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Platelet and Coagulation Factors in Proliferative Diabetic Retinopathy

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Platelet and Coagulation Factors in Proliferative Diabetic Retinopathy J Clin Pathol: first published as 10.1136/jcp.37.6.659 on 1 June 1984. Downloaded from J Clin Pathol 1984;37:659-664 Platelet and coagulation factors in proliferative diabetic retinopathy DQ BORSEY,* CV PROWSE,t RS GRAY,* J DAWES,t K JAMES,§ RA ELTON, | BF CLARKE* From the *Diabetic and Dietetic Department; tBlood Transfusion Service, Royal Infirmary, Edinburgh; tMRC Immunoassay Team, Edinburgh; §Department ofSurgery and || Medical Computing and Statistics Unit, University ofEdinburgh, Scotland SUMMARY Plasma ,8-thromboglobulin, platelet factor 4, fibrinogen, fibrinopeptide A, antithrom- bin III, factor VIII related antigen, a2-macroglobulin, platelet count, and total glycosylated haemoglobin were measured in three well matched groups of subjects: non-diabetic controls, diabetics without retinopathy, and diabetics with proliferative retinopathy. /8-thromboglobulin and platelet factor 4 concentrations were significantly higher in the diabetics with retinopathy than in the controls and platelet factor 4 was also increased in the diabetics without retinopathy compared with controls. Fibrinogen concentration was raised in diabetics without retinopathy compared with controls, diabetics with retinopathy compared with controls, and diabetics with retinopathy compared with those without. Fibrinopeptide A concentration did not differ significantly between groups. Antithrombin III levels were increased in diabetics with retinopathy compared with controls, and in diabetics with retinopathy compared with those without. Factor copyright. VIII related antigen values were higher in both the diabetic groups when compared with the controls. Fibrinopeptide A concentration correlated with both /8-thromboglobulin and platelet factor 4 in each of the three groups. Haemostatic abnormalities in diabetes have been shown, although a hypercoagulable state has not been confirmed. These changes in platelet and coagulation function may be secondary to the development of microvascular disease and their role in the pathogenesis of retinopathy remains http://jcp.bmj.com/ uncertain. Patients with diabetes mellitus have significantly We have measured plasma concentrations of increased morbidity and mortality as a consequence ,8-thromboglobulin, platelet factor 4, fibrinogen, of specific microvascular disease, which results in fibrinopeptide A, antithrombin III, factor VIII conditions such as retinopathy and nephropathy. related antigen, a2-macroglobulin, platelet count, on September 28, 2021 by guest. Protected Although the precise cause of these vascular compli- and total glycosylated haemoglobin in three well cations remains uncertain, evidence is accumulating matched groups of subjects: non-diabetic controls, that an imbalance of the haemostatic mechanisms insulin dependent diabetics without retinopathy, may be entailed in their initiation or propagation. and insulin dependent diabetics with proliferative Various abnormalities of platelet function and of retinopathy. coagulation in diabetes have been identified and recently reviewed.' The contradictory findings and Patients and methods conclusions of different research groups may be par- tially attributed to the heterogeneity of their diabe- PATIENTS tic patients, the poor matching of control groups, the We studied 20 healthy non-diabetic controls, 20 non-standardisation of coagulation and platelet insulin dependent diabetics without ophthalmos- assays, and the uncertain importance of abnormal in copic evidence of retinopathy, and 20 insulin vitro tests. dependent diabetics with proliferative retinopathy. Subjects in each group were individually matched Accepted for publication 20 February 1984 for age, sex and, where applicable, duration of diab- 659 J Clin Pathol: first published as 10.1136/jcp.37.6.659 on 1 June 1984. Downloaded from 660 Borsey, Prowse, Gray, Dawes, James, Elton, Clarke Table 1 Details ofthe subjects studied rations was included on every plate. The standard Normal Diabetics was a human a2-macroglobulin reference obtained subjects from Meloy Laboratories Inc (Springfield, Virginia, without with USA). a2-macroglobulin was measured only in the retinopathy retinopathy diabetic subjects. No of subjects 20 20 20 % male 60 60 60 Glycosylated haemoglobin-measured by a mod- Age (yr) 35 ± 9 34 9 35 ± 11 ification of the short column chromatographic Duration of diabetes (yr)- 21 + 7 19 ± 7 method of Kynoch and Lehmann.9 Daily insulin dose (units)- 62 + 18 68 ± 29 % ideal body weight* - 101 + 11 100 + 11 Platelet count-electronically determined using the Thrombocounter (Coulter Ltd). Values given as mean ± 1 SD. *Metropolitan Life Assurance Company Tables 1960. The assay methods for 3-thromboglobulin, platelet factor 4, fibrinopeptide A, antithrombin III activity, antithrombin III antigen, and factor VIII etes. Details of the subjects studied are given in related antigen gave within batch coefficients of var- Table 1. All diabetics had presented with the clinical iation between 4 and 6% with between batch features of type I diabetes before the age of 35 and coefficients of variation of 8-12%. The between been treated with insulin since diagnosis. batch coefficient of variation for glycosylated All subjects had normal serum creatinine con- haemoglobin was 3 5%. Fibrinogen levels and centrations and absence of proteinuria on testing platelet count were determined by non-batch with Albustix. None had chronic liver disease or was methods with coefficients of variation of 5 % and 3% receiving drugs (for example, oral contraceptives, respectively. Previous studies by one of us have salicylates) known to interfere with haemostasis. shown that the assay procedure for a2- Only two of the diabetics studied had signs or symp- macroglobulin is highly reproducible (100 + toms, or both, of macrovascular disease (one angina 22 %).8 Further details of the above assays are and the other peripheral vascular disease) and they available in the quoted references. both had retinopathy. Laboratory staff were unaware of the identities ofcopyright. the samples. METHODS All diabetic fundi were examined ophthalmoscopi- STATISTICAL METHODS cally through dilated pupils by one observer (BFC). Non-parametric methods were used to analyse the Proliferative retinopathy was defined as the pres- data in view of the presence of skew and outliers in ence of neovascularisation. A single, 2 h postpran- at least some of the measurements. Median and dial venous blood sample was withdrawn into a range were used as summary statistics within groups, http://jcp.bmj.com/ polypropylene syringe, with minimal venous stasis, paired measurements were tested between groups from resting subjects. The samples were analysed as by Wilcoxon's signed rank tests, and associations detailed below: between measurements within groups by Kendall /3-thromboglobulin-radioimmunoassay of samples rank correlation coefficients. collected into anticoagulant mixture containing prostaglandin E,.23 Results Platelet factor 4-radioimmunoassay.4 on September 28, 2021 by guest. Protected Fibrinopeptide A-radioimmunoassay of samples Table 2 details the medians and ranges of the meas- collected into aprotinin-heparin.5 ured platelet and coagulation factors within the Fibrinogen (factor 1)-clot turbidity assay of Ellis three groups and also the results of a comparison of and Stransky.6 paired measurements between groups. Antithrombin III /3-thromboglobulin concentrations were higher (p (a) Antithrombin III activity-bioassay with < 0.01) in the diabetics with retinopathy than in the chromogenic substrate using a commercially avail- non-diabetic controls, and the platelet factor 4 val- able kit (Boehringer-Mannheim Ltd). ues were significantly increased in both the diabetic (b) Antithrombin III antigen-electroimmunoassay groups when compared with the controls (diabetics method of Laurell using commercial antisera with retinopathy p < 0-01; diabetics without (Behringwerke).' retinopathy p < 0.05). A highly significant correla- Factor VIII related antigen-electroimmunoassay tion between /8-thromboglobulin and platelet factor method of Laurell using commercial antisera 4 were seen in each of the three groups (controls and (Behringwerke).' diabetics with retinopathy p < 0.001; diabetics ar2-macroglobulin-standard Mancini gel diffusion without retinopathy p < 0-01). method.8 Standard antigen in four different concent- The fibrinogen concentrations were higher in the J Clin Pathol: first published as 10.1136/jcp.37.6.659 on 1 June 1984. Downloaded from Platelet and coagulation factors in proliferative diabetic retinopathy 661 Table 2 Platelet and coagulation factors (median and range) in the three study groups and a comparison ofpaired measurements between groups Non-diabetic Diabetics Controls v Controls v Diabetics with controls diabetics without diabetics with retinopathy v those without retinopathy with retinopathy retinopathy retinopathy without ,S-thromboglobulin (ng/ml) 31-5 (19-1-68-5) 44-9 (22-2-96-3) 51-5 (24-1-127-5) NS t NS Platelet factor 4 (n$/rni) 10-4 (10.0-52-0) 17-5 (8.5-72.6) 19-6 (6-7-63.7) * t NS Fibnnogen (gil) 1-70 (1-04-2-93) 2-46 (1-56-3-46) 2-93 (1-85-5-70) t * Fibrinopeptide A (nglml) 1-64 (1-60-19-20) 2-82 (2-00-16,60) 2-80 (2-00-17-80) NS NS NS Antithrombin III activity (%) 103 (82-119) 104 (92-122) 116 (92-132) NS t Antithrombin HI antigen (%) 100 (76-126) 100 (87-121) 113 (87-126) NS * * Factor VW1 related antigen (%) 98 (72-140) 252 (126-460) 267 (142-400) t NS a2macroglobulin (mc%o - 378 (192-641) 480 (187-696) - - NS Platelet count (x109/l) 226
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