Chemistry: analytical and immunological testing – Sedimentation rate or hematocrit
Reexamination Certificate
1999-06-25
2001-03-20
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Sedimentation rate or hematocrit
C436S069000, C436S177000, C422S073000, C073S061650
Reexamination Certificate
active
06204066
ABSTRACT:
TECHNICAL FIELD
This invention relates to a method for determining the erythrocyte, or red blood cell, sedimentation rate in a sample of anticoagulated whole blood which is contained in a transparent tube. More particularly, this invention relates to a method for rapidly determining the erythrocyte sedimentation rate from successive erythrocyte layer/plasma interface position readings taken at known time intervals during centrifugation of the blood sample in the tube.
BACKGROUND ART
The erythrocyte sedimentation rate (ESR) is a widely used test which can help determine if a patient has a condition which is causing sub-acute or chronic inflammation. It Is non-specific, but It is still very helpful, particularly in following the course of some inflammatory diseases. Some of the problems with the test are that it uses a relatively large amount of blood (about one-half ml or more); it normally requires at least sixty minutes to complete, and thus is somewhat impractical for immediate patient care; and it requires the operator to handle bloody equipment. Additionally, the test requires a relatively high degree of skill and care in setting it up if the results are to be trusted.
The Westergren ESR test, which is the “Gold Standard” reference method for the ESR, is performed by placing a diluted sample of anticoagulated blood in a tall, perfectly vertical tube and measuring how far the plasma/erythrocyte (P/E) interface has settled under the influence of gravity after sixty minutes. The test works because the proteins associated with inflammation, particularly fibrinogen, counteract the zeta potential of red blood cells, which is a negative surface charge on the erythrocytes that serves to repel the individual erythrocytes from each other and thus prolong erythrocyte sedimentation. When systemic inflammation is present, the fibrinogen content of the blood increases, and the erythrocytes tend to aggregate, and thereby decrease their surface-to-mass ratio, and thus increase their rate of sedimentation.
Various approaches to automation have been attempted, notably the use of electronic means to track the sedimentation of the erythrocytes and provide a result in less than the usual sixty minutes, but these methods require at least fifteen minutes, and their results do not correlate well with the “reference” ESR method, i.e., that of Westergren.
A centrifugal method for packing blood so as to determine the approximate ESR was invented by Brian S. Bull et al, and is described in U.S. Pat. No. 3,824,841. The Bull et al method is, however, undesirable since it requires a large amount of blood and subjects the clinician performing the test to the possibility of exposure to infectious aerosols.
Another centrifugal method for measuring the ESR has been proposed by James W. Winkelman et al and is described in International Patent Application No. WO 96/39618. The Winkelman et al technique solves many of the problems of previous ESR techniques in that it uses a smaller quantity of blood and provides results within a few minutes. The Winkelman et al method involves the centrifugation of a quantity of blood in a disc-shaped rotor chamber and the observance of position of the P/E interface as the blood cells pack under centrifugal force. The total time from the start of centrifugation to the cessation of movement of the interface is related to the ESR. This device also requires a modest amount of blood, and the results moderately correlate with the Westergren method.
It would be desirable to be able to perform a rapid ESR measurement using a conventional blood sample container, such as a capillary tube, or the like, so as to derive an ESR from a small amount of blood in a matter of minutes
DISCLOSURE OF THE INVENTION
The method of this invention indirectly measures the repulsion or zeta potential of the erythrocytes, which is the main contributor to the sedimentation rate. The erythrocytes are caused to settle gravimetrically in a discrete layer in a capillary tube under modest centrifugation, and the movement of the plasm/erythrocyte interface is mathematically analyzed to yield an ESR number which closely correlates to the ESR obtained by the method of Westergren.
I have observed that in an anticoagulated blood sample which is centrifuged in a capillary tube, the position of the red blood cell-plasma interface in the capillary tube descends in the capillary tube, over time, initially in a linear fashion until such time that the repulsion force zeta potential of the red blood cells becomes the controlling factor in the red blood cell layer compaction, after which the position of the red blood cell-plasma interface descends in the capillary tube in a non-linear fashion. Thus the red blood cell layer in a centrifuged sample of anticoagulated whole blood sample compacts in an initially linear fashion and in a subsequent non-linear fashion to substantial completion.
I have determined that the initial linear portion of the red blood cell layer compaction in a capillary tube is due to the patient's hematocrit, and the subsequent non-linear portion of the red blood cell layer compaction in a capillary tube is due primarily to the strength of the repulsion or zeta potential of the subject's red blood cells. Thus, the compaction of the red blood cell layer, over time, can be plotted as a curve having an initial linear portion and an subsequent non-linear portion.
The method of this invention involves the detection of the beginning of the non-linear portion of the red blood cell compaction curve; the determination of the slope of a mazthematically linearized transformation of the non-linear portion of the red blood cell compaction curve; and correction of the length of the packed red blood cells resulting from the linear hematocrit-related part of the curve. I have found that after the initial linear compaction of the red blood cell layer, there occurs a “knee” in the compaction curve wherein the curve becomes non-linear. The non-linear portion of the red cell compaction curve continues after occurrence of the knee to a theoretical point of ultimate erythrocyte compaction which would occur if the blood sample were centrifuged for an endless period of time.
This invention involves the linearization of the non-linear portion of the curve which follows the knee, and the determination of the slope of the linearized portion of the curve. The linearization step can be performed by mathematically transforming the non-linear portion of the curve to a linear representation by means of a hyperbolic or exponential fit of successive erythrocyte/plasma interface locations derived during centrifugation of the blood sample. The method also involves the determination of a corrected starting time for the red cell compaction which negates the initial linear portion of the red cell compaction curve. This corrected starting time, which I refer to in this description as “t
c
”, can typically be a time that is about midway between the elapsed time that the knee is formed, and the elapsed time that the erythrocyte compaction is substantially completed.
It is therefore an object of this invention to provide an ESR measurement within a few minutes after drawing a blood sample so that this useful diagnostic data will be available while the patient is still in the physician's office.
It is a further object of this invention to provide the ESR measurement results from a tiny quantity of blood, so that that a venipuncture may be avoided, although venous blood may also be used.
It is yet another object of this invention to provide ESR measurement results which closely match those obtained by the traditional Westergren method.
These and other objects and advantages of the invention will become more readily apparent from the following detailed description of a specific embodiment of the invention when taken in conjunction with the accompanying drawings, in which:
REFERENCES:
patent: 3824841 (1974-07-01), Bull
patent: 4558947 (1985-12-01), Wardlaw
patent: 5731513 (1998-03-01), Bull
patent: 5827746 (1998-10-01), Duic
patent:
Jones William W.
Levine Robert A.
Wallenhorst Maureen M.
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