Automatic Ultra-filtration (AUF)

Introduction

The Company’s AUF technology will allow the preparation of ultra-filtrates of serum and plasma samples, for measurement of free therapeutic drugs and hormones to be done quickly on an automated system.

Plasma & Serum

When blood in a tube containing an anti-clotting agent (anti-coagulant) is allowed to stand, the red cells fall to the bottom, and the yellow liquid at the top is called plasma.  If the tube does not contain an anti-coagulant, the blood clots and the yellow liquid at the top is called serum. 

Bound, Free  & Total Drug

Shown above in (a) is an illustration of free drug molecules (blue dots) and bound drug or drug-protein complex (Protein “P” with blue dots attached).  Shown above in (b) are the free drug & bound drug (i.e., the total drug) in plasma flowing through small blood vessels (capillaries).  The walls of the capillaries have holes, which allow substances to move from the blood to body tissues that are targeted by the drug.  Only the free drug can get to the tissues and exert their effect.  In other words, only the free drug can exert its therapeutic or biological action on body tissue.  The same concept applies to hormones, for example Testosterone.

Measurement of Free and Total Drug

Serum or plasma is used to determine the concentration of therapeutic drugs in a patient’s blood.  Tests for hormone and therapeutic drug levels are usually performed using immunoassays performed on laboratory immunoanalyzers.  With the exception given below, immunoassays are incapable of discriminating the free drugs/hormones from the bound drugs/hormones, and measure total drugs/hormones.  Dilantin is a common drug use to treat epilepsy, and will be used as an example.  About 90-95 % of Dilantin binds to proteins in the blood system, the protein rendering most of the drugs inactive.  There are a variety of conditions (e.g., pregnancy and certain diseases) that changes the protein concentration in a patient’s blood, whereby the total drug concentration can change dramatically.  However, the change in free drug concentration could be very small.  Some drugs have a narrow therapeutic range.  In other words, the drug is ineffective if it is below the lower limit of the therapeutic range, and toxic if it is above the upper limit of the therapeutic range.  In a patient with low total protein, the total drug concentration may be low due to the low total protein, but the patient’s free drug concentration could be above the upper limit of the therapeutic range and therefore in the toxic level.  In order to measure the free drug/hormone, an additional manual step of separating the protein-free fraction from the sample must be taken. This is done by a process that incorporates either equilibrium dialysis or centrifugation.  About eighteen hours are required to perform equilibrium dialysis, and thirty minutes are required for centrifugation to separate protein-free serum/plasma, referred to as a serum/plasma ultra-filtrate.  

In 1976, the first commercial immunoassay designed specifically to measure free thyroid hormone became available. The availability of free thyroid hormone measurement revolutionized the way thyroid disorders (a common medical disorder) are diagnosed and treated.  Today it is difficult to find a laboratory that measures total thyroid hormone, since measurement of free thyroid hormone has become standard practice. To date, very few new assays are available for measuring free drugs or free hormones.  Development of immunoassays for free therapeutic drugs and free hormones are difficult and expensive. The ChroMedX alternative is to rapidly modify the sample in a disposable cartridge that can be presented to the existing immunoanalyzer. 

Unless the free drug is measured, it is more likely that a patient will be misdiagnosed if the total drug concentration is used, but free drug measurement is usually not available due to cost and long result turnaround time.  The ChroMedX AUF (Automated Ultra-filtration) cartridge (pictures shown below) provides a rapid inexpensive way of preparing samples for analysis of free hormones and therapeutic drugs.  The cartridge can be part of a fully automated immunoassay system, a stand-alone processing workstation or as a simple manual sample preparation procedure.

The ChroMedX Alternative

Shown below is an example of the ChroMedX patented AUF cartridge.  The first picture shows the intact cartridge, which is designed to fit in a standard test tube rack.  The second picture is an exploded view showing the components of the cartridge.  The red component is a bundle of hollow fiber membranes and the yellow component is dead-end air chamber. When a serum/plasma sample is injected into the cartridge by a positive displacement pipette (through the off-center hole), the dead-end chamber acts as a spring and allows multiple passes of the serum/plasma over the hollow fiber membranes, thereby unplugging the membrane pores.  Ultra-filtrate can be removed through the center hole.  Ultra-filtrates prepared using the AUF cartridges are free of drugs/hormones bound to proteins and thus the measurements yield only the free drug/hormone concentrations.  The rapidity and automation of the AUF technology promises to alter the way therapeutic drugs are monitored, and the way hormone-related disorders are diagnosed and treated.

Automatic Ultra-filtration (AUF) Disposable Cartridge The center hole is for retrieving Ultra-filtrate. The off-center hole is for injecting the serum/plasma into the filtration chamber.

Automatic Ultra-filtration (AUF) Disposable Cartridge

The center hole is for retrieving Ultra-filtrate.

The off-center hole is for injecting the serum/plasma into the filtration chamber.

Table below shows results of proof of concept testing of the AUF cartridge. It can be seen from the table that the key electrolytes sodium, potassium and chloride, which do not bind to proteins are present close to their original levels prior to ultrafiltration. Proteins are shown to have been completely eliminated from the ultrafiltrate and both calcium and cortisol, which are partially bound to proteins, are at their predicted values in the ultrafiltrate.