CONTINUOUS HEMODIAFILTRATION WITH BLOOD RECIRCULATION PREVENTS BLOOD CLOTTING

Continuous renal replacement therapy (CRRT), including continuous hemodialysis (CHD), continuous hemofiltration (CHF) and continuous hemodiafiltration (CHDF), is advantageous for patients with unstable hemodynamics because it gradually removes fluid and solute without altering the fluid levels or distribution. However, CRRT is associated with frequent filter clogging, especially in cases of severe sepsis with high circulating levels of cytokines, coronavirus disease 2019 (COVID-19), systemic coagulation activation, or catheter dysfunction. Because conventional CRRT involves a single pathway from the body to the hemofilter, the rate of blood flow from the body and the rate of flow to the filter are inevitably the same. Consequently, blood flow slows, resulting in a lower shear rate at the membrane surface and a greater accumulation of rejected solute, which shortens hemofilter life. In addition, a lower blood flow rate from the body is required for dialysis of neonates and infants, again resulting in a lower shear rate. To address this issue, outside-in hemofiltration has been proposed for long-term operation of hemofiltration. As yet, however, no trial with low blood flow rate has been carried out. One solution to the slow blood flow rate is the use of blood recirculation around the hemofilter, that enables separation of the blood flow from the body and to the hemofilter. Previous studies of such blood recirculation primarily focused on dialysis efficiency; they did not focus on clogging or longevity of dialysis filters. For instance, Mohamad used blood recirculation to observe the efficacy of hemodialysis with accelerated blood flow through a partially controlled feedback segment and measured the postdialysis-to-predialysis ratios of blood urea nitrogen (UN) and creatinine; neither were affected by the feedback. Another study evaluated “double path dialysis” with blood recirculation and measured its effect on the clearance of low- and high-molecular-weight solutes. Interestingly, one patient who was treated using CHDF, with and without partial blood recirculation, experienced improved filter life with recirculation. In that case, however, nafamostat mesylate, which is available only in Japan, was used as the anticoagulant, and no tests of coagulation, hemolysis, or dialysis performance were conducted. The aforementioned studies also did not measure transmembrane pressure (TMP), which is the pressure difference between the inside and outside of the hollow fiber membrane. A higher TMP at the same ultrafiltration rate indicates more clogging of the filter. The present study aimed to evaluate the beneficial effects of increasing the blood flow rate to the hemofilter using blood recirculation to reduce the TMP and material deposition on the filter membranes.

Flow settings

The flow rates of bovine plasma and blood were set at 30 mL/min in this study. In clinical practice, one session of intermittent hemodialysis for children lasts less than 6 h. The initial blood flow rate is 3 mL/kg per min, which is eventually increased to 5 mL/kg per min. When the body weight of the child is 10 kg, the blood flow rate is set to 30 mL/min. In the present study, the flow rate setting mimicked the conditions in the most severe cases encountered in clinical practice. The blood recirculation flow rate was set at 170 mL/min using pump P2 (Figure 1B), which achieved a rate of 200 mL/min to the hemofilter.

Test samples and anticoagulants

Bovine plasma was purchased from Funakoshi Co. Ltd. (Tokyo, Japan). The quality control criterion was a protein concentration of 6.5 ± 0.5 g/dL. After starting hemodiafiltration, heparin was injected for 1 h at a rate of 500 U/h upstream of chamber A to prevent clotting (Figure 1A and B). Bovine blood samples were purchased from Funakoshi Co. Ltd. The quality control criteria were a protein concentration of 6.5 ± 0.5 g/dL and hematocrit of 35% to 40%. The blood also included 10,000 U of sodium heparin. The temperature of the samples was maintained at 37 °C using a hot bath, which was mixed continuously with a magnetic stirrer. To prevent coagulation, 10% sodium citrate was injected at a rate of 40 mL/h upstream of chamber A (Figure 1A and B).

Calculation of the transmembrane pressure and sample collection

TMP was recorded and 10-mL blood samples were collected 10 min, 1 h, 2 h, 3 h and 4 h after starting hemodiafiltration. A video monitor was used to record the TMP for 60 s at each time point, and the average TMP was calculated using values recorded every 1 s during the 60-s period. Four hemodiafiltration trials were performed. Continuous hemodiafiltration with blood recirculation (CHDF-R) using bovine blood were performed five times. Experiments with bovine plasma were performed using a polysulfone hemofilter (SHG-1.0; Toray Medical Company Ltd.) with a membrane area of 1.0 m^2 and maximum allowable pressure of 500 mmHg. Experiments with bovine blood were conducted using a polymethyl methacrylate hemofilter (CH-0.6W; Toray Medical Company Ltd., Chiba, Japan) with a 0.6-m^2 membrane area and maximum allowable pressure of 500 mmHg. The rate of dialysate flow and filtration flow was set at 7 mL/min. A 1-L container mimicked the body, and samples were dialyzed by drawing blood from and returning the blood to the same container. The concentration obtained by ultrafiltration at 170 mL/h was corrected with an equivalent amount of saline to ensure that the total volume of the container was constant. CHDF and CHDF-R were performed under the same conditions, and 10-mL blood samples were collected from the sampling ports upstream of chamber A (Figure 1A and B), frozen, and stored at -80 °C until assays were performed.

Electron microscopy

A scanning electron microscope (Gemini®; Carl Zeiss Co. Ltd., Tokyo, Japan) was used to examine the surface of the hemofilter membranes after hemodiafiltration with blood plasma. The hollow fiber membranes were sampled 5 cm from the inlet. The membranes were dehydrated using an ethanol series consisting of 5 min each in 20%, 50%, 80%, 90%, and 100% ethanol. After drying, the membranes were frozen in liquid nitrogen. The areas where clogging was observed on the electron micrographs were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Coagulation analysis

The contact system is believed to be the main trigger for the coagulation cascade during extracorporeal circulation. We therefore, measured levels of D-dimer, thrombin-antithrombin III complex (TAT), and plasmin-alpha 2 plasmin inhibitor complex (PIC) to estimate the effects of CHDF-R on coagulation. D-dimer levels were measured using a latex photometric immunoassay with CP3000 (Sekisui Medical Corporation, Tokyo, Japan). TAT levels were measured using a time-resolved fluoroimmunoassay with a STACIA automatic coagulation analyzer (LSI Medience Corporation, Tokyo, Japan). And PIC levels were measured using a latex photometric immunoassay with a STACIA automatic coagulation analyzer (LSI Medience Corporation).

Hemolysis analysis

Hemoglobin (Hb) levels were measured using the sodium lauryl sulphate-Hb detection method with a XN-9000 instrument (Sysmex Corporation, Hyogo, Japan). Lactate dehydrogenase levels were measured using the International Federation of Clinical Chemistry and Laboratory Medicine method with a Toshiba 2000FR analyzer (Toshiba Medical System Corporation, Tokyo, Japan). Total bilirubin levels were measured using the bilirubin oxidase method with a Toshiba 2000FR analyzer (Toshiba Medical System Corporation).

Statistical analysis

Statistical analyses were performed using EZR statistical software version 1.55 (Saitama Medical Center, Jichi Medical University, Saitama, Japan). The TMP, D-dimer, Hb, lactate dehydrogenase, total bilirubin, and Kt/V values for CHDF and CHDF-R were compared using the Mann-Whitney U test. Statistical significance was set at p < 0.05.

FIGURE LEGENDS

Figure 1: Hemodiafiltration circuits. (A) Standard circuit (continuous hemodiafiltration, CHDF). (B) Novel blood recirculation circuit (continuous hemodiafiltration with blood recirculation, CHDF-R). P, pump; PG, pressure gauge. The ultrafiltration rate was 170 mL/h. Sodium citrate (10%) was injected at a rate of 40 mL/h upstream of chamber A. To maintain the same total volume in the container, the flow rate of normal saline was set at 130 mL/h.

Transmembrane pressure during hemodiafiltration

It has been suggested that a secondary protein layer forms during ultrafiltration. As hemodiafiltration proceeds, the protein layer accumulates on the surface of the hollow fibers, resulting in an increase in TMP. In the present study, that increase in TMP occurred over the course of hemodiafiltration as the membrane became increasingly clogged (Figure 2A and B). By the end of dialysis, TMP was significantly lower for CHDF-R than CHDF with bovine plasma (median CHDF: 23.7 mmHg; median CHDF-R: 18.1 mmHg; p = 0.029) or bovine blood (median CHDF: 151 mmHg; median CHDF-R: 96.5 mmHg; p = 0.016).

Membrane clogging

Because coagulation and the protein layer would be expected to impact membrane clogging, 6 macroscopic views and electron microscopy were used to evaluate clogging. Macroscopic views of the hemofilter membrane after the tests using bovine blood showed significantly less deposition due to thrombosis after CHDF-R than with CHDF (p = 0.009) (Figure 3A-C). Similarly, electron microscopic analysis of the internal surface showed that more substance was present on the internal surface of the hollow fiber after CHDF than after CHDF-R (p = 0.004) (Figure 4A-D).

Coagulation and hemolysis

Coagulation within the two circuits was compared by measuring D-dimer, TAT and PIC levels. D-dimer levels did not significantly differ between the two circuits (median CHDF: 0.555 µg/mL; median CHDF-R: 0.515 µg/mL; p = 0.081), and all TAT and PIC levels were below the detection limits of 1.0 ng/mL and 0.5 mg/mL, respectively. The higher flow rate of 200 mL/min to the hemofilter during CHDF-R did not appear to affect the coagulation pathway.

To assess hemolysis, we compared the 4-h levels of Hb, lactate dehydrogenase, and total bilirubin for CHDF and CHDF-R. None of these three indicators significantly differed between the two circuits. Neither the higher flow to the hemofilter nor the blood recirculation appeared to cause hemolysis.

FIGURE LEGENDS

Figure 2: (A) Transmembrane pressure (TMP) during hemodiafiltration with bovine plasma. Box plots show the TMP at the indicated times during continuous hemodiafiltration (CHDF) and continuous hemodiafiltration with blood recirculation (CHDF-R) (n = 4 in each group). Blue circles represent the TMP with CHDF, red circles with CHDF-R. The TMP at 3 h with CHDF-R was significantly lower than with CHDF (p = 0.021). (B) TMP during hemodiafiltration with bovine blood. Box plots show the TMP at the indicated times during CHDF and CHDF-R (n = 4 in CHDF, n = 5 in CHDF-R). Blue circles represent the TMP with CHDF, red circles with CHDF-R. The TMP at 4 h with CHDF-R was significantly lower than with CHDF (p = 0.016). 

Figure 3: Macroscopic view showing deposition due to thrombosis on the hemofilters after 4 h of hemodiafiltration. Photographs of the dialysis membranes were converted to grayscale images, and the degree of deposition was measured. (A) Representative hemofilter after continuous hemodiafiltration (CHDF). (B) Representative hemofilter after continuous hemodiafiltration with blood recirculation (CHDF-R). (C) Statistical comparison of deposition on the hemofilter after CHDF and CHDF-R (p = 0.009).

Figure 4: Internal surface of the hollow fiber membranes. (A) Scanning electron micrographs of unused hollow fiber membranes supplied by Toray Medical Co. Ltd. The left side of the image shows the internal surface of the filter, while the right side shows a cross-section of the filter. (B and C) Scanning electron micrographs of the internal surface of the hollow fiber membranes after continuous hemodiafiltration (CHDF) (B) and continuous hemodiafiltration with blood recirculation (CHDF-R) (C) with bovine plasma. (D) Statistical comparison of substance on the hollow fiber membranes after CHDF and CHDF-R (p = 0.004). Scale bars = 1 µm.

The shear stress, t, can be calculated using the following formula: t = 4∙h∙Qb / πri^3. (t: shear stress, h: blood viscosity, Qb: blood flow rate, ri: inner radius of the hollow fibers) The shear stress is proportional to the blood flow rate. The blood flow rates to the hemofilter were set at 30 mL/min during CHDF and 200 mL/min during CHDF-R. Therefore, shear stress during CHDF-R was, theoretically, 6.7-times greater than that during CHDF. This suggests CHDF-R appears to suppress accumulation of clogging substances by producing higher shear stress within hollow fiber membranes. CHDF-R enables separate settings for the blood flow from the body and the flow to the hemofilter. CHDF-R could prolong the life of the hemofilters, resulting in lower costs. The abstract with similar content was submitted for the 70th annual conference of American Society for Artificial Internal Organs.