Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
2001-03-28
2002-10-22
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C204S401000, C435S001200, C435S004000, C435S005000, C436S017000, C436S018000
Reexamination Certificate
active
06468732
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to stable reagent compositions, and storage containers therefor, for use as blood diluents and for assaying blood cells and platelets in an aliquot of a whole blood sample. The invention further relates to the long-term storage of such reagent compositions in packaging designed to maintain MCV-assays of blood samples aeration-insensitive and to maintain reagent stability over time.
BACKGROUND OF THE INVENTION
The analysis of blood samples using semi-automated and fully-automated hematology analyzer instruments requires the use of specialized reagents in which blood samples are mixed and diluted. Such reagents allow the practitioner to obtain accurate, sensitive and precise measurements of a variety of blood cell parameters and to analyze the different blood cell types in a blood sample. As a particular example, hematology analysis of blood cells, especially red blood cells, platelets and reticulocytes, on automated instruments frequently involves the use of reagents comprising components which have limited stability overtime in storage.
For the analysis of blood samples containing red and white blood cells and platelets, blood samples are routinely drawn into containers (e.g., glass tubes and the like) for subsequent use and/or storage. When a blood sample is to be analyzed or tested, the container carrying the blood sample is opened and the sample is, in turn, exposed to the air. As a rule, venous blood contains about 100 times more dissolved carbon dioxide (as bicarbonate) than is present in the air. This is because red blood cells carry metabolic waste carbon dioxide from tissues to the lungs. When the container housing a venous blood sample is opened, the blood sample begins to be aerated and the surface of the red blood cell begins to be exposed to air-saturated plasma. This results in a driving force for the cells, i.e., red blood cells, to unload carbon dioxide to the air. Carbon dioxide diffuses out of the red cell membrane causing a measurable decrease in intracellular bicarbonate, and therefore, a decrease in osmolality. The cell simultaneously loses water to maintain the original osmolality. As a result, the cell shrinks during aeration. This is known as a sample handling “aeration effect”. (Dacie and Lewis, Practical Haematology, fifth edition, p.38, Churchill Livingstone, 1975). With repeated blood samplings in air, the red blood cell MCV decreases and the sample handling effect is evidenced such that about 2-5% of the sample's red blood cell volume is lost in a fully aerated sample of venous blood.
Virtually all hematology methods using conventional reagents and analyzer instrumentation are susceptible to the problem of sample handling effects, in which aeration of a blood sample induces red cell shrinkage, known as aeration-induced mean cell volume (MCV) shrinkage. The extent of such MCV shrinkage can be as high as 5%. As a blood sample is sampled repeatedly for various assays and mixed, the headspace (i.e., the volume of air above the blood) in the sample storage tube increases. As the tube is opened, there is contact between air and the plasma surrounding the blood cells in the sample, which can have a detrimental effect on the blood sample, particularly after repeated uses. The severity of such a sample handling aeration effect increases if the sample is sampled repeatedly with frequent remixing and opening of the tube to the air.
In addition to the above-described sample handling effect, another serious problem related to blood sample analysis is the problem of storage effect, which is also detrimental to the integrity and quality of the cells in a stored blood sample. Indeed, the actual storage of whole blood samples in closed containers (e.g., tubes) at room temperature for 24 hours may cause the MCV to increase as much as 7%, due to the metabolism of both the red and white blood cells in the closed container of blood. The metabolism leads to the accumulation of carbon dioxide and other osmotically active products which tend to increase the internal osmolality of the cells, thereby producing swelling during storage.
More specifically, to provide a theoretical mechanism not intended to be in any way limiting, during the storage of whole blood, carbon dioxide (CO
2
), a major metabolic product, diffuses from the white blood cells into the plasma and then into the red blood cells. Once inside the red cells, carbon dioxide is enzymatically hydrated to carbonic acid, which then dissociates to an osmotically active bicarbonate ion and a buffered proton. The dissociation is forced by the difference between the intracellular pH of the cell (approximately pH 7.4) and the pK
a1
of carbonic acid. As a result of these steps, cellular osmolality increases. Water therefore simultaneously enters the red cells to restore the original internal osmolality of approximately 290 milliOsmoles/kg, to match the extracellular osmolality, and the cells swell. Carbon dioxide, which is generated within red cells, produces a similar effect. Storage of whole blood for 24 hours at room temperature (i.e., approximately 25° C.) causes an approximately 5-7 fL (femtoliter) increase in MCV, at least part of which is due to the described process.
Thus, a goal of the present inventors was to develop reagents, which are stable over time, for use in blood sample analysis. The reagents of the present invention can reverse, decrease, or eliminate the above-described sample handling and storage effects on blood cells, i.e., by reducing aeration-induced-shrinkage or swelling from storage at room temperature. Correlative to this goal is the added need for novel storage devices and materials which provide storage packaging that maintains the stability of the newly developed reagents. Long-term stability provides considerable economic advantages to those in the art, since such reagents can be used, stored and re-used for longer periods of time before new reagents need to be purchased and used. In addition, stability of the reagent components over time is required to provide assurance to the user that the results obtained after use of the reagents with cells in hematology analysis will remain accurate, sensitive and precise following repeated use and long-term reagent storage.
Prior to the present invention, bicarbonate-containing reagents, e.g., blood diluents, that partially or completely reversed the effects of sample handling, e.g., aeration-induced MCV shrinkage, were not described. The art also fails to describe flexible, collapsible CO
2
barrier packaging for such bicarbonate-containing diagnostic reagents, particularly for the purposes of long-term storage of such reagents, for hematology cell analysis and measuring the properties of red blood cells and other blood cells in a whole blood sample.
To address shrinkage of MCV due to aeration of blood, Bryner et al., (1997, “The Spun Micro-Hematocrit and Mean Cell Volume are Affected by Changes in the Oxygenation State of Red Blood Cells”,
Clin. Lab. Haem
., 19:99-103), proposed fully oxygenating blood samples by treatment with an equilibrating gas mixture containing oxygen, carbon dioxide and nitrogen and the use of a layer of mineral oil in the tube as a barrier to the loss of both oxygen and carbon dioxide from the blood samples equilibrated with the gas mixtures. However, because both gases are more soluble in mineral oil than in water, mineral oil is a very poor barrier for these gases. Another drawback of the approach proposed by Bryner et al. is that their treatment of opened tubes of blood with the equilibrating gas mixture is both time consuming and a potential biohazard. The approach of Bryner et al. involves the bubbling of air through blood which generates aerosols when the bubbles break. If the blood sample should contain pathogenic agents or substances, such aerosoling could be harmful to the handler, or others nearby, due, for example, to the inhalation of such pathogenic agent or substances.
In general, diluents employed for red blood cell analysis (e.g., Bayer H
Malin Michael J.
Ornstein Leonard
Bayer Corporation
Morgan & Finnegan L.L.P.
Srivastava Kailash C.
Tate Christopher R.
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