Abstract
Background
Hemodialysis patients who are subject to increased risk of hemorrhage may need specific dialysis regimes to avoid bleeding. The aim of this study was to determine in vitro which of various anticoagulation options were most beneficial.
Materials and Method
60 in vitro hemodialyses (HD) were performed in parallel using blood from healthy donors. The dialysis circuits were rinsed with either 1 L of 0.9% NaCl alone (n = 6), or with 1 L saline and the addition of either 5 mL 20% albumin (Alb, n = 6), 5,000 U of heparin (Hep, n = 6), Hep and Alb in combination (HA, n = 30), 20,000 U of Hep and Alb (4H-A, n = 6), and finally Hep and 20 mL 20% albumin (H-4A, n = 6). The blood was recirculated for a maximum of 192 min. Clotting was graded.
Results
A 192 min dialysis was completed with all series of HA, 4H-A, and H-4A, all with a slight grade of clotting. In contrast to the above settings (p = 0.002, Fisher's test), a total clotting of the dialysis circuit occurred for all series using the NaCl rinsing alone (median time to stop: 21, range: 18–27 min, p = 0.026 compared to the HA setting) and for the Alb rinsing (median 26, range: 19–35 min, p = 0.028).
Conclusions
Priming using HA, Hep, 4H-A, and H-4A reduced clotting and allowed 192 min of HD. Clinical studies need to confirm these data in vivo.
Introduction
Acute and chronic renal disease (end-stage renal disease) in most instances is treated with hemodialysis (HD). Each HD session lasts from a few hours up to 24 hours per day. During HD the activation of extrinsic and intrinsic pathways of coagulation results in more or fewer clotting problems with the dialysis circuit (1, 2). In order to prevent clotting, the most common method is to administer anticoagulants such as heparin as a bolus at the start and as a subsequent infusion during the hemodialysis (HD). Another option is to administer a bolus with only low molecular weight heparins such as tinzaparin at the start of HD (3). The disadvantage of using systemic anticoagulation is the risk for bleeding in vulnerable patients such as those who are pre- or postoperative. To avoid systemic anticoagulation, one method consists of intermittent flushes of saline into the dialysis circuit just before the dialysis filter (4). The dilution reduces the hematocrit in the dialyzer and thereby the risk for clotting (5). However, the fluid flushed into the patient in most cases needs to be removed by ultrafiltration (6), which again increases the hematocrit. Another method is to infuse a citrate solution into the circuit (regional administration) before the dialyzer. The anticoagulant effect is reversed, by adding calcium ions after the filter (7–9). This method might increase the risk of metabolic alkalosis as the citrate follows the blood back into the patient (7, 9, 10). A lower risk for alkalosis is presented by the use of citrate in the dialysis fluid. This results in local anticoagulation in the dialyzer (11). However, a significant proportion of these patients need additional anticoagulation (11).
Another method is to rinse or flush the dialysis circuit with a solution of heparin, as it can provide a local anticoagulant effect on the surface of the filter and tubes while not being administered systemically into the patient. In a six-month patient study, heparin-flushed membranes permitted a 50% reduction in the standard UFH and LMHW doses. The feasibility of producing a 50% reduction in heparin dose with heparin-flushed AN69 ST membranes was shown again in a six-month study comparing this technique to non-heparin-binding dialyzers subjected to full-heparin dosing by Chanard and others (12, 13). However, some authors showed negative results (14) while others found a reduction of thrombogenicity (15, 16). When rinsing the dialysis circuit with a combination of heparin and albumin (HA) the extent of anticoagulation used, in clinical practice, could be reduced in patients with an increased risk for bleeding (17). This method, however effective, likely has room for improvement as there has not been any study to determine the optimal amounts of the components in the priming solution.
The aim of this study was to design a controlled in vitro environment to systemically examine the potency of the components in the priming solution. This would enable investigation of different priming methods to clarify which of them have a beneficial outcome, motivating their use in hemodialysis patients prone to bleeding.
Materials and Methods
In total, 70 in vitro hemodialyses were performed. Blood from five donors was used for quality assessment and to develop the models used in the studies (5 parallel series, n = 10 hemodialyses). Blood from 30 other healthy donors was collected and divided equally, to be used in parallel between two dialysis settings and machines (30 parallel series, n = 60 hemodialyses). The healthy donors were not taking any anticoagulant medications. Studies were performed according to Helsinki declarations, after information and consent was obtained. The study was approved by the local ethics committee (Dnr 2012-232-31M, March 6, 2012).
The whole blood was collected during blood donation (if the capillary screening test of the blood hemoglobin was above 120 g/l) by the Blood Center at the University Hospital, using the standard method. The collection bags were modified, excluding the white blood cell filter and the fourth bag, which was not necessary for our experiment. A total of 450 mL of whole blood was collected in the first bag and mixed with 63 mL of citrate fluid as anticoagulation, already present in the bag. The blood was then divided equally between the second and third bag using a scale. Two dialysis circuits were built using the same type of tubing system; the dialyzers were placed in parallel with two Fresenius 4008 dialysis machines (Fresenius Medical Care, Bad Homburg, Germany). Each circuit was connected to the respective blood bag. The two dialysis circuits were rinsed/primed differently. After rinsing the circuits with various rinsing/priming solutions, the blood bags were connected and the blood was pumped through each dialysis circuit. This allowed the remaining rinsing/priming fluid to be pushed out into a waste-container. When the rinsing fluid was eliminated (blood visible at the end of the circuit), each system was closed for recirculation and the dialysis process initiated.
One liter of saline (0.9% NaCl) priming solution was used as the basis for the various rinsing solutions employed for the dialysis circuits. The dialysis solution contained potassium in a final concentration of 3 mmol/l, calcium 1.25 mmol/l, glucose 5 mmol/l, Na 136 mmol/l, and bicarbonate 32 mmol/l. Dialysate flow was 500 ml/min.
The study was divided into two parts. In the first part we investigated the efficacy of various priming solutions at maintaining patency of the dialysis circuit. An in vitro model was set up. In the second part, the ratio between heparin and albumin was changed in contrast to that used in the first part of the study.
No systemic anticoagulation was used. As a control series we used saline alone as one priming option (n = 6). With the concept of coating the dialysis circuit with a protective layer, we perfused the dialysis circuit with a solution of saline alone or 1 L saline containing either 1) 5 mL of 20% albumin (Alb, n = 6); 2) 5,000 U of heparin (Hep, n = 6); or 3) with a combination of heparin and albumin as above (HA, n = 18; Tab. I). In these experiments we used the dialyzer F4HPS (n = 36 dialyses, Fresenius Medical Care) with a blood pump speed of 200 ml/min. The F4HPS dialyzer contains low-flux membranes that eliminate heparin molecules only to a negligible extent.
Priming Methods
In the second part of the study, again with the concept of covering the material of the dialysis circuit, we perfused the dialysis circuit with a solution of saline containing either 3) a combination of heparin and albumin as above (HA, n = 12); 4) four times 5,000 U of heparin and 5 mL albumin (4H-A, n = 6) or V): 5,000 U of heparin and four times 5 mL of albumin (H-4A, n = 6). These series 4 and 5 were also compared with the HA solution (placed in parallel; in total n = 12 runs). In the first series a recirculation time of less than five minutes was used before blood was connected. In clinical practice and in the second series, the priming solution was perfused through the dialyzer only, without recirculation. To facilitate removal of free heparin (not fixed or loosened from the surface of the material) from the recirculating system we used a high-flux dialyzer FX-50 (n = 24, Fresenius Medical Care) in the second part of the study. For this phase, we used the tubing setting of the model AV-Set FMC Pead/Baby-R (Fresenius Medical Care). With these narrow tubes the speed of the blood pump was kept at 100 ml/min. This setting of the dialysis circuit contained less than half of the blood volume collected, enabling more frequent blood sampling.
The whole blood was recirculated and dialyzed for a maximum of 192 min. Clotting was evaluated by objective grading of the dialyzers and venous air trap, ranging from Grade 0: no visible clotting, to Grade 1: lightly striped capillaries of the dialyzer, Grade 2: moderately striped, Grade 3: severely striped, and Grade 4: fully clotted (resulting in premature stopping of the dialysis).
Separate grading of the venous airtrap was investigated for a slight-to- extensive rim of clot at the fluid level, slight-to-moderate clots at the bottom, to fully-clotted chamber. Grading of clotting was performed by the investigating person. If the finding was not evident, a photo helped to evaluate the finding by another staff member.
Blood samples were collected before start and after 12 min and every 30 min until 192 min or until clotting of the system occurred. The samples were collected using citrate tubes. All samples were taken on the arterial side of the circuit. Ionized calcium was analyzed to clarify how long time it took until citrate was dialyzed out of the circuit (free ionized calcium close to 1.25 mmol/l, as present in the dialysate).
The bioactivity of UFH was clinically monitored by measuring activated partial thromboplastin time (APT), the increased time taken for clotting to occur (18, 19). Normal APT time at the laboratory was set between 24 s to 36 s. Dialysis was performed until it had either reached the final end of the experiment (192 min) or until total clotting of the device prevented further dialysis. After each fulfilled series and when clotting occurred, the system was flushed with a NaCl solution to rinse unclotted blood from the dialysis filters before performing the visual scaling of the clotting of the capillaries of the dialyzer. This rinse was continued until the dialyzer alarm was activated due to the fluid/blood being too diluted, thanks to the Fresenius device 4008 and its optical sensor, located after the air guard on the venous side.
During the preparatory studies to establish the model, it was found that the dialysis machines withdraw fluid differently from the 30–60 ml/h dialysis circuit, despite the device being set at zero for fluid removal. This loss of fluid from the system was compensated for by adding the same amount of saline per hour with an infusion pump into the venous air chamber after the dialyzer, before the return to the blood bag.
Another preparatory experiment was performed to evaluate how long the model took to neutralize the effect of citrate on calcium (citrate was included in the blood bags used for collection). We found out that this took approximately 12 min (Fig. 1). Therefore the experiments were set for 3 h + 12 min (192 min). When using the FX-50 the question was if the leakage of albumin through the membrane was extensive. In separate experiments, this loss of albumin was found to be less than 100 mg/3 h of HD.
Level of ionized Ca after start of dialysis procedure.
In another pre-preparation experiment we tested adherence/loss of albumin from the fluid by analyzing albumin concentration in mg/l before and during the dialysis.
Albumin concentration was analyzed with immunoturbidimetric analysis using Cobas 6000/8000 (ALBT2; Roche, Rotkreuz, Switzerland). This was performed to establish if there was any loss of albumin by attachment to or by filtration through the dialysis membrane of the dialyzer. In the same way we prepared our standardized H-A priming solution into a 2000 mL saline, as mentioned above, and primed the HD apparatus while the entire system was connected to a re-circuit. Before priming, the albumin concentration was measured directly from the 2000 mL bag. Then repetitive measurements of albumin were done every 15 min throughout an entire HD run of 180 min. The same type of High Flux FX-50 dialyzers were used. But an adult tubing system, from the AV-Set SRB-R 2008/4008 model (Fresenius Medical Care) was used instead of the AV-Set FMC Pead/Baby-R that was used during the blood experiment. Another difference was that the analyzed samples were taken from the venous injection port instead of from the arterial injection port.
Statistical analyses were performed using the Wilcoxon paired non-parametric rank test. Comparisons between groups were made using Fisher's test. A two-tailed significance value less than 0.05 was considered significant.
Results
Series 1
During hemodialysis all 18 treatments with HA as well as all 6 with Hep procedures were completed to the maximum length of 192 treatment minutes (see Tab. II). All HA and Hep treatments exhibited a slight grade of clotting (see Tab. III). This maintenance of hemodialysis function was longer than the median dialysis time of 21 min until total systemic clotting occurred that was found for the NaCl setting (n = 6, range: 18–27 min, p = 0.026 compared to the HA setting) and for the albumin priming (n = 6, median 26 min range: 19–35 min, p = 0.028). All dialyses using HA and Hep were able to be completed, in contrast to none of the ones with priming solution alone or with the addition of Alb alone (p = 0.002 Fisher's test).
Mean Dialysis Time Using Various Priming Techniques in Series 1
Extent of Clotting Seen with the Priming Techniques in Series 1
In addition to the results summarized above, other, more preliminary data was gathered at the early stages of designing the method used in this study. This data includes free calcium in the blood, which we used to evaluate when the anticoagulant effect of the citrate was reversed. The citrate was necessary while collecting blood to avoid uncontrolled clotting while setting up the dialyzers (Fig. 1).
We noted an increase in APT time in the preliminary data with the dialyzer. As this increase in APT time was not easily explained, we decided not to use this test to evaluate the priming solutions. More studies of these preliminary data are needed for them to be conclusive, but they served as an indicator when designing the model for our study. As these preliminary data are not complete, they are not shown in detail in this report.
The F4HPS filter that was used is a low-flux membrane that eliminates the heparin molecules only to a negligible extent.
Series 2
All dialysis runs using priming with HA, 4H+A, and H+4A were completed (192+ min) with grade 1 clotting. The HD runs that were performed with standardized H-A did not show any macroscopic clotting in the venous bubble catcher of the tubing system, and nor did the six HD runs with the 4H-A priming. On the other hand, the six HD runs with H-4A priming did show a slight suspicion of a macroscopic visible ring on the venous bubble catcher that could be washed away when flushing the system with saline. This clotting tendency was worse than the two other options (Fisher's test p<0.003).
Priming with H-A and H-4A showed that the APT time was maximal at 10 min and then decreased over time until 192 min in both models (p = 0.008 and p = 0.043, respectively). For 4H-A, the level was above 180 s for the whole series. The APT-time was significantly more reduced during HD with the setting H-A versus 4H-A (p = 0.010) while there was no difference in the change of APT time for H-A versus H-4A. There was no significant difference in change of platelets (B-TPK) during the 192 min of dialysis period between the series, even when adjusting for changes in blood hemoglobin or hematocrit.
The mean baseline values for blood hemoglobin were 145 (1SD = ±11 g/l), erythrocyte volume fraction 0.42 (±0.03), and platelets 217 × 10E9 (±47).
Using an albumin solution containing 1 g/l and perfusing the dialysis circuit for 60 min resulted in a loss of 60 mg albumin using the FX50 dialyzer and a 50 mg loss of albumin using the APS-18U dialyzer (Fig. 2).
Change in albumin concentration (mg/l) during recirculation of the dialysis circuit for 60 min.
Discussion
These in vitro models allowed easy estimation of clotting with various dialysis settings. The results indicate that no systemic anticoagulation or a limited amount of it is necessary using a priming solution containing heparin or heparin in combination with albumin. In contrast, priming with albumin alone or saline alone will cause more problems with clotting. In this setting, the HA and Hep-priming solutions can prevent the dialysis circuit from total clotting during a three-hour HD session. In clinical practice there have been controversial data associated with using heparin alone as a priming solution (12–16). Our clinical experience using HA priming showed that this concept allows a reduction and, in approximately 20% of patients, even total withdrawal of regional anticoagulation (17). Additional results in this study indicate that an increase in the ratio of heparin may result in an increased risk for bleeding compared to when using the HA and H+4A options. The somewhat increased tendency to clot using the H+4A option has to be confirmed in a clinical setting. A clinical comparative study should also clarify if flushing using a solution containing heparin alone has the same effect as one including both heparin and albumin, since heparin alone is the most routinely employed form of priming solution.
It is noteworthy that the APT time was significantly increased above the normal limit during the experiments. This may be due to partly retained heparin in the priming fluid mixed with the blood, and also due to leakage of heparin from the surface over time. Therefore it is important to consider the type of dialyzer in relation to the clinical setting of the patient. However, in clinical practice the amount of heparin retained in the dialysis circuit after priming will be considerably diluted. For this reason, the effect in 500 mL blood in the experimental circuit, for example, should be considered in relation to the dilution of at least 3,500 mL blood in a patient. In vivo an additional metabolic breakdown of heparin is present.
The dialyzer used through these first experiments was a low-flux type and therefore did not have the capacity to dialyze heparin out of the dialysis circuit. We therefore performed the second part of the study using a high-flux dialyzer where heparin could partly be dialyzed out of the circuit. Using that setting, the HA priming solution seemed to be the best, since the APT time was within the range of determination and lowered significantly during the observation period, while 4H-A exhibited an APT time above 180 s throughout the whole experimental dialysis. The latter would constitute a greater risk for bleeding than HA priming in patients at risk. The H-4A priming fluid resulted in a smaller rim of clots in the venous bubble catcher, indicating an increased tendency for clotting compared to HA priming.
In this in vitro model, albumin alone was not significantly better than saline alone, although the patency of the dialyzers was maintained, at a mean, a few more minutes. Therefore, we feel that the concept of using albumin alone as priming, as suggested by Tan et al (20) is not supported by this study. In their concept, anticoagulant-free albumin dialysis was considered effective despite frequent circuit clotting that was described (20). However, the Tan study was performed using the molecular adsorbents recirculating system (MARS) in patients with hepatic failure. These patients also might have had more of an increased risk for bleeding and reduced ability for clotting due to liver failure.
In the present model we strived to come as close to the clinical setting as possible. The blood collection process was designed to not allow any blood-air contact, while still having the necessary anticoagulants in the blood bag. The serial setup allowed the same blood to be divided between the two dialyzers with the same mixture of citrate so as to obtain as accurate a comparison as possible between the different priming solutions.
Our tests of ionized calcium levels showed that the effect of the citrate was definitely eliminated from the blood within 12 min of dialysis (Fig. 1). This guided us to decide when to perform blood sampling.
The choice of dialyzer has to be taken into account when performing various experiments, but also in clinical practice. The F4HPS filter is a low-flux membrane and not very good at eliminating heparin, while the FX-50 does have this capacity.
A limitation of this study was that blood from dialysis patients was not used. In a clinical setting, dialysis patients would have less of a tendency toward clotting than blood donors. This is because in patients with renal impairment, uremic toxins and even anemia facilitates bleeding and therefore they require very minimal or no anticoagulation at all during their first HD session (21). Another limitation may be that recirculation was not used in the second series. Such recirculation could eventually increase the extent of coating. However, a priming volume of 2000 mL corresponds to approximately 10 dialysis circuit volumes. For future studies, it would be beneficial to have a study design in which the HD sessions continue until the circuit clotting has occurred. In this way, the difference between heparin and the solutions containing albumin could have been better highlighted.
In conclusion, this study shows two models that may be used for comparative experiments to investigate developments of dialysis procedures such as various priming modes. The data from this study indicate the benefit of having a priming solution containing heparin or a combination of heparin and albumin to allow hemodialysis without the addition of further anticoagulation. This can be helpful if other options are not possible when patients are at risk for bleeding. Since the in vivo situation also involves metabolic processes besides those present in only one isolated blood bag, clinical evaluation of these beneficial treatment concepts are necessary.