Assiut Med. J. Vol. (38), No. (3), March, 2015

Assiut Med. J. Vol. (38), No. (3), March, 2015

Role of Haemodiafiltration in Reducingβ2-microglobulin and Phosphorus in Patients with ESRD versus Conventional Haemodialysis

Essam Mohamed Abd-El Aziz Ali*Ashraf Anwar Al-Shazly *Refaat Fathy Abd-El Aal*, Samir Kamal Abdu-El Hamid*,Shabaan Radwan Helal**Madleen Adel A. Abdou**

The Department of Internal Medicine*,The department of Clinical Pathology**, Faculty of Medicine-Assiut University.

ABSTRACT

Online haemodiafiltration (HDF) offers the best renal replacement therapy (RRT) option for end stage renal disease (ESRD) patients. By combining diffusive and convective transfer in the same exchange module, HDF provides the highest clearances of both small and large solutes .Objective:To assess Β2-Microglobulin level in patients undergoing haemodiafiltration compared to those undergoing conventional haemodialysis, To asses the role of haemodiafiltration in clearing of phosphorus in patients with ESRD and To introduce a new technique of dialysis in Dialysis Unit of Assiut University Hospital.Methods:47 patients with chronic renal failure on regular hemodialysis for at least six months,non diabetic ,aged 18-60 years,were classified into two groups:group(A),were 23 patients shifted from conventional HD to post-dilution HDF and group(B),were 24 patients continue their conventional HD rigmen(control group) and and the two groups were on 3 sessions/week 4 hours each, rigmen and subjected to full history and clinical examination,routine investigations in addition to measurement of serum Β2-Microglobulin and Phosphorus level at the beginning of the study and after six months(the duration of the study) irrespective to the days of HD or HDF.Results:The mean ± SD of β2-Microglobulin before HDF was 14.00± 3.07 ug/ml while after HDF was 11.45 ± 2.51 with significant reduction, the mean ± SD of β2-Microglobulin before HD was 12.37 ± 2.30 ug/ml while after HD was 13.72 ± 3.87 with insignificant rise and The mean ± SD of phosphorus before HDF was 6.84 ± 1.59 mg/dl while after HDF was 5.36 ± 1.11 mg/dl withsignificant reduction the mean ± SD of phosphorus before HD was 6.00 ± 2.04mg/dl while after HD was 6.02 ± 1.96 mg/dl with insignificant rise.Conclusion:Online HDF is an ideal HD technique for reducing Β2-microglobulin and phosphorus levels in HD patients.

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Assiut Med. J. Vol. (38), No. (3), March, 2015

Introduction and aim of the work

ß2-M (MW approximately 12,000D) is a component of the major histocompatibility antigen. Uremia-related amyloid is to a large

extent composed of ß2-M, and is essentially found in the osteo-articular system and in the carpal tunnel, although deposition can be systemic as well (Campistol J.M et al., 1990).

β2-microglobulin is a precursor in the formation of dialysis related amyloidosis and carpal tunnel syndrome and was a significant predictor for all-cause mortality, particularly for non-cardiovascular mortality ( Fry AC et al., 2007) in HD patients. Also,hyperphosphataemia is the main factor for inducing secondary hyperparathyroidism in dialysis patients,hence itching, metastatic calcifications and increased risk of all cause mortality, including cardiovascular mortality (Block GA et al., 2004) and these two important problems were commonly seen in our patients in the dialysis unit of Assiut University Hospital.So, we started studying the effect of post-dilution HDF in clearing these two harmoful substances (β2-microglobulin and phosphorus) and to introduce a new modality of HD in our dialysis unit.

HDF is a blood purification therapy combining diffusive and convective solute transport using a high-flux membrane characterized by an ultrafiltration coefficient greater than 20 mL/h/mm Hg/m2 and a sieving coefficient (S) for β2-microglobulin of greater than 0.6. Convective transport is achieved by an effective convection volume of at least 20% of the total blood volume processed. Appropriate

fluid balance is maintained by external infusion of a sterile, non-pyrogenic solution into the patient’s blood (Canaud B et al., 2006).

Uraemia is a pathological condition caused by the retention of solutes that are normally excreted by the kidneys. The aim of haemodialysis (HD) is to remove these solutes, but standard HD is not very efficacious, and patient morbidity and mortality rates are still very high (15–25% per year). (Eknoyan G et al., 2008).At present, online hemodiafiltration (HDF) offers the best renal replacement therapy (RRT) option for end stage renal disease (ESRD) patients (Canaud et al., 1993).

HDF methods require specific technical options and careful clinical surveillance to ensure safety and optimal performance (Canaud B et al., 2010).

• Patients should be equipped with a vascular access, regularly providing an extracorporeal blood flow of 400ml/min.

• A high-flux dialyser is required to perform HDF. Several dialysers made of synthetic polymers are available on the market. High-flux dialysers share in common a dialysis membrane with high hydraulic permeability (Kuf≥50ml/h/mmHg), high solute permeability (KoA urea>600 and B2MG>60 ml/min) and large surface of exchange (1.5–2.1m2).

• Certified HD/haemodiafiltration (HDF) machines (blood monitoring and dialysate proportioning system), specifically designed to safely achieve the dialysis session and to monitor treatment, are required. Bicarbonate-based dialysate solutions and ultrafiltration controllers have been generalised in dialysis machines, rending dialysis a very smooth process.

• Ultrapure dialysate is a standard for HDF machines. This is currently achieved by feeding the dialysis machine with ultrapure water. The production of sterile and non-pyrogenic dialysis and substitution fluids is achieved by online ‘cold sterilisation’ of the dialysate, based on two sterilizing ultrafilters interposed on the inlet dialysate line.

• low resistance (arteriovenous pressure drop less than 300 mmHg). Moreover, the patient’s vascular access must be able to permit blood flow rates in the 400 ml/minute realm and the HDF machine must be able to generate 600–800 ml/minute of dialysate fluid. Online HDF machines have the capacity to divert a fraction of the proportioned dialysate (varying from 50 to 200 ml/minute) to be used as the substitution fluid.

There are four common types of HDF according to the site of infusion of the substitution volume in extracorporeal blood circuit (post-dilution,pre-dilution,mid-dilution and mixed-dilution HDF).

The removal of B2-microglobulin has been proved to be superior with convective techniques in numerous studies. In fact, whether looking at reduction ratios ( Krieter DH et al., 2005). or at mass removal, HDF has consistently shown superior results by a factor of more than two to those obtained with high-flux hemodialysis. Once again, this improved removal translates into clinical benefits for patients. On the one hand, pre-dialysis B2-microglobulin levels progressively decrease in patients switched to high-efficiency HDF and this difference is all the more amplified when limiting the analysis to ESRD patients without residual kidney function. On the other hand, in the long term, convective therapies can nearly halve the risk of developing B2-microglobulin amyloidosis ( Susantitaphong P et al., 2010).

Phosphate (a small solute that behaves like a middle substance owing to a surrounding hydration shell) appears to be well cleared by convective modalities. Indeed, studies have shown that phosphate mass removal is 20–30 % higher with HDF than with high-flux hemodialysis.This improved phosphate elimination with HDF translates into increased pre-treatment phosphorus control, less phosphate-binder use and better achievement of therapeutic targets (Penne EL et al., 2010).

Multiple prospective studies have demonstrated better hemodynamic stability with convective renal replacement modalities. The beneficial impact of convective therapies on the prevention of intradialytic hypotensive episodes appears to reside in thermoneutral balance(Locatelli F et al., 2010) use of a substitution fluid with a high sodium concentration and better removal of mediators with vasodilating and negative inotropic properties.Finally,online HDF provides a multipurpose RRT.

SUBJECTS & METHODS

StudyDesign:

Prospective,randomised,double blinded clinical trial.

Study populations and Sampling:

47 patientswere eligible for inclusion criteria, if they were aged 18-60 years with ESRD receiving thrice-weekly 4 hours each ,standard hemodialysis for at least 6 months in the dialysis unit of Assiut University Hospital. Exclusion criteria:

1-Patients age below 18 yr and above 60 years.

2- Treatment with HDF in the 6 months preceding randomization.

3-Diabetes mellitus, active systemic diseases, liver-cirrhosis, malignancy andimmunosuppressive therapy.

4-Single needle dialysis, and temporary nontunnelized catheter.

5- A life expectancy less than 3 months because of another cause than kidney disease.

6-Participation in another clinical intervention trial.

7-Severe incompliance regarding frequency and duration of dialysis treatment.

The patients were classified into two groups:group(A),were 23 patients shifted from conventional HD to post-dilution HDF and group(B),were24patients continue their conventional HD rigmen(control group).

We choose this modality because it presents the advantage of providing a higher convective clearance at a given ultrafiltration rate since solutes are not diluted by the substitution fluid before entering thefilter as the substitution fluid is administered after the filterin this modality.

Technical Design:

I- Field Work:

The field work of this study was carried out from February 2012 till December 2013.

All candidates were subjected to:

1- Full clinical history and clinical examination.

2- Routine investigations:

-Laboratory: Complete blood count, sodium, Potassium, Calcium and Magnsium.

- ECG.

-Radiological investigations:

*Abdominal ultrasound.

*Chest X-ray.

3- Specific investigations:

-Serum Β2-microglobulin and phosphorus levels.

Sample Collection:

The blood samples were drawn at the beginning of the study and after six months(the duration of the study) irrespective to the days of HD or HDF.

1. 2 ml of blood were drawn on K3EDTA vaccutainer for complete blood count and, reticulocyte count were done using Caulter Hmx, USA.

2. 4 ml of blood for separation of serum for routine kidney function tests and, serum phosphorus.The rest of the serum was aliquited and stored refrigerated under -20C for the estimation of serum beta2-microglobulin by ELISA

Kidney function tests were done using colourimetric analysis by (Diamond, Egypt),serum phosphorus,calcium and Magnsium were done using autoanalyser (Dimension R x L, Germany).

Sodium and Potasium were done using (Easy lyte lithium, USA), Na/ K/ Li analyser.

Beta-2-Microglobulin:

Using ELISA from ORGENTEC Diagnostica ,Germany.

Principle:

Highly purified anti-human-beta-2-microglobulin antibodies are bound to microwells. Beta-2-microglobulin, if present in diluted serum, plasma or urine, binds to the respective antibody. Washing of the microwells removes unspecific components. Horseradish peroxidase(HRP) conjugate anti-human beta-2-microglobulin immunologically detected bound patient beta-2-microglobulin forming a conjugate beta-2-microglobulin/antibodycomplex. Washing of the microwells removes unbound conjugate.An enzyme substrate in the presence of bound conjugate hydrolyzes to form a blue color. The addition of an acid stops the reaction forming a yellow end-product. The intensity of this yellow colour is measured specrtophotometrically at 450nm.The amount of colour is directly proportional to the concentration of beta-2-microglobulin present in the original sample.

Sample preparation

Serum samples were diluted 1:100 with sample buffer,(10μl of urine with 990 μl of sample buffer) in polystyrene tubes. They were mixed well. Calibrators and controls are ready to use and do not need to be diluted.

Test Procedure:

1. 100μl of the control and prediluted patient samples were pipetted in to the assigned wells.
2. Incubation was done for 30 minutes at room temperature.
3. The contents of the microwells were discarded and wash 3 times with 300μl of wash solution.
4. 4 100μl of enzyme conjugate was dispensed into each well.
5. Incubation was done for 15 minutes at room temperature.
6. The contents of the microwells were discarded and wash 3 times with 300μl of wash solution.
7. 100μl of TMB substrate was dispensed into each well.
8. Incubation was done for 15 minutes at room temperature.
9. 100μl of stop solution was dispensed into each well and the plate was left untouched for 5 minutes.
10. The absorbance was read at 450nm (using a reference wavelength of 600-690nm) in a microplate reader.
11. The absorbance obtained is printed out.

Expected normal range:

Serum samples: 0-3.0μg/ml

Urine samples: 0-0.3μg/ml

II-Statistical Analysis:

• Revision of data and coding of variables was done and was entered in the computer.
• Data entry and data analysis were done using SPSS version 19 (Statistical Package for Social Science). as follows:
• Descriptive statistics (number, percentage, mean and standard deviation) were done.
• Chi-square test was used to compare qualitative variables between groups.
• Mann-Whitney test was used to compare quantitative variables between groups.
• Wilxocon Signed Rank test was done to compare between before and after treatment in each group.

• R-test (correlation co-effeciant) was used to rank different parameters against each others either direct or indirect.
• P value>0.05 is non significant(NS), <0.05 is significant(S) and <0.01 is highly significant(HS).

RESULTS

Table (1): Showed that the mean age of the patients on HDF was 38.24 ± 11.09 years while the mean age of the patients on HD was 41.76 ± 9.25 years old. Males on HDF were 15 patients, females on HDF were 8 patients, while males on HD were 14 patients and females on HD were 10 patients.

Table (2):Showed that36 patients were hypertensive ,18 on HD and 18 on HDF while only 11 patients were not hypertensive of the total patients.

Table (3): Showed that the mean ± SDof β2-Microglobulin level before HDF was 14.00± 3.07 ug/ml while after HDF was 11.45 ± 2.51 ug/ml with significant reduction (p-value 0.002).While the mean ± SDof β2-Microglobulin level before HD was 12.37 ± 2.30 ug/ml while after HD was 13.72 ± 3.87 ug/ml withinsignificant rise (p-value 0.088).

Table(4): Showed that the mean ± SD of phosphorus level before HDF was 6.84 ± 1.59 mg/dl while after HDF was 5.36 ± 1.11 with significant reduction (p-value 0.000).While the mean ± SD of phosphorus level before HD was 6.00 ± 2.04mg/dl while after HD was 6.02 ± 1.96 with insignificant rise (p-value 0.819).

The mean ± SD of calcium level before HDF was 8.72 ± 1.09mg/dl while after HDF was 8.94 ± 0.57 with non significant reduction (p-value 0.484).Also, the mean ± SD of Calcium level before HD was 8.44 ± 0.94mg/dl while after HD was 8.46 ± 1.18 with non significant reduction (p-value 0.649).

The mean ± SD of blood urea level before HDF was 156.40 ± 38.20mg/dl while after HDF was 146.72 ± 35.31 with significant reduction (p-value 0.014*).Also, the mean ± SD of urea before HD was 147.04 ± 169.71 ± 39.85mg/dl while after HD was 8.46 ± 1.18 with significant reduction (p-value 0.010*).

The mean ± SD of creatinine before HDF was 89.20 ± 1.79 mg/dl while after HDF was 8.31 ± 1.86 with significant reduction (p-value 0.002).While the mean ± SD of Creatinine before HD was 8.70 ± 1.97 mg/dl while after HD was 8.92 ± 1.88 with non significant reduction (p-value 0.382).

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Assiut Med. J. Vol. (38), No. (3), March, 2015

Table (1): Distribution of the studied sample according to demographic criteria:-

Personal characteristics / HDF
(n= 23) / HD
(n= 24) / P-value
No. / % / No. / %
Sex: / 0.627
Male / 15 / 65.2 / 14 / 58.3
Female / 8 / 34.8 / 10 / 41.7
Age: (years) / 0.281
Mean ± SD / 38.24 ± 11.09 / 41.76 ± 9.25
Range / 16.0 – 60.0 / 24.0 – 59.0

Table (2): Frequency of risk factor (hypertension) among patients included in this study:-

Hypertension / HDF
(n= 23) / HD
(n= 24) / P-value
No. / % / No. / %
HTN: / 0.792
Yes / 18 / 78.3 / 18 / 75.0
No / 5 / 21.7 / 6 / 25.0

Table (3): β2-Microglobulin removal by HDF vs. HD:-

β2-microglobulin
(ug/ml) / HDF / HD / P-value
Mean ± SD / Mean ± SD
Before / 14.00 ± 3.07 / 12.37 ± 2.30 / 0.341
After / 11.45 ± 2.51 / 13.72 ± 3.87 / 0.021*
P-value / 0.002* / 0.088

*Significant

Table (4): Phosphorus removal by HDF vs. HD:

Phosphorus (mg/dl) / HDF / HD / P-value
Mean ± SD / Mean ± SD
Before / 6.84 ± 1.59 / 6.00 ± 2.04 / 0.527
After / 5.36 ± 1.11 / 6.02 ± 1.96 / 0.027*
P-value / 0.000* / 0.819

*Significant

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Assiut Med. J. Vol. (38), No. (3), March, 2015

DISCUSSION

Online HDF constitutes a very attractive method of RRT. On one hand,it has the capacity to eliminate a wider range of molecular-weight solutes through the added convective-base clearance it provides. On the other hand, its use of ultrapure water and better biocompatibility profile make it a less inflammatory technique. Clinical studies have demonstrated a positive impact of these technical aspects in lowering B2-microglobulin amyloidosis, improving phosphate control and diminishing the rate of symptomatic hypotensive episodes (Maduell et al., 2011).

In this study, the distribution of studied sample among different age groups is nearly close, with the largest number in the age group less than 40 years old ,may be because most of them were educated and they could understand the importance of our study.The distribution of gender in the studiedsample was not equal, males are more than females and this conforms with gender distribution in CKD which is attributed to increased rates of hypertension, bilharziasis,stones and UTI in males.

Because of its size, removal of B2-MG is negligible during low-flux HD. In contrast, significant removal of B2-MG can be established with high-flux HD, because of convective transport by internal filtration within the dialyzer. The Hemodialysis (HEMO) and the Membrane Permeability Outcome (MPO) study showed lower serum B2-MG levels in high-flux HD as compared with low-flux HD patients (Locatelli E et al., 2009). In addition, it has been shown that removal of B2-MG is further increased with online hemodiafiltration (HDF) by using excess ultrafiltration to provide increased convective transport.

The main finding from our study is in agreement with these observations as, after switching from HD to OL-HDF, there was a significant reduction in pre-dialysis of B2MG concentrations,as compared to patients treated only with Low-efficiency-HD (control group).This suggests better long-term amyloidosis control by OL-HDF compared to Lf-HD. as (table3) show that B2-MG concentrations increased after 6 months of Lf-HD in Control group, but after the switchover from Lf-HD to post dilutional OL-HDF, in Study group, there was a significant reduction in B2-MG concentrations, and hence bettermineral metabolism control and B2- MG induced amyloidosis reduction in HDF patients.

Failure to adequately clear phosphate during intermittent haemodialysis is now well established.More recent work seems to indicate that adding a convective component to the standard diffusion-based treatment can improve phosphate control (Davenport et al., 2010).

In the(Ezio Movilli et al,2010) study showed that , sPO4 and sCa concentrations remained stable during the 6 months of Lf-HD in both the study group and in controls, but after the switchover from Lf-HD to post dilutional OL-HDF, in study group was associated with a significant reduction (24% ) in pre-dialysis sPO4 concentrations, while no change occurred in controls. During the same period of time, sCa concentrations remained remarkably stable in both groups of patients(with no significant reduction).

Minutolo et al.,2002,have shown, in acute study, that HDF was associated with larger intradialytic removal of phosphate compared to standard HD, and more recently, (Penne et al.,2010) reported the bone mineral parameters of patients participating in the Convective Transport Study (CONTRAST) showing that the proportion of patients reaching phosphate treatment targets increased significantly in patients treated with HDF, while this was stable in patients treated with standard HD (with no significant reduction) even after adjustment for phosphate binders use.