Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.¹ The assay still requires clinical confirmation.

By way of introduction, we refer here to published work on the time course of the plasma levels of the low molecular weight heparin Enoxaparin in human subjects. We are demonstrating today our results with a rapid point of care assay for low molecular weight heparin. In this case, the entity is Enoxaparin.

Results shown in this slide depict plasma concentration in patients who have been receiving the drug chronically once a day, 40 milligrams per day, and the time cost of that last administration followed to further disappearance can see then that at approximately four hours after administration, a peak concentration of 0.4 international units per ML is achieved, and that level is within the therapeutic range of the drug. A point of particular interest also, however, is how much remains 12 hours after administration because there is a guideline that is clinically accepted that one should wait at least 12 hours after the administration of low molecular weight heparin, in this case Enoxaparin before doing some particularly invasive procedure, which might result in a risk of bleeding. It is of interest to us that there is still a clearly detectable amount present at that time, and we have focused on that in some of our studies as we will demonstrate.

Also presented in this work by sender in etal is the fact that individuals with renal impairment have considerably higher levels of the drug throughout the time course, and we see that the 12 hour guideline might be very inadequate in the case of a patient with renal impairment or as is known in a woman who is pregnant or in patients who are obese. I'd like to introduce the investigators that have been involved in this project at New York Medical College. My name is Mario Osa.

I am professor of pharmacology here. I'm Dr.Surya Patula, associate professor of Clinical anesthesiology, New York Medical College. My name is Keshar Kal.

I'm a third year medical student. This is the Apparatus that we use for these assays. The apparatus is produced by the Omicron Corporation.

It can be used either in line or battery operated, and so it is truly point of care in that sense. The apparatus has two thermostatically controlled compartments and we use these compartments for a pre incubation of the samples in the preparation for the actual assay as well as for the coagulation assay itself. A major feature of the assay is the use of a highly purified, commercially available preparation of heparinase.

We purchased this from the Siemens corporation. It is provided in a very stable form and is readily soluble in saline or any aqueous solution. The use of heparin ACE is particularly valuable in our approach because it permits us to be able to obtain the patient's baseline coagulation value.

In the absence of any influence of heparin, heparin, ACE is able to completely degrade low molecular heparin and unfractionated heparin, but our particular interest here is in low molecular heparin and to degrade to inactivity and produce a value for coagulation time in the absence of any influence of heparin. What we are illustrating here is the time course of the degradation of low molecular weight heparin, in this case Enoxaparin by heparinase. The data are plotted as a percent of the baseline cutting time, so what we see here is that Enoxaparin added at one anti 10.

A international units per ML causes approximately a 50%extension in the clotting time of the blood. Then after varying periods of incubation with heparinase at 37 degrees centigrade, we see the time cost for the recovery of the baseline clotting times. In other words, the complete degradation of Enoxaparin to inactivity.

In terms of anticoagulant effect, we see that already by one and a half minutes. There is essentially a 100%recovery of the baseline clotting time. After inspection of this time course, we elected to use five minutes of incubation as our standard pre incubation of the blood before measurement of clotting time.

The assay tubes for this point of care assay have been slightly modified from the way we receive them from the manufacturer. These tubes originally contained in activator kaolin, which is present for a measurement of activated clotting time. We have found that when we use the tube without the activator, it does increase its sensitivity quite substantially.

We have termed this our minimally activated assay because we feel that the only possible source of activation would now be the glass surface of the tube and perhaps the magnet. The magnet is the essential part of the automated assay. These tubes rotate in the tube wells and when the magnet is entrapped by a clot of density to result in its loss of movement, then that is detected and signals the completion of coagulation.

We also studied the assay using the omicron activated partial thrombo Platin time cartridge. The assay is conducted in the identical fashion. There is slightly more activation present in this system.

Our first step is to reconstitute the lily heparinase as we receive it in its vial. We add 0.25 ml of saline to the vial and then allow approximately 20 seconds for complete dissolution. We next aspirate the entire contents of the vial into a tuberculin syringe, usually recovering about 0.2 ml in this next step.

Additions for pre incubation are added to freshly drawn blood samples containing enoxaparin in three ml vacutainer tubes. The first edition represents the heparinase and that is being mixed at this time. Point following, this is the addition of 0.2 ml of saline to the tube containing enoxaparin but no heparinase for a comparable VO total volume.

We use the built-in timer of the instrument to count down the 300 seconds. That is five minutes of pre incubation. The vacu tubes are removed when the pre incubation is complete.

The Final step is the transfer of two ml of the pre incubated samples to our minimally activated assay tubes. These tubes are prefilled with a hundred microliters of 0.25 molar calcium chloride to initiate coagulation. This first tube is the one pre incubated with heparinase.

The second sample is that containing enoxaparin but no heparinase. The Timers are expressing the expired time since the addition of calcium chloride, and we can see now that the first sample is complete. That is the upper vial at 200 seconds required for completion of coagulation.

The sample that did not contain heparinase is proceeding longer and that has finished now. This is a simulation of our typical result with blood spiked with 0.4 international units of Enoxaparin per ml. That is approximately a 45 second shortening of clotting time at this concentration of enoxaparin or an 18%decrease in clotting time.

As can be seen, the clotting detection is automated and the times are preserved on the instrument until inspection is complete. We should mention that we have actually tested this assay at as lower concentration as 0.1 international units of Enoxaparin per ml. As we noted in the introduction, this is the concentration that might be present 12 hours after a typical therapeutic dose of Enoxaparin.

We have been able to reliably detect at this concentration as well.