Renal Research Laboratories, Manchester Institute of Nephrology and Transplantation, The Royal Infirmary, Manchester, and 1 Laboratory Medicine Academic Group, University of Manchester, UK
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Abstract |
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Methods. The effect of two different venepuncture procedures on plasma TGFß1 was compared in 10 healthy volunteers, one procedure designed to minimize platelet activation and the other representing standard venepuncture practice in a clinic situation. Blood samples from 52 renal transplant recipients on either cyclosporine or tacrolimus immunosuppression were taken by standard venepuncture to investigate the effect of immunosuppressive drugs on plasma TGFß1. Plasma TGFß1 and ß thromboglobulin were measured by ELISA.
Results. Among 10 healthy volunteers who underwent two different methods of venepuncture, eight of 10 had undetectable levels of TGFß1 (<100 pg/ml) under conditions that minimize platelet activation. In contrast, all 10 paired plasma samples collected by vacutainer had measurable TGFß1 (median 7.70 ng/ml, interquartile range 5.8713.64 ng/ml) following acid/ urea activation. The median ßTG level (a measure of platelet degranulation) was 0.71 µg/ml (interquartile range 0.531.19 µg/ml) in the special collections compared with 3.39 µg/ml (interquartile range 2.274.33 µg/ml) in the vacutainer samples (P=0.0029).
Among 52 allograft recipients there was a significantly higher mean TGFß1 level in plasma from patients on cyclosporine therapy compared with patients on tacrolimus (28 090±26 860 pg/ml vs 7173±10 610 pg/ml, respectively; P<0.002). Mean plasma ßTG levels were also significantly higher during cyclosporine therapy compared with tacrolimus (8.14±5.54 µg/ml vs 3.66±3.32 µg/ml, respectively; P<0.002). However, when TGFß1 values were corrected for the degree of platelet activation (by factoring with ßTG) there was no significant difference between TGFß1 levels on cyclosporine or tacrolimus (4117±2993 pg/µg ßTG vs 2971±658 pg/µg ßTG, respectively; P=0.294).
Conclusions. To avoid erroneous hypotheses concerning TGFß1 and perpetuating confusion in the literature over levels in health and disease, it is imperative that proper internal controls for platelet activation are used. The effects of experimental treatments and drugs on platelet biology must be rigorously controlled when attempting to measure and interpret plasma levels of TGFß1 in clinical practice.
Keywords: immunosuppression; platelets; renal allografts; TGFß1; immunoassay
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Introduction |
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Clinical studies of fibrotic disease have measured circulating blood levels of TGFß1 in patients and healthy individuals to investigate the link between TGFß1 expression and the clinical presentation and prognosis of disease. A highly confused literature on blood levels of TGFß1 has developed with claims that values in normal controls are low [3] or high [4], that certain drugs induce high levels of circulating TGFß1 [5], and that patients in end-stage renal failure (ESRF), in particular African Americans [6], have high levels of circulating TGFß1.
Attention has been drawn recently to the difficulties involved in interpretation of data on TGFß1 blood levels [7]. The complex interaction of factors including the choice of blood sample (serum or plasma), the deliberate sample activation, the technical measurement of TGFß1 by ELISA, and the presence of latent and active pools of TGFß1 have a profound influence on the interpretation of TGFß1 levels in a clinical context. The fundamental problem in estimating a real in vivo circulating level of TGFß1 is how to avoid the enormous platelet pool of readily releasable TGFß1. TGFß1 was originally isolated from platelets [8], where it is stored at high concentration as a latent complex. Most of the confusion in the literature relates to whether or not researchers adopt a strategy to avoid, minimize or control for platelet-released TGFß1 occurring in the sample ex vivo.
The choice of serum as the medium in which to quantitate TGFß1 seems particularly inappropriate for two reasons. First, there is a significant correlation between the total platelet count and TGFß1 levels [9] (measured after activation of the latent molecule, see below), which is common to other platelet-stored proteins e.g. ß-thromboglobulin [10]. Secondly, it has been demonstrated that 95% of the TGFß1 in serum is artefactually derived from platelets on blood clotting [3]. This accounts for serum TGFß1 levels being reported as 10- to 20-fold [11], 20-fold [3] and 3.84-fold [9] higher than plasma levels.
TGFß1 levels in plasma (measured after activation of the latent molecule and often referred to as total level) have also been reported by workers in clinical studies of TGFß1 expression as representing a circulating blood level [3]. The recommendation that plasma TGFß1 levels should be related to the level of a recognized platelet release protein (ß thromboglobulin or platelet factor 4) has unfortunately not been adopted as standard practice [3]. Workers who recognize the danger of platelet-released TGFß1 recommend the use of platelet poor plasma [12], but this does not address or control for the variable platelet release of proteins that occurs during the trauma of venepuncture.
This study is therefore designed (i) to illustrate the effect of venepuncture on the non-specific release of TGFß1 from platelets at the point of blood collection, and (ii) to demonstrate how a failure to correct for platelet activation can lead to erroneous conclusions about the effect of different immunosuppressive drugs on plasma TGFß1 expression. This study is not designed to recommend the optimum method of venepuncture or quantitation of plasma TGFß1, but is investigating the effects of two important modulators of platelet activation (trauma at venepuncture and drug-platelet effects) on the subsequent measurement of plasma TGFß1 after deliberate sample activation and how these impact on our interpretation of TGFß1 biology in renal transplantation.
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Subjects and methods |
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Special venepuncture procedure
This procedure is recommended to minimize platelet activation during venepuncture [12]. Blood samples were collected from healthy volunteers without the use of a tourniquet by introducing a wide bore (16 gauge needle) into the antecubital vein with minimal trauma and allowing blood to flow slowly without suction from syringe or vacutainer into a pre-chilled EDTA bottle. Samples were separated immediately by centrifugation at 1200 g for 10 min and the plasma aliquots were stored at -70°C.
Standard venepuncture
Blood samples were collected from healthy volunteers using a tourniquet by venepuncture with a 21 gauge needle and vacutainer blood collection system (Becton and Dickenson, Oxford, UK). Blood was collected into EDTA tubes and plasma was immediately separated by centrifugation at 1200 g for 10 min, and plasma aliquots were stored frozen at -70 °C. Thus, trauma during venepuncture was the only difference in the procedure for obtaining plasma samples from normal volunteers.
The effect of immunosuppressive drugs: the allograft recipients
Blood samples from 52 patients (mean age±SEM 44.2±2.0 years; 35 males, 17 females; 44 Caucasion, 8 non-Caucasians) undergoing renal transplantation at Manchester Royal Infirmary were obtained by standard venepuncture procedure as part of normal clinical follow up and monitoring of graft function. Samples were separated within 3 h by centrifugation at 1200 g and storage at -70°C. Thirty-two patients were receiving cyclosporine-based immunosuppression with a mean drug trough level of 156 ng/ml, range 64275 ng/ml (monotherapy or combined with steroids and azathioprine), and 20 patients received tacrolimus monotherapy with a mean drug trough level of 13 ng/ml, range 836 ng/ml. In the total patient group, the methods of venepuncture and sample preparation were kept constant (and were typical of those employed widely in clinical practice) to allow comparison of the effects of immunosuppressive drugs on TGFß1 levels.
Acid/urea activation of plasma TGFß1
The method was based on the R&D Systems (Abingdon, UK) procedure from the Quantikine Immunoassay kit. To 100 µl of plasma, 100 µl of 2.5 N acetic acid/10 M urea were added, mixed and incubated for 10 min at room temperature. The samples were neutralized by adding 100 µl of 2.7 N NaOH/1 M HEPES and added to the assay plate within 15 min of activation.
Acid activation of plasma TGFß1
One-and-a-half microlitres of 6 M HCl was added to 100 µl of plasma and incubated for 15 minutes at room temperature. Samples were neutralized to pH 7.07.4 with 3.0 µl of neutralizing buffer (50% vol/vol 1 M HEPES/6 M NaOH).
Immunoassay for TGFß1
The in-house assay has been described in detail [13]. Briefly, the capture antibody was a monoclonal to TGFß1,2,3. (Genzyme, Framingham, USA, clone 1D11) and the detection antibody was a chicken polyclonal antibody specific to TGFß1 (R&D Systems), followed by peroxidase conjugated donkey anti-chicken Ig (Jackson Labs, Stratch, Luton, UK). Signal was generated by the addition of Amerlite substrate (Johnson and Johnson Clinical Diagnostics, Ascot, UK) and read on a Microlumat LB96P luminometer (EG&G Berthold, Leeds, UK). Data was analysed using Mikrowin software.
Assay for ß thromboglobulin (ßTG)
An ELISA for ßTG was employed using the principle of competition between soluble ßTG in plasma and solid phase ßTG coated onto an ELISA plate for a limited amount of anti-ßTG antibody added to the samples. Purified ßTG (Novabiochem Ltd, Nottingham, UK) was coated onto ELISA plates at 200 ng/ml in 100 µl of 0.1 M sodium bicarbonate/carbonate buffer, pH 9.6 for 16 h. After washing, the plates were blocked with 5% bovine serum albumin (BSA) for 1 h. Plasma samples were diluted 1:25 with assay buffer (5% BSA in PBS with 0.05% Tween 20) and mixed with an equal volume of rabbit anti-ßTG antibody (at a dilution of 1:2000) and added to the plate. ßTG standards covering the range 3.0600 ng/ml were included and the assay was calibrated against the WHO 1st International Standard Preparation for ßTG (83/501). After incubation for 4 h, the plates were washed and incubated with peroxidase-conjugated anti-rabbit IgG (Jackson Labs) (dilution 1:8000) for 2 h. Following washing, ABTS substrate was added and the absorbance was measured at 405 nm in a multiwell spectrophotometer and results calculated using Softmax software.
Statistical methods
Non-parametric data sets were analysed by MannWhitney U-test for differences in median. Data sets showing a normal distribution were analysed for differences in mean by students t-test or paired t-test. A correlation between TGFß1 and ßTG was assessed using linear regression and Pearson's correlation test. Significance was attributed to analyses with P<0.05.
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Results |
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The effect of immunosuppressive drugs on platelet-released TGFß1 and ßTG
TGFß1 and ßTG were measured in plasma samples taken by standard venepuncture from renal transplant patients on either cyclosporine- or tacrolimus-based immunosuppressive therapy and the results are shown in Figure 2. There was a significant relationship between TGFß1 and ßTG in the sample population when analysed by Pearson's correlation test, r=0.751, P<0.0001.
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There was a significantly higher mean concentration of TGFß1 in samples from patients on cyclosporine therapy compared with patients on tacrolimus (28 090±26 860 pg/ml vs 7173±10 610 pg/ml, respectively; students t-test P<0.002) (Figure 3a). Mean ßTG values were also higher in samples from patients on cyclosporine therapy compared with those on tacrolimus (8.14±5.45 µg/ml vs 3.66±3.32 µg/ml, respectively, students t-test P<0.002) (Figure 3b
). However, when the TGFß1 values were corrected for the degree of platelet activation by factoring with ßTG, there was no significant difference in plasma TGFß1 between patients on cyclosporine or tacrolimus (4117±2993 pg/µg ßTG vs 2971±658 pg/µg ßTG, respectively; P=0.294) (Figure 3c
).
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Discussion |
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The one study carefully established to avoid platelet activation during venepuncture reports 4.1±2.0 ng/ml (range 2.012.0 ng/ml) [3]. However, we have demonstrated that this plasma TGFß1 level is artefactually generated by activating platelets during the standard venepuncture procedure and therefore does not represent the in vivo circulating pool of TGFß1. Eight of 10 paired samples from healthy volunteers show <100 pg/ml TGFß1 when precautions to reduce platelet activation are introduced. The two normal samples with the detectable level of TGFß1 have the highest level of ßTG, an accepted measure of platelet granule secretion [12], consistent with either in vivo platelet activation or activation despite the special procedure. Furthermore, dual measurement of TGFß1 and ßTG in 52 plasma samples from allograft recipients taken by standard venepuncture shows a significant positive correlation, indicating that the degree of platelet activation either in vivo or during venepuncture determines the level of detectable TGFß1 following activation of the sample.
Thus, deliberate sample activation prior to measurement of TGFß1 simply reveals the quantity of latent TGFß1 released from platelets during venepuncture. For each subject, the TGFß1 level represents the product of their intrinsic platelet stability, the degree of trauma that these platelets suffer during venepuncture and their total platelet count. In addition to these variables, further modulation of platelet biology in vivo by disease, drug therapy, surgery, haemodialysis or ethnic influences may need to be controlled. We contend that the evidence we have provided in this study supports the hypothesis that normal plasma TGFß1 levels are undetectable (<100 pg/ml) if procedures are taken to minimize platelet activation.
It is valid to ask whether immunosuppressive drugs used in transplantation, in particular cyclosporine and tacrolimus, promote the expression of TGFß1. It is conceivable that part of their immunosuppressive action may be mediated through the potent immunosuppressive properties of TGFß1. Additionally, their chronic nephrotoxicity may be driven through the profibrotic expression of TGFß1. Therefore, we quantitated the levels of plasma TGFß1 in transplant patients on cyclosporine or tacrolimus using routine venepuncture and deliberate activation of the samples prior to measurement. Under these conditions, patients on cyclosporine appear initially to have higher plasma levels of TGFß1, compared with patients on tacrolimus. However, there are clearly increased levels of platelet activation as evidenced by increased ßTG levels in the cyclosporine samples; the apparent high plasma TGFß1 levels in patients on cyclosporine merely result from greater platelet degranulation during venepuncture. This is not surprising since cyclosporine augments platelet reactivity [15,16] whereas tacrolimus does not [17,18]. Therefore, expressing the plasma results as TGFß1 per unit of ßTG identifies no difference in the plasma total TGFß1 levels in patients on cyclosporine or tacrolimus. Thus, any study claiming to show a difference in TGFß1 levels between groups or treatments that relies on deliberate activation of the latent TGFß1 must control for the level of platelet activation in the sample as has been previously recommended [3]. Failure to include proper controls for platelet activation as a source of circulating TGFß1 has resulted in a claim that cyclosporine stimulates in vivo TGFß1 expression [5]. Yet this effect is arguably no more than that demonstrated in this paper, i.e. that cyclosporine renders platelets more sensitive to secretion of granule proteins such as ßTG and TGFß1 when subjected to the stress of venepuncture. Similarly, a study suggesting that ethnic differences in rates of ESRF are dependent on overexpression of TGFß1 has not controlled for differential platelet activation between the groups, resulting in artefactual secretion of TGFß1. It is established that patients in ESRF on chronic haemodialysis show enhanced platelet activation [19]. Therefore, this apparent ethnic difference in expression of TGFß1 may simply reflect uncontrolled effects on platelet activation caused by different patterns of haemodialysis and prescription of anti-hypertensive drugs [20].
In order to ensure meaningful data on circulating TGFß1 levels, the ideal strategy is the adoption of the rigorous procedures necessary to avoid platelet activation during venepuncture as described by workers in the field of platelet biology [12]. To date, only one study of TGFß1 has adopted these precautions [3] and they claim that there is a low level of circulating TGFß1 from a non-platelet derived pool. These authors advocate strongly the use of a platelet release marker correction factor, which we have demonstrated does control for spurious effects of drugs causing release of TGFß1 into the blood sample. Concentrating on producing platelet poor plasma overlooks the variable platelet secretion of TGFß1 that has already happened during venepuncture.
We suggest that the quality and validity of interpretation of most of the clinical data on circulating TGFß1 levels is seriously flawed due to the failure to control for the level of platelet activation in the blood samples. The future for measurement of clinically relevant pools of TGFß1 must be in the development of assays that quantitate the products of in vivo activated TGFß1 which may circulate as TGFß1 complexes. Stable complexes of TGFß1endoglin have already been described and similar complexes with other natural antagonists may exist. Understanding the role of TGFß1 in disease pathology is an important goal that is not being helped currently by the assay of a spuriously high platelet-derived pool of TGFß1, as if it represented a real in vivo circulating pool of TGFß1.
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Notes |
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References |
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