JP2011521251A - Method for measuring hemoglobin ratio, method for counting and identifying white blood cells, and suitable medium - Google Patents

Abstract

The present invention is a method for the determination of the hemoglobin ratio in a blood sample and for the counting and identification of white blood cells, comprising the steps of: at least one quaternary ammonium in the absence of cyanide ions Bring it into an isotonic or hypertonic medium with a pH that does not exceed 10 including salt and preservatives; detect the complex formed between hemoglobin and quaternary ammonium groups spectrophotometrically; Counting and discriminating into at least three subpopulations, said method being performed in the absence of all other ligands. The invention also relates to a medium for carrying out the method.
[Selection figure] None

Description

  The present invention relates to a method and reaction medium for simultaneously measuring hemoglobin levels and counting and discriminating white blood cells (WBCs) in the same dilution of a blood sample without any toxic products such as cyanide. .

  A conventional method for measuring hemoglobin was based on detecting a chromogenic complex formed between cyanide ion, oxidized hemoglobin, and methemoglobin by spectroscopic measurement at 540 nm. The blood sample to be analyzed had been exposed to an aqueous reaction medium containing a cyanide salt (potassium cyanide) and a phylicyanide salt (potassium ferricyanide or sodium ferricyanide). In aqueous media, red blood cells (RBCs) are lysed and hemoglobin is oxidized to methemoglobin (also called hemoglobin) by ferricyanide and cyanide ions, which have a strong affinity for heme; Ligand for methemoglobin heme, forming cyan methemoglobin complex, its maximum absorbance is 540 nm. The hemoglobin is measured by spectroscopic measurement.

  Thereafter, a quaternary ammonium salt was added to the reaction medium at a low concentration to promote RBC dissolution. However, this resulted in the problem of complex aggregation between ferricyanide ions and quaternary ammonium groups; these aggregations show turbidity that impedes the photometric analysis of hemoglobin. For this reason, the ferricyanide salt has been abandoned while the cyanide salt has been maintained.

  Automated devices have been developed to rapidly analyze hemoglobin and the number of RBCs and WBCs in blood samples. The reaction medium has also evolved at the same time; changes were made from aqueous diluents to isotonic or slightly hypertonic diluents to count RBCs and WBCs. Under these conditions, it has become necessary to increase the concentration of quaternary ammonium to dissolve RBCs.

  These techniques have significant drawbacks including toxic cyanide ions.

  No method has been developed that requires cyanide ions anymore.

  I. Oshiro et al., Clin. Biochem. 15 (1) 83-88 (1982), describes a method of using sodium lauryl sulfate (SLS) in the absence of any cyanide ions to measure hemoglobin levels in blood samples. is suggesting. SLS contacted with the sample causes hemolysis of RBC, leading to the conversion of hemoglobin to methemoglobin by oxidation of iron atoms. The formed stable complex is detected by absorption spectrometry. SLS causes lysis of all cells, including WBC, even at the lowest concentrations indicated, which reduces the state of the nucleus. This method uses strong dilution, which is acceptable inaccuracy with limited counting time and makes it impossible for WBC to be measured. In addition, it cannot be used for differential counting.

  Document US5468640A discloses a rapid method for measuring hemoglobin in a blood sample, according to which the sample comprises a surfactant at a pH of 11.3-13.7, and Contacted with a reaction medium that does not contain any cyanide ions. The surfactant is also a strong base such as stearyl trialkylammonium hydroxide and it gives the required pH in the medium or it is not a strong base such as, for example, cetyltrimethylammonium bromide (CTAB) Are either strong bases such as alkali metal hydroxides must be added. After hemolysis of RBCs with detergents, the latter activity and the activity of strong bases result in hemoglobin denaturation, and exposure of hemoglobin heme to oxygen in the air results in oxidation of iron atoms. Complexes formed between ferric heme and hydroxide ions are detected by absorption spectroscopy. Like the previously described method, this method does not make it possible to measure WBC discrimination, because extreme conditions inevitably cause the WBC to dissolve into the nuclear state. .

  Document US6740527A discloses a method for measuring hemoglobin levels in a blood sample, lacking any cyanide ions, which also makes it possible to obtain a WBC count. The sample is first diluted and then brought into contact with a reaction medium comprising a solubilizer comprising 0.1 to 20% by weight of at least quaternary ammonium salt and 0.1 to 15% by weight of hydroxylamine salt. . This method allows measurement of hemoglobin levels and WBC counting, but cannot allow the latter to be identified.

  Hemoglobin level measurement, WBC number and WBC identification with one lysing agent in an automated hematology analyzer can reduce the number of reagents used and simplify the fluid configuration of the analyzer become. These are factors that lower the manufacturing and operating costs to harden the system.

  Contrary to all expectations, the authors of the present invention have determined that hemoglobin measurements can be made by linking the quaternary ammonium salt to heme, where the only presence of one or more quaternary ammonium salts is sufficient to dissolve WBC. Found to stabilize. It is therefore sufficient to measure hemoglobin levels and to count leukocytes and identify the latter in the absence of any other lysing agent and any other hemoglobin heme ligand in the blood sample.

  Thus, the present invention provides a simple method that does not have any toxic products, which allows the measurement of hemoglobin levels and the counting of WBC simultaneously in the same blood sample. This method involves conventional reading and measurement techniques used in the complete blood count field and does not include any toxic chemical entities such as cyanide ions or hydroxylamine salts.

The method of the present invention comprises the following steps:
The blood sample is provided in an isotonic or hypertonic medium that does not exceed pH 10, is free of cyanide ions, includes at least one quaternary ammonium salt and at least a preservative;
Complexes formed between hemoglobin and quaternary ammonium salts are detected spectrophotometrically; and WBC are counted and distinguished into at least three subpopulations;
The method is performed in the absence of any ligand other than the quaternary ammonium salt.

  The medium containing the blood sample is conventionally prepared by one or two steps; in the preparation of one step, the components of the dilution and lysis medium and the blood sample are mixed in one step; In the preparation, the blood sample is brought into the dilution medium, and the resulting medium and the dissolution medium are then mixed.

  The term “isotonic or hyperosmotic medium” is intended to mean a medium that is preferably isotonic or slightly hyperosmotic, which is any of the WBCs that would impair their identification. This is to prevent excessive shrinkage.

For carrying out the method of the invention, the quaternary ammonium salt or salts advantageously correspond to the characteristics considered alone or in combination:
One or more quaternary ammonium salts are compounds of formula (I):
NR1R2R3R4 ⁺ X ⁻
put it here,
R1 represents a hydrocarbon-based chain containing 1 to 18 carbon atoms;
R2, R3 and R4 are each independently, represent an alkyl radical containing from 1 to 6 carbon atoms, and X represents a group selected from halogen or OH, _CH 3 SO ₄ and PO _4;
Selected from.

  R1 represents a hydrocarbon-based chain containing 10 to 18 carbon atoms, so that the salt is singular or plural, preferably dodecyltrimethylammonium, myristyltrimethylammonium, palmityltrimethylammonium and stearyltrimethyl Selected from ammonium salts.

  R1 also represents a hydrocarbon-based chain containing 1 to 6 carbon atoms, the salt being one or more, preferably selected from tetraethylammonium salts.

  Of course, according to the present invention, the medium may contain some quaternary ammonium salts, especially some salts of formula (I), for example one or more salts of formula (I), wherein R1 is C10-10. C1 chain), and / or one or more salts of formula (I), wherein R1 is a C1-C6 chain.

  Particularly suitable salts are selected from myristyltrimethylammonium bromide, dodecyltrimethylammonium chloride and tetraethylammonium hydroxide used alone or as a mixture.

  The proportion of the quaternary ammonium salt ranges from 2% to 4% by weight with respect to the weight of the medium.

  As before, the medium contains one or more preservatives. This agent or these agents contributes to optimizing each step of the method, but most particularly contributes to optimizing WBC identification. This agent or these agents are in particular selected from bacteriostatic agents, preferably dimethylurea and sodium pyridineethiol-1-oxide.

The medium may also contain other components:
Stabilizers such as ethylenediaminetetraacetic acid (EDTA),
At least one non-ionic surfactant, such as Triton®.

  The above components are not essential for carrying out the method, but they will allow their optimization.

  Hemoglobin levels can be measured by conventional spectroscopic techniques. The WBC count is performed by itself or in a customary manner, for example according to the method of the present invention, by a method based on the principle of impedance, and the WBC is classified into at least the following three subpopulations: lymphocytes, A) monocytes and granulocytes.

  According to one variant of the process of the invention, the medium is at least one quaternary ammonium salt of the formula (I), in which R1 represents a hydrocarbon-based chain containing 12 carbon atoms and A quaternary ammonium salt of formula (I), wherein R1 represents a hydrocarbon-based chain containing 14 carbon atoms. Under these conditions, the combination of impedance biparametric methods and wide-angle laser measurements, which are also conventional, at least the following four subpopulations: lymphocytes, monocytes, eosinophilic granulocytes and neutrophils Granulocytes can also be identified.

  The medium may also contain up to three quaternary ammonium salts, or even more.

  According to one preferred practice of the invention, the blood sample is slightly hypertonic and diluted in a medium buffered at a neutral pH. A pH above 10 causes partial dissolution of WBC and prevents its differential counting.

  The invention also relates to a medium for measuring hemoglobin levels and for counting and identifying white blood cells, said medium being free of cyanide ions and comprising at least one quaternary ammonium salt and a preservative. And the medium does not contain any ligand agent other than the salt. Thus, the medium according to the invention comprises a solubilizer consisting of one or more quaternary ammonium salts, the latter constituting the only ligand agent (s) that binds to the hemoglobin heme.

This salt or these salts are preferably compounds of the formula (I);
NR1R2R3R4 ⁺ X ⁻
put it here,
R1 represents a hydrocarbon-based chain containing 1 to 18 carbon atoms;
R2, R3 and R4 are each independently, represent an alkyl radical containing from 1 to 6 carbon atoms, and X represents a group selected from halogen or OH, _CH 3 SO ₄ and PO _4;
Selected from.

  This medium is advantageously as previously described and is considered alone or in combination and has the same characteristics.

  Preferred media include the following quaternary ammonium salts: myristyltrimethylammonium bromide, dodecyltrimethylammonium chloride, tetraethylammonium hydroxide. Advantageously, the proportion of myristyltrimethylammonium bromide is in the range 20-40 g / L, the proportion of dodecyltrimethylammonium chloride is in the range 1-10 g / L and the proportion of tetraethylammonium hydroxide is 1- The range is 5 g / L.

  It may also contain at least one bacteriostatic agent selected as preservative, preferably selected from dimethylurea and sodium pyridineethiol-1-oxide. The proportion of dimethylurea is in the range of 1 to 10 g / L, and the proportion of pyridine ethiol-1-oxide sodium is in the range of 1 to 10 g / L.

  It can also be used as a stabilizer, for example ethylenediaminetetraacetic acid (EDTA), preferably in a proportion ranging from 1 to 10 g / L.

  The subject matter of the present invention is described and illustrated in more detail in the following examples.

<Example 1: Composition of medium according to the present invention>
Suitable media include diluents and dissolution and reactants as follows:
As a diluent, an aqueous solution containing:
Organophosphate buffers such as anhydrous disodium phosphate or sodium hydrogen phosphate:
Membrane stabilizers such as sulfate chloride or sodium sulfate
Bacteriostatic agents, such as dimethylurea or sodium pyridineethiol-1-oxide,
Stabilizers such as disodium EDTA (EDTA.Na ₂ );
And, as a solubilizer, an aqueous solution comprising:
A mixture of myristyltrimethylammonium bromide, dodecyltrimethylammonium chloride and tetraethylammonium hydroxide,
Triton (Triton (registered trademark)), as a nonionic surfactant,
As a membrane stabilizer, sodium chloride, which may be the same as or different from that of the diluent, and
Deionized water.

  The diluent has an osmotic pressure between 320-360 mOsm / Kg at neutral pH, and the solubilizer has a pH of 10.5 +/− 0.4. The resulting reaction medium has a pH close to 8.

<Example 2: Implementation of the method in the “3 population” analysis>
Blood Sample Preparation Approximately 16 μL of blood is removed from the test tube from which the sample was taken. The needle is rinsed inside and outside with diluent on the rinse basin. Next, 3.5 mL of the diluent described in Example 1 is added to obtain the first dilution (1/219). A second dilution is made with 31.2 μL and 4 mL of diluent in this first dilution (1/128). The dilution is transferred to the counting chamber by aspiration. During the RBC dilution, 0.41 mL of lysing agent is added to the first dilution in the WBC counting chamber. The final dilution is approximately 1/244 for WBC and 1/28200 for RBC / platelet. Once the transfer has been performed, the dilution basin is filled with diluent and thereby rinsed.

Count:
A series of counts is performed as follows:
180 μL of the first dilution is measured volumetrically for WBC counting, while approximately 100 μL of the second dilution is measured for RBC / platelets.

  The volume of the WBC dilution is adjusted by two optical barriers in the volumetric tube. During the volumetric counting, the data is divided into 4 tables of 2 seconds.

  The RBC / platelet count starts with the WBC count and stops after exactly 8 seconds.

Identification into three WBC populations:
Different WBC subpopulations are identified by adjusting dissolution conditions (diluent, solubilizer, duration of action) based on size criteria. When these conditions were brought together, the resulting chemical reaction was divided into three WBC populations: lymphocytes, granulocytes and average sized cells (basophils + monocytes + part of eosinophils + young granulocytes). Makes it possible to identify

  After lysis treatment, lymphocytes are the smallest cells with their small nuclei, matching cells between 20-90 ± 5 fL (parameterizable) on the WBC discrimination histogram (in human mode) (shown) )

  Average cells are in the area located between lymphocytes and granulocytes.

  After lysis has taken effect, they match between 90 ± 5 and 140 ± 5 fL of cells on the (changeable) WBC discrimination histogram (not shown). Some eosinophils, however, can exceed 220 fL.

  Neutrophilic granulocytes are the largest cells with their multiple segmented nuclei that retain a portion of the cytoplasm after the action of lysis. Neutrophils correspond to cells between 140 ± 10-449 fL in the WBC discrimination histogram (not shown).

The hemoglobin light source, green light emitting diode (LED) (555 Nm) allows the calculation of absorbance, which is proportional to the concentration of hemoglobin in the sample. The optical track was measured with two optical paths in the WBC chamber. The Hb reference is stored during start-up or when the chamber is filled with diluent.

<Example 3: Implementation of the method in at least four population analyzers>
Blood Sample Preparation 150 μL of blood is removed from a test tube from which blood has been previously collected. The blood column is pulled up by the analyzer through a ceramic valve, 25 ± 1 μL is sampled, and the first dilution is formed approximately 1/72 with 1800 μL of weak hyperosmotic diluent at neutral pH. It was.

  A second dilution is prepared in a cascade with 25 ± 1 μL and about 2 mL of diluent, thereby measuring RBC and platelets at 1/6000.

  475 μL of the pre-dilution is dissolved with 210 μL of lysing agent and approximately 1440 μL of diluent, thereby obtaining the final dilution 1/251.

Measurement:
Hemoglobin is measured at 560 nm directly after 15 seconds in the cuvette for preparing WBC.

  This 1/251 WBC / Hb dilution is then injected through a 75 μm microaperture and a near focus laser beam in a dual hydrodynamic flow architecture, 267 μL. Go to a hemocytometer. This dual device for electrical impedance measurements and wide-angle laser measurements allow for the measurement of WBC counts and at least four leukocyte subpopulations: lymphocytes, monocytes, erythrocyte granulocytes and neutrophilic granulocytes: However, it is also possible to measure the signaling of modified neutrophilic granulocytes, platelet aggregation or nucleated RBCs in granulocytes of malaria (malaria) plasmodium.

  67 μL of the 1/6000 dilution is transferred in sequence and injected into the dual hydrodynamic fluidic structure through a 75 μm micro-opening, thereby counting RBCs and platelets.

Claims (26)

A method for measuring hemoglobin levels and for counting and identifying white blood cells in a blood sample, said method being performed in the absence of any other ligand and comprising the following steps;
Feeding the blood sample to an isotonic or hypertonic medium that does not exceed ph, does not contain cyanide ions, and contains at least one quaternary ammonium salt and one preservative;
Spectroscopic detection of complexes formed between hemoglobin and quaternary ammonium groups; and counting white blood cells and distinguishing into at least three subpopulations. The method of claim 1, said quaternary ammonium salt is one or more in the formula (I) NR1R2R3R4 ⁺ X ^- wherein the selected from the compounds;
put it here,
R1 represents a hydrocarbon-based chain containing 1 to 18 carbon atoms;
R2, R3 and R4 each independently represent an alkyl group containing from 1 to 6 carbon atoms, and X represents a group selected from halogen or OH, _CH 3 SO ₄ and PO _4.   3. A method according to claim 2, characterized in that R1 represents a hydrocarbon-based chain containing 10 to 18 carbon atoms.   4. The method according to claim 3, wherein the ammonium salt is selected from one or more of dodecyltrimethylammonium, myristyltrimethylammonium, palmityltrimethylammonium and stearyltrimethylammonium salts.   3. A method according to claim 2, characterized in that R1 represents a hydrocarbon-based chain containing 1 to 6 carbon atoms.   6. The method according to claim 5, wherein the ammonium salt is selected from one or more tetraethylammonium salts.   7. The method according to claim 4 or 6, wherein the ammonium is selected from one or more of myristyltrimethylammonium bromide, dodecyltrimethylammonium chloride and tetraethylammonium hydroxide.   The method according to any one of claims 1 to 7, wherein a ratio of the quaternary ammonium salt is in a range of 2 wt% to 4 wt% with respect to a weight of the medium. how to.   The method according to any one of claims 1 to 8, wherein the preservative is a bacteriostatic agent, preferably dimethylurea.   10. A method according to any one of claims 1 to 9, wherein the medium further comprises a stabilizer.   11. A method according to claim 10, wherein the stabilizer is ethylenediaminetetraacetic acid (EDTA).   12. A method according to any one of the preceding claims, wherein the medium further comprises at least one nonionic surfactant.   13. The chain method according to claim 12, wherein the nonionic surfactant is Triton (registered trademark).   14. The method according to any one of claims 1 to 13, wherein the leukocytes are counted by impedance and the following three subpopulations are recognized: lymphocytes, monocytes other than lymphocytes and granulocytes. A method characterized by that.   15. A method according to any one of claims 2 to 14, wherein the medium is at least a quaternary ammonium salt of the formula (I), wherein R1 containing 12 carbon atoms is based on hydrocarbons. A chain) and another quaternary ammonium salt of the formula (I), wherein R1 represents a hydrocarbon-based chain containing 14 carbon atoms.   16. The method of claim 15, wherein the white blood cells are counted by a biparametric method of impedance and a wide angle laser measurement, and the following four subpopulations: lymphocytes, monocytes, eosinophils and neutrophils; A method characterized by being identified.   17. A method according to any one of the preceding claims, characterized in that the medium comprises three quaternary ammonium salts. A medium for measuring hemoglobin levels and for counting and identifying white blood cells, wherein said medium does not contain cyanide ions and is at least one formula (I);
NR1R2R3R4 ⁺ X ⁻
here,
R1 represents a hydrocarbon-based chain containing 1 to 18 carbon atoms;
R2, R3 and R4 each independently represents an alkyl group containing from 1 to 6 carbon atoms; and X represents a group selected from halogen or OH, _CH 3 SO ₄ and PO _4;
Quaternary ammonium salts and preservatives,
A medium characterized in that the reagent does not contain any other ligand agent.   19. The medium of claim 18, wherein the ammonium is one or more selected from myristyltrimethylammonium bromide, dodecyltrimethylammonium chloride and tetraethylammonium hydroxide.   20. The medium of claim 19, wherein the concentration of the myristyltrimethylammonium bromide is in the range of 20-40 g / L, the concentration of dodecyltrimethylammonium chloride is in the range of 1-10 g / L, and tetraethylammonium A medium having a hydroxide concentration in the range of 1 to 5 g / L.   21. A medium according to any one of claims 18 to 20, characterized in that the preservative is a bacteriostatic agent, in particular selected from dimethylurea and sodium pyridineethiol-1-oxide. Medium.   The medium according to claim 21, wherein the concentration of the dimethylurea is in the range of 1 to 10 g / L, and the concentration of the pyridineethiol-1-oxide sodium is in the range of 1 to 10 g / L. Feature media.   The medium according to any one of claims 18 to 22, further comprising a stabilizer.   24. The medium of claim 23, wherein the stabilizer is ethylenediaminetetraacetic acid (EDTA).   25. The medium according to claim 24, wherein the EDTA concentration is in the range of 1 to 10 g / L.   26. A medium according to any one of claims 18 to 25, wherein the quaternary ammonium salt of formula (I), wherein R1 represents a hydrocarbon-based chain containing 12 carbon atoms, And a further quaternary ammonium salt of the formula (I), wherein R1 represents a hydrocarbon-based chain containing 14 carbon atoms.