Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Particle count or volume standard or control
Reexamination Certificate
2001-03-23
2003-02-04
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Particle count or volume standard or control
C436S008000, C436S017000, C436S063000, C252S408100
Reexamination Certificate
active
06514763
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a hematology blood control and, more particularly, to a hematology control made from human leukocytes and a method for the preparation of the control.
BACKGROUND OF THE INVENTION
Instruments for the analysis of blood components and chemistry have been used for many years. The accuracy and sensitivity of these hematology instruments have steadily advanced. The early forms of hematology instrumentation have evolved to relatively complex machines that analyze the discreet components of blood based upon the intricate and subtle characteristics of its components.
The most recent iteration in automated hematology instrumentation has been the multi-part analysis of human white cells, in addition to the detection of red blood cells and platelets. The white cell populations typically include lymphocytes, monocytes, neutrophils, basophils, and eosinophils. The methods for blood cell analysis involve the electrical and optical properties of each type of blood cell. The Beckman-Coulter™ five-part white cell analysis instrument uses three different types of technologies, which include electrical impedance, a DC mathematical manipulation called conductance, using a low voltage DC (direct current) measurement, Rf (radio frequency modulation), and laser technology which includes light scatter and light absorption. The Rf measurement is typically used with the DC low frequency measurement to create a parameter called opacity which is a calculation of Rf divided by DC. Instruments by other manufacturers, such as Abbott Diagnostics™, Technicon™, and TOA™, use a combination of electrical impedance, DC conductance and/or laser technology, Rf, depolarized 90° angle light scatter, and/or light absorption. Although the basic types of electronic technology may appear the same, each manufacturer has a unique implementation for the instrument hardware and software that is required to analyze blood cells. The individual implementations of this technology by the various manufacturers have resulted in a wide array of reagents and methodology for each specific instrument of each manufacturer, thereby increasing the complexity and expense of their use. There is no one reagent or methodology that can be used with a plurality of instruments.
In order to ensure that a hematology instrument is working properly, it has been mandated by governments that there be a method to verify the integrity of the instrument using a blood control. The control should contain particles that represent all of the cellular elements of fresh blood, as well as a liquid component that serves as a suspending media similar to the function of human plasma. This synthetic plasma usually contains components that are the same as or function the same as native plasma. The components of synthetic plasma include inorganic salts, organic and/or inorganic buffers, and a viscous material for maintaining homeostasis similar to the plasma proteins. The manufacturer of a control provides all of the critical values such as cell count, cell size, and cell type. The control material should have sufficient shelf life to be used for days, weeks, or months to ensure the consistency of instrument performance over time.
The method for preparing a hematology control is dependent on the hardware and software design of the specific instrument in which the control is to be used, as well as the requirements for extended shelf life. Particles in blood control products that work like human white cells, red blood cells, or platelets on a Coulter™ type instrument may not work effectively on other instruments, such as instruments manufactured by Abbott Laboratories™, Technicon™, or TOA™ instruments.
Moreover, because these particles are usually modified from various types of blood cells, they do not behave like living native fresh blood cells. Consequently, human white cells fixed with a cross-linking agent like glutaraldehyde may behave like a neutrophil on an Abbott™ instrument, but behave like cellular debris on a Coulter™ instrument. Specially treated and cross-linked red blood cells from non-mammalian vertebrates may look like mononuclear cells on one type of hematology instrument and look like lymphocytes on another.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method for preparing white blood cells from a vertebrate source for use in a hematology blood control product. The method provides sufficient flexibility to compensate for the different technologies employed by instrument manufacturers. Thus, it is an object of the present invention to customize a process using the described method, resulting in a hematology control unique to a hematology instrument specification.
It also is an object of the present invention to provide a method to prepare a hematology blood control from human white cells without the need for plasma cholesterol to maintain the electrical and/or optical characteristics of the cells.
It is a further object of the present invention to provide a hematology blood control that can be utilized on a plurality of instruments made by different manufacturers.
It is yet another object of the present invention to provide a hematology blood control that can be prepared easily and inexpensively.
To accomplish these and other related objectives, the control of the present invention is achieved using a gentle chemical removal of red cells, leaving an intact white cell preparation. The preservation steps, namely the use of cross-linking agents as aldehydes, involves a step-wise process starting with very low concentrations of a cross-linking agent. The fixation part of the process outlined in this disclosure has also been applied to non-mammalian blood cells and therefor would appear to be a universal procedure for preparing all types of vertebrate blood cells. The examples below include small as well as large volume processes. It has been found that the concentration and timing elements must be adjusted based on the volume of the reaction mixture. Thus, a final volume of 15 mL will require different proportions of elements and different timing steps compared to a 1 liter batch. For example, it is possible to perform a 15 second centrifugation with a 15 mL tube process but this 15 second step is essentially impossible with a 1 L bottle batch. The selective use of lytic agents and the unique fixation process eliminates the need for supportive reagents as cholesterol, which have been found to be necessary for the successful display of preserved human white cells.
DETAILED DESCRIPTION
The basic components for the preparation of the control of the present invention include the following:
A quantity of source leukocytes between 0-5 days old, preferably one day old, stored in a styrofoam or other insulated container with sufficient cold packs to insure the units are cool but not cold. Overnight storage at 4° C. will sufficiently alter the quality of the white cells or potentially require modifications in the processing of the blood.
A lytic agent including of any one or a combination of the following:
an organic acid such as formic, acetic, and propionic acids. The preferred acid is propionic acid.
a quantity of saponin in water. Suitable types of saponin include, but are not limited to, Sapindus and Quillaja. The preferred saponin is Sapindus.
A quench made with inorganic salts including one or a combination of the following:
Carbonate buffered, similar to that used in certain commercial products, such as Hematronix, Inc Diff Pak™ containing sodium carbonate, sodium sulfate, and sodium chloride.
Buffer free sodium sulfate and sodium chloride salts.
Buffer free sodium sulfate only
Buffer free sodium chloride only
Sodium phosphate only
Any of the above salt solutions with cross-linking agent.
A post-lytic hypotonic fixing reagent including one or more of the following:
Low osmolarity salt, such as diluted M-Ringers, an &agr;-naphtol based salt solution, or diluted mammalian balanced salt solution as Osmocel®.
a low carbon number glycol, such as propylene glycol
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Carver Franklin J.
Lapicola James D.
Hematronix, Inc.
Stinson Morrison & Hecker LLP
Wallenhorst Maureen M.
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