Abstract: A reagent blood coagulation that causes an increase in turbidity changes when added to a plasma sample containing a substance activating coagulation factor activating such as tissue thromboplastin, phospholipid and thrombin, calcium ion, and molecular substance such as high molecular vinyl and a high molecular polysaccharide. By adding a molecular substance to the reagent, the turbidity change due to blood coagulation is increased, and hence the changing quantity of transmitted light or scattered light increases, thereby making it possible to achieve a more accurate detection. Besides, by adjusting the electric conductivity, pH and osmotic pressure to proper values, the turbidity change due to blood coagulation may be further amplified.

Abstract: Disclosed is a blood sampling apparatus containing an anticoagulant composition which allows for the accurate analysis of blood electrolyte concentrations. The claimed apparatus comprises a receptacle, a blood inlet and the disclosed anticoagulant composition which does not unduly bind sodium, potassium or calcium ions from the sampled blood.

Abstract: Reagents for enabling leukocytes to be classified and counted lyse erythrocytes and act on leukocytes to stabilize them are disclosed. The reagents contain polyoxyethylene-based surfactants, and may contain hyperosmotic or hypoosmotic agents and solubilizing agents. One embodiment of the invention contains surfactants selected from the anionic and nonionic polyoxyethylene-based surfactants having 18-30 repeating oxyethylene units per molecule. A second embodiment of the invention contains nonionic polyoxyethylene surfactants only, having 20-100 repeating oxyethylene units per molecule, does not employ either saponin or quaternary ammonium compounds, and provides for adjustment of the pH of the resulting solutions. The invention allows leukocytes to be classified into as many as five types, each type to be quantified, and enables the detection of abnormal cells, when used in conjunction with particulate analysis flow cytometers employing the RF method and the DC method.

Abstract: An antithrombin III is described comprising a lyophilized powder of antithrombin III and a polyoxyethylene glycol sorbitan alkyl ester as well as a kit composed of a container wherein a lyophilized powder of antithrombin III is placed and a solvent for the powder, characterized in that a polyoxyethylene glycol sorbitan alkyl ester is added to either or both of the powder and the solvent. The preparation has a high solubility in water by retaining its activity.

Abstract: This invention primarily is directed to a hematology reference control solution, the three separate white cell control portions thereof consisting of three types of fixed red cells of determined size distribution for checking the operation of a particle analyzing instrument, including its predetermined lower and upper threshold settings for each class or subclass of leukocytes.For preparing a human granulocyte analogue, nurse shark erythrocytes are altered and fixed in a chilled solution to simulate in number, size and distribution the granulocytes in human whole blood.

Abstract: The invention concerns a method of detecting proteins, especially HIV and CMV antibodies in body fluids using solid-phase immune reaction with a buffer containing an animal serum and/or a non-ionic surfactant in a phosphate or carbonate buffer together with further additives selected from inorganic salts, water-soluble polymers, organic compounds, and propionic acid and their derivatives and mixtures. The buffer makes it possible to reliably eliminate false positive results.

Abstract: A composition, method and test device for determining the divalent cation concentration or estimating the specific gravity of a test sample are disclosed. The method utilizes a test device comprising a test pad, wherein the test pad includes a carrier matrix incorporating a reagent composition capable of interacting with divalent cations present in the test sample to produce a detectable and measurable response that correlates to the divalent cation concentration or to the specific gravity of the test sample. The reagent composition, comprising a metal-sensitive triphenylmethane dye; a buffer; an optional chelating agent; and suitable carrier, is incorporated into a carrier matrix to provide a test pad of a device useful in a divalent cation assay or a specific gravity assay of a test sample.

Abstract: Compositions, such as liquid therapeutic or diagnostic compositions, and methods for their preparation and involving their use. The compositions comprise an effective amount of gelatin from cold water fish skin as a protein base. The indicated gelatin provides many significant advantages and improvements, including for instance its high stabilizing effect on labile organic substrates included in the compositions, and its low temperature gelling properties which provide improved compositions which do not substantially gel during refrigeration. Further representative advantages relate to its behavior as a zwitterion thus reducing or eliminating needs for buffers, and its surprising behavior similar to human serum protein in protein analyses such as the biuret procedure.

Abstract: Assay methods are provided for determining the presence of lithium in high sodium content liquids such as serum, plasma, urine or other sample without deproteinization. The novel compounds are water soluble derivatives of TMC-crownformazans and provide signal enhancement by increased absorbance of the dye-lithium complex over the dye anion.

Abstract: A standard solution containing thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine dissolved in a buffer solution is used for calibration in a method for the determination of thyroxine (T.sub.4) or triiodothyronine (T.sub.3) in serum, wherein both human and bovine TBG can be used.

Abstract: A stabilized, aqueous composition containing FK506. FK506 degrades rapidly in most aqueous matrices. The rate of degradation is decreased in the presence of unfixed blood cells or fragments of blood cells from human or animal sources. A number of matrices using blood components are possible. Blood can be used directly or the blood cells can be lysed. The blood is diluted with a solution of an alkali halide.

Abstract: A process is disclosed for the production of an anticoagulant composition which allows for the accurate analysis of blood electrolyte concentrations. Also disclosed is the anticoagulant composition produced in accordance with the disclosed method and an apparatus containing said composition useful in the selection and sampling of blood.

Abstract: The present invention provides an aqueous buffer solution and method and solution for hydrating and calibrating a sensor membrane of a fiber-optic catheter useful for measuring an analyte other than chloride in a physiological milieu. The temperature, gaseous atmosphere, and aqueous buffer solution of the sensor component are controlled throughout the calibration process in order to ensure that the device is calibrated in accordance with its intended use. The solution is chemically compatible with the intended use of the device and provides compensation for evaporative loss, contaminants, or changes in the solution occurring during storage especially in plastic containers.

Abstract: A stable reference solution for calibrating and monitoring blood gas instrumentation is disclosed. The solution comprises an aqueous mixture containing a hemoglobin solution derived from a mammalian source which comprises at least about 95% reduced hemoglobin. The solution additionally contains a bicarbonate buffer and a metal catalyzed methemoglobin reducing system and an organic buffer. To provide a control element having a variety of properties similar to fresh, whole, human blood, the reference solution is stored in a sealed ampule under an inert atmosphere containing CO.sub.2 until just prior to use. The ampule is subsequently opened and the solution is equilibrated with a gas mixture having components which provide gasses in a physiological range to the solution. The solution is characterized by exhibiting properties similar to fresh blood subsequent to equilibration and an extended storage life prior to equilibration.

Abstract: A hematology control product comprising leukocyte analogs is described. The analogs comprise red blood cells which simulate at least two physical properties of human leukocytes. A method for making leukocyte analogs from blood cells having desired physical properties is also described. The process comprises expanding the cell volume, changing the hemoglobin content of the cell and fixing the cell. Generally, the monocyte and lymphocyte analogs leak hemoglobin from the cell while the eosinophil analog has the hemoglobin precipitated in the cell. A further method is described to use the control product to determine whether an automatic instrument is operating within manufacturer's specification.

Abstract: A lytic reagent composition highly selective in its interactions with the cell membranes of white blood cells and also provides a method of using the reagent composition in a particle analyzing system to effect a significantly improved white blood cell differential determination. The lytic reagent composition is characterized by an ability to selectively shrink the white blood cells into the increasing size order of lymphocytes, basophils, monocytes, eosinophils and neutrophils, and effect a five component separation of these major subpopulations of white blood cells on the histogram of an automated blood analyzer when used in conjunction with a suitable blood diluent.

Abstract: A composition which reduces the turbidity of samples of artificial or naturally occurring biological fluids comprising:a buffering agent;a nonionic surfactant;a lipolytic enzyme; and water.The composition provides a catalyst for and agents which hydrolyze triglycerides present in the sample to glycerol and free fatty acids. Also present in the composition is a fatty acid scavenging agent or agents to render the catalyzed fatty acids water-soluble. The fatty acid scavenging agent may be cyclodextrin, albumin or mixtures thereof.

Abstract: A blood serum sample useful as a calibration baseline comprising a natural blood matrix is disclosed. This serum sample contains less than an HPLC-detectable amount of cortisol. Also disclosed is a serum sample containing less than HPLC-detectable amounts of cortisone and corticosterone and less than RIA-detectable amounts of testosterone, androstenedione, progesterone, DHEA sulfate and 17-OH progesterone. A method of preparing such a serum sample is also disclosed.

Abstract: A stable reference solution for calibrating and monitoring blood gas instrumentation is disclosed. The solution comprises an aqueous mixture containing a hemoglobin solution derived from a mammalian source which comprises at least about 95% reduced hemoglobin. The solution additionally contains a metal catalyzed methemoglobin reducing system, reference concentrations of selected electrolytes and an organic buffer. To provide a control element having a variety of properties similar to fresh, whole, human blood, the reference solution is stored in a sealed ampule under an inert atmosphere containing CO.sub.2 until just prior to use. The ampule is subsequently opened and the solution is equilibrated with a gas mixture having components which provide gasses in a physiological range to the solution. The solution is characterized by exhibiting properties similar to fresh blood subsequent to equilibration and an extended storage life prior to equilibration.

Abstract: The present invention is concerned with a combination for the preservation of diagnostic tests, characterized by a content of at least two components selected from the group comprising 2-methyl-4-isothiazolin-3-one hydrochloride, 2-hydroxypyridine-N-oxide, chloroacetamide, (N,N-methylene-bis-(N-(1-hydroxymethyl-2,5-dioxo-4-imidazolidinyl))-urea and 5-bromo-5-nitro-1,3-dioxan.The present invention is also concerned with a preserved diagnostic test kit, comprising test reagents and at least two preservation agents selected from the group comprising 2-methyl-4-isothiazolin-3-one hydrochloride, 2-hydroxypyridine-N-oxide, chloroacetamide, (N,N-methylene-bis-(N-(1-hydroxymethyl)-2,5-dioxo-4-imidazolidinyl))-urea and 5-bromo-5-nitro-1,3-dioxan.

Abstract: Control reagents an oxidant or a hydroxylamine compound together with other ingredients, the most important of which is a known amount of the analyte for which the reagent acts as a control.

Abstract: A kit including a reagent containing a buffer suitable for maintaining the pH of the reagent between 3-7, sulfanilic acid or sulfanilamide, a cupric soluble salt and an alkaline metal nitrite, for the detection and determination of bilirubin in the urine providing precise results and ready automation of the procedure, characterized in that it is a stable combination of all the substances used for such analysis.

Abstract: An eliminating agent used for the measurement of the amount of glycosylated hemoglobins in a blood sample is provided. The eliminating agent comprises condensed phosphoric acids and/or the salts thereof as the main ingredient, wherein the agent eliminates labile glycosylated hemoglobin A.sub.1c into non-glycosylated hemoglobin and glucose. There is also provided a reagent comprising the eliminating agent and a hemolysis agent, which is used for the measurement of the amount of glycosylated hemoglobins; and an eluent comprising the eliminating agent, which is used for the separation of glycosylated hemoglobins in blood samples by ion-exchange chromatography to measure the amount of the glycosylated hemoglobins. A method for measuring the amount of glycosylated hemoglobin A.sub.1c in a blood sample involves the use of the eliminating agent, the reagent comprising the eliminating agent and the hemolysis agent, and/or the eluent.

Abstract: An indirect potentiometric method and diluent for the analysis of lithium are disclosed. The diluent includes effective amounts of a pH buffer and a non-cationic surfactant comprising at least one hydrophobic group, at least one hydrophillic group and being substantially free of polyoxyethylene groups. Most preferably, the pH buffer is tris-(hydroxymethyl) aminomethane-phosphate, and the surfactant is 2, 4, 7, 9-tetramethyl-5-decyn-4,7 diol. An indirect potentiometric determination of lithium in a clinical sample comprises the steps of mixing the sample with a diluent, contacting an aliquot of the diluted sample with a lithium specific ion selective electrode and at least one ion selective elctrode specifically responsive to a monovalent interfering ion, and measuring both the response of the lithium specific ion selective electrode and the monovalent interfering ion specific ion selective electrode, the responses being an indication of the concentration of lithium in the sample.

Abstract: Methods for blood sample preparation using reagent compositions which include a zwitterionic surfactant for sphering of cells to eliminate orientational noise. When the reagent composition including a for staining a subset of cells is reacted with an anticoagulated blood sample, and the reaction mixture is passed through the sensing region of a flow cytometer, the light scattered and absorbed by each cell is measured, the stained cells can be distinguished from the unstained cells, and when the cells are reticulocytes and mature erythrocytes, respectively, the volume and hemoglobin concentration of each reticulocyte or erythrocyte, and the hemoglobin content, mean cell volume, mean corpuscular hemoglobin concentration, and mean cell hemoglobin of the reticulocytes or erythrocytes are calculated from the measured cell-by-cell volume and hemoglobin concentration.

Abstract: Reference controls comprised of aldehyde-fixed white blood cells stabilized red blood cells and simulated blood platelets exhibiting a white blood cell histogram profile that is substantially that of whole blood are obtained by the addition of a lipoprotein to the control and an antioxidant to inhibit lysis of stabilized red blood cells by said lipoprotein.

Abstract: Diluent and wash compositions are useful in a rapid and sensitive assay for detecting antibodies, and especially retroviral antibodies, in a biological specimen. The diluent composition is buffered to a pH of 6 to 10 and includes a protein or carbohydrate, a surfactant and a negatively-charged organic compound. The wash composition is buffered to a pH of 5 to 10 and includes a surfactant. These compositions can be included in a diagnostic kit. The method of this invention includes mixing the biological specimen with the diluent composition, forming an immunological complex between ligand and antibodies in the specimen and separating complexed materials from uncomplexed materials using a filtration membrane and a washing step. An enzyme labeled anti-antibody is added to form a ligand-antibody-antibody complex followed by its detection using suitable reagents.

Abstract: A lytic reagent composition highly selective in its interactions with the cell membranes of blood cells and also provides a method of using the reagent composition in a semi-automated or an automated system to effect a significantly improved white blood cell differential determination. The lytic reagent composition is characterized by an ability to selectively shrink the white blood cells into the increasing size order of lymphocytes, a midregion consisting mainly of monocytes, other mononuclear cells, some basophils and eosinophils, and neutrophils and can readily be optimized to effect a significantly improved three component separation on the histogram of commercially available semi-automated and automated blood analyzers when compared to the reagents conventionally employed on such instruments.

Abstract: A method and apparatus for calibrating or checking the calibration of vertical photometers. The invention may also provide easy and convenient assay results for diagnostic profiles. The invention comprises pre-dispensed amounts of a suitable reagent, chosen for the particular use, disposed in a vessel with an amount of a wetting agent or other means to yield a reproducible meniscus in the resulting control solution. The control solution is dried to leave a precise amount of dried reagent and other constituents in the vessel which can then be stored and distributed until subsequent use. The dried control solution can then be reconstituted to provide a standard having known absorbance and physical characteristics.

Abstract: An aqueous alcohol buffer solution for substantially ambient, in vitro preservation of mammalian cells for a selected duration. The solution generally contains a water-miscible alcohol in an amount sufficient to fix the sample cells without coagulation, an anti-clumping agent, and a buffer agent to maintain the solution at a pH within a range of four to seven.

Abstract: A new method for separation of white cells in differential hematology instruments is described. In the method described, white cell shrinkage is controlled by the use of diazolidinyl urea and accelerated with alcohol thereby providing a reproducible method for easy manipulation of the white cells. In addition, it was found that adsorption of blood platelets and red blood cells in the instruments can be prevented by use of a surfactant such as the polyols.

Abstract: A reagent for measuring the hemoglobin concentration and counting the number of leukocytes in a blood sample, which contains at least one of a quaternary ammonium salt or a pyridinium salt, having a concentration capable of hemolyzing erythrocytes in the blood and denaturing hemoglobin and at least one of cationic, nonionic or amphoteric surfactants or an oxidant capable of oxidizing heme in hemoglobin, the reagent being capable of dividing leukocytes into two or three fractions, for example, a fraction of lymphocytes, a fraction of monocytes, eosinophils and basophils, and a fraction of neutrophils.

Abstract: The method of stabilizing a carbon dioxide sensor involves formulation of a bicarbonate buffer solution in the sensor with a bicarbonate ion concentration of from about 100 mM to about 200 mM bicarbonate. The method also involves treatment of the sensor to reduce the instability that may occur in carbon dioxide sensors when such sensors are exposed to either very low or very high carbon dioxide levels for extended periods of time. The sensor is treated by exposing the sensor to an aqueous solution containing at least 2 weight percent carbon dioxide, for from several days to several months. The solution may be prepared in advance, or may be dynamically infused with carbon dioxide to provide the desired carbon dioxide content.

Abstract: A reagent, satisfying the undermentioned conditions, is useful for counting the number of leukocytes and measuring the hemoglobin concentration in blood samples. The reagent is free of cyanides and it is able to maintain hemoglobin in a stable state for a prolonged time. The reagent contains at least one cationic surfactant selected from the group consisting of a quaternary ammonium salt of a concentration capable of hemolyzing erythrocytes in the blood and oxidizing hemoglobin and a composition of pyridinium salts; at least one cationic, nonionic or amphoteric surfactant; and at least one hemoglobin stabilizer.

Abstract: A method for producing a liquid phase substance having a pre-established concentration of CO.sub.2 suitable for calibrating analytical instruments such as hemogasanalyzers consists in:preparing an aqueous solution of precisely known strength of a water-soluble salt of carbonic acid,then transferring said solution to a flow reactor containing a cation exchange resin in hydrogenionic form,passing the aforesaid solution of carbonic acid salt through the aforesaid cation resin,thereby achieving the reaction of producing a pre-established quantity of CO.sub.2, andtransferring the eluate thus obtained directly to the analytical instrument for calibration.

Abstract: The invention relates to a procedure and agent for the colorimetric determination of nitrate ions in aqueous solutions by mixing the sample which is to be investigated with resorcinol and a mixture of sulfuric acid and phosphoric acid, characterized in that at least 2 g/l, preferably about 10 to 30 g/l chloride ions are also added.

Abstract: The present invention relates to a reagent and to a method of using the same for automatically determining at least one leukocyte sub-population from a total blood sample. Using this reagent, it is possible, in a single step, to lyse the erythrocytes through the action of saponin and SDS, as well as to ensure the differential staining of the leukocyte sub-populations, at the same time preserving the integrity and morphology of the cells, through the combined action of various components including chlorazol black, an alcohol, a surfactant and preserving agents. The reagent according to the invention can be applied to different types of automatic flow cytometry analyzers.

Abstract: A formulation of combined-composition electrolyte and pH buffers for use as standardization solutions in analyzers for clinical chemistry which simultaneously use more than one ion-selective electrode plus more than one standard solution. Each ion is present in the standard solution in stoichiometric quantity such as to compensate the effect due to the incomplete dissociation of the salt used, and to obtain an activity coefficient and liquid junction potential such as to reduce the error generated by the use of standardization solutions which do not take account of these quantities. Buffer solutions are described having a pH within the range of 6-8 and an electrolyte compositions (Na.sup.+, K.sup.+, Ca.sup.+ and Cl.sup.-) which reflect the composition of the biological liquid to be analyzed, namely whole blood, plasma or serum, in equilibrium with different partial pressures of oxygen and CO.sub.2.

Abstract: An improved multi-purpose blood diluent for use with a gentle lysing agent and improved detergent reagent system are disclosed which are especially suitable for use in routine electronic enumeration and volumetric differentation of blood cells. The preferred imidazole stabilizer used in the diluent reagent is found to be an excellent cell stabilizing agent and buffer for maintaining cell morphology during operation. A synergistic combination of a superior antimicrobial agent, the preferred Triadine-10, used in the diluent and the detergent reagents, not only prevents bacterial or fungal growth, but also helps to stabilize cells and to obtain distinct volumetric differentiation of certain leukocyte subpopulations. The preferred Brij 35 in a balanced salt solution has proved to be an efficient and cost effective detergent to ensure accurate results and trouble-free operation of the analyzers.

Abstract: A method for determination of biological activity of AT III which comprises mixing a specimen, AT III free-extrinsic coagulation factor-containing plasma, heparin and a prothrombin time-measuring reagent or a factor X-activating reagent and then measuring a coagulation time, and a reagent and plasma to be used therefor.

Abstract: The solution for storage and calibration of an intravascular blood sensor is an aqueous solution of from 10-35 mM of a first buffer component of bicarbonate, and at least one additional buffer component selected to maintain the pH of the solution substantially at or slightly below a neutral pH, and preferably below approximately pH 8. The first and additional buffering components preferably are selected to act in two separate pH regions with substantially no overlap of the buffering capacity of the different buffering components, and the concentration of the first buffer component in the solution is selected to permit a two point calibration of the intravascular multiparameter blood sensor by carbon dioxide and oxygen gas tonometry.

Abstract: A method for quantifying in vivo ascorbate concentration in body fluids uses gas chromatography/mass spectrometry (GC/MS). Further provided is a method for measuring redox potentials of body fluid by determining the ratio of in vivo concentration of ascorbate to one or more of its metabolites.

Abstract: A method of stabilizing the glucose content of a blood sample applied to a sorbent for drying is provided. A solution of sodium fluoride or other glycolysis inhibitor is applied to a sorbent. A blood sample for glucose determination is then applied to the sorbent whereupon glycolysis is immediately inhibited. The blood sample may then be dried for shipping without loss of glucose content during the drying period.

Abstract: Magnetic separation apparatus and methods for separating colloidal magnetic particles from a non-magnetic test medium in which the magnetic particles are suspended. The separator comprises a container holding the non-magnetic test medium, one or more magnetic wires disposed substantially within the test medium in the container and an external magnet for producing a magnetic field gradient within the test medium. According to the method of the invention, the container holding the test medium is positioned in the separator, producing a magnetic field gradient operative to cause the magnetic particles to be attracted to the areas surrounding the magnetized wires and to adhere to the wires. The non-magnetic test medium is separated from the magnetized particles, which may conveniently be washed while adhered to the wires, and subjected to further analysis, preferably while on the wires.

Abstract: An antimicrobial composition for the preservation of milk samples required for analysis. The composition comprises 2-bromo-2-nitropropane-1,3-diol and natamycin, in effective amounts to prevent spoilage of the milk for a period of at least 5 to 10 days. The amount of 2-bromo-2-nitropropane-1,3-diol for a 50 ml milk sample is preferably 6-12 mg and the amount of natamycin is preferably 0.3-0.6 mg. The composition is preferably employed in the form of a tablet containing a suitable filler and usually other ingredients. In a method of preserving milk samples, the 2-bromo-2-nitropropane-1,3-diol and natamycin may be added separately or simultaneously (e.g. in admixture). The composition and method provide a way of preserving milk samples without the health problems associated with conventional preservatives such as dichromates and without the need to refrigerate the milk.

Abstract: A buffer composition which can be used to dilute assay components and which also can be used when performing immunohematological procedures. The buffer composition includes effective concentrations of phosphate buffer, polyvinylpyrrolidone, cholic acid, bovine serum albumin, a biological detergent and sodium hydroxide, at a pH of from about 5.6 to 7.2. The method of using the buffer composition and a kit containing the buffer composition also is provided.

Abstract: A heavy metal-free composition and its method of use in an ion selective electrode-based assay are disclosed. The heavy metal-free composition includes a borate compound and an alkalinity adjusting compound, and is used to dilute a test sample; buffer the test sample; and substantially reduce the effects of interferents present in the test sample on the ion selective electrode-based assay. The heavy metal-free composition and its method of use are especially useful in an ion selective electrode-based assay for carbonate ion concentration in a test sample, like blood serum, blood plasma, urine or cerebrospinal fluid.

Abstract: A process for the removal of non-specific turbidities in the carrying out of determinations according to the immuno-assay principle, wherein an inorganic boron compound is added to the sample solution in combination with a buffer system.

Abstract: The treatment of blood or body fluid and body fluid samples to inactivate or destroy infective viruses, such as the cytomegalovirus, by mixing the sample with an effective amount of glycyrrhizic triterpenoid-detergent, glycyrrhizic triterpenoid-glycerol or glycyrrhizic triterpenoid-EDTA, combination followed by analysis or testing for diagnostic or other purposes is disclosed.

Abstract: A method and reagent system are disclosed for the rapid isolation, identification and/or analysis of leukocytes from a whole blood sample. The method and reagent system of this invention has application to any environment in which the study and/or analysis of the leukocyte fraction of whole blood requires their isolation in their native or near native state. One of the environments in which this invention can be used to advantage is in the performance of white cell differentiation on automated instrumentation designed for that purpose. In one of the preferred embodiments of this invention, the lytic reagent can contain a mixture of both formic and acetic acid, with the formic acid comprising the major component thereof and the acetic acid being present in only minor quantities, (if at all). This reagent system is used to selectively effect stromatolysis of red blood cells and create subtle modifications to the leukocyte population to enable their automated differentiation into five (5) sub-populations.