HO-1), a stress-response protein, has been implicated as a protective agent against MARF. We hypothesized that hyperbaric oxygen therapy (HBOT) may alleviate MARF by inducing renal HO-1 expression. Wistar-Albino rats were randomly assigned into three groups: Control (n = 4), MARF (n = 8), MARF + HBO (n = 8). MARF was induced by intramuscular glycerol (50%, 8 mL/kg) injection. Saline (8 mL/kg) was injected into the hind limb of the animals in the control group. Animals in the MARF + HBO group received two sessions of HBO therapy (90 min at 2.5 atm) 2 and 18 h after glycerol injection. Serum and tissue samples were taken at 24 h. Serum urea and creatinine levels increased in the MARF and MARF + HBO groups confirming the development of MARF. But, serum urea and creatinine levels were similar in MARF and MARF + HBO groups. Oxidative stress parameters were similar among all groups. Histological renal injury score was similar in MARF and MARF + HBO groups. HO-1 level, determined by immunohistochemistry, was significantly higher in MARF and MARF + HBO groups, compared to the control group. Although HO-1 level in MARF + HBO group was higher than MARF group, it was not statistically significant. We found that HBOT did not reduce renal injury in experimental MARF model. HBOT is used to reduce the muscle damage after crush injury, which may be accompanied by MARF. Therefore, more studies are needed to understand the effects of HBO treatment on renal functions after MARF.

Neuropsychological Evaluation of Children and Adolescents With Acute Carbon Monoxide Poisoning.

OBJECTIVES: Carbon monoxide (CO) poisoning is a worldwide health problem. We have limited information regarding psychological adversities of CO poisoning in children and adolescents. The aim of this study was (1) to investigate the effects of severe CO poisoning on cognitive functions, mood, and behaviors in children and adolescents and (2) to identify factors related to occurrence of neuropsychological symptoms. METHODS: This study included pediatric patients, who were evaluated after CO poisoning at the Department of Child and Adolescent Psychiatry between January 2012 and April 2013. The patients were evaluated at 2 time points. The first evaluation was done when they were discharged from emergency department, and the second evaluation was done 1 month after CO poisoning. Turkish versions of internationally recognized tests were used to evaluate anxiety, depressive symptoms, attention, visual-spatial skills, memory, and behaviors of patients. RESULTS: Twenty-seven patients were analyzed. The mean age of the patients was 11.8 ± 2.7 years (range, 6-18 years). The mean carboxyhemoglobin level was 31.5% ± 7.8% (range, 19%-51%) dir. Delayed neurological sequel was observed in only 1 patient, who had headache and tinnitus. We found that carboxyhemoglobin level was not correlated with later neuropsychiatric test scores. However, we found a correlation between history of loss of consciousness and anxiety symptom level, hyperbaric oxygen (HBO) therapy session and behavioral problems, and time to HBO therapy and attention problems. CONCLUSIONS: We suggest that CO exposure duration, history of loss of consciousness, time to HBO therapy, and the number of HBO therapy session affect neuropsychological symptom levels and occurrence of attention and behavioral problems.

Hyperbaric oxygen therapy in the treatment of osteonecrosis of the femoral head: a review of the current literature.

OBJECTIVE: To review the current literature on the use of hyperbaric oxygen (HBO2) therapy in the treatment of osteonecrosis of the femoral head (ONFH). SEARCH STRATEGY: We searched PubMed, Directory of Open Access Journals (DOAJ), EMBASE, Web of Science, Academic Search Complete, CINAHL and MEDLINE through April 2015. We hand-searched relevant textbooks, conference proceedings and the reference lists of review articles and clinical studies STUDY ELIGIBILITY CRITERIA: Randomized controlled trials (RCT) and observational studies (cohort study, case-control study, case series) that reported the outcome of patients who received HBO2therapy for ONFH were included. Only English-language articles were included. Study quality was not used as an exclusion criterion. STUDY APPRAISAL AND SYNTHESIS METHODS: Two authors independently assessed trials for inclusion, extracted data and presented to other authors. Disagreements were resolved by consensus. RESULTS: We identified eight clinical studies; two randomized controlled trials (RCTs); one historically controlled study; and five case series. The majority of the studies were small-scale, heterogeneous and methodologically weak. In four of the studies HBO2therapy was combined with other treatment modalities, making it impossible to draw firm conclusions on the specific effects of HBO2therapy. Hip survivorship in studies wherein HBO2therapy was used alone was 95.5% in Steinberg Stage I lesions, 89% in Steinberg Stage II lesions and 100% in Ficat Stage II lesions. CONCLUSIONS: There is a room for HBO2therapy in the management ONFH. Further RCTs, however, are required to better elucidate the role of HBO2therapy in the treatment of ONFH.

The use of hyperbaric oxygen therapy for carbon monoxide poisoning in Europe.

BACKGROUND: The aim of this survey was to identify practice differences in the treatment of carbon monoxide (CO) poisoning with hyperbaric oxygen (HBO2) therapy among centers in Europe. MATERIALS AND METHODS: Using a commercial online survey website (surveymonkey.com), we created a questionnaire and invited the medical directors of hyperbaric centers in Europe by email to complete the survey. RESULTS: Sixty-eight centers from 23 countries participated in the survey. While transient or prolonged unconsciousness was unanimously recognized as an indication for HBO2 therapy, positive neurological findings, ECG suggesting acute ischemia and pregnancy were considered important indications of HBO2 therapy in more than 95% of the centers. Twenty-three (44%) centers reported that they used carboxyhemoglobin (COHb) levels as a criterion for patient selection. Among responders, 39% (18/46) reported delivering a single session within the first 24 hours of CO poisoning, and nine (19%) reported delivering three sessions in the first day. The majority of the centers (52%) replied that they initially gave a single session per patient, which was repeated if symptoms persisted. We identified a total of 21 different HBO2 profiles used in European centers. CONCLUSION: Our results showed that the indications of CO poisoning for HBO2 therapy are still not universally recognized. Additionally, HBO2 therapy protocols used at European hyperbaric centers varied significantly, suggesting a need for more education regarding the published guidelines.

Early treatment response predicts outcome in patients with idiopathic sudden sensorineural hearing loss treated with hyperbaric oxygen therapy.

Hyperbaric oxygen (HBO2) therapy is used in the treatment of idiopathic sudden sensorineural hearing loss (ISSNHL). Some patients, however, do not benefit from HBO2 therapy despite several weeks of treatment. We hypothesized that early treatment response could predict treatment failure. We conducted a retrospective chart review and identified patients diagnosed with ISSNHL and recorded the pure tone average (PTA) levels prior to, after the first week and at completion of HBO2 therapy. Early treatment response (ETR) was assessed after the first week of HBO2 therapy, and an increase ⟨10 decibels (dB) in mean PTA level was defined as a negative ETR. Factors associated with no recovery were determined using logistic regression analysis. Overall, 39 patients (22 males; mean age (±SD) 41.9 ±14.3 years) were assessed. At treatment completion, the improvement in hearing was 'very substantial' (≥20 dB) for 21 patients (53.8%), 'substantial' (10-19 dB) for eight (20.5%), 'minimal' (⟨10 dB) for five (12.8%) and absent for five (12.8%) patients. After the first week of HBO2 therapy, 18 (46.2%) patients displayed negative ETR. Multivariate logistic regression analysis revealed 'negative ETR' as the only significant predictor of treatment failure (odds ratio (95% confidence interval): 6.98 (1.59-30.61), p=0.010) in patients with ISSNHL treated with HBO2. In conclusion, we found that patients who fail to benefit from an initial set of HBO2 sessions were less likely to benefit from additional HBO2 sessions. Further prospective studies are necessary to confirm our findings.

Changes in pulmonary function in hyperbaric chamber inside attendants: a case-control study.

BACKGROUND: Inside attendants (IAs) [are] exposed to high pressure during hyperbaric oxygen (HBO2) therapy. The aim of this study was to evaluate the alterations of pulmonary functions in IAs over time. METHODS: IAs in our hyperbaric center constituted the IA group (n=11). A sex- and age-matched control group (n=15) was constituted from hospital staff who had not dived or been exposed to hyperbaric environments before. We measured the respiratory function of all subjects at two time points: 1) at the start of the study; and 2) 12 months after the first measurement. The following parameters were recorded: forced vital capacity (FVC), forced expiratory volume in one second (FEV1), rate of FEV1 to FVC (FEV1/FVC%), forced expiratory flow at 25% to 75% vital capacity (FEF25-75%), forced expiratory flow at 50% vital capacity (FEF50%), forced expiratory flow at 25% vital capacity (FEF25%). RESULTS: Both groups were similar in terms of age, sex, smoking and body mass index. We found that FEV1, FEV1/FVC%, FEF25-75% and FEF50% significantly reduced in both groups after 12 months (p⟨0.05). However, the rate of change in all parameters was similar in both groups (p⟩0.05). In the IA group, the total number of exposures within the 12 months was positively correlated with the rate of reduction in FEF25% (r=0.788, p=0.004). CONCLUSION: Working as an IA does not deteriorate pulmonary function in the short term. However, there is a need for long-term follow-up studies.

Efficiency of hyperbaric oxygen and steroid therapy in treatment of hearing loss following acoustic trauma.

INTRODUCTION: Most commonly used treatment modalities for acute acoustic trauma (AAT) include steroid and hyperbaric oxygen (HBO2) therapy. The aim of this study is to investigate the effectiveness of combined steroid and HBO2 therapy in patients who develop AAT during firearms training and the effect of delay to treatment on treatment success. MATERIALS AND METHODS: Patients admitted with the complaint of hearing loss after firearms training between January 2011 and April 2013 were evaluated retrospectively. Patients were grouped according to date of admission; patients admitted within the first 10 days were included in Group A and those admitted between Days 11 and 30 in Group B. RESULTS: A total of 48 patients (73 ears) with AAT were included. There were 37 ears in Group A and 36 ears in Group B. The number of ears with complete treatment response, partial treatment response and treatment failure (unchanged) were one (2.7%), 7 (18.9%) and 29 (78.4%) in Group A and 0 (0%), 3 (8.3%) and 33 (91.7%) in Group B, respectively. There was no statistically significant difference between the groups (p = 0.095). Late-term results (at Week 6) demonstrated Group A showed higher hearing gain on high frequencies than Group B (p < 0.05), but this result was not consistent with clinical outcome results. CONCLUSION: The success rate of combined HBO2 and steroid therapy was very low in our study. However, early initiation of treatment results in better outcomes. Protective measures have great importance in preventing AAT.

A case of tension pneumothorax during hyperbaric oxygen therapy in an earthquake survivor with crush injury complicated by ARDS (adult respiratory distress syndrome).

Pneumothorax (PTX) is rarely reported in patients receiving hyperbaric oxygen (HBO2) therapy. Patients with air-trapping lesions in the lungs and those with a history of spontaneous PTX, lung disease, mechanical ventilation or chest trauma are at an increased risk for PTX during HBO2 therapy. A 28-year-old male earthquake survivor was referred to our center for multiple wounds 21 days after being rescued from the debris. He had been intubated and put on mechanical ventilation for three days because of adult respiratory distress syndrome (ARDS). At initial presentation, he was conscious, well-oriented and hemodynamically stable. The initial six HBO2 treatments were uneventful. On the seventh HBO2 treatment, the patient lost consciousness and developed cardiopulmonary arrest near the end of decompression. The HBO2 specialist accompanying the patient inside the chamber immediately initiated CPR. A diagnosis of tension PTX was made. After the patient was removed from the chamber, a chest tube was inserted, which improved the symptoms. Although rare, tension PTX can occur during HBO2 therapy. Early diagnosis and intervention are crucial for saving a patient's life. Increased vigilance is required during treatment of patients with risk factors for PTX.

Decompression sickness cases treated with recompression therapy between 1963 and 1998 in Turkey: review of 179 cases.

The purpose of this study was to review the cases diagnosed as decompression sickness (DCS) with recompression therapy treament between 1963 and 1998 in Turkey. The records of 179 cases were analyzed for age, gender, type of DCS, presenting signs and symptoms, time to onset of symptoms, time to recompression therapy, recompression table used, total number of treatments and outcomes. The diving depth on the day of injury ranged between 60 and 215 fsw (18 and 65 msw). The symptoms developed during ascent or within 10 minutes after surfacing in 47% of divers and within the first hour in 87% of the divers. The most frequent symptom was sensory loss in the extremities. The dive before the symptoms was a repetitive dive in 50% of the cases and the diagnosis was Type II DCS in 79% of these divers. Most of the divers (84%) received recompression therapy after a delay of more than 12 hours. Healing rate was 68% with the air recompression tables and 86% with the oxygen tables. Repetitive dives were associated with a higher incidence of Type II DCS than single dives (79% and 66%, respectively). The results using recompression tables with oxygen were more successful than the treatment with air tables.

Antibiotherapy with and without bone debridement in diabetic foot osteomyelitis: A retrospective cohort study.

BACKGROUND AND OBJECTIVE: The treatment of diabetic foot osteomyelitis (DFO) is a controversial issue, with disagreement regarding whether the best treatment is surgical or conservative. The purpose of this study was to compare the outcome of patients with DFO who were treated with antibiotherapy alone and those who underwent concurrent minor amputation. METHODS: Hospital records of patients who were diagnosed as having DFO within a 2-year study period were retrospectively reviewed. Patients were divided into two groups: those who received antibiotherapy alone and those who underwent concurrent minor amputation. Groups were compared in terms of duration in hospitalization, antibiotherapy, and wound healing. RESULTS: Thirty seven patients were included in the study. These comprised patients who received antibiotherapy alone (ABG, n=15) and patients who underwent concurrent minor amputation (AB-MAG, n=22). Hospitalization duration was 37.2 (± 16.2) days in ABG and 52.8 (± 40.2) days in AB-MAG (p = 0.166). Mean duration of antibiotherapy was 45.0 (± 21.7) days in ABG and 47.7 (± 19) days in AB-MAG (p = 0.689). Wound healing duration was 265.2 (± 132.7) days in ABG and 222.6 (± 85.9) days in AB-MAG (p = 0.243). None of the outcome measures were significantly different between ABG and AB-MAG. CONCLUSIONS: Our results have shown similar outcomes for both patient groups who received antibiotherapy alone and who underwent concurrent minor amputations. Considering the small sample sizes in this study, it is important to confirm these results on a larger scale.