Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Particle count or volume standard or control
Reexamination Certificate
1999-02-18
2002-02-26
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Particle count or volume standard or control
C436S008000, C436S063000, C436S164000, C436S172000, C435S002000, C422S051000, C422S051000, C356S039000, C356S244000, C356S246000
Reexamination Certificate
active
06350613
ABSTRACT:
TECHNICAL FIELD
This invention relates to an apparatus and method for analyzing a quiescent sample of anticoagulated whole blood. More particularly, this invention relates to an apparatus and method for analyzing the blood sample in a quiescent state in order to provide a white blood cell differential count, a reticulocyte count analysis, an enumeration of nucleated red blood cells, and the ability to detect abnormal nucleated circulating cells, such as cancer cells, which are rare events.
BACKGROUND ART
Recent advances in analytical hematology have increased the quantity and quality of information available from a patient's blood sample. As a result, the medical community's interest in using patients' blood samples as a diagnostic tool has also increased, with the most commonly performed test performed on anticoagulated whole blood being the complete blood count, or CBC, which is a suite of tests which may include, in addition to the enumeration of the cellular components and platelets, red blood cell metrics; reticulocyte counts; and the leukocyte differential count (LDC or “Diff”) which is the classification of the types of white blood cells present in the blood sample. The general physical properties of the sample, namely various cell or counts must be analyzed using quantitative methods relating to the entire sample. In conventional instrumentation and methods, this requires accurate sample metering and dilution, followed by specialized measurement apparatus. Additionally, the instrument must measure quantitative aspects of the individual cells, which usually involves providing a high dilution of the sample with a subsequent passage of the diluted material through a flow cell which measures the cells using electrical or optical means. Still further, qualitative measurements are used to classify the percentage of the total white blood cells which are composed of specific sub populations. The number of sub-populations depends upon the sophistication of the instrument involved, which may be as little as two or more than seven classifications.
Historically, the differential aspects of the CBC have been performed using separate methods from those used for enumeration. For example, the LDC portion of a CBC was traditionally performed by smearing a small amount of undiluted, blood on a slide, staining the dried, fixed smear, and examining the smear under a microscope. Reasonable results can be gained from such a smear, but the accuracy and reliability of the data depends largely on the technician's experience and technique. One problem with such smears is that the cells must be killed and fixed, and this precludes many types of supravital stains and analyses whose results depend upon the living cell, such as some cytochemical analyses. In addition, the use of blood smears is labor intensive and cost prohibitive, and is therefore generally not favored for commercial applications.
Another method of performing an LDC uses electrical impedance or optical flow cytometry. Flow cytometry involves passing a diluted blood sample through a small vessel wherein electrical impedance or optical sensors can evaluate the constituent cells as they pass serially through the vessel. The same apparatus may also be used to simultaneously enumerate and provide cell metric data. To evaluate WBC'S, the blood sample must be diluted, and the sample must be treated to mitigate the overwhelming number of the RBC's relative to the WBC'S. Although more expedient and consistent than the above described smear methods, flow cytometry also possesses several disadvantages. One disadvantage of flow cytometry is the plumbing and fluid controls that are necessary for controlling the flow rate of the diluted blood sample past the sensors. The plumbing in current flow cytometers can, and often does, leak, thus potentially compromising the accuracy and the safety of the equipment. These analyses are also generally incapable of providing any type of morphometric analysis, since an actual image of each cell is not obtained; only the total signal from any given cell may be analyzed. Another disadvantage of many current flow cytometers relates to the accuracy of the internal fluid flow controls and automated dilution equipment. The accuracy of the flow cytometer depends upon the accuracy of the fluid flow controls and the sample dilution equipment, and their ability to remain accurately calibrated. Flow controls and dilution equipment require periodic recalibration. The need for recalibration illustrates the potential for inaccurate results and the undesirable operating costs that exist with many presently available flow cytometers. An article authored by John L. Haynes, and published in
Cytometry Supplement
3: 7-17 in 1988 describes the principles of flow cytometry, both impedance and optical, and the application of such a technology to various fields of endeavor. Blood samples being examined in flow cytometers are diluted anywhere from 10:1 to 50,000:1.
Another approach to cellular analysis is volumetric capillary scanning as outlined in U.S. Pat. Nos. 5,547,849; 5,585,246 and others, wherein a relatively undiluted sample of whole blood is placed into a capillary of known volume and thickness and is examined while the blood is in a quiescent state. This technique deals with the presence of the red blood cells by limiting the scanning wavelengths to those to which the red blood cells are relatively transparent, and it requires that the sample be treated so that the red blood cells do not aggregate during the measurement process. Thus, this technique is limited to the use of longer wavelength fluorescence, and there is no provision for the enumeration of reticulocytes or nucleated red blood cells. Additionally, as with flow cytometry, no morphologic information is available from the scans. There are a number of commercial instruments available for performing a CBC or related tests, but those which provide more than a few of the CBC tests quickly become complex, expensive and prone to malfunction. In addition, there are a number of methods proposed for specific hematological tests, but these do not provide all of the clinically useful information which is expected in a CBC.
All of the above methods are generally limited to a single mode of analysis, in that a combination of histochemical staining and cellular morphology is not possible. Having the capability to perform both of these types of tests expands the number of groups which can be recognized by the method.
It would be desirable to have a method and apparatus for examining a quiescent sample of anticoagulated whole blood, which method and apparatus are capable of providing accurate results for a LDC, reticulocyte enumeration and detection of nucleated red blood cells and abnormal circulating nucleated cells, such as cancer cells, and does not require sample fluid flow through the sampling chamber during sample analysis.
DISCLOSURE OF THE INVENTION
This invention relates to a method and apparatus for use in examining and obtaining information from a quiescent substantially undiluted anticoagulated whole blood sample which is contained in a chamber. The phrase “substantially undiluted” as used in connection with this invention describes a blood sample which is diluted by no more than about 1:1, and preferably much less. Generally the only reagents that will be used in performing the method of this invention are dyes, stains and anticoagulants, and these reagents, even if liquid, are not designed to dilute the specimen. The analysis may be performed within a chamber having a fixed depth, as long as the depth supports the formation of red blood cell aggregations and lacunae, which form in a layer having a thickness from about seven to about forty microns, depending upon the hematocrit of the sample. However, having a fixed depth makes it more difficult to analyze samples having a widely varying white cell count, and the simultaneous enumeration of reticulocytes is impossible in this type of chamber. Preferably, a chamber is used whi
Levine Robert A.
Rodriguez Rodolfo R.
Wardlaw Stephen C.
Belton Dickinson & Co.
Jones William W.
Wallenhorst Maureen M.
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