US 3558522 A
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United States Patent Olhce 3,558,522 Patented Jan. 26, 1971 US. Cl. 252-408 6 Claims ABSTRACT OF THE DISCLOSURE A hematology control standard for the calibration of blood cell counting apparatus and a method for the preparation thereof by admixing predetermined amounts of washed red blood cells and synthetic latex particles having a to 15 micron particle size range in a fluid suspension of a known amount of serum albumin.
This invention relates to a control standard for use in the counting of blood cell particles. More particularly, this invention relates to a composite and unitary hematological control standard for calibrating and checking the accuracy of automatic or manual red blood cell and white blood cell counts, hematocrit and hemoglobin determinations.
Quality control has long been a necessary and routine procedure in clinical chemistry and coagulation laboratories. Accuracy in the counting of red and white blood cells and in the making of hematocrit and hemoglobin determinations of the patients serum is dependent, in part, upon the use of adequate control standards. Thus, the accuracy of the manual technic of particle counting, such as by the classical method of microscopy, can be checked by giving the technician a so-called blind sample, or control standard, containing a known concentration of particles for comparison with the unknown samples for which he is to make a determination.
Modern technology has provided numerous types of automatic equipment for particle counting which is gradually replacing the older and more laborious manual techniques. But even these automatic methods of particle counting require constant quality control by the use of control standards since the possibility of malfunctioning of the instrument is ever present. Consequently, the importance of accurate and reliable checks on hematological determinations that may be used in the diagnosis of disease speaks for itself and needs no further amplification here.
The traditional method of calibrating automatic particle counting equipment has consisted of providing a whole blood standard by repeatedly counting such blood by manual hemacytometer techniques to establish its value.
The disadvantage of this method is that the standard is usable for only one day and each time a fresh whole blood standard is prepared, the manual counts must be repeated.
Other conventional methods consist of providing a red blood cell standard in which the red cells have been stabilized by one means or another to prolong their shelf life. However, these methods are not usable for white cell counts in a system which provides for the destruction of the red cells by a lysing agent prior to counting of the white cells.
Still other methods consist of substituting simulated cell particles in suspension, for example, polystyrene latex particles, in place of natural blood cells. These methods have not been very satisfactory, however, since it has been too difficult heretofore to suitably suspended sulficient particles in the carrier liquid.
Various other approaches to the problem of providing control standards for blood cell particle counting are described in recent US. Pats. 3,406,121 and 3,412,037, and in recent British Pats. 1,129,873 and 1,131,690.
It is an object of the present invention to provide a new and improved control standard for use in the counting of blood cell particles.
It is another object of the present invention to provide a composite and unitary hematological control standard for calibrating and checking the accuracy of automatic or manual red blood cell counts, white blood cell counts, hematocrit and hemoglobin determinations.
These and other objects will be apparent to the person skilled in the art after reading the disclosure hereof.
Briefly stated, the objects of the present invention are achieved by providing a hematological control standard which comprises a fluid suspension of a known amount of serum albumin containing predetermined amounts of washed red blood cells and synthetic latex particles having a particle size ranging from about 5 to about 15 microns, said fluid suspension having a specific gravity and viscosity similar to normal blood serum.
The hematological control standard of this invention provides users of automated instruments with control for red blood cell counts, white blood cell counts, hematocrit and hemoglobin determinations all in a single product, thereby eliminating the need for separate control standards for red blood cell counts and white blood cell counts as used heretofore. The control standard has a shelf life of at least four Weeks, which can be extended by fixing or stabilizing the viable red cells by known methods. It has been found that in this control standard there is only about a one percent hematocrit decrease per week, due to the natural shrinkage of the red blood cells, and virtually no hemolysis.
The synthetic latex particles used in the control standard of this invention are generally spherical in shape, they have a relatively uniform size of from about 5 to about 15 microns, which approximates the relative size of the normal leukocytes, or White cells, and are preferably employed in the fluid suspension at a concentration of from about 5,000 to about 10,000 particles per cubic millimeter,, which is the approximate count in normal blood.
These latex particles can comprise polystyrene, polyvinyltoluene and/or styrene-divinylbenzene copolymer latex and the like synthetic polymeric latex materials of suitable particle size.
The styrene-divinylbenzene copolymer latex particles are preferred for use in this invention. These latex particles are visible under the microscope under conventional magnifications at 10X and 40x, they are inert to the usual red cell lysing agents, such as acetic acid and various detergent substances, and otherwise provide suitable simulation of the white blood cells in the control standard of this invention.
The washed red blood cells are preferably employed in the control standard at a concentration of from about 3 million to about 5 million per cubic millimeter, which approximates the count in normal blood. These washed cells are preferably obtained from 1 to 24 day old human whole blood, although any fresh red cells or viable red cells which have not been lysed can be used. Careful and thorough washing of the blood cells after separation from the plasma, such as by saline washing and/ or washing in Alsevers solution, followed by filtering to remove any residual white blood cells, is desirable. As used herein, the term washed red blood cells refers to red blood cells which have been separated from the other constituents of whole blood by the above methods or by any other conventional method of separating red blood cells from contaminating substances.
The serum albumin is employed in the control standard of this invention to provide a proteinaceous medium closely resembling plasma in consistency. Serum albumin also has been found to provide a suitable medium for suspension of the latex particles in the control standard of this invention without necessity of employing any of the other normal serum proteins. The serum albumin can be obtained from whole plasma by alcohol fractionation, ammonium sulfate precipitation, and any other such conventional procedures for preparing serum albumin.
From about 2 percent to about 5 percent, and preferably about 3 percent, by Weight of albumin is generally contained in the control standard fluid suspension of this invention. When the washed red cells and latex particles, at approximately the normal particle counts of red and white cells as herein'before stated, are suspended in an aqueous solution of the albumin at a pH of about 6, the control standard fluid suspension is found to very closely resemble normal blood serum in specific gravity and viscosity.
It is preferable to additionally include in the control standard a small but effective amount of antibiotic or preservative, for example, T erramycin, neomycin, sodium azide, and the like antibiotics or preservatives, for their biocidal effects. About one percent by weight of antibiotic or preservative in the final product is suitable for this purpose.
The hematological control standard of this invention can be used for both automatic and manual particle counting techniques as hereinbefore stated. The automatic equipment to which the control standard is adapted is of great diversity. In general, one or the other of two types of particle size analysis equipment is employed. In one type, each particle is counted and its discrimination property is measured directly. In the other type, the particles are measured in bulk and particle behavior is recorded through a series of measurements of the magnitude of the bulk, in terms of the count, combined surface area or combined mass. The type of measurement used then determines the basis of the size distribution,
Optical and electrical properties are two of the most prevalent types of size discriminating properties employed in the particle size analysis equipment to which the control standard of this invention is adapted. The optical equipment can employ imaging, spectral transmission, scattering and diffraction mechanisms, while the electrical equipment can employ resistance, capacitance, and charge mechanisms.
A specific suitable example of an instrument useful with the control standard of this invention is the Coulter Electronic Blood Cell Counter and similar such equip ment as described, for example, in US. Pats. 2,656,508, 2,869,078, 2,985,830 and 3,340,470. This particular in strument discriminates among particles by how they affect the electrical resistance of the fluid medium containing the particles in suspension as they pass through an orifice.
Other examples of apparatus which can be calibrated by the control standard of this invention are the Technicon Instruments Auto Analyzers and similar such equipment as described, for example, in US. application Ser. No. 427,593, filed Jan. 25, 1965. This apparatus provides a support for a plurality of whole blood samples which are sequentially diluted, a flow cell of small crosssection through which the volume of diluted blood ispassed, illuminated optical means coupled to the flow cell for detecting the passage of individual cells therethrough and providing an output pulse signal in response thereto, and electronic means for receiving and totaling the number of pulses per unit of time and providing an output signal.
Various other conventional types of particle counting instruments which employ the metering of a sample of the particle-containing suspension past a scanning point in the detecting system will be apparent to the person skilled in the art.
In the use of the control standard in calibrating or checking the accuracy of the above or similar such equipment, the fluid suspension is mixed well prior to its use to ensure good dispersion and prevent the particle sizes from being indicated too large and size distribution too wide. The consistency of the fluid suspension of the control standard of this invention as described hereinbefore is capable of retaining the particles in suspension without appreciable loss of particles by settlement during the usual calibration procedures on equipment such as described herein. Excellent calibration can be achieved when every fifth sample used in these instruments is the control standard of this invention. The employment of this control standard in place of an unknown blood sample at other predetermined periodic and regular intervals will similarly provide suitable calibration of various other instruments.
After making the cell counts, calculation of the red blood cell indices can be made by the following formulas:
(a) Mean corpuscular volume (MCV) is the average of the individual red blood cell.
Hematocrit (percent) X 10 Red blood cell count (in millions) =MCV (in cubic microns) (b) Mean corpuscular hemoglobin (MCH) is the average weight of hemoglobin in the individual red blood cell.
Hemoglobin (g./l00 ml.) X 10 Redbloodcellcount (in millions) =MCH (in micrograms) (c) Mean corpuscular hemoglobin concentration (MCHC) is the percent of hemoglobin in the average red cell.
Hemoglobin (g./ ml.) X 100 Hematocrit (percent) The following example further illustrates the invention herein although the invention is not limited to this specific example. All parts and percentages herein are by weight unless otherwise specifically stated.
EXAMPLE Whole human blood containing anticoagulant is centrifuged and the supernatant plasma is aspirated. Saline (an aqueous solution of 1.2% NaCl) is added to the packed cells in an amount sufiicient to replace the volume of separated plasma. The packed cells are thoroughly mixed with saline and centrifuged again. This saline washing and centrifugation is repeated two more times.
The packed cells are then similarly washed three times with a modified Alsevers solution, pH 7.0, made-up by dissolving in one liter of Water, 20.5 grams of anhydrous dextrose, 8.0 grams of sodium citrate-211 0, 4.2 grams of sodium chloride and 5.2 ml. of an aqueous one percent citric acid solution. After the third washing with the modified Alsevers solutions, the red cells are again spun down and the supernatant is extracted.
About 40-50% by volume of an aqueous solution of 6 percent by weight human serum albumin in modified Alsevers solution (as described above) is then added to the washed red blood cells. The fluid suspension is then adjusted to a concentration of about 5 million red cells per cubic millimeter and 3 percent by weight of albumin.
Latex particles of styrene-divinylbenzene copolymer having a particle size range of 6 to 14 microns and an average particle size of 7.6 microns are then added to a count of about 10,000 particles per cubic millimeter. The final product is then filled into bottles of 10, 20 and 50 ml. size and is ready for use in calibration and checking the accuracy of automatic and manual blood cell counting instruments as described hereinbefore. The specific gravity of the final product is similar to that of normal blood .serum (about 1.03) and its viscosity, as determined by its flow and other handling characteristics, very closely resembles that of normal blood serum.
= MCHO (in percent) Various other examples and modifications of the foregoing example will be apparent to the person skilled in the art after reading the invention described herein and defined in the appended claims without departing from the spirit and scope of the invention. All such further examples and modifications of the foregoing example are included Within the scope of the invention.
What is claimed is:
1. A fluid suspension of a known amount of serum albumin containing predetermined amounts of washed red blood cells and synthetic latex particles having a particle size ranging from about 5 to 15 microns, said fluid suspension having a specific gravity and viscosity similar to normal blood serum,
2. The fluid suspension of claim 1 in which the concentration of serum albumin is from about 2 percent to about 5 percent by weight, the red blood cell count is from about 3 million to about 5 million per cubic millimeter and the latex particle count is from about 5,000 to about 10,000 per cubic millimeter.
3. The method of making a hematological control standard for the calibration of blood cell counting apparatus comprising the steps of admixing predetermined amounts of washed red blood cells and synthetic latex particles having a particle size range of from about 5 to about 15 microns in a fluid suspension of a known amount of serum albumin.
4. The method of claim 3 in which the concentration of serum albumin in the fluid suspension is from about 2 percent to about 5 percent by weight, the red blood cell count is from about 3 million to about 5 million per cubic millimeter and the latex particle count is from about 5,000 to about 10,000 per cubic millimeter.
5. The method of calibrating automatic blood cell counting apparatus comprising substituting the fluid suspension of claim 1 in place of an unknown blood sample in said apparatus at predetermined periodic and regular intervals. 6. The method of claim 5 in which the concentration of serum albumin in the fluid suspension is from about 2 percent to about 5 percent by weight, the red blood cell count is from about 3 million to about 5 million per cubic millimeter and the latex particle count is from about 5,000 to about 10,000 per cubic millimeter.
References Cited UNITED STATES PATENTS 3,406,121 10/1968 Jones 23-230X 3,412,037 11/1968 Gochman et al 252-408 3,436,187 4/1969 Ferro et a1 252408X 3,466,249 9/ 1969 Anderson 23230X FOREIGN PATENTS 690,849 7/1964 Canada 252-408 HAROLD ANSHER, Primary Examiner J. C. GIL, Assistant Examiner US. Cl. X.R.