Classifications

• • 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
  • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
  • G01N33/721—Haemoglobin
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
  • G01N33/56972—White blood cells

Definitions

• the invention relates to a method and a reaction medium for both determining, without a toxic product such as cyanide, the hemoglobin level, counting and differentiating white blood cells (GB) on the same dilution of a sample. blood.
• a toxic product such as cyanide
• GB white blood cells
• red blood cells In an aqueous medium, red blood cells (RBCs) are lysed, hemoglobin is oxidized to methemoglobin (also called hemoglobin) by ferricyanide and cyanide ions, which have a strong affinity for the subject; they are methemoglobin theme ligands forming a cyanmethemoglobin complex, whose maximum absorbance is at 540 nm.
• methemoglobin also called hemoglobin
• ferricyanide and cyanide ions which have a strong affinity for the subject; they are methemoglobin theme ligands forming a cyanmethemoglobin complex, whose maximum absorbance is at 540 nm.
• the measurement of hemoglobin is made by spectrophotometry.
• a quaternary ammonium salt was added in low concentration to the reaction medium.
• US5468640A discloses a rapid method for determining the level of hemoglobin in a blood sample, wherein said sample is contacted with a reaction medium at a pH of 11.3-13.7 comprising a surfactant ionic, also free of any cyanide ion.
• a surfactant ionic also free of any cyanide ion.
• Either the surfactant is also a strong base, such as stearyltrialkylammonium hydroxide, and it imparts the required pH to the medium, or it is not a strong base, for example cetyltrimethylammonium bromide (CTAB), and a strong base must to be added, for example an alkali metal hydroxide.
• CTAB cetyltrimethylammonium bromide
• US6740527A discloses a method for determining the hemoglobin level of a blood sample, still devoid of cyanide ion, which further allows to give a count of GB.
• the sample is first diluted and then contacted with a reaction medium which comprises a lysing agent consisting of 0.1-20% by weight of at least one quaternary ammonium salt and 0.1-15% by weight. % by weight of a hydroxylamine salt.
• a reaction medium which comprises a lysing agent consisting of 0.1-20% by weight of at least one quaternary ammonium salt and 0.1-15% by weight. % by weight of a hydroxylamine salt.
• Determining the hemoglobin level, the number of GB and the differentiation of GB with a single lysis agent on an automatic hematology analyzer would reduce the number of reagents used and simplify the fluid organization of the analyzer. These are factors of robustness of the system and reduction of manufacturing and operating costs.
• the inventors of the present invention have unexpectedly discovered that the mere presence of one or more quaternary ammonium salts is sufficient to lyse GBs and stabilize the measurement of hemoglobin by binding quaternary ammonium groups. on Theme. It is therefore sufficient to determine the hemoglobin level, count the white blood cells and differentiate them, in a blood sample, in the absence of any other lysis agent and other ligand of the subject. hemoglobin.
• the invention provides a simple method, free of toxic product, allowing, in the same blood sample, both to determine the hemoglobin level, and to count and differentiate the GB. This method involves the reading and measuring techniques conventionally employed in the field of blood formulation and does not involve any toxic chemical entities, such as cyanide ions and hydroxylamine salts, for example.
• the method of the invention comprises the following steps:
• the blood sample is placed in an isotonic or hyperosmolar medium, at a pH not exceeding 10, devoid of cyanide ions, and comprising at least one quaternary ammonium salt and at least one preservative agent,
• the complex formed between hemoglobin and the quaternary ammonium groups is detected by spectrophotometry and
• GB is counted and is differentiated into at least three sub-populations, said process being carried out in the absence of any other ligand than said quaternary ammonium salt (s).
• the medium in which the blood sample is typically is prepared in one or two steps; in one step, the constituent ingredients of the diluent and lysis media and the blood sample are mixed in one step; in two stages, the blood sample is placed in the diluent medium, and the medium thus obtained and the lysis medium are mixed.
• isotonic or hyperosmolar medium is meant a preferably isotonic or slightly hyperosmolar medium, to avoid any excessive contraction of the GB which would hinder their differentiation.
• the quaternary ammonium salt or salts advantageously correspond to the following characteristics, taken alone or in combination:
• the quaternary ammonium salt or salts are chosen from the compounds of formula (I) NR1R2R3R4 + X " , in which
• R1 represents a hydrocarbon chain having from 1 to 18 carbon atoms
• R2, R3 and R4 each independently represents an alkyl group having 1 to 6 carbon atoms
• X represents a halogen or a group selected from OH, CH 3 SO 4 and PO 4 .
• R1 may represent a hydrocarbon chain having from 10 to 18 carbon atoms and then the said salt or salts are preferably chosen from the salts of dodecyltrimethylammonium, myristyltrimethylammonium, palmityltrimethylammonium and stearyltrimethylammonium.
• R1 may also represent a hydrocarbon chain having from 1 to 6 carbon atoms, and said salt or salts are preferably chosen from tetraethyl ammonium salts.
• said medium may comprise several quaternary ammonium salts, in particular several salts of formula (I), and for example one or more salts of formula (I) in which R1 is a C10-C18 chain. and / or one or more salts of formula (I) in which R1 is a C1-C6 chain.
• Particularly suitable salts are chosen from myristyltrimethylammonium bromide, dodecyltrimethylammonium chloride and tetraethylammonium hydroxide, used alone or as a mixture.
• the proportion of the quaternary ammonium salt or salts varies from 2 to 4% by weight.
• the medium comprises one or more preservatives.
• This or these agents contribute to an optimization of each of the steps of said process, but particularly to that of the differentiation of GB.
• This or these agents are in particular bacteriostatic agents preferably chosen from dimethylurea and 2-pyridinethiol-1-sodium oxide.
• the medium may also contain other ingredients: a stabilizing agent, for example ethylene diamine tetraacetic acid (EDTA), at least one nonionic surfactant, such as Triton ® .
• EDTA ethylene diamine tetraacetic acid
• Triton ® nonionic surfactant
• the hemoglobin level can be measured by standard spectrophotometric techniques.
• the enumeration of GB is carried out by a conventional method also based for example on the principle of impedance which, according to the method of the invention, also makes it possible to differentiate GB in at least the following three sub-populations: lymphocytes, monocytic cells (other than lymphocytes) and granulocytes.
• the medium contains at least one quaternary ammonium salt of formula (I) in which R 1 represents a hydrocarbon chain of 12 carbon atoms and another quaternary ammonium salt of formula (I ) in which R 1 represents a hydrocarbon chain of 14 carbon atoms.
• R 1 represents a hydrocarbon chain of 12 carbon atoms
• another quaternary ammonium salt of formula (I ) in which R 1 represents a hydrocarbon chain of 14 carbon atoms under these conditions, and associated with a biparametric method of impedance and laser measurement at large angles, also conventional, we can differentiate at least the following four sub-populations: lymphocytes, monocytes, eosinophilic granulocytes and neutrophilic granulocytes .
• the medium may also comprise up to three quaternary ammonium salts, or even more.
• the blood sample is diluted in slightly hyperosmolar medium and buffered at neutral pH.
• a pH greater than 10 causes partial lysis of GBs preventing their differential counting.
• the invention also relates to a medium for determining the hemoglobin level and for counting and differentiating white blood cells, said medium being free of cyanide ions, and comprising at least one quaternary ammonium salt and a preservative, said medium being devoid of any other ligand agent than said salt or salts.
• the medium according to the invention comprises a lysing agent which consists of one or more quaternary ammonium salts, this or these latter constituting the only ligand binding agent (s) to the hemoglobin theme.
• This salt or these salts are advantageously chosen from the compounds of formula I 1 NR1 R2R3R4 + X " in which
• R 1 represents a hydrocarbon chain having from 1 to 18 carbon atoms
• R2, R3 and R4 each independently represents an alkyl group having from 1 to 6 carbon atoms
• X represents a halogen or a group selected from OH, CH 3 SO 4 and PO 4 ,
• This medium is advantageously that described above and has the same characteristics, considered alone or in combination.
• a preferred medium comprises the following quaternary ammonium salts: myristyltrimethylammonium bromide, dodecyltrimethylammonium chloride, tetraethylammonium hydroxide.
• myristyltrimethylammonium bromide ranges from 20-40 g / l, that of dodecyltrimethylammonium chloride of 1-10 g / l and that of tetraethylammonium hydroxide from 1-5 g / l.
• It may further comprise at least, as a preservative, a bacteriostatic agent preferably selected from dimethylurea and 2-pyridinethiol-1-sodium oxide.
• a bacteriostatic agent preferably selected from dimethylurea and 2-pyridinethiol-1-sodium oxide.
• the proportion of dimethylurea varies from 1 to 10 g / l and that of 2-pyridinethiol-1-sodium oxide from 1-10 g / l.
• EDTA ethylene diamine tetraacetic acid
• EXAMPLE 1 Formulation of a Medium
• the appropriate medium comprises a diluent and a lysing and reaction agent, as follows:
• a diluent an aqueous solution composed of: an organic phosphate buffer, for example hydrogen phosphate sodium or disodium phosphate anhydrous a membrane stabilizer, for example chloride or sodium sulfate bacteriostatic agents, for example dimethylurea and
• 2-pyridinethiol-1-sodium oxide a stabilizing agent for example disodium EDTA (EDTA.Na2) and as an agent for lysis, an aqueous solution consisting of: a mixture of bromide myristyltriméthyl ammonium chloride, dodecyltrimethyl ammonium and tetraethylammonium hydroxide, Triton ®, as nonionic surfactant, sodium chloride as a membrane stabilizer, the same or different from the diluent, and deionized water.
• EDTA.Na2 disodium EDTA
• Triton ® as nonionic surfactant
• sodium chloride sodium chloride as a membrane stabilizer, the same or different from the diluent, and deionized water.
• the diluent has an osmolarity of 320-360 mOsm / Kg at neutral pH and the lysing agent has a pH of 10.5 +/- 0.4.
• the reaction medium obtained has a pH close to 8.
• Example 2 Implementation of the process on a "3 populations" analyzer Preparation of the blood sample:
• a counting sequence is performed as follows:
• 180 ⁇ l of the first dilution are measured volumetrically to count the GB whereas approximately 100 ⁇ l of the second dilution are measured for RBCs / platelets.
• the dilution volume of the GB is controlled by two optical barriers in a volumetric tube. During the volumetric count, the data is divided into 4 tables of 2 seconds each for statistical use. GR / Platelet counting starts with GB counting and stops exactly after 8 seconds.
• the different GB subpopulations are differentiated according to size criteria by controlling the lysis conditions (diluent, lysing agent, time of action). When these conditions are met, the chemical reaction that takes place makes it possible to distinguish three populations of GB: lymphocytes, granulocytes and medium-sized cells (basophils + monocytes + a part of eosinophils + young granulocytes). After the lysis process, the lymphocytes are the smallest cells with their small nucleus and correlate with the cells between 25 and 90 ⁇ 5 fL (parameterizable) on the histogram of distribution of GB (in human mode) not shown.
• the average cells are in the region between lymphocytes and granulocytes.
• eosinophils After lysis action, they are correlated with the cells between 90 ⁇ 5 and 140 ⁇ 5 fL (modifiable) on the GB distribution histogram, not shown. Some eosinophils may, however, exceed 220 fL.
• Neutrophilic granulocytes after the action of lysis, are the largest cells with their polysegmented nuclei retaining part of the cytoplasm. Neutrophils are correlated with cells between 140 ⁇ 10 and 449 fL on the GB distribution histogram, not shown.
• the light source a green light emitting diode (LED) (555 nm) calculates the absorbance that is proportional to the hemoglobin concentration of the sample.
• the optical path is determined by two optical paths in the GB chamber.
• the reference Hb is stored during start-up or when the chamber is filled with thinner.
• Example 3 Implementation of the process on an analyzer at least 4 populations
• 150 ⁇ L of blood is taken from the tube where the blood was collected beforehand.
• the blood column is sucked by the analyzer through a valve ceramic where 25 + 1 ⁇ L are sampled to form a first dilution at about 1/72 with 1800 ⁇ L of low-hypertonic diluent at neutral pH.
• a second dilution is done in cascade with 25 ⁇ 1 ⁇ l and about 2 ml of diluent for measurement at 1/6000 GR and platelets. 475 ⁇ L of the predilution are taken up with 210 ⁇ L of lysing agent and
• Hemoglobin is measured at 560 nm directly in the GB preparation tank after 15 seconds. This 1/251 GB / Hb dilution is then transferred to a cytometer where 267 ⁇ L are injected through a 75 ⁇ m micro-orifice and a nearby focused laser beam in a hydrodynamic double-flow architecture.
• This double device for measuring electrical impedance and laser measurement at large angles makes it possible to count the GB and to determine at least 4 sub-leukocyte populations: lymphocytes, monocytes, erythrocyte granulocytes, neutrophil granulocytes, but also the detection of neutrophil granulocytes. hypogranules, plasmodium vivax, reported platelet agglutinates or nucleated GR.
• 67 ⁇ L of the 1/6000 dilution are in turn transferred and injected through a 75 ⁇ m micro-orifice into a hydrodynamic double-flow architecture for the counting of GRs and platelets.

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• 2008
  • 2008-05-20 FR FR0802729A patent/FR2931557A1/fr not_active Withdrawn
• 2009
  • 2009-05-20 WO PCT/FR2009/000593 patent/WO2009150327A2/fr active Application Filing
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