M.P. van Dieijen-Visser

The value of HbA1 and fructosamine in predicting impaired glucose tolerance-an alternative to OGTT to detect diabetes mellitus or gestational diabetes

Glycosylated haemoglobin and glycosylated serum protein concentrations (fructosamine) have been monitored in patients suspected of diabetes mellitus (n = 183), in pregnant women suspected of gestational diabetes (n = 250) and in control groups (n = 184). The response to the standard 75 g oral glucose tolerance test was used to confirm or reject the diagnosis, using different criteria for detection of diabetes mellitus compared to detection of gestational diabetes. A slightly higher sensitivity was observed for fructosamine compared to glycosylated haemoglobin to detect impaired glucose tolerance (52 vs 44%) or gestational diabetes (17% vs 8%). For detection of diabetic oral glucose tolerance no difference was observed between glycosylated haemoglobin and fructosamine; sensitivity for both parameters was 67%. The results suggest that fructosamine is slightly more sensitive in detecting borderline-abnormal glucose tolerance, whereas no differences are observed for detection of clearly abnormal oral glucose tolerance tests.

Glycosylated serum proteins and glycosylated haemoglobin in normal pregnancy

Glycosylated haemoglobin (HbA1) and glycosylated serum protein concentrations (fructosamine) were monitored in 162 normal pregnancies. The response to the standard 75 g oral glucose tolerance test (OGTT) was used to exclude gestational diabetes. A significant increase in glucose intolerance, measured from the area under the OGTT-curve (r=0.29, P<0.0005) and a highly significant decrease in serum albumin concentration (r=−0.79, P<0.0005) due to the redistribution of body water, as occurs during pregnancy, resulted in a statistically significant decrease in serum fructosamine concentration (r=- 0.23, P<0.01) when related to gestational age in weeks.

Spectrophotometric Method for the Assay of Human Blood Coagulation Factor VIII

A spectrophotometric method for the assay of human blood coagulation factor VIII in plasma is presented. The chromogenic assay for factor VIII:C in plasma is performed in 3 steps: activation of factor VIII by thrombin; activation of factor X in a mixture of factor X, factor IXa, phospholipids/Ca2+ and plasma containing activated factor VIII, and determination of the rate of factor Xa formation with the chromogenic substrate S2337. Within-assay variation was between 5 and 6.9% for factor VIII:C activities between 20 and 150%. Clotting and chromogenic factor VIII:C activities were compared in plasma of 50 normal healthy donors (coefficient of correlation r = 0.83).

Time-Dependent Instability of Cardiac Troponins in Human Plasma Spiked with NIST Reference Material 2921

An important prerequisite for guaranteeing comparability of results among cardiac troponin I (cTnI) assays is the availability of a reference system, including a secondary, matrix-based reference material (1). Presently only the NIST Standard Reference Material (SRM) 2921 is available (2). This purified primary material has partial commutability characteristics, making it inadequate for directly calibrating commercial cTnI assays (3). External quality assurance organizers, such as the Dutch SKML (Dutch Foundation for Quality Assessment in Clinical Laboratories), have a policy of monitoring, for their participants, the effect of ongoing harmonization and standardization efforts, with the goal of having commutable and value-assigned cTnI control materials. We created 2 plasma pools, one with plasma from healthy controls (C), and one with fresh-frozen heparin plasma from acute coronary syndrome patients (P). Immediately after thawing, a portion of these pools were supplemented with NIST SRM 2921, a human cardiac troponin ternary complex–based reference material with a certified cTnI concentration of 31.2 mg/L and a reference cTnT concentration of 36.9 mg/L. To minimize exposure to light and avoid contact …

Characterization of the perfusate proteome of human donor kidneys

Background Preservation of deceased donor kidneys by hypothermic machine perfusion results in superior transplant outcomes as compared with static cold storage and provides the opportunity to measure biomarkers of cellular injury in perfusate samples. Identification of biomarkers predicting early graft dysfunction so far has met with limited success. Methods Two-dimensional difference gel electrophoresis and mass spectrometry were used to explore the proteome of perfusate samples from machine-perfused human donor kidneys (N = 18) and to discover novel biomarkers of ischaemic acute kidney injury. Results Thirty-two protein spots were successfully identified, representing 19 unique proteins that were derived from renal tissue and from residual plasma in the renal microcirculation. Two unidentified protein spots were significantly up-regulated, whereas one protein spot - identified as haptoglobin - was significantly down-regulated in the perfusate of ischaemically injured kidneys from donors after cardiac death as compared with kidneys from brain-dead donors who had not suffered warm ischaemic injury. Furthermore, two protein spots were up-regulated in kidneys that never functioned after transplantation, whereas one spot was up-regulated - identified as α1-antitrypsin - in kidneys with delayed graft function. Conclusions We provide the first description of the renal perfusate proteome and present preliminary evidence of differentially expressed biomarkers in human donor kidneys with different levels of acute ischaemic injury. Their diagnostic value for the selection of marginal kidneys in clinical transplantation should be determined in future studies.

Multicentre Study of a New Test for TSH-Screening in Blood Spots

A multicentre study for the detection of congenital hypothyroidism by enzyme-immunoassay of thyrotropin in filter paper blood spots is described. The method is accurate, with good precision and adequate sensitivity. No interference due to common blood constituents has been observed. Moreover, the TSH determination can easily be performed by photometric measurement of enzyme activity. Large series of samples can be processed by automation using common laboratory analysers. Values show good agreement with those obtained by RIA methods.

Significance of serum troponin T in patients with kidney disease.

Lamb et al. recently reviewed the signi¢cance of raised cardiac troponin T (cTnT) concentrations in patients with kidney disease.1 Their clearly written paper addressed a multitude of possible factors that might in£uence these cTnT measurements in uraemic patients. We recently published an article2 that answers some speci¢c questions raised by Lamb et al. By identifying several fragments of cTnT that are small enough to accumulate in the serum of dialysis patients, and by showing the absence of intact cTnT, we can explain the elevated cTnT levels in patients without detectable acute coronary syndromes. The main conclusion of our article was that, in most dialysis patients, two simultaneous processes occur at the same time. In previous articles, authors used cTnT concentrations for group classi¢cation in the assessment of mortality, thereby concentrating the group of patients with additional cardiovascular problems. From this, it is obvious that more cardiac damage leads to a worse outcome. In contrast, we used treatment duration instead of cTnT concentration for group classi¢cation. Despite constant CK-MBmass values indicating an even distribution of patients with additional (minor) ischaemic events, cTnT concentrations still increase with increasing duration of dialysis therapy, revealing an underlying phenomenon. As well as this increase in cTnT concentrations due to the accumulation of cTnT fragments, a further increase can of course be caused by additional release of cTnT in cases of (minor) myocardial ischaemia.This additional release then accounts for the useful predictive value of cTnT with respect to mortality in dialysis patients. We thus clearly demonstrated that part of the increase in cTnTconcentration is caused by impaired renal clearance of the fragments without a¡ecting the predictive value of cTnT. In our opinion, cTnT can only be used in dialysis patients suspected of acute myocardial infarction whena second sample is drawnafter 3-6 h. In cases of myocardial injury, the cTnT concentration in this second sample should be signi¢cantly elevated compared to the ¢rst. Another possibility, although less speci¢c for myocardial tissue, is the use of CKMBmass in these cases. To gain a better understanding of the mechanisms of cTnT fragmentation and accumulation, we are currently investigating the time-dependent changes in release pattern of cTnT fragments in patients with acute myocardial infarction with and without decreased renal function. Perhaps themeasurement of speci¢c cTnT fragments can di¡erentiate between accumulated cTnTand recently released cTnT.

The Authors Reply as Follows

We read Dr Colleys comments with much interest and agree that statistical significance does not always cover physical reality. However, for the detection of gestational diabetes more subtle changes in glucose intolerance have to be registered than would be noted in type I or type II diabetes. Therefore. we carefully examined different parameters to deseribe glucose metabolism and related them to gestational age. Figures containing all the data points arc included to show the effect of gestational age on the different parameters especially because we are aware of the difficulties in using the coefficient of correlation for scattered points. In accordance with all studies in the literature relating parameters on glucose metabolism to gestational age. we used coefficients of correlation to express the relation between gestational age and glucose metabolism. In most studies however, a limited number of patients has been examined. We agree with Dr Colley that from the present data the quantitative effect of variations in serum albumin concentrations on the fructosamine concentration cannot be estimated because of simultaneous effects of changes in glucose tolerance and of changes in albumin concentrations during pregnancy. Therefore, we also examined the effect of ehanging albumin concentrations in groups of hospitalised vs nonhospitalised patients, where no ehanges in glucose tolerance occurred, but where there were large differences in albumin concentrations. I An evident effect of variation in albumin eoncentrations on fructosamine concentrations has been observed (r=O·66) confirming our former conclusion.

Heparin-Dipyridamole (Persantin®) Treatment in Pregnancy: Effect on Blood Coagulation Parameters

13 patients suspected of intra-uterine growth retardation (IUGR) were treated with a combination of heparin and dipyridamole. The effect of this anticoagulant therapy on platelet count, clot retraction, fibrinogen degradation products, antithrombin III (AT-III), plasminogen, fibrinogen and platelet aggregation induced by epinephrine, ADP, collagen and ristocetin has been examined. All parameters were measured both before and after treatment with heparin-dipyridamole. The results were compared with the results from a control group consisting of pregnant women of the same gestational age using no medication and not suspected of IUGR (n = 17). A significant decrease in the clot retraction was observed in patients with IUGR as compared to pregnant controls (p less than 0.05). A significant decrease in the AT-III level (p less than 0.01) and a significant increase (p less than 0.01) in the fibrinogen level were observed after heparin-dipyridamole treatment of patients suspected of IUGR. We conclude that the combination of a high fibrinogen and a low AT-III level will cause an increased risk for the development of thrombosis upon termination of treatment. We also observed that heparin clearance is increased during pregnancy.