PLATELET COUNT RATIO 1 Introduction

The PCR is a method giving an indirect quantitation of platelet aggregates in blood, and was first thought to provide a measure of in vivo platelet aggregates. 325,809

Normal individuals have a PCR close to unity. 809 A lower PCR occurs in various

forms of vascular disease compared to a "normal" control group. 325 However, a

large overlap in the distribution of the PCR is present within these groups. 325 In

addition, there appears to be a greater variability within patients than within a normal population.325,584 This broad distribution limits the sensitivity of the method

for use within individuals. The nature of the test and the diversity of stimuli affect­ ing platelets makes the technique a diagnostically non-specific functional test. For the study of a population group how ever, the m ethod appears to be satisfactory.325,327,810,811

7.2.1.1 Limitations of the PCR measurement methods. Initially there were a number of reports that the PCR was significantly lower in patients with vascular dis­ ease and thrombotic disorders.325,327,810 However, doubts as to the internal reprodu­

cibility of the method and the problems of the large between group and within group variance in study populations have been raised, 584,812 leading to concern about the

validity of results using the method.327,812 In fact, the methodology used in this assay

is highly operator dependent.328,813 Nevertheless, reproducible PCR measurements

have been obtained with narrow normal ranges,328 as a result of changes in method­

ology including very accurate pipetting of blood samples or direct collection of blood into the buffered EDTA solutions, using a standardized region of the supernatant for counting, collecting a fixed volume, and reducing the amount of EDTA and formalin used.328

The technique to measure platelet aggregates is clearly susceptible to inaccuracies due to artifacts caused by sample collection and processing. 810,812,814 Moreover,

evidence has emerged that the platelet aggregates measured by this method are probably not present in the circulation of the patients.327,810,812,815 Indeed, there is

evidence that the platelet aggregates are formed during the venipuncture.327,810,814

Others have found that the PCR in normal subjects is unaffected by the method of blood collection and processing when the platelet counts are measured in whole blood and not PRP.327 On the other hand, in patients with diseases associated with

thrombosis, the PCR was found to be significantly lower when blood was collected through a needle and tubing when compared to when the PCR is measured in whole blood, suggesting that aggregates form during collection.327

Athough it appears unlikely that platelet aggregates are commonly formed in vivo in vascular disease states,327,812 the hypothesis that the circulating platelet has the poten­

tial to express greater reactivity in certain disease states remains tenable. That is, the formation of platelet aggregates during sampling is a measure by which platelets are determined to be more reactive.327

The degree of tourniquet stasis, age, sex and the time of food intake does not appear to influence the test results.584 A very small variance within normal subjects corn-

pared to that among individuals with various disease states has been docum ented.584 This would support the concept that there are indeed factors influencing the PCR in patients w ith vascular disease that are absent in norm al individuals. The m arked overlap betw een "normals" and "abnorm als"334 how ever, m akes any clinical inter­ pretation difficult.

O ther doubts about the presence of circulating platelet aggregates arise from the lack of direct evidence that platelet aggregates are present in the p atient’s circulation.810 C irculating platelet aggregates larger than the capillary diam eter would be expected to be filte re d out in the m icro circu latio n , w ith the p o ssib ility o f im p aired organ perfusion.327

7.2.1.2 Platelet count ratio in disorders involving vascular disease. In the original study in which the m ethod was applied,325 a significantly low er PCR was found in a num ber o f disorders involving vascular disease. The study population included patients with transient cerebral ischaemic attacks, AMI, stable angina, acute p erip h eral vascular insufficiency and chronic peripheral vascular disease.325 This group was com pared to a "normal" and a "patient" control group. The norm al con­ tro l g ro u p had a PC R o f 0 .9 0 + /-0 .0 2 an d th e p a tie n t c o n tro l g ro u p a PC R o f 0.88+/-0.01 (m ean+/-SEM ). H ow ever, in the vascular disease groups, apart from those patients with a PCR less than 0.70, there was a considerable overlap in values betw een groups, obviously lim iting the sensitivity in individuals. In addition, the nature o f the m ethod and the div ersity o f potential stim uli affecting the platelets during processing make the method intrinsically non-specific.

The utility o f the PCR for clinical or research purposes is further confused by the failure to support the original finding of a decreased PCR in patients with chest pain, A M Is, com pleted strokes and transient ischaem ic attacks.584 A num ber o f research groups have observed no decrease in the PCR in individuals with stable angina com ­ pared w ith norm al control groups.325,500,584,811,814,816 H ow ever, with different m eth­ ods, an increase in reversibly aggregated platelets in patients with unstable angina and AM I, and an increase in irreversibly aggregated platelets in patients with stable angina compared with controls has been docum ented.817 Scanning electronm icrosco- py also reveals the presence of irreversible aggregates in the blood o f patients with throm botic episodes.814 The incorporation of red and white blood cells into "platelet aggregates" was used to demonstrate more irreversible aggregates.814

I n c r e a s e d p l a t e l e t a g g r e g a te s d u rin g A M I h a v e b e e n o b s e r v e d by m o st in v e stig a to rs.325,327,803,811,818'822 although not all.584 P atients w ith unstable angina have also been shown to have increased p latelet ag g re g ate s.811 F u rth erm o re, in ­ creased aggregates have been observed in other vascular conditions associated with intrav ascu lar throm bosis.325,327,819 M ost studies indicate that patients with chronic stable CHD do not have a lower PCR, and by inference either circulating aggregates or hypersensitive platelets. However, those with unstable CHD do have a lower PCR which w ould indicate that they either have circulating platelet aggregates or that they have hypersensitive platelets.

7.2.1.3 Platelet Aggregates in Normal Subjects. G enerally it is believed that agg reg atio n does not usually occur in vivo in norm al subjects.325,496,812 H ow ever,

some investigators have found platelet aggregates in normal population groups. Possibly up to 15% of p latelets in the norm al population may circu late as aggregates.584,816 Less than 10% of circulating platelets in the normal population may be present as reversible aggregates, while others have observed that about 6% of platelets circulate as aggregates which are irreversibly aggregated.817 Platelets sensitised in the circulation can more easily undergo activation and aggregate upon contact with a thrombogenic surface.823

7.2.1.4 Summary. Normal groups have a broad variation in the PCR in most studies, although the PCR in normals may be narrower than originally thought when a modified technique is used.328,584 Attention to detail to control confounding factors in the methodology may allow differences to be more easily detected, or confirmed as being absent. Unfortunately it is difficult to compare studies since there has been a wide variation in techniques used, differences in the patient characteristics of the populations studied and small numbers in the study groups. Furthermore, when comparing a normal population group to a patient study group, the population groups have not always been well defined. In addition, drug treatment has not always been well documented.

When appropriate methods are used to calculate the necessary sample size to detect a true difference between two groups, one finds that at least 30 individuals or greater are needed to detect a significant difference in the PCR. There is no documented dif­ ference in the PCR due to age or sex in normal individuals,584 but this does not necessarily apply to the patient population with premature CHD. Therefore it is still appropriate to have relatively homogeneous study and control groups.

The wide variation in PCR found in patients with stable CHD may be related to other factors, or there may even be a bimodal distribution present. None of the previous studies have satisfactorily evaluated the PCR in patients with premature CHD. Therefore this study has evaluated such a group using methodology that has been demonstrated to produce a narrow range for the PCR in a normal population group.328 Strict controls to limit confounding methodological factors influencing aggregate formation were applied.

7.2.2 Methods

The PCR was determined by a modification of the method of Wu and Hoak325'328 and is described in Chapter 2.

7.2.3 Results

7.2.3.1 Group comparisons. The case group had a significantly lower PCR than the control group (0.81+/-. 14 versus 0.87+/-0.08 respectively, p<0.05), although there was a large overlap in the values for both groups.

1232 Correlation with blood cell indices and CHD risk factors. In the case group, the PCR did not correlate consistently with any of the major risk factors, although a significant but weak correlation existed with scapular skinfold thickness (Table 7.7). In addition, the PCR did not correlate with any of the blood cell indices

except for a negative correlation with the W CC in both groups (Table 7.7). How ev­ er, in the control group there was a positive association betw een the PCR and the approxim ate am ount o f alcohol regularly used, as w ell as w ith the blood glucose level (Table 7.7).

7.2 .3 3 Correlation with other platelet function tests. The PCR had a border­ line relationship with m easures o f in vitro aggregation induced by collagen (Table 7.8). The more reactive that platelets were in vitro, the more that platelet aggregates w ere form ed during blood collection in both groups, the PCR being low er. In the control group, the greater the serum TXB2 production the more that platelet aggre­ gates form ed during blood collection, a circum stance which was not present in the case group (Table 7.7).

7 .2 3 .4 Correlation with plasma fatty acids. Some plasm a FA had a significant correlation w ith the PCR in the case group only. Palm itic acid ( C l 6:0) positively correlated with the PCR w hilst oleic (C 1 8 :lw 9 ), docosapentanoic (C22:5w 3) and docosahexanoic (C22:6w3) acids negatively correlated with the PCR (Table 7.9). In the control group, alpha-linolenic acid (C 18:3w 6) had a positive association, and C22:5w 3 had a negative association w ith PCR, both o f w hich w ere o f borderline significance (Table 7.9).

7.23.5 Regression analysis. Those variables w hich may have in flu en ced the PCR or have had a significant correlation with the PCR were entered into the step­ wise regression model to determine the most predictive variable for the PCR (Table 7.10). In the case group, only oleic acid (C 18:lw 9), retained a significant predictive value for the PCR, whereas in the control group only the blood glucose level and the approximate alcohol intake retained a significant (and opposite) association with the PCR (Table 7.10).

7.2.4 Discussion

As observed in other studies, the norm al group had a narrow er range o f values for the PCR than the group with CHD.328,584 How ever, the observation o f a low er PCR in the case group com pared to the normal control group differs from some previous studies,325,584,816 although not a ll.817 These oth er studies evaluated older and m ore heterogeneous CHD groups. O ur results infer that platelets from males with prem a­ tu re CHD are m ore lik ely to form p la te le t a g g re g ate s d u rin g b lo o d c o lle c tio n . N e v e r th e le s s , a s y s te m a tic b ia s d u e to c o lle c tio n p r o c e d u r e s c a n n o t be excluded810,812,814 since the method is highly operator dependent.328,813

W hether circu latin g platelet aggregates are p resen t or absent in vivo in norm als is controversial.325,496,812,816,817 Our normal group did not have a PCR o f unity, indicat­ ing the presence o f aggregates in the blood samples. M ost available evidence would indicate that these aggregates formed during the blood collection.327,810,812,814,815 The case group had m ore platelet aggregates in the blood samples collected. This study was not designed to determine when such aggregates appeared, in vivo or during the blood collection.

ed, although not proven, on a number of grounds. Platelets from normal subjects are less influenced by blood collection methods than platelets from patients with vascular disorders,327 there is a smaller variance for the PCR in normal subjects328 than in patients with vascular disorders,325,584 platelets sensitised in the circulation more readily undergo activation and aggregation,823 and the PCR is decreased in condi­ tions where in vivo platelet activation has been proven to occur.325,818"820 In the present study, a modest relationship between the PCR and in vitro measures of plate­ let aggregation, but not in vivo measures of platelet activation, was demonstrated. These observations further support the proposition that the PCR is a measure of ex vivo platelet reactivity, rather than a measure of the presence of in vivo platelet aggregates.

S pecific facto rs, such as age and sex, w hich are asso ciated with in vitro

aggregation,331 have not been shown to be associated with the PCR.584 Furthermore, we have shown no relationship between the fibrinogen level and the PCR, fibrinogen being a factor associated with in vitro aggregation measures.331

A similar relationship has been documented between the amount of alcohol used and the PCR, as well as a borderline relationship with previous tobacco intake for normal subjects with the in vitro aggregation results.331 In fact, the relationship with alcohol in the normal control group maintained a predictive association independent of the other variables. It is not possible to make any interpretation concerning the influence of alcohol in the case group since a major proportion of the subjects had changes in their pattern of alcohol use (see Chapter 5). The results, however, sup­ port the proposal of a favourable effect of alcohol on platelet function, and that the changes could conceivably reduce thrombotic CHD events.164

Abnormalities of platelet function in vascular disorders have been consistently demonstrated and by various methods of evaluation.46,141,824"826 Increased platelet sensitivity may be a factor in the development of vascular disease in diabetic pa­ tients.141,824 Although a relationship between the PCR and measures of glucose metabolism has not been systematically evaluated in a normal population, a relation­ ship between decreased PCR and diabetes has been documented.825 Our study groups were selected on the criteria of not having diabetes or overt glucose intolerance. Nevertheless, in the normal group, but not the CHD group, an independent associa­ tion was demonstrated between the PCR and the fasting blood glucose level. The significance of this observation is uncertain at present and may simply be due to chance from multiple comparisons.

7.2.5. Summary

1. A difference between the control and case groups has been demonstrat­ ed, the PCR being lower in young males with CHD, most of whom have had a previous AMI.

2. This difference in the PCR (a functional measure of platelet reactivity) appears to be predicted by different factors in the two groups. Whether this is due to intrinsic platelet factors, influences within the blood and/or environmental effects, remains uncertain.