Chemistry: analytical and immunological testing – Clotting or clotting factor level tests
Reexamination Certificate
2000-02-14
2002-07-09
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Clotting or clotting factor level tests
C436S149000, C436S150000, C436S157000, C422S073000, C073S064410
Reexamination Certificate
active
06417004
ABSTRACT:
BACKGROUND
During cardiopulmonary bypass (CPB) for open-heart surgery, the patient's blood is circulated into an extracorporeal circuit. The non-biological surfaces of the bypass circuit are known to exhibit a strong procoagulant effect on the circulating blood. To offset this biological reaction, anticoagulants are routinely administered; the most common of which is heparin. Heparin is usually administered in high concentrations during periods of extracorporeal circulation. During these procedures, the Activated Clotting Time (ACT) and other endpoint based coagulation assays are frequently used to monitor these high levels of heparin and other coagulation parameters.
Blood clot formation is a complex phase. Several principles are useful in understanding fluid-phase coagulation. In general, the clotting proteins circulate normally as inactive precursors. Coagulation involves a series of activation reactions that in turn act as the catalysts for the next level of reactions and hence, the frequent term Coagulation Cascade. During the reaction(s) process, these proteins and the fibrin mass itself, is highly unstable and water-soluble. This will remain until the very final aspects of coagulation. In addition, without (or in limited quantities) those clotting proteins (or in the presence of anticoagulants, i.e., heparin), clotting becomes delayed or prolonged. Eventually, however, fibrin (the foundation of a blood clot) will be formed. This occurs with the cleaving of fibrinogen, one of the coagulation proteins. Finally, Factor XIII (stabilizing factor) is activated by thrombin to yield cross-linked fibrin, which is highly insoluble and stable in formation.
In 1966, Dr. Paul Hattersley, a physician from California, outlined the design and usage of a fresh whole blood clotting test utilizing a particulate for contact activation. This was to facilitate rapid test conclusion in a clinically meaningful timeframe. The test Hattersley described included placing 1 ml or more of blood into a tube prefilled with 12 mg of activator (diatomaceous earth, Celite®). This tube was prewarmed to 37 degrees C. prior to administration of the patient blood sample. A timer was started when blood first entered the test tube. The tube was filled, and inverted a few times to accommodate mixing. The tube was then placed into a 37 degree C. water bath. At one minute and at every 5 seconds thereafter the tube was removed from the water bath and tilted so that the blood spread the entire length of the tube. The timer was stopped at the first unmistakable signs of a clot.
Shortly thereafter, automated methods based on the original Hattersley ACT were developed. Using several different techniques, all offered automated temperature management, automated end point detection, and automated timing counters. All of these automated ACT systems terminated the testing cycle when a significant clot mass was formed. This clot mass needed to be of sufficient size and strength to displace a ceramic magnet in a test tube and the displacement was detected by actuation of a magneto-mechanical switch. Of note, almost thirty years has elapsed with no fundamental improvement to this clot detection principle.
Over those thirty years, surgical techniques have changed greatly. Many complicating factors now exist that may induce artificial error in ACT test systems that require a strong blood clot to trigger the clot detection mechanism. These variables include deep hypothermia, new platelet preservation/enhancement drugs (aprotinin, DDAVP, transexamic acid), thrombolytics, antiplatelet compounds, and a series of new opiates, benzodiazapenes, and analgesics.
For almost three decades, electromechanical ACT clot detection mechanisms have been the mainstay of ACT testing systems. Throughout that time, they have undergone no fundamental change.
In the early 1960's, a saline-rinsed ACT test tube was manufactured to work on an automated instrument for determining the ACT on routine heparinized patients. This was designed to specifically supercede the manual Lee-White Clotting time (a non-activated whole blood clotting test that predated Hattersley's ACT). The instrument, called HEMOCHRON® (International Technidyne Corporation, Edison, N.J.) employed a single point electromechanical clot detection mechanism for identifying clot end-point in these heparinized samples. The mechanics behind this clot detection system were simple, yet effective. They are disclosed in U.S. Pat. No. 3,695,842, issued Oct. 3, 1972 and a further development thereof is disclosed in U.S. Pat. No. 5,154,082, issued Oct. 13, 1992.
Inside each HEMOCHRON unitized test tube (along with the reagent or activator) is a magnetized ceramic bar. In order to minimize the change from the original Hattersley hand-held ACT to the automated ACT system, the magnetized ceramic bar was coated with a substance as to make it “invisible” to the blood. In other words, the magnet was intentionally treated to minimize thrombogenic capabilities. The ACT test tubes are inserted into the analyzer. and are held in the detection well at a 30-degree angle. This angle is used to proctor the reagent, patient sample and magnet placement to rest in the bottom of the test tube in close proximity to the tube center post. This center post provides the main adhesion point for the clot to assist in magnet displacement. In addition, at the top of the center post is a spindle. This spindle is designed to grab fibrin formation forming in the top of the blood sample. The spindle is placed well above the detection magnet in the test tube. It is important to note that this tube is designed to utilize large blood sample volumes, not reduced or micro volumes more accepted in medicine today. Once the tube is inside the well, the sample is heated to 37 degrees C. (body temperature) and the tube is rotated at a speed of 1 revolution per minute. As the blood in the tube remains liquid (unclotted), the magnet is free to roll in the bottom of the tube independent of the 1 revolution per minute tube rotation (FIG.
1
). As the clot mass forms and solidifies, the clot mass binds the magnet and the tube center post and no longer allows the magnet to roll independently of the test tube. When the magnet reaches a fixed distance from the detector, the magnetic field is no longer sensed by the detector, the switch opens, and the instrument detects a clot. In traditional electromechanical clot detection systems, the detection distance is approximately 90 degrees from the bottom of the tube (FIG.
2
).
The detection distance is an important measurement. This measurement has direct correlation with the instrument's clot detection sensitivity, especially when a stable clot mass cannot be formed reliably. In reference to single detection point mechanisms, as described above, these are very susceptible to prolonged end point detection during high heparinization (i.e., bypass surgery) procedures. In these highly heparinized procedures, large amounts of anticoagulants are given to prevent fibrin formation. Despite the strong clot activators in the ACT tubes (celite, kaolin), this large amount of heparin (and subsequent hemodilution) usually prevents stable clot masses from forming consistently. As the clot begins to form in the tube, the clot mass, and its attachment to the center post, must be stable (strong or tight) enough to displace the magnet the minimal detector distance. As the magnet/clot mass travels this large distance along the rotation of the outer wall of the tube, the stability/strength of the clot mass must have the ability to bear the weight of the clot mass/magnet as it rotates to the 90-degree point (FIG.
3
). As the mass approaches 90-degrees from the bottom of the test well, gravity begins to put stress on the mass. If the mass is not “strong” enough, or properly adhered to the wall of the test tube, the mass will “slide” back, and the detector will not signify the proper end of the clotting cycle despite the obvious formation of clot.
As the clot mass/magnet continues to rotate to the detec
Brady Terry E.
Corsello Michael F.
Helena Laboratories Corporation
Piper Rudnick LLP
Schneider Jerold I.
Wallenhorst Maureen M.
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