Increased reticulated platelets in dialysis patients

Kidney International, Vol. 51 (1997), pp. 834—839 Increased reticulated platelets in dialysis patients JONATHAN HIMMELFARB, DIE HOLBROOK, ELLEN MCMON...
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Kidney International, Vol. 51 (1997), pp. 834—839

Increased reticulated platelets in dialysis patients JONATHAN HIMMELFARB, DIE HOLBROOK, ELLEN MCMONAGLE, and KENNETH AULT Division of Nephrology, Maine Medical Center, Portland, and Maine Medical Center Research Institute, South Portland, Maine, USA

associated with a transient worsening of platelet dysfunction

Increased reticulated platelets in dialysis patients. The purpose of this study was to measure the percent reticulated platelets and platelet counts

[22—26]. A repetitive activation of platelets that is caused by the dialysis procedure may induce platelet refractoriness to further stimulation, thereby contributing to the clinical bleeding sometimes observed temporally related to the dialysis procedure. While there are conflicting studies examining platelet function

in patients on chronic hemodialysis, peritoneal dialysis and normal volunteers. The relationship between the percent reticulated platelets and

the platelet count can then be used to determine the rate of platelet turnover. Platelet rich plasma was obtained, platelets were fixed and incubated with thiazole orange and analyzed for the percent reticulated platelets by flow cytometry. Normal controls had a mean of 2.77 0.17% reticulated platelets while peritoneal dialysis patients had a mean percent reticulated platelets of 6.92 0.68 (P < 0.00001). Chronic hemodialysis

in dialysis patients, there are no studies examining platelet survival in this patient population. This is primarily due to the technical difficulties involved in measuring the survival of radiolabeled platelets in patients who are undergoing intermittent extracorporeal procedures. Recently a new approach to measuring in vivo platelet turnover has been developed that utilizes the nucleic acid content of platelets to identify reticulated platelets that have been recently released into the circulation [27—29].

patients had a mean percent reticulated platelets of 8.21 0.36 (P < 0.00001 vs. normal controls and P = 0.05 vs. peritoneal dialysis patients). Platelet counts did not differ significantly among the three groups. The identity of reticulated platelets was confirmed in experiments measuring platelet specific glycoproteins, experiments using RNase, and in mixing experiments with normal and uremic platelets and plasma. We conclude that dialysis patients have a marked increase in circulating reticulated platelets compared to normal controls, indicating accelerated platelet

Reticulated platelets, similar to reticulated erythrocytes, lose their

turnover. Increased platelet activation and turnover may contribute to the qualitative platelet dysfunction observed in dialysis patients.

RNA content in the process of aging and senescence. Because reticulated platelets are detectable in the circulation for only 24 hours [30, 31], they are an excellent measure of platelet production and turnover. We measured the percentage of reticulated platelets in patients

It has been recognized since at least 1907 that a bleeding diathesis is a major cause of morbidity and mortality in patients with uremia [1]. Clinical bleeding in patients with uremia is due to an acquired qualitative platelet defect and has best been correlated with a prolonged bleeding time [2, 3]. However, at present there is no single unifying pathogenetic mechanism to explain the acquired platelet dysfunction of uremia. Numerous studies have also failed to determine a consistent single intrinsic defect in platelet dysfunction in uremia. Studies of acquired platelet dysfunction in patients with endstage renal disease are also complicated by the potentially conflicting effects of dialysis on platelet dysfunction. Although some investigators have found that both hemodialysis and peritoneal dialysis partially improved the hemostatic abnormality of uremia [4—11], both forms of dialysis can potentially produce adverse effects on hemostasis. Peritoneal dialysis has been reported to cause platelet hyper-reactivity which may be related in some cases to the development of hypoalbuminemia [10]. Hemodialysis is frequently accompanied by transient platelet activation due to interactions of platelets with both dialyzer membranes [12—191

with end-stage renal disease on hemodialysis or peritoneal dialysis

and in revised form September 13, 1996 Accepted for publication September 16, 1996

maintained using heparin at an initial bolus of 100 UIg with

compared to normal controls and have demonstrated increased reticulated platelet levels in dialysis patients despite normal platelet counts, suggesting increased platelet turnover compared to controls. We have further validated these measurements by demonstrating platelet specific glycoproteins on reticulated platelets to confirm that these cells are platelets and by using RNase to confirm the specificity of platelet reticulation. Methods Patient populations

Twenty-eight patients on chronic maintenance hemodialysis, 22 patients with end-stage renal disease on chronic peritoneal dialysis, as well as 20 normal volunteers were chosen at random. No patients or controls were on aspirin, NSATDS or other medications known to interfere with platelet function. Seven of the 22 patients on chronic peritoneal dialysis and 10 of the 28 patients on chronic maintenance hemodialysis were diabetic. Of the chronic and the vascular access itself [20, 21]. Several studies have hemodialysis patients, 12 were being dialyzed with cellulosic suggested that platelet activation during hemodialysis may be membranes and sixteen with polysulfone membranes. All patients were on a hemodialysis reuse program using 1% formaldehyde incubated at 40°C for at least 24 hours as a sterilant. Bleach was Received for publication June 26, 1996 not used in the processing of these dialyzers. Anticoagulation was

© 1997 by the International Society of Nephrology

additional doses if activated clotting times were less than 1.5 times predialysis values.

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Fig. 1. Identification of platelet reticulation with flow cytometly. The analysis of data from a normal control subject (A) and a hemodialysis patient (B) are illustrated. In each panel the horizontal axis is forward angle light scatter (a measure of particle size) and the vertical axis is thiazole orange fluorescence (a measure of RNA).

Measurement of reticulated platelets

Treatment of platelets with RNase

Reticulated platelets were measured according to previously described methods. Briefly, whole blood was drawn into EDTA vacutainer tubes, predialysis and prior to heparinization in chronic hemodialysis patients, or at the time of routine monthly visits in chronic peritoneal dialysis patients. Platelet-rich plasma was prepared by centrifugation of blood anticoagulated with EDTA. The platelets were then washed, fixed in 1% paraformaldehyde,

Samples of washed platelets with high levels of reticulated platelets were fixed in 1% paraformaldehyde and then treated with 1 mg/ml RNase (Sigma Chemical, St. Louis, MO, USA) for 30 minutes at 37°C. The treated platelets were then washed and labeled with thiazole orange, as described above.

washed again, and resuspended at 107/ml in Tyrode's buffer

Statistics

containing 10 mmol/liter EDTA, One hundred microliters of this Comparisons of platelet count and percent of reticulated suspension was mixed with 900 microliters of thiazole orange solution (Becton-Dickinson, San Jose, CA, USA) and incubated platelets between normal controls, peritoneal dialysis patients and at room temperature for one hour. The samples were then hemodialysis patients were made using an F test to determine analyzed on the flow cytometer. In some experiments washed variance followed by a two-tailed t-test. Comparisons of relationplatelets were also labeled with phycoerythrin conjugated mono- ship of the platelet count to the percent of reticulated platelets

clonal anti-GPIIb/IIIa or GPIb (AMAC Inc., Westbrook, ME, within each group was made using linear regression analysis. Comparisons of the percent of reticulated platelets before and USA), after RNase treatment were made using a paired t-test. Flow cytometric data analysis

Analysis of the samples was carried out using a BectonDickinson FACScan flow cytometer. Analysis of thiazole orange positivity was done by measuring both forward light scatter and green (540 nm) fluorescence using logarithmic amplification. A plot of forward light scatter versus fluorescence was generated, showing the expected positive correlation between forward light scatter, which is a measure of particle size and fluorescence. Large platelets containing more nucleic acid were assumed to represent reticulated platelets. To define the frequency of reticulated platelets, a standardized bitmap was created using a line with the same

slope of green fluorescence as the platelet cluster set (Fig. 1). Platelets in the bitmap above this line were considered reticulated platelets (such as expressing more thiazole orange positivity).

Results Measurement of reticulated platelets in normal human volunteers, peritoneal dialysis patients and hemodialysis patients

Figure 2 illustrates the results of studies of 20 normal volun-

teers in whom we measured both the platelet count and the percentage of reticulated platelets. The mean platelet count was 248,000 9,100/pA (range 192,000 to 327,000/1id). The mean 0.17 (range 1.39 to percent reticulated platelets was 2.77 4.48%). Figure 3 represents results of studies of 22 patients on

chronic peritoneal dialysis. The mean platelet count was 283,000

17,000/,.d (range 120,000 to 43O,000/pJ), which was not

Himmelfarb et al: Reticulated platelets in dialysis

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Fig. 4. Percent reticulated platelets and platelet counts in chronic hemodialysis patients. E E :3

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Hemodialysis Fig. 5. Percent reticulated platelets in diabetic (LI) and non-diabetic () dialysis patients.

statistically significantly different than normal controls. In contrast, the mean percent reticulated platelets was 6.96 0.68 (range 1.98 to 11.67), which was significantly different (P < 0.00001) compared to normal controls. Figure 4 illustrates the results of studies in 28 patients on chronic maintenance hemodi-

for chronic peritoneal dialysis patients and 0.140 for normal controls. Because previous studies have indicated a relationship between

diabetes mellitus and platelet hyperreactivity, we examined the relationship between the presence or absence of diabetes mellitus alysis. In this patient group the mean platelet count was 278,000 and the percent reticulated platelets in both chronic peritoneal 23,000/pA (range 94,000 to 703,000/pA), which was not statistically dialysis patients and chronic hemodialysis patients (Fig. 5). The significantly different from either peritoneal dialysis patients or percent reticulated platelets did not differ in the chronic hemodi0.70) and nonnormal volunteers. The mean percent of reticulated platelets was alysis population between diabetics (8.32 8.21 0.36 (range 4.97 to 10.36%), which was significantly diabetics (8.15 0.41). Similarly, there were no differences in the different from normal controls (P < 0.00001) and also significantly percent reticulated platelets between diabetics (6.77 0.96) and non-diabetics (7.05 0.91) among patients on chronic peritoneal different from peritoneal dialysis patients (P = 0.05). To determine if there was a relationship between the platelet dialysis. Platelet counts also did not differ between diabetics and count and the percent of reticulated platelets for chronic hemo- non-diabetics either in the chronic hemodialysis patients (288,000 dialysis patients, peritoneal dialysis patients and normal controls, 33,000 .t/liter vs. 260,000 27,000 pr/liter) or in the patients on linear regression analysis was performed for each group. In each chronic peritoneal dialysis (280,000 3100 p/liter vs. 285,000 case there was no significant relationship between the platelet 22,000 p/liter). Thus, the presence or absence of diabetes mellitus count and the percent reticulated platelets. The R value for the does not account for the differences in the percent reticulated relationship between the platelet count and the percent reticu- platelets between patients on chronic dialysis therapy and normal lated platelet was 0.195 for chronic hemodialysis patients, 0.234 controls.

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Himmelfarb et al: Reticulated platelets in dialysis

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Fig. 6. Effect of RNase on measurement of platelet reticulation. Symbols are: (E2) samples without RNase; (LI) samples with RNase. Results are from two experiments with four healthy subjects and seven hemodialysis patients. P < 0.05 versus samples without RNase.

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Fig. 7. Platelet and plasma mccing expenments. Percent reticulated platelets were measured in samples in which either normal () or uremic (LI) platelets were incubated with normal or uremic plasma. N = 3 experiments. p < 0.05 normal versus uremic platelets.

Measurement of platelet specific glycoproteins on reticulated platelets

To confirm the identity of reticulated platelets as RNA bearing

platelets, platelets were simultaneously stained with thiazole orange and phycoerythrin conjugated monoclonal antibodies to glycoprotein lb or glycoprotein lib-Illa. In all cases, both reticulated and nonreticulated platelets exhibited greater than 94% positivity for both glycoprotein lb and glycoprotein Jib-lila

either with normal plasma (4.3 0.5%) or uremic plasma (3.9 0.4%). These experiments confirm that the increase in thiazcle orange positive reticulated platelets seen in dialysis patients is indeed due to increased levels of platelet RNA and not due to any interfering substance from uremic plasma.

Discussion staining (data not shown). This experiment confirms the identity of flow cytometrically labeled reticulated platelets as platelets In this study we have demonstrated that patients with end-stage because of their expression of platelet-specific cell surface pro- renal disease who are treated with either chronic peritoneal teins. dialysis or hemodialysis have a markedly increased percentage of

circulating reticulated platelets compared to normal controls. Since the platelet counts were similar among the three groups, To demonstrate that thiazole orange positivity in reticulated these results strongly suggest that both peritoneal dialysis and platelets was due to the presence of RNA, samples were incu- hemodialysis patients have markedly increased in vivo platelet bated with RNase. The percent reticulated platelets were then turnover compared to normal controls. determined in the same samples with or without RNase (Fig. 6). Although there have been many studies of platelet function in In all cases there was a significant reduction in percent of thiazole patients with renal disease, there are, to our knowledge, no orange positive reticulated platelets after the addition of RNase. previous studies examining platelet turnover. Previous techniques Identification of reticulated platelets as RNA containing platelets

As we and others have previously noted, the use of RNase before labeling reticulated platelets does not completely abolish thiazole orange positive staining in the platelet preparation. The residual

RNase resistant labeling may be due to ribosomal RNA or

to quantify platelet production and turnover were limited to bone

marrow examination, mean platelet volume and platelet radio labeling techniques. Bone marrow aspiration is subjective in its

interpretation and may vary between aspirates in the same

possibly be due to other platelet components that take up thiazole subject. The mean platelet volume is a parameter that is also subject to a number of technical variables, including type of orange dye. anticoagulation, time of measurement following venipuncture, Mixing experiments with normal and uremic platelets and plasma and the type of technology used to determine the platelet size Mixing experiments were performed to confirm that chronic [32]. Platelet radiolabeling techniques arc difficult to standardize

renal failure is not associated with a plasma factor or retained and are also subject to technical difficulties when patients are uremic toxin that affects the thiazole orange positive staining of undergoing intermittent extracorporeal treatments. Recent studplatelets. Plasma from patients on chronic dialysis was mixed with ies have also emphasized that radiolabeling techniques can have normal platelets, while plasma from normal volunteers was also problems related both to in vivo platelet compartmentalization mixed with platelets from chronic hemodialysis patients and the (especially if platelets are injured or activated in the collection or percent of thiazole orange positive staining was determined (Fig. radionuclidc labeling process 133, 34]) as well as elution of 7). Platelets from chronic hemodialysis patients expressed a radionuclide from platelets in vivo [35]. higher level of thiazole orange positivity whether they were Karpatkin suggested more than two decades ago that platelets, like erythrocytes, decrease in size, reduce their rate of protein assayed in normal plasma (8.5 0.9%) or in uremic plasma (8.6 0.6%) when compared to platelets from normal volunteers mixed

synthesis and decrease their RNA content as they age [36]. Since

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Himmelfarb et al: Reticulated platelets in dialysis

platelet turnover may contribute to the acquired platelet defect acid content of platelets can be used to identify those platelets associated with renal failure. that have recently been released into the circulation. Kienast and Schmitz first proposed using the dye thiazole orange and flow Reprint requests to Jonathan Himmelfarb, MD., Division of Nephrology, that time, considerable evidence has accumulated that the nucleic

cytometry to measure individual platelet nucleic acid content [37]. Thiazole orange is an ideal dye for measuring intracellular nucleic acid content and has become the standard for the flow cytometric analysis of reticulated erythrocytes. Thiazole orange enters living cells without any pretreatment, and displays a very large increase in fluorescence emission on binding to RNA and DNA [381. Using a mouse model of in vivo biotinylation we have recently

demonstrated that reticulated platelets are indeed the youngest platelets in circulation, circulating for an estimated 1.8 days prior to losing RNA [30]. Dale and colleagues have also recently used an in vivo biotinylation technique in a dog model [31] to demonstrate that thiazole orange positive platelets are less than 24 hours old. The measurement of circulating reticulated platelets has been utilized to evaluate platelet turnover in thrombocytopenic disorders [28, 29]. The use of reticulated platelets can detect consumptive platelet disorders with a positive predictive value of 96% and a negative predictive value of 100% [29]. In patients undergoing

stem-cell transplantation a rise in the percent of reticulated

Maine Medical Center, 22 Bramhall Street, Portland, Maine 04102, USA.

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