In order to determine their value for estimating the heparin concentration in plasma, we established the relationship between test result and heparin concentration in plasma from various individuals, for five assays used with heparin treatment. Only assays which can be carried out routinely in clinical laboratories were considered. The thrombin time and the whole blood recalcification time give pointless and ambiguous information respectively, concerning the heparin level. The activated partial thromboplastin time with and without heparin neutralisation give only a rough estimate. The spectrophotometric method using a chromogenic substrate gives the best information. The latter can be improved by using a non-linear (parabolic) equation for the calculation of the reference curve. Current heparin therapy, controlled with the aid of a clotting assay, may result in plasma heparin concentrations that vary widely from one patient to another.

The applicability was investigated of automated spectrophotometric heparin assays and three clotting assays for determination of two low molecular weight (LMW) heparin fractions: Org 10172 and DxN10 and two infractionated commercially available heparins. The relative activity of the two commercially available heparins was similar in the anti-Xa assay, in the anti-IIa assay and in 3 clotting assays. The LMW heparins showed markedly different relative activity in all 5 assays. The activities of those heparin preparations relative to the standard heparin were compared in the 5 assays, but standardization against a standard heparin preparation appeared impossible. Methods of heparin determination can be used to monitor treatment with a heparin preparation only if the same preparation is used as a reference substance.

Three automated spectrophotometric heparin assays were investigated. The day-to-day reproducibilities in routine laboratory use were compared with two commercial manual kits for heparin determination. Regression analysis of the activated partial thromboplastin time (APTT) on results of any of the heparin assays shows that the heparin concentration cannot be deduced from the APTT values found in patients receiving heparin. The automated heparin assays that employ thrombin and Chromozym-Th or S-2238 were found to be most suitable for routine heparin determination. Heparin concentrations obtained from assays based on factor Xa inactivation were not significantly different from those employing thrombin (p < 0.01), but revealed a wider standard deviation. The relationship between APTT and heparin level found was not related to the plasma antithrombin III concentration. The extra antithrombin III that is added in the assays had to be freed of heparin neutralising activity to obtain reliable estimates of the heparin concentration in the low range (0–200 U/l).

Spectrophotometric heparin assays are expected to be independent on clotting factors, either activated or nonactivated, but could be sensitive to heparin neutralization during blood collection. Is was shown that more than 200 U of heparin/1 plasma can completely be neutralized during blood processing. Because the heparin neutralization is not constant but dependent on the sample as well as on the type of sample handling, one cannot beforehand compensate and correct the results for possible heparin neutralization. We tested the use of pyridoxal 5’-phosphate (PLP) to prevent heparin neutralization. As the use of PLP in the citrate tube decreased the heparin neutralization to a negligible effect, PLP-citrate tubes are to be preferred for all plasma heparin determinations.

Spectrophotometric heparin assays which are based on the catalytic effect of heparin on either the inactivation of thrombin or that of factor Xa by antithrombin III, were adapted for use in a laboratory batch analyzer. Optimal conditions were determined for assays using the chromogenic substrates Chromozym-Th and S-2238 with thrombin, and S-2222 with factor Xa. Inactivation of the clotting enzyme by antithrombin III was stopped by addition of chromogenic substrate. Assays thus obtained appeared to be applicable in a wider range of heparin concentrations and were less dependent on plasma antithrombin III concentration that known manual spectrophotometric methods. The best results were obtained with the methods based on thrombin inactivation and applying a logarithmic reference curve.