Complement activation negatively affects the platelet response to thrombopoietin receptor agonists in patients with immune thrombocytopenia: a prospective cohort study

Abstract Increased platelet destruction is central in the pathogenesis of immune thrombocytopenia. However, impaired platelet production is also relevant and its significance underlies the rationale for treatment with thrombopoietin receptor agonists (TPO-RAs). Previous studies have associated enhanced complement activation with increased disease severity. Additionally, treatment refractoriness has been demonstrated to resolve by the administration of complement-targeted therapeutics in a subset of patients. The association between complement activation and the platelet response to TPO-RA therapy has previously not been investigated. In this study, blood samples from patients with immune thrombocytopenia (n = 15) were prospectively collected before and two, six and 12 weeks after the initiation of TPO-RA therapy. Plasma levels of complement degradation product C4d and soluble terminal complement complexes were assessed. Patients with significantly elevated baseline levels of terminal complement complexes exhibited more often an inadequate platelet response (p = .04), were exclusively subjected to rescue therapy with intravenous immunoglobulin (p = .02), and did not respond with a significant platelet count increase during the study period. C4d showed a significant (p = .01) ability to distinguish samples with significant terminal complement activation, implying engagement of the classical complement pathway. In conclusion, elevated levels of complement biomarkers were associated with a worse TPO-RA treatment response. Larger studies are needed to confirm these results. Biomarkers of complement activation may prove valuable as a prognostic tool to predict which patients that potentially could benefit from complement-inhibiting therapy in the future. Plain Language Summary What is the context? Primary immune thrombocytopenia (ITP) is a potentially serious illness associated with an increased risk of bleeds. Manifestations range from confined skin bruising to life-threatening intracranial hemorrhages. It is an acquired immune disorder characterized by increased destruction and impaired production of platelets. Treatments aim at suppressing the destruction and supporting the production of platelets. Thrombopoietin receptor agonists (TPO-RA) are medically approved platelet growth factors that contribute to the generation of new platelets. The complement system is an evolutionary preserved part of innate immunity. Previous studies have indicated that complement activation may be an important contributor to disease and that the administration of complement-inhibiting therapy improves the platelet count in a subset of patients with primary ITP. What is new? The potential association between complement activation and a poor platelet response to TPO-RA therapy in primary ITP has not been previously studied. In fifteen patients with primary ITP starting TPO-RA therapy, we prospectively followed the platelet response and levels of complement biomarkers for 12 weeks. We showed that patients with high levels of complement biomarkers exhibited a worse treatment response during the study period. What is the impact? Our results suggest that levels of complement biomarkers may be valuable to predict which patients with treatment-refractory ITP that potentially could benefit from complement-inhibiting therapy in the future Larger studies are needed to confirm our results.


Plain Language Summary
What is the context?
• Primary immune thrombocytopenia (ITP) is a potentially serious illness associated with an increased risk of bleeds.Manifestations range from confined skin bruising to lifethreatening intracranial hemorrhages.• It is an acquired immune disorder characterized by increased destruction and impaired production of platelets.
• Treatments aim at suppressing the destruction and supporting the production of platelets.
• Thrombopoietin receptor agonists (TPO-RA) are medically approved platelet growth factors that contribute to the generation of new platelets.• The complement system is an evolutionary preserved part of innate immunity.
• Previous studies have indicated that complement activation may be an important contributor to disease and that the administration of complement-inhibiting therapy improves the platelet count in a subset of patients with primary ITP.

Introduction
Primary immune thrombocytopenia (ITP) is an acquired immune disorder characterized by isolated thrombocytopenia [1].It is distinguished from other thrombocytopenias, and from secondary ITP, as a diagnosis of exclusion.The response to treatment and the severity of bleeding manifestations vary in patients with equal platelet counts (PLC) [2].Consequently, primary ITP has been suggested to be not a single entity but rather a heterogeneous group of disorders sharing common denominators of altered primary hemostasis and lost immune tolerance toward cells derived from megakaryopoiesis [3].Initially, increased platelet destruction due to opsonization by anti-platelet antibodies was considered the only evident pathogenetic cause of thrombocytopenia identified in cases of primary ITP [3].Indeed, an up-regulated humoral response resulting in the production of anti-platelet antibodies that target membrane glycoproteins still is considered the main contributor of disease [1].However, in 30-40% of patients, no autoantibodies are detected [4] and not all patients respond to treatments directed at platelet autoantibodies.These findings have brought into question whether autoreactive T cells contribute to disease beyond the T cell influence on plasma B cells [1] or whether limitations related to the reliability of laboratory assays and available assay antigens [5] falsely suggest the presence of other mechanisms.In addition, it is reported that both cytotoxic T cells and autoantibodies impair megakaryocyte maturation [1], thus suppressing the generation of new platelets.While most ITP therapies aim to modulate the immune response that contributes to platelet destruction, the introduction of thrombopoietin receptor agonists (TPO-RA) has aimed to counteract impaired platelet production [6].To date, orally-administered eltrombopag or avatrombopag and subcutaneously-administered romiplostim are approved for the treatment of ITP [7,8].
The potential contribution of complement activation in patients with primary ITP has not been fully explored [9].However, enhanced complement activation has been shown to correlate with the presence of severe and refractory disease [10,11].The contribution of complement, at least in a subset of patients, is now recognized and studies have proposed that primarily the classical pathway is involved [10][11][12][13][14]. Existing evidence so far suggests that increased complement activation mainly depends upon the complement-fixing capacity of anti-GPIIb/IIIa and anti-GPIb/IX antibodies [13][14][15].However, increased levels of soluble terminal complement complexes (sTCC; the final product of the terminal complement pathway, also termed as C5b-9 or the membrane attack complex [16]) have been observed in autoantibody-negative cases during acute disease as compared to the corresponding type of cases in remission [12].Thus, it is unclear whether complement activation may occur in an autoantibody-independent manner or whether this is also potentially a result of a limited ability to detect all antiplatelet antibodies.
The temporal development of complement biomarkers in TPO-RA-treated ITP patients has not been previously studied.It is yet to be shown whether complement activation may impair the platelet response in this setting.Further, previous studies on ITP and complement activation have primarily relied on plasma levels of complement components and not so much on levels of complement degradation products and sTCC.Complement degradation product C4d has been proven a stable and valuable biomarker in various conditions associated with classical pathway-mediated complement activation [17][18][19][20].The associations between plasma levels of sTCC and C4d, the severity of disease, and therapy response to TPO-RA remain unclear.Consequently, we designed this prospective cohort study with the aim to investigate in patients with primary ITP the relationship between the platelet response and levels of sTCC and C4d before and sequentially after initiation of TPO-RA therapy.We hypothesized that i) cases with increased levels of sTCC and C4d would be associated with a worse platelet response and that ii) levels of C4d would positively correlate with terminal complement pathway activation in these patients.

Methods
Written informed consent was obtained from all participating subjects.The Institutional Review Board at Weill Cornell Medicine of Cornell University (protocol number 1106011743), the South-Eastern Norway Regional Committee for Medical and Health Research Ethics (diary number 2011/1747a), and the Swedish Ethical Review Authority (diary number 2021-06757-01) approved the study.The study was conducted in compliance with the Declaration of Helsinki and the manuscript was prepared according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for observational studies [21].

Recruitment and study design
Eligible subjects were prospectively recruited from four centers in Norway and one center in the USA during the years 2012-2019.Inclusion criteria were a diagnosis of primary ITP, an age of ≥18 years, and planned initiation of TPO-RA therapy (eltrombopag or romiplostim).Concomitant ITP therapies and rescue therapy with intravenous immunoglobulin (IVIg) were not considered as exclusion criteria.
Samples acquired from age-and gender-matched healthy hospital employees (n = 14) collected at one of the Norwegian centers during the enrollment period (Table I) were used to determine the cutoff value for post hoc classification of the cohort into two study groups based on levels of sTCC at baseline: (1) sTCC-high: significantly elevated baseline concentration of sTCC compared to healthy controls, defined as sTCC levels > (mean controls +2SD).
Blood samples were collected at study visits before initiation of TPO-RA therapy (baseline) and sequentially at weeks two, six, and 12 of therapy.Baseline data including clinical characteristics and concomitant ITP therapies were acquired from all subjects at study entry (Table II).
Intra-study data on administered IVIg cycles and platelet transfusions were obtained.The number of individual subjects exhibiting an inadequate platelet response after the initiation of TPO-RA therapy was assessed.An inadequate platelet response was defined as a PLC <30 (x 109/L) or ≥ 30 (x 109/L) without a twofold increase in the baseline count [22].Both the number of individual subjects per study group with one or more inadequate platelet responses during one or more study visits and the total number of inadequate platelet responses per group during the study period were assessed.

Blood sampling and preparation
Venous blood was peripherally collected into two ethylenediamine-tetraacetic acid (EDTA) vacuum tubes (Becton-Dickinson, Plymouth, UK/Greiner Bio-One, Kremsmünster, Austria) per sampling occasion and study subject.Samples used for the measurement of the PLC were analyzed the same day according to the local routine at the accredited laboratory in each center.Samples used for complement analyses were centrifuged at 2.000 × g for 15 minutes and aliquots were stored at −80°C.

Complement analyses
Levels of C4d (mg/L) and sTCC (mg/L) were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits: Complement C4d -RUO [22] and Complement TCC -RUO [23] (SVAR Life Science, Malmö, Sweden).All complement analyses were processed centrally at one center.Assays were performed according to the manufacturer's instructions.The absorbance was measured at 450 nm with the correction wavelength set at 620 nm using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Winooski, VT, USA).Samples were analyzed in duplicate and concentrations were extrapolated relative to standard curves generated by fourthorder polynomial equations in GraphPad Prism version 8.0.0 (GraphPad Software, San Diego, CA, USA).Final concentrations were determined as the mean of duplicates.

Statistical analyses
All continuous variables had a non-Gaussian distribution and were descriptively presented as medians with lower and upper quartiles (q1, q3).Categorical variables were presented with counts and percentages.Statistical differences for binary variables between the sTCC-high and sTCC-normal groups were evaluated using Fisher's exact test.Statistical differences for continuous variables between the sTCC-high and sTCC-normal groups were evaluated using the Mann-Whitney U test.The absolute platelet count (PLC), change in PLC (as compared to baseline; ΔPLC) and the concentrations of sTCC and C4d were compared at each sampling occasion and as study period aggregates (measurements including all sampling occasions throughout the study period per variable and group).Comparisons of study period aggregates were utilized due to the low number of observations per sampling occasion.Statistical differences for continuous variables between the time points (sampling occasions) within each study group were evaluated using the Friedman test followed by a post hoc Dunn's multiple comparisons test.Significant p-values were adjusted by the Bonferroni correction for multiple tests.Spearman's rank-order correlation test was used to assess bivariate correlations between variables with non-normally distributed residuals.A post hoc receiver operating characteristic (ROC) curve was applied to evaluate the ability of C4d to distinguish samples with significantly elevated concentrations of sTCC.All pvalues were two-tailed and values <.05 were considered significant.All analyses were performed using IBM SPSS Statistics version 25.0 (IBM, Armonk, NY, USA).

Enrollment and study group classification at baseline
Fifteen patients (ten females) and fourteen healthy controls were included in the study.Median age at study entry was 61 (44, 66) years.Baseline concentrations of sTCC and C4d in the whole patient cohort did not differ significantly from age-and gendermatched healthy control subjects.Based on the concentration of sTCC for the control subjects, the cutoff for significantly elevated baseline concentration of sTCC was determined to be >0.11mg/L (Table I).Five subjects had a baseline concentration of sTCC greater than the cutoff value and were labeled sTCC-high.The remaining 10 subjects were labeled sTCC-normal.

Clinical characteristics according to study group classification
Table II provides specifics for the clinical characteristics.No significant differences regarding gender (p = .60)and age (p = .25)were observed between the sTCC-high and sTCC-normal groups.

Complement activation and TPO-RA therapy in ITP 3
Prior to study entry, three subjects (60%) in the sTCC-high group had undergone splenectomy as compared to one subject (10%) in the sTCC-normal group (p = .08).At study entry, three (60%) subjects in the sTCC-high group received concomitant ITP drugs as compared to three (30%) subjects in the sTCC-normal group (p = .33).Third-line therapy [24] was only utilized in the sTCC-high group with two (40%) subjects treated with cyclosporine.Types of TPO-RA (romiplostim vs. eltrombopag) were equally distributed between the study groups (p = 1.0).During the study period, three subjects (60%) in the sTCC-high group but none in the sTCCnormal group received IVIg rescue therapy (p = .02).All subjects (n = 5) in the sTCC-high compared to four (40%) subjects in the sTCC-normal group had an inadequate platelet response on at least one study visit after treatment initiation (p = .04),which corresponded to a total of eight (62%) compared to six (25%) sampling occasions (p = .04),respectively.No subject suffered from thromboembolism during the study period.

Intragroup developments of the platelet response and complement levels during TPO-RA therapy
Significant differences in the PLC and the concentrations of sTCC and C4d were assessed between the sampling occasions within each group during the study period (Figure 1).For a detailed visualization of the platelet response, Figure 2 represents the temporal development of the platelet count for each subject in the two study groups.
A significant difference in the PLC (p = .016)was observed between the mean ranks of at least one pair of time points for the sTCC-normal group.Dunn's multiple comparisons test indicated a significant increase (adjusted p = .01)between week 0 (26 [10,35] x 10 9 /L) and week 2 (161 [31, 310] x 10 9 /L) after Bonferroni correction.No significant differences in the PLC were observed for the sTCC-high group during the study period.
A significant difference (p = .029)in the concentration of sTCC was observed between the mean ranks of at least one pair of time points for the sTCC-high group.Dunn's multiple comparisons test indicated a significant decrease (adjusted p = .027)between week 0 (0.14 [0.13, 0.77] mg/L) and week 12 (0.11 .04 Sampling occasions: week 0 (baseline, i.e., before the initiation of TPO-RA therapy), week 2, week 6 and week 12.All p-values were calculated using Fisher's exact test for categorical variables and the Mann-Whitney U test for continuous variables.a sTCC-high: significantly elevated baseline concentration of sTCC compared to healthy controls.b sTCC-normal: non-significant difference in baseline concentration of sTCC compared to healthy controls.c romiplostim vs. eltrombopag.ITP: immune thrombocytopenia; IVIg: intravenous immunoglobulin therapy; N.A.: not applicable; PLC: platelet count; sTCC: soluble terminal complement complexes; TPO-RA: thrombopoietin receptor agonist [0.08, 0.14] mg/L) after Bonferroni correction.No significant differences in the concentration of sTCC were observed for the sTCC-normal group during the study period.
A visually decreasing trend in the concentration of C4d (p = .08)was observed for the sTCC-high group.No significant differences in the concentration of C4d were observed for the sTCC-normal group during the study period.

Associations between levels of complement degradation product C4d and terminal complement activation
Spearman's rank-order correlation test indicated a significant positive correlation (r s = 0.52, p = <.001) between the concentrations of C4d and sTCC (both study groups and all time points included; n = 52).The post hoc ROC analysis confirmed a significant (p = .01)ability of C4d to distinguish samples with significant terminal complement activation as indicated by the cutoff for significantly elevated sTCC concentration (sTCC > 0.11 mg/L).The mean area under the curve was calculated to 0.74 (CI 95%: 0.59-0.90).

Discussion
This study evaluated the effects of complement activation on the platelet response in patients with primary ITP during TPO-RA therapy.To the best of our knowledge, this has not been investigated before.The cohort was divided into two study groups based on plasma levels of sTCC before the initiation of TPO-RA therapy.From a pathogenetic perspective, the cutoff was arbitrary as the effect of complement activation cannot be dichotomized.However, the cutoff enabled us to compare subjects with very high levels of sTCC at baseline to the remaining cohort.This was regarded as the most feasible approach considering the low number of subjects and observations as the small cohort setting limited the utilization of mixed effects models otherwise more advantageous in larger cohort designs.
A higher percentage of subjects in the sTCC-high (60%) compared to the sTCC-normal (10%) group were splenectomized prior to study entry.Only subjects in the sTCC-high group (40%) received third-line therapy options.Further, the sTCC-high group was significantly more prone to an inadequate platelet response and IVIg rescue therapy was exclusively administered to these patients during the study period.The main reason to assess the number of individual subjects with an inadequate platelet response (at any study visit) per study group in addition to the total number of study visits with an inadequate platelet response per study group, was that the resulting thrombocytopenia in primary ITP depends on multifactorial and dynamic immunogenic processes that potentially could affect the severity of thrombocytopenia differently in different patients during an observed treatment period.Thus, choosing one single time point to assess the platelet response, e.g. at the end of the study period, would risk to ignore clinically relevant information.However, merely Study sampling occasions: week 0 (baseline, i.e., before the initiation of TPO-RA therapy), week 2, week 6 and week 12. Due to the low number of observations per sampling occasion, particularly in the sTCC-high group, study period aggregates per variable and group are presented to show general non-temporal differences between the study groups.The proportion of sampling occasions missing for analysis per study group was similar.
All p-values were calculated using Fisher's exact test for categorical variables and the Mann-Whitney U test for continuous variables.a sTCC-high: significantly elevated baseline concentration of sTCC compared to healthy controls.Complement activation and TPO-RA therapy in ITP 5 presenting the total number of inadequate platelet responses per group would have introduced a presentation bias in our small cohort setting.That is, particularly treatment-refractory individuals would influence the total number of inadequate responses per group disproportionally.The sTCC-high group was, however, shown to exhibit a proportionally higher number of both individual subjects and study visits with inadequate platelet responses as compared to the sTCC-normal group.In summary, clinical differences between the study groups suggested that subjects with higher levels of sTCC at baseline required more intensive treatments before study entry and that they had more refractory disease.
Comparisons of the PLC revealed a lower PLC in the sTCChigh compared to the sTCC-normal group at baseline.However, comparisons for the succeeding sampling occasions only revealed significantly lower numbers in the sTCC-high group at week six (when there were two cases of missing data for the sTCC-high group).Given the small sample size, the temporary and partial boosting of the PLC following the administration of IVIg therapy in the sTCC-high group may have potentially obscured further actual differences.However, the comparison of study period aggregate values, reflecting general non-temporal differences between the groups, indicated a significantly lower PLC in the sTCC-high compared to the sTCC-normal group.Additionally, intragroup analyses showed that the sTCC-normal group responded with a significantly increased PLC after the initiation of TPO-RA therapy whereas the temporal trend observed for the sTCC-high group was insignificant.In summary, differences in the PLC between and within the two study groups jointly suggested a worse platelet response during TPO-RA therapy for the sTCC-high compared to the sTCC-normal group.
In the sTCC-normal group, levels of sTCC and C4d were consistently low throughout the study period.For the sTCChigh group, however, it is uncertain whether the declining trends observed for sTCC and C4d were attributed to effects of TPO-RA therapy.Rather, the repeated administration of IVIg among these Figure 1.Temporal developments of the platelet response and complement levels.Week 0 equals baseline, i.e., before the initiation of thrombopoietin receptor agonist therapy.The Friedman test, followed by Dunn's multiple comparisons test and Bonferroni correction, was used to estimate statistical differences between the sampling occasions per variable within each study group.(A) Platelet count (PLC).In the sTCC-normal group, a significant increase was indicated between week 0 and week 2. No significant differences were observed for the sTCC-high group.(B) Soluble terminal complement complexes (sTCC).In the sTCC-high group, a significant decrease was indicated between week 0 and week 12.No significant differences were observed for the sTCC-normal group.(C) Complement degradation product C4d.A trending decrease was observed for the sTCC-high group (p = .08).No significant differences were observed for the sTCC-normal group.a sTCC-high: significantly elevated baseline concentration of sTCC compared to healthy controls.b sTCC-normal: non-significant difference in baseline concentration of sTCC compared to healthy controls.c One outlier related to the week 2 boxplot of the sTCC-high group is not shown (PLC 1369 x 10 9 /L) in order to maintain the graphical resolution of the boxplots.

d
Two outliers related to the week 2 boxplot of the sTCC-high group are not shown (sTCC concentration 0.54 mg/L and 1.2 mg/L, respectively) in order to maintain the graphical resolution of the boxplots.subjects likely influenced the trend.It has been shown that IVIg binds to complement fragments C3b and C4b, ultimately preventing the formation of TCC [25,26].Whether these interactions solely result in declining plasma levels of sTCC and C4d or whether it also reflects a temporary reduction of complement depositions on the membranes of platelets (and megakaryocytes) remains to be investigated.The inconsistent platelet response following IVIg therapy among subjects in the sTCC-high group was not supportive of either hypothesis.
Complement-mediated opsonization may contribute to enhanced splenic clearance of platelets via complement receptors on macrophages in the mononuclear phagocyte system [14], thus supporting the Fc receptor-mediated clearance of platelet-antibody complexes.Patients with refractory ITP have a 60% response rate (complete remission) to splenectomy [1], emphasizing the role of splenic clearance as an important cause of platelet destruction in ITP.Interestingly, a study by Peerschke et al. reported a trend toward an increased response rate to splenectomy in patients with a positive plasma complement activation/fixation capacity (CAC) test compared to CAC negative cases [14].These results provide support for the hypothesis that complementmediated phagocytosis is pathogenetically important in a subset of patients.Evidently, the involvement of the splenic mononuclear phagocyte system was not a contributing factor for the splenectomized patients in the present study.However, complement activation may possibly contribute to disease by means of other mechanisms, e.g., due to direct platelet lysis secondary to increased membrane formation of TCC.In some patients with ITP, a loss of fluid-phase complement inhibition has been observed [15].
As titers of detected autoantibodies do not correlate with disease severity, other biomarkers for disease monitoring are needed.For the subset of patients affected by enhanced complement activation, C4d is a biomarker candidate that is theoretically superior to the measurements of other complement proteins previously reported [10][11][12]27].C4d is a degradation product Complement activation and TPO-RA therapy in ITP 7 generated by factor I from C4b during complement activation and it reflects the engagement of the classical and lectin pathways.It has been utilized to monitor for antibody-mediated allograft rejections [17] and, more recently, the risk of flare-ups in lupus nephritis [18,28].Its feasibility as a biomarker is supported by its stability.Its plasma levels are independent of the elevated synthesis of complement components caused by acute-phase responses [17].Hence, it accurately reflects the true magnitude of classical and lectin pathway-mediated complement activation.The potential need for a reliable complement biomarker is highlighted by results from a recent study by Broome et al.where seven patients diagnosed with chronic ITP, and who were unresponsive to two or more conventional therapy options, were treated with the monoclonal anti-C1s antibody sutimlimab.Treatment resulted in a rapid increase in the PLC, however, a sustained response was conditioned by retreatment after the washout period of four weeks [29].Thus, a reliable complement biomarker would possibly aid in the identification and monitoring of patients with ITP who would be eligible for complementtargeted therapeutics.In the present study, there was a significant positive correlation between levels of C4d and sTCC.Additionally, the ROC analysis demonstrated a significant ability of C4d to distinguish samples with significant terminal complement activation.Thus, our study agreed with the understanding that engagement of the classical pathway is pathogenetically important and our results propose that C4d and sTCC may prove valuable as future plasma biomarkers.
The present study was obviously burdened with several important limitations.First, no sample size estimations were conducted prior to study inclusion.Thus, the low number of participating subjects evidently made the statistical analyses hypothesis-generating at best.Consequently, the low sample size ruled out the ability to reliably adjust for confounders.The comparisons of platelet responses were likely prone to type II statistical errors owing to the exclusive administration of IVIg in the sTCC-high group.Additionally, no information on adjustments of TPO-RA dosages was included.Practically, however, patients who lacked an initial response routinely had their doses increased.This likely resulted in higher a proportion of adjustments carried out in the sTCC-high group, again making the statistical analyses prone to type II errors.Thus, we hypothesize that differences in the platelet response would be more evident in a larger cohort setting that could be more optimally adjusted for confounding factors.Secondly, the anti-platelet antibody status among the subjects was unknown, preventing comparison of the association between the platelet response and complement biomarkers for confirmed autoantibody-positive vs. -negative cases.Thirdly, in the C4d assay, we identified a tendency toward higher concentrations among the healthy control subjects and in the sTCC-normal group as compared to historical data from in-house healthy control samples used in previous studies.This aberration may be attributed to ex vivo complement activation resulting from repeated freeze-thaw cycles.However, as the control samples had been handled simultaneously with the study samples, they were similarly treated.The unexpectedly high levels of C4d among the control subjects were also the reason to choose biomarker levels of sTCC, and not C4d, to determine the cutoff for study group classification.The finding obviously constitutes a central limitation, particularly regarding the general feasibility of C4d as a biomarker candidate, as our study did not include a priori stability analyses assessing ex vivo shifts in biomarker concentrations influenced by repeated freeze-thaw cycles and storage time in a controlled test sample setting.However, we remain confident that the internal comparisons of C4d and sTCC still are reliable.To support this statement, Spearman's rank-order correlation test showed a significant correlation between levels of C4d and sTCC.Moreover, the post hoc ROC analysis indicated a significant overall ability of C4d to distinguish samples with significantly increased sTCC levels.Consequently, internal ratios at the top-end concentrations of the two biomarkers were not skewed: levels of C4d were expectedly elevated in samples with significantly increased sTCC concentrations.Finally, although the studied complement biomarkers showed an association between the classical pathway and terminal complement activation, we did not investigate the alternative pathway.Thus, we cannot exclude the potential contribution of simultaneous amplification generated by the alternative pathway.Given our restricted sample volumes and previous studies of ITP supporting classical pathway-mediated complement activation, we anticipated that C4d was the most relevant upstream complement biomarker to analyze.
In conclusion, for the first time, an inverse relationship was prospectively shown between biomarkers of complement activation and the platelet response during TPO-RA therapy in a small cohort of patients with primary ITP.This calls for further studies to confirm the results and the reliability of C4d and sTCC as plasma biomarkers in this setting.The implementation of routine complement testing in patients with primary ITP may prove clinically valuable as a subset of patients with difficult or refractory disease could benefit from complement-targeted therapeutics.

Figure 2 .
Figure 2. Temporal development of the platelet count per subject.Week 0 equals baseline, i.e., before the initiation of thrombopoietin receptor agonist therapy (TPO-RA).Timing of administered intravenous immunoglobulin (IVIg) therapy in the sTCC-high group is illustrated.Illustrations include the platelet overshoot registered for one subject at week 2. No subjects in the sTCC-normal group were treated with IVIg during the study period.Interpolated missing values are provided for graphics only.a sTCC-high: significantly elevated baseline concentration of soluble terminal complement complexes (sTCC) compared to healthy controls.b sTCC-normal: non-significant difference in baseline concentration of sTCC compared to healthy controls.

Table I .
Cohort characteristics at baseline.: https://doi.org/10.1080/09537104.2022.2159019 Baseline equals week 0, i.e., before the initiation of TPO-RA therapy.The study cohort was divided into two study groups.Healthy control subjects were used to determine the cutoff value for the classification.Cutoff for significantly elevated baseline concentration of sTCC was determined to be >0.11mg/L.Five and ten study subjects were classified as sTCC-high and sTCC-normal, respectively.All p-values were calculated using Fisher's exact test for categorical variables and the Mann-Whitney U test for continuous variables.PLC: platelet count; sTCC: soluble terminal complement complexes; TPO-RA: thrombopoietin receptor agonist DOI

Table II .
Clinical characteristics according to study group classification.

Table III .
Temporal development of the platelet response and complement levels.