Classifications

• • C—CHEMISTRY; METALLURGY
  • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
  • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
  • C23C16/00—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes
  • C23C16/44—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the method of coating
  • C23C16/455—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the method of coating characterised by the method used for introducing gases into reaction chamber or for modifying gas flows in reaction chamber
  • C23C16/45502—Flow conditions in reaction chamber
  • C23C16/4551—Jet streams
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • C—CHEMISTRY; METALLURGY
  • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
  • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
  • C23C16/00—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes
  • C23C16/01—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes on temporary substrates, e.g. substrates subsequently removed by etching
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations

Definitions

• Antimicrobial agents and uses thereof are antimicrobial agents and uses thereof.
• This invention relates to antimicrobial agents and uses thereof, for example in methods for diluting blood and blood diluents, especially suitable for use in enumeration and sizing of blood cells, including white blood cell
• Analytical data of blood is important as an index of health and well being.
• WBC white blood cell count
• RBC red blood count
• PHT platelet count
• HTC hematocrit
• HGB hemoglobin
• MCV corpuscular volume
• MCH mean corpuscular hemoglobin
• MCHC mean corpuscular hemoglobin concentration
• the diluent comprises a stable water solution of chemical salts providing an electrolytic solution capable of conducting current to which a blood sample can be added so as to dilute the red blood cells, white blood cells, platelets and other blood components to be measured, counted and evaluated. See, for example, US Patent Nos. 4,213,876, 4,244,837, 4,521,518 and 4,745,071.
• an automated, blood-analysis instrument or other analytical method including manual microscopic evaluations
• the blood diluent may shrink or expand.
• the diluent should not adversely affect the blood analysis itself.
• the diluent must also be free from particles which may interfere with the analysis of the blood. While the diluent is normally filtered to remove particles larger than 0.2 micron in diameter at the time of its manufacture, the diluent may be susceptible to the support of microorganism grown after the manufacture and packaging of the diluent. The presence of microorganisms may result in inaccurate and non-reproducible results.
• an antimicrobial agent in blood diluents to retard the growth of microorganisms.
• the antimicrobial agent as with other components of the diluent, must not adversely affect the blood cells or
• an antimicrobial agent which has been employed for many years in blood diluents is sodium azide. Its use has not, however, been without significant problems.
• sodium azide has been found to influence the formation of cyanmethemoglobin, a chromogen formed for determining hemoglobin in a blood sample. For instance, without sodium azide in the diluent, the hemoglobin as determined photometrically may be significantly different than that with the azide present.
• 2-phenoxyethanol is used as an antimicrobial agent. There are disadvantages to the use of phenoxyethanol as well. First, phenoxyethanol is a rather weak antimicrobial agent. It has been found to be more effective when used in conjunction with other preservatives, such as the
• Another object of the present invention is to provide an antimicrobial agent for a blood diluent that does not
• Still another object of the present invention is to provide an antimicrobial agent for a blood diluent that is potent but relatively non-toxic.
• an antimicrobial agent e.g. a fungistatic and/or fungicidal agent comprising an effective concentration of EDTA, typically a concentration of between approximately 0.02 grammes/litre and 1.0 grammes/litre.
• the antimicrobial agent also comprises an
• effective concentration of sodium fluoride typically a concentration of between approximately 0.02 grammes/litre and 0.5 grammes/litre.
• the antimicrobial agent of the invention may be in the form of a blood diluent.
• the invention also provides a method for determining the cell number and/or size of a cell subpopulation in a blood sample, comprising the steps of:
• the invention also encompasses the general use of EDTA, preferably together with sodium fluoride, as an
• the invention therefore generally comprises an
• the invention is directed to a method for diluting blood, a blood dilutent, and antimicrobial agent or reagent for use in a blood
• the improvement comprises the use of EDTA alone, or EDTA and sodium fluoride together as an
• antimicrobial reagent More specifically, in a method of determining the number and/or size of cells or cell
• the improvement comprises employing EDTA alone, or EDTA and sodium fluoride together, as an antimicrobial and/or fungistatic reagent.
• a blood diluent according to the invention generally comprises an organic buffer adjusted to an appropriate pH, a cell stablilising agent, an inorganic salt to adjust
• Figures 1A, 1B and 1C are histograms showing the number of cells versus cell volume analysed on an automated
• Figures 2A, 2B and 2C are histograms showing the number of cells versus cell volume analysed on an automated
• Figures 3A, 3B and 3C are histograms showing the number of cells versus cell volume analysed on an automated
• Figures 4A, 4B and 4C are histograms showing the number of cells versus cell volume analysed on an automated
• the present invention is directed to an antimicrobial agent for use in a diluent for analysing population and subpopulation differentiation of blood cells in a sample of blood. Routinely, such analysis can be performed on an automated, blood-analysis instrument, such as, eg, a Sequoia-Turner CELL-DYN 2000 blood-analysis
• control diluent has been used extensively and successfully for the analysis of blood cell populations on an automated, blood-analysis instrument, such as, eg, a
• 1-hydroxypyridine-2-thione is an effective bacteriostatic or bacteriocidal agent but not a fungistatic or fungicidal agent.
• the present invention prevents microbial growth, and in particular fungal growth, in general circumstances, for example in a blood diluent.
• the blood diluent utilizing the invention comprises an aqueous solution of a cell stabilising agent, an antimicrobial agent, an organic buffer adjusted to an appropriate pH, a fungistatic agent or reagent(s), and additional ionic components to provide a suitable ionic strength, and osmolality to maintain the normal volume of the cells.
• the present diluent is a solution which includes various components well known in the art of blood cell analysis.
• water is routinely used as the solvent, as it is inexpensive, safe and generally compatible with the sample to be analysed.
• the presently preferred cell stabilising agent is
• 1,3-dimethylurea and the presently preferred organic buffer is N-(2-acetamido)-2-iminodiacetic acid (ADA). While these are the preferred compounds, either presently known or equivalent compounds hereafter developed, might be
• the pH of the buffered diluent is conveniently adjusted to approximately 6.9 with, eg, sodium hydroxide, but other bases may be substituted and the pH may vary between at least 6.5 and 7.5 without
• sodium chloride is routinely used to adjust the osmolality of the diluent to approximately 320 milliosmoles, which is known to be appropriate for
• the antimicrobial and/or fungistatic agents or reagents of the present invention are 1) ethylenediaminetetraacetic acid (EDTA) alone or 2) EDTA in combination with sodium fluoride.
• EDTA ethylenediaminetetraacetic acid
• Table 1 The presently preferred composition of the diluent employing the former is illustrated in Table 1, while the preferred composition of the diluent containing the mixture of agents is illustrated in Table 1, while the preferred composition of the diluent containing the mixture of agents is illustrated in Table 2.
• Sodium fluoride is known as an excellent preservative blood (Med J. Aust. 1:1939, 1968). Sodium fluoride is classified as a toxic substance (toxicity level 4-5 on a maximum scale of 6). See Gosselin et al., supra (Section IID, position 100).
• EDTA on its own is relatively non-toxic; this acid and many of its salts bind metal cations tightly and so render them essentially innocuous. See
• the calcium salt of EDTA is used intravenously to detoxify and enhance the renal excretion of lead.
• the calcium derivative (chelate) has a low toxicity in experimental animals
• WBC white blood cell
• RBC red blood cell
• PHT platelet
• Figures 1-4 show the impact of the antimicrobial agents of the present diluent on the analysis of population and subpopulation differentiation of blood cells. Because the cell volume of RBCs and WBCs overlap considerably, it is not possible to count one in the presence of the other by size discrimination alone. Thus, the diluent of the present invention must also serve as a diluent for a lytic reagent.
• a standard lytic reagent and method, such as that described in US Patent No. 4,745,071 is used to analyse each population separately and is hereby incorporated by reference.
• WBC subpopulations can be examined.
• subpopulations are (in order of increasing size) lymphocytes, monocytes, and granulocyutes.
• Granulocytes are a
• Pathological conditions may give rise to at least three types of variations in blood cell profile.
• the size of existing populations and/or subpopulations may be altered (eg larger WBCs).
• the number of normal sized cells of an existing population or subpopulation may be altered (eg more monocytes).
• a completely different population or subpopulation may emerge (eg appearance of promyelocytes).
• Figure 1A shows the WBC histogram achieved on the instrument using the preferred diluent of Table 1, except that EDTA was not included. Three WBC subpopulations can be identified.
• the lymphocyte subpopulation peak (10) shows a mean cell volume of approximately 62 cubic microns.
• the monocyte subpopulaiton (11) ranges in size between
• the granulocyte subpopulation peak (12) shows a mean cell volume of
• Figure 1B shows the RBC histogram using the same diluent as in Figure 1A.
• Figure 1C shows the PLT histogram using the same diluent as in Figures 1A and IB.
• the mean cell volume for the PLT population peak (15) is approximately 7 cubic microns.
• Figures 2A-2C show the results of an experiment designed to assess the impact on the analysis of sodium fluoride alone.
• the diluent is the preferred diluent of Table 2, except that EDTA was not included. It was hoped that sodium fluoride could be used to prevent microbial growth.
• the addition of sodium fluoride causes the lymphocyte subpopulation peak (20) to shift to the right relative to the (control diluent) WBC population peak (10) of Figure 1A; the mean cell volume is approximately 85 cubic microns. In this manner, the lymphocyte subpopulation peak (20) intrudes into the monocyte subpopulation range (21).
• Figure 3A shows that, in the presence of EDTA, the addition of sodium fluoride does not cause the lymphocyte subpopulation peak (30) to intrude into the monocyte subpopulation range (31); the mean cell volume for the lymphocyte peak (30) is approximately 61 cubic microns. There is a very low level of cell stroma (33).
• Samples were also used to inoculate thioglycollate broth. If growth was observed in the thioglycollate, samples from the broth were then used to inoculate Sabouraud Dextrose and Mycosel slants as a "subculture".
• Lactophenol Cotton Blue prep was carried out according to Laboratory Methods in Medical Mycology. US Dept. of HEW, Public Health Service, CDC, June 1978, pp. 29-30 and a slide culture was performed. The slide culture involved gently removing and mounting the coverslip of the Lactophenol Cotton Blue prep to observe the fruiting bodies of the fungus. Under a hood, two square plugs from a cornmeal agar plate are placed on the surface of the same plate with a sterile wood stick. A small portion of unknown fungus colongy, again with a sterile wooden stick, is then placed inside of the square "holes" and the top edges of the square plugs.
• the subculture technique appears from the data of Table 3 to measure a different level of growth. This is probably due to the lower concentration of the antimicrobial agents when the diluent is put into the thioglycollate (1 ml into 15 ml). Interestingly, the combination of EDTA and sodium fluoride is effective in stopping growth in the subcultures when used in higher concentrations.
• the present invention provides a novel and improved method for determining the cell number and size of cell populations and subpopulations in a blood sample wherein the blood is diluted in an osmotically
• the invention also provides a novel blood diluent containing an antimicrobial agent for a blood diluent that does not interfere with the blood analysis, is potent, but relatively non-toxic and inexpensive.
• both EDTA and EDTA in combination with sodium fluoride in effective concentrations may provide heretofore unknown antimicrobial agents and/or fugistatic agents, of wide general utility, but of particular utility in blood diluents.

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• 1989
  • 1989-11-28 WO PCT/GB1989/001421 patent/WO1990006055A1/en unknown
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