# US 5,705,739 A · Detecting specific medical conditions from erythrocyte density distrubition in a centrifuged anticoagulated whole blood sample

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- Number: US 5,705,739 A
- Document ID: US5705739A
- Status: Granted
- Kind: A
- Application number: 08/703,509
- Filed: 1996-08-27
- Granted: 1998-01-06
- Earliest claimed: 1996-08-27
- Inventors: Robert A. Levine; Stephen C. Wardlaw
- Web page: https://patentgrep.com/patent/US5705739A

## CPC

- G01N 15/042: {by centrifuging and investigating centrifugates (centrifuges per se B04B)}
- G01N 33/491: {by separating the blood components (G01N15/05 takes precedence)}
- Y10T 436/25375: Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

In most mammals studied, a histogram of the erythrocytes' density of a healthy individual normally has a Gaussian distribution. Gaussian or near-Gaussian distributions may be characterized by their mean, by their standard deviation, and their absence of skewness. A measurement of the erythrocyte density distribution (EDD) in mammalian whole blood which has been anticoagulated with heparin can yield information which is indicative of certain physiologic and pathophysiologic conditions that are characterized by various EDD abnormalities. These abnormalities may include abnormalities in the standard deviation of the EDD; the mean erythrocyte density; and any skewness of the EDD curve. The hematocrit or percent packed red cell volume, which is obtained at the same time as the EDD can also yield information. The EDD measurement is made by providing a sample of heparin anticoagulated whole mammalian blood to which is added a plurality of density markers having known individual specific gravities that are within the range of specific gravity values of mammalian erythrocytes. The blood sample-density marker mixture is centrifuged, and measurements are taken of the location of the density markers within the centrifuged erythrocyte cell pack. These measurements are analyzed to determine the standard deviation of the EDD in the sample; the mean erythrocyte density in the sample; the hematocrit value of the sample; and any skewness in the EDD curve in the sample. The resultant data is compared to empirical data obtained from patient populations with known normal and abnormal medical histories in order to determine whether there is any variation from norm of one or more of the parameters measured which are characteristic of abnormal medical conditions. Alternatively since an erythrocyte's hemoglobin concentration (HC) is the main determinant of an ethrocyte's density, HC measured on a cell counter that determines each erythrocyte's HC may be used as a surrogate for erythrocyte density. Suitable cell counters can also derive HCT from a blood sample.

## Claims

### Claim 1

A method for detecting abnormal mammalian physiologic conditions which are characterized by deviations from norm of certain erythrocyte parameters in a donor blood sample, said method comprising the steps of:
a) providing a sample tube containing a sample of mammalian anticoagulated whole blood from a blood donor and a plurality of artificial density markers which are operable to form gravimetrically separated discernible reference lines in the blood sample, each reference line forming an upper boundary for one erythrocyte band and a lower boundary for a next adjacent erythrocyte band, the density of each density marker being within an expected range of densities of erythrocytes;
b) centrifuging the sample tube so as to gravimetrically separate the erythrocytes into an erythrocyte column which is separate from the remaining components of the blood sample, and so as to embed the density markers in the erythrocyte column whereby erythrocyte subsets in an erythrocyte density distribution (EDD) gradient are demarcated by the reference lines created by the embedded density markers in the erythrocyte column;
c) measuring and recording distances between adjacent density marker reference lines in the erythrocyte column to provide a plurality of EDD values;
d) defining an EDD curve from said EDD values;
e) determining the erythrocyte density standard deviation (EDSD), mean erythrocyte density (MED), and curve skewness (R.sup.2) in said defined EDD curve; and
f) comparing any noted differences in two or more of the determined EDSD, MED and R.sup.2 with empirical EDSD, MED and R.sup.2 EDD curve data which have been derived from physiologically abnormal mammalian populations in order to determine whether any such differences are indicative of an abnormal physiologic condition in the blood donor.

### Claim 2

A method for detecting abnormal mammalian physiologic conditions which are characterized by deviations from norm of certain erythrocyte parameters in a donor blood sample, said method comprising the steps of:
a) providing a sample tube containing a sample of mammalian anticoagulated whole blood from a blood donor and a plurality of artificial density markers which are operable to form discernible bands in the blood sample, each density marker in each band having a different density from density markers in any other band, and the density of each density marker being within an expected range of densities of erythrocytes;
b) centrifuging the sample tube so as to gravimetrically separate the erythrocytes into an erythrocyte column which is separate from the remaining components of the blood sample, and so as to embed the density markers in the erythrocyte column whereby erythrocyte subsets in an erythrocyte density distribution (EDD) gradient are demarcated by density markers which are embedded in the erythrocyte column;
c) measuring and recording distances between adjacent density markers in the erythrocyte column to provide a plurality of EDD values;
d) determining an EDD curve from said EDD values;
e) measuring and recording a hematocrit (HCT) value from said erythrocyte column to provide an HCT value for the centrifuged blood sample;
f) defining the erythrocyte density standard deviation (EDSD), mean erythrocyte density (MED), and curve skewness (R.sup.2) in said determined EDD curve;
g) comparing any noted differences in one or more of EDSD, MED, R.sup.2 and HCT with empirical EDSD, MED and R.sup.2 EDD curve data, and with HCT data, which have been derived from physiologically abnormal mammalian populations to determine whether any observed differences, or any combination thereof, are indicative of an abnormal physiologic condition in the blood donor.

### Claim 3

A method for detecting abnormal mammalian physiologic conditions which are characterized by deviations from normal erythrocyte density standard deviation (EDSD) in a donor sample of blood, said method comprising the steps of:
a) providing a sample tube containing a sample of mammalian anticoagulated whole blood from a blood donor and a plurality of artificial density markers which are operable to form discernible bands in the blood sample, each density marker in each band having a different density from density markers in any other band, and the density of each density marker being within an expected range of densities of erythrocytes;
b) centrifuging the sample tube so as to gravimetrically separate the erythrocytes into an erythrocyte column which is separate from the remaining components of the blood sample, and so as to embed the density markers in the erythrocyte column whereby erythrocyte subsets in an erythrocyte density distribution (EDD) gradient are demarcated by density markers embedded in the erythrocyte column;
c) measuring and recording distances between adjacent density markers in the erythrocyte column to provide a plurality of EDD values;
d) determining a donor EDD curve from said EDD values;
e) defining the erythrocyte density standard deviation (EDSD) in said donor EDD curve; and
f) comparing any noted differences in said defined EDSD with empirical EDSD curve data which have been derived from physiologically abnormal mammalian populations to determine whether any such differences are indicative of an abnormal physiologic condition in the blood donor.

### Claim 4

A method for detecting abnormal mammalian physiologic conditions which are characterized by deviations from norm of mean erythrocyte density (MED) in a sample of blood from a blood donor, said method comprising the steps of:
a) providing a sample tube containing a sample of donor mammalian anticoagulated whole blood and a plurality of artificial density markers which are operable to form discernible bands in the blood sample, each density marker in each band having a different density from density markers in any other band, and the density of each density marker being within an expected range of densities of donor erythrocytes;
b) centrifuging the sample tube so as to gravimetrically separate the donor erythrocytes into a donor erythrocyte column which is separate from the remaining components of the donor blood sample, and so as to embed the density markers in the donor erythrocyte column whereby erythrocyte subsets in a donor erythrocyte density distribution (EDD) gradient are demarcated by density markers embedded in the erythrocyte column;
c) measuring and recording distances between adjacent density markers in the erythrocyte column to provide a plurality of donor EDD values;
d) determining a donor EDD curve from said donor EDD values;
e) determining the MED in said donor EDD curve; and
f) comparing any noted differences in donor MED with empirical abnormal MED curve data which have been derived from physiologically abnormal donor populations to determine whether any such noted differences are indicative of an abnormal physiologic condition in the blood donor.

### Claim 5

A method for detecting mammalian physiologic conditions which are characterized by deviations from norm of curve skewness (R.sup.2) of an erythrocyte density distribution (EDD) curve in a donor blood sample, said method comprising the steps of:
a) providing a sample tube containing a sample of mammalian anticoagulated whole blood from a blood donor and a plurality of artificial density markers which are operable to form discernible bands in the blood sample, each density marker in each band having a different density from density markers in any other band, and the density of each density marker being within an expected range of densities of erythrocytes;
b) centrifuging the sample tube so as to gravimetrically separate the erythrocytes into an erythrocyte column which is separate from the remaining components of the blood sample, and so as to embed the density markers in the erythrocyte column whereby erythrocyte subsets in an EDD gradient are demarcated by density markers embedded in the erythrocyte column;
c) measuring and recording distances between adjacent density markers in the erythrocyte column to provide a plurality of EDD values;
d) determining a donor EDD curve from said donor EDD values;
e) defining the R.sup.2 in said donor EDD curve; and
f) comparing any noted differences in R.sup.2 with empirical R.sup.2 EDD curve data which have been derived from physiologically abnormal mammalian populations in order to determine whether any such differences are indicative of an abnormal physiologic condition in the blood donor.