| Abstract|| |
Hepatitis-C virus (HCV) infection remains highly prevalent among hemodialysis (HD) patients, but there is a notable paucity of information on aspects of bio-incompatibility in those infected patients. This study aimed to answer the following question: In HD patients, does chronic HCV infection attenuate the acute inflammatory response that results from contact of patient's blood with the dialyzer membrane? We elected to investigate the contact response in the initial 15 min of a standardized single dialysis session in a before-after design. Thus, we compared magnitude of dialysis-induced changes in total leukocyte counts, platelet counts, and C3a levels in a cohort of HCV-infected and a cohort of non-infected dialysis patients (57 patients in each group). Distribution of gender, age groups, and hypertension was comparable in both HCV-infected and non-infected patients. Furthermore, the baseline pre-dialysis measurements of the studied biocompatibility markers showed statistically equivalent values in the two groups with the exception of a marginally lower platelet count among HCV-infected patients. After 15 min of HD, the total leukocyte count dropped by 16% in HCV-infected patients and by 21.5% in the non-infected group (P <0.01). However, in both groups of patients, all values remained within the customary warm zone of normal distribution of these cells in the general population. There was no statistically significant difference between the two groups in dialysis-induced thrombocytopenia or C3a levels. The magnitude of cell count reduction and complement activation in both the HCV-infected and non-infected groups of HD patients was modest and unlikely to symbolize any clinical relevance. A valid answer to our research question may be only distinctly obtained if novel molecular and sub-molecular biomarkers for detection of micro-inflammation are used.
|How to cite this article:|
Ahmed S, El-Sherif A. Interaction between blood and dialysis membrane in hepatitis-C virus-infected patients: A comparative study. Saudi J Kidney Dis Transpl 2016;27:677-84
|How to cite this URL:|
Ahmed S, El-Sherif A. Interaction between blood and dialysis membrane in hepatitis-C virus-infected patients: A comparative study. Saudi J Kidney Dis Transpl [serial online] 2016 [cited 2021 Oct 18];27:677-84. Available from: https://www.sjkdt.org/text.asp?2016/27/4/677/185223
| Introduction|| |
Despite the major advance in quality of the dialysis membranes, it remains true that some of the important adverse effects of hemodialysis (HD) technology emanate from exposure of blood to the HD membranes. Such exposure triggers inflammatory reactions with subsequent abundant alterations in blood elements. The various adverse interactions between the blood elements and the membrane are referred to as biocompatibility disorders, and these continue to be an important area for both research and technology. , Indeed, the HD procedure provides an excellent experimental model where components of inflammatory response may be studied different ways in patients with end-stage renal disease.
Hepatitis-C virus (HCV) infection is associated with several cellular and molecular alterations that influence the patient's inflammatory response to other infections and vaccination, well described in their review by Yao and Moorman.  For example, co-infection of hepatitis-B virus (HBV) in HCV patients leads to more severe liver disease with higher rates and faster progression to liver cirrhosis and liver cancer.  Furthermore, response to standard HBV-vaccine is often blunted in HCV-infected individuals when compared with the healthy subjects.  This phenomenon also occurs to other vaccines in adults.  Persistent activation during chronic HCV infection may lead to an exhaustion as well as senescence of immune resources. Essential features of immune senescence include reduced number and function of APCs in blood; reduced natural killer cell cytotoxicity; and decreased naive T-and B-cells with an increase in terminally differentiated lymphocytes. , Many of these features have been observed in HCV-infected patients. ,
The prevalence of HCV infection among HD patients is several folds greater than that of general population, and chronic HCV infection is associated with significant morbidity and mortality among those patients.  However, there is a paucity of information on whether HCV-infected patients react differently to the dialysis membrane, compared to non-infected HD patients. Indeed, we failed to identify any information concerned with that in the English-written literature. If such information becomes available, it may throw additional lights on understanding aberrations of inflammatory response of HCV patients to noxious stimuli and vaccination.
| Patients and Methods|| |
Fifty-seven HD patients infected with HCV and 57 non-infected patients were recruited from the dialysis units of Suez Canal University and Ismailia General Hospitals in Ismailia, Egypt. HCV infection was documented following the condition of having positive HCV antibody (HCV-Ab) test confirmed repeatedly over a period of six months or a positive HCV-Ab test plus a positive virus RNA test. Informed consent has been obtained from all subjects.
Patients with any of the following conditions were not enrolled in the study: chronic inflamatory diseases, acute infections, pregnant women, HBs Ag-positive patients, and patients who received blood transfusion within the preceding two weeks. Patients received drugs known to alter the acute phase response or to alter blood cell counts were also excluded from the study. [Table 1] compares the two groups for baseline demographic and clinical characteristics.
|Table 1: Comparison of the two groups (hepatitis-C virus antibody-positive and hepatitis-C virus antibody-negative patients) for the baseline clinical characteristics and laboratory investigations.|
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Each patient served his own control in a before-after design of a standardized single dialysis session. Test dialysis session was planned to follow a minimum of two weeks of adequate dialysis (i.e. three-dialysis sessions/ week, each for more than 3.5 h). The test session was scheduled to be preceded by a regular session done no longer than 48 h earlier, with the targeted ultra-filtration not exceeding 4.5% of the dry body weight.
The test session was commenced as a heparin-free one until blood sampling was completed. The dialysis procedure was performed each time using a new polysulfone (PS) membrane dialyzer of a fixed surface area of 1.3 m 2 (F6HPs dialyzer, Fresenius, Wendel GmbH, Frankfurt, Germany) and bicarbonate dialysate. Blood flow rate was kept at ≈250 mL/min and the dialysate flow around 500 mL/min with a temperature of 37°C during the 15 min sampling period. Water for the dialysate was purified by reverse osmosis.
Blood sampling and laboratory studies
Blood samples were collected just before commencing dialysis (pre HD sample), and at min "15 th" of dialysis. The 15 th min time point was selected after reviewing several studies. Majority of studies concerned with dialysis biocompatibility has shown that the peak changes in inflammatory markers are observed 15 min after commencement of dialysis and wane progressively thereafter.  The pre-HD samples were collected from the afferent sampling port before connecting to the dialyzer. The second sample was collected from the efferent sampling port without changing the blood flow rate. Samples for C3a and liver function tests were separated immediately after centrifugation and stored at −20°C for one to two months, whereas platelet counts, total leukocyte counts, and prothrombin time (PT) measurements were obtained immediately.
Total leukocyte and platelet counts were measured on automated hematology analyzer (Sysmex KS-21 N; Sysmex Corporation, Kobe, Japan). Serum albumin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined on Cobas6000 (Roche Diagnostics, Indianapolis, IN, USA). PT was determined using automated coagulometer; CDX-II (Diamed, Canada). C3a was determined with human complement C3a ELISA kit (WKEA MED Supplies Corp., Changchun, China). This kit is designed to provide a solid phase biotin-avidin assay to detect human C3a in the tested samples. It uses a biotinylated purified human C3a antibody coated microtiter plate wells. The concentration of C3a in the samples was determined by comparing the optical density of the samples with the standard curves (assay range 7-150 μg/mL).
| Statistical Analysis|| |
The paired t-test was used for comparison of continuous variables for differences induced by the test dialysis session within the same group. Comparisons between the two groups were done using the unpaired t-test. Chi-square test was used for comparison of qualitative data. A P <0.05 was considered statistically significance.
| Results|| |
Fifty-seven HD patients infected with HCV and 57 non-infected patients on maintenance HD therapy for at least three months were recruited from the HD units of two liaising hospitals for this study. [Table 1] shows that at commencement of the study, distribution of gender, age (by group), hypertension, and liver function tests were comparable in the two groups but duration on dialysis was significantly longer in HCV-infected patients. We elected not to match for the duration on dialysis because majority of patients generally acquires HCV infection related to the vintage on dialysis and hence matching for that variable can distort clinical representation and allow for effects of some innate biases. Nevertheless, more than half of patients in each group were on dialysis for longer than four years.
The baseline (pre-dialysis) measurements of the three studied biocompatibility markers were comparable in the two groups. Values in both groups were collectively within the dense central zones of normal reference ranges and the differences between HCV Ab-positive and Ab-negative patients were generally small and of no clinical importance. This was evident, even though the difference for platelet counts was statistically marginally significant (P = 0.048), favoring true lower platelet counts among the HCV Ab-positive patients compared with HCV-Ab negative patients (155.35 ±50.04 and 176.84±63.96/mm 3 , respectively). Of note, the variability in pre-dialysis C3a levels among patients of each group was substantial. Coefficients of variation for C3a distribution were 1.47 and 1.1 for HCV-positive and negative patients, respectively.
[Table 2] demonstrates the differences induced by HD (measurements obtained before dialysis and at 15 th min of dialysis) for each of the three studied measurements in the two groups. Both HCV Ab-positive and Ab-negative patients displayed an analogous response to dialysis. Specifically, dialysis has induced modest, but statistically significant, reductions in counts of the two cell lines, more in leukocyte than platelet counts. On the other hand, C3a levels did not significantly change as a result of blood-membrane interaction.
|Table 2: Before-after dialysis measurements of the studied biocompatibility markers for each group (paired t-test).|
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The changes observed as a result of this interaction in the experimental session are displayed in [Figure 1]. In HCV-Ab-positive and negative patients, respectively, the dialysis-induced reductions in platelet counts (−6.3±20.8 and −10.07±21.81/mm 3 , P = 0.35) and the increase in C3a levels (0.52±16.32 and 6.239±54.6 μg/mL; P = 0.45) were comparable. Reductions in total leukocyte counts (TLCs) in the HCV-infected patients were less remarkable than those in non-infected patients (−1.049±0.8 and −1.5±0.99/mm 3 , respectively); here differences between the two groups attained the level of statistical significance. [Figure 1] also demonstrates a trend of predominantly small changes in cell counts in patients of the two groups, whereas C3a levels tended to cluster in the majority of patients below 50 μg/mL and remained so 15 min after dialysis.
|Figure 1: Platelet counts, leukocyte counts, and serum C3a levels, measured before and 15 min after hemodialysis in hepatitis-C virus antibody positive patients (a) and hepatitis-C virus antibody-negative patients (b); (n = 57 patients per group). TLC: Total leukocyte count.|
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| Discussion|| |
Substantial improvement in quality of synthetic material used for dialysis membrane has been witnessed during the past decades.  However, the problem of bio-incompatibility of artificial dialyzer membranes (which get in contact with the patient's blood for 12 h per week) still remains an important concern in HD practice.  Many markers of blood-membrane interaction have been employed during the past decades but these markers have not been evaluated in patients with altered immunological profiles. Herein, we attempted to investigate whether the consequences of that interaction differed between HCV-infected and non-infected HD patients.
The prevalence of HCV infection among patients of the two studied dialysis units was slightly higher than 40% (unpublished observation). Despite the fact that in many areas around the world HCV infection is still highly prevalent in dialysis units, there is a notable paucity of information on aspects of biocompatibility in those infected patients. In non-dialysis patients, HCV infection has been shown to be associated with several cellular and molecular alterations that influence immune response. Natural killer cell dysfunction has been observed in chronic HCV-infected patients  and function of dendritic cells was reported to be impaired earlier, even in patients with normal ALT.  Furthermore, HCV infection was shown to induce an alteration on neutrophil oxidative burst function. 
In this study HCV-infected patients had a longer duration on dialysis therapy and were older than non-infected patients. This and the other differences observed in pre-dialysis baseline features between the two groups concur with the corroborated information that incidence of HCV infection steadily increases with advance of time on HD. ,,, For this reason, we elected not to match the two groups for "duration on dialysis;" favoring to assert clinical generalizability of results rather than to support non-representative but age-comparable cohorts.
The overwhelming majority of patients of both groups had ALT, AST, and PT levels clustered in the inner zones of the normal reference ranges of these variables, reflecting quiescent disease activity among the infected patients. Differences in baseline cell counts between the two groups of patients were modest and coherent with those reported in the literature. Similar to non-dialysis patient, the prevalence of leukopenia and thrombocytopenia was observed to be higher in patients on HD with HCV infection than those without. 
In our study, there was no statistically significant difference between the two groups in dialysis-induced thrombocytopenia. After 15 min of dialysis, the platelet counts dropped by 3% and 5.6% of the pre-dialysis levels in HCV-infected and non-infected patients, respectively. Differences in percent reduction between the two groups of patients were not statistically significant. Even though the change induced by dialysis in TLC was statistically higher among the non-HCV-infected patients, the magnitude of count reduction in both groups was modest and far from symbolizing any clinical relevance.
In HD patient at large, studies using PS or other synthetic dialysis membranes have yielded variable but frequently tenuous results. Stefoni et al  found that PS membrane led to low leukocyte count at 15 th min of dialysis but had no effect on platelets. Daugirdas et al  reported that a substantial degree of platelet activation could be observed with PS and other synthetic membranes, but the amount of activation differed substantially among different brands. A recent study by Nasr et al  using a newer generation of Fresenius PS membrane demonstrated that there is no significant effect on platelet counts, using this membrane. In a review of recent literature pertaining to the effects of HD on platelet count and function, authors  pointed out that more attention needs to be paid to potential synergistic effects between dialyzer membrane manufacturing processes and methods of sterilization, and especially to determine whether and how the use of electron beam sterilization may impact the interaction between certain PS membranes and platelets.
The reduction of leukocyte count observed 15 min after starting dialysis has been generally attributed to the sequestration of the leukocytes to the pulmonary circulation. Conventionally, that has been ascribed to complement activation and the up-regulation of the adhesion-promoting integrins of these cells, resulting in their binding to the endothelial cells. , An increased formation of platelet-neutrophil aggregates was observed during dialysis with cellulose diacetate and PS but not with ethylene-vinyl membranes.  Authors of the latter study have indicated that cellular activation depends largely on the membrane material. However, the mechanism of direct membrane activation of neutrophils is not fully clarified. 
Pertinent to our study is the observation that complement C3a is elevated in non-dialysis patients with chronic hepatitis C and HCV-related hepatocellular carcinoma.  Furthermore, it has been found that the C3 complement component is significantly reduced in serum and liver biopsy specimens from chronically HCV-infected patients, but C3 reduction did not appear to be related to the viral load or the stage of liver disease. 
The complement activating potential of biomaterial is one of the important biocompatibility features. However, in an old observation, Hakim and Lowrie  found that patients dialyzed with the same membrane have different levels of C3a and C5a. In our study, the pre-dialysis levels of C3a were highly variable among all patients. Such high variability remained obvious after 15 min of dialysis; attenuating the probability of detection of dialysis-induced differences in C3a levels between patients of our two groups.
| Conclusion|| |
To conclude, in our study neither the basal levels nor the magnitude of dialysis-induced changes in the above-mentioned three measurements was importantly different in infected patients from the non-infected ones. The study supports the evidence that the currently used generation of dialyzers is far more biocompatible than the previous generations. Such higher biocompatibility should have diluted the cogency of the used conventional markers and thwarted the detection of any significant differences between our two groups of patients. Hence, it remains true that contact of blood with any synthetic membrane does certainly induce micro-inflammatory phenomena that could have been difficult to verify by the crude measurements adopted by this and some other biocompatibility studies.
Given the above, it seems reasonable that a valid answer to our research question may be more distinctly obtained if novel more sensitive biomarkers for detection of micro-inflammation are used. Candidate markers may incorporate transcellular and intracellular machinery events at a molecular level such as immune transcriptomic studies of blood cells, markers of cell apoptosis, or the mitochondrial respiratory system. Probably then, it could be possible to find out if hepatitis-C patients present inflammatory response different from that of non-hepatitis dialysis patients.
Conflict of interest: None declared.
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Department of Medicine, Renal Unit, Suez Canal University, Ismailia
[Table 1], [Table 2]