MXPA99003779A - Method and elaboration of a hemolizant without cyanides, for obtaining the leukocytes, differentiation in the hemaglobin concentration in automated apparatus - Google Patents

Abstract

Se describe un procedimiento en la elaboración de un reactivo hemolizante, para usarse en aparatos electrónicos automatizados que proporcionan la cuenta de leucocitos, su diferenciación en sus tres poblaciones principales (linfocitos, monocitos, granulocitos) y simultáneamente la concentración de hemoglobina sanguínea. Los objetivos de esta investigación son:proporcionar un hemolizante;al agregar a una cetrimida (Bromuro de Cetildimetiletil amonio) citrato de sodio, que mezclada con Cloruro de Dodeciltrimetilamonio, no dependa de sales de cianuro para medir la concentración de hemoglobina sanguínea, darle una estabilidad al reactivo a bajas temperaturas al añadirle glicerina y que sea, un reactivo que cuide nuestro medio ambiente.

Description

METHOD AND PREPARATION OF A HEMOLYZANT WITHOUT CYANURES, TO OBTAIN THE LEUCOCITE ACCOUNT, ITS DIFFERENTIATION AND THE CONCENTRATION OF HEMOGLOBIN IN AUTOMATED APPLIANCES.

BACKGROUND OF THE INVENTION For 5 decades, the concentration of blood hemoglobin is measured by the cyanohemoglobin technique, using a solution called Drabkin, consisting of: potassium cyanide, potassium ferrocyanide, sodium bicarbonate and water.

There are hemolytic reagents of foreign technology, some being manufactured in Mexico City and all of them in their formulation depend on cyanide salts; for example: Coulter with its product LYSE (Pat.4,485,175) or Ficher (Pat 5,180,677) see appendices.

We describe a method to differentiate leukocytes in three populations: lymphocytes, monocytes and granulocytes; with the use of an automated electronic device. The system includes a blood thinner and a hemolyzing reagent.

The blood thinner is an osmotically balanced aqueous solution with a preferential pH, its components do not modify the morphology of the erythrocytes, stabilize the leukocytes, act as a boffer solution and do not allow bacterial growth. The lytic reagent is a mixture of an aqueous solution with quaternary ammonium salts, this reagent is added together with the diluent the blood, which is hemolyzed, leaving the leukocytes in suspension, to be differentiated by their different volumes in three populations.

In order to suppress the cyanide salts, it was thought to develop a hemolyzing reagent that is intended to be protected by means of the present application.

DESCRIPTION OF THE INVENTION The present invention consists in the production of a haemolysing reagent that, when used in automated devices (Coulter series T, JT, S plus, STK), determines the count of leukocytes (white blood cells), their differentiation in their three main populations: lymphocytes (LY), monocytes (MO) and granulocytes (GR); simultaneously measuring the concentration of hemoglobin (the main protein that forms red blood cells or red blood cells) without depending on its preparation of cyanide salts (potassium cyanide, potassium ferrocyanide, potassium nitroferrocyanide).

The technical field of the hemolysate is in any laboratory of Clinical Analysis, Laboratory of Hematology or Research, that is, any laboratory that has an automated device to measure hematological parameters of the series indicated.

You can analyze bloods of humans or animals such as dogs, cats, sheep, horses, chickens, etc., that is, anyone who wishes to quantify hematological parameters. The analysis is useful to determine anemia states, inflammatory processes and as an aid in the diagnosis of some type of leukemia.

The investigation refers to a hemolyzing reagent, capable of rapidly lysing the red blood cells, releasing the hemoglobin and leaving the leukocytes in suspension. The hemoglobin released is quantified by colorimetric method (wavelength 540 nm), while leukocytes are counted electronically and differentiated by their different volumes, in the series devices already described.

The reagent consists mainly of the mixture of two quaternary ammonium salts. The first is dodecyltrimethyl ammonium chloride (this salt is constant). One aspect of the investigation was to analyze which of the following quaternary ammonium salts was the best to be mixed with the first: - Benzylimethyltetradecyl ammonium chloride - Cetylpyridine chloride - Cetyldimethylethyl ammonium bromide - Cetyltrimethyl ammonium bromide - Tetradecyltrimethyl ammonium bromide Any of the aforementioned salts can be mixed with the dodecyltrimethylammonium chloride; The one that behaved better and we consider more suitable is Cetrimide Cetildimetiletil Bromide, this when mixed with Sodium Citrate, provides less overlap of residual particles and good conductivity.

This haemolysing reagent is capable of rapidly lysing (as well as or better than the other previously used hemolysates) the red blood cells, releasing the hemoglobin and leaving the leukocytes in suspension. The hemoglobin released is quantified by colorimetric method (wavelength 540 nm), while leukocytes are counted electronically and differentiated by their different volumes, as previously indicated.

Cetyl dimethyl ethyl ammonium bromide in concentrations ranging from 1.0 to 1.5% w / v as water diluent, at refrigeration temperature (4 to 8 ° C) undergoes crystallization at the time. In this investigation, we added glycerin to give the formulation stability, avoiding the crystallization process. Said in passing that the reagent should remain at room temperature; but there are regions where the ambient temperature is from 0 to 8aC. Semi automated equipment (ZF Coulter Counter), require to hemolyze of these concentrations, when coming into contact with low temperatures, the reagent is separated from its components; by adding glycerin, this problem is greatly reduced.

By adding the first salt (dodecyltrimethyl ammonium chloride) to our mixture of sodium citrate Cetrimide (Cetyldimethyl ethyl ammonium bromide), we obtain the quantification of hemoglobin and leukocytes, the differentiation of these into its three main populations: lymphocytes, monocytes and granulocytes. .

Figure 1 shows the leukocyte histogram obtained by introducing a sample of anticoagulated total blood in the electronic instrument Coulter Counter JT, where we can observe the relationship in number of leukocytes (ordinates), with leukocyte globular volume (abscissas) and differentiation of leukocytes in their three main populations: Lymphocytes (LY), Monocytes (MO) and granulocytes (GR).

In order not to depend on cyanide salts in the preparation of the hemolytic reagent, a chemical equilibrium in the mixture of quaternary ammonium salts, sodium citrate and water is important.

In the investigation of the hemolytic reagent, the automated equipment of the Coulter Counter Company series T-890 and JT were used. These require mainly two reagents, one of which is the hemolysate with which the following parameters are obtained: Hemoglobin concentration (HGB), leukocyte count (WBC) and its differential: Lymphocytes (LY), monocytes (MO), granulocytes (GR) Figure 2 shows the leukocyte histogram as well as the parameters that depend for their quantification of the hemolyzing reagent, which are: Total leukocytes (WBC), Hemoglobin concentration (HGB), Lymphocytes (LY), Monocytes (MO) and granulocytes (GR) .

The other is a diluent (Buffer), the parameters that are obtained directly from it are: Red cell count (RBC), mean globular volume (MVC), platelet count (PLT) and platelet volume (MVP).

Figure 3a shows the erythrocyte histogram, which is obtained by introducing whole anticoagulated blood in the Coulter Counter JT., Where the ordinates indicate the ratio of the number of erythrocytes and in the abscissa the erythrocytic globular volume; as well as the parameters obtained from the diluent reagent: total red cell count (RBC), mean globular volume (MVC).

Figure 3b shows the platelet histogram, which is obtained by introducing whole blood anticoagulated in the Coulter Counter JT., Where the ordinates indicate the ratio of the number of platelets and abscissas, the globular platelet volume; as well as, the parameters obtained from the diluent reagent: total platelet count (PLT) and platelet globular volume (MVP). Other parameters are obtained by mathematical relationships: Average concentration of hemoglobin (MCH) MCH = Hemoglobin No. of erythrocytes Average corpuscular concentration of hemoglobin (MCHC) MCHC = Hematocrit Hematocrit Hematocrit (HCT) = Average globular volume x No. of erythrocytes.

HEMOLYZING INGREDIENTS Sodium Citrate.- It is a harmless salt, it is used as an anticoagulant to trap calcium from the blood. Citrate when mixed with a cetramide (Cetyl dimethyl ethyl ammonium bromide), provides the hemolyzing power; it also acts as an electrical conductor.

Cetildimethylethyl ammonium bromide .- It is a quaternary salt of ammonium and biological detergent, soluble in water, is itself hemolyzing, adding sodium citrate, this activity is considerably increased.

Dodecyltrimethyl Ammonium Chloride.- It is a quaternary salt of ammonium and a biological detergent soluble in isopropyl alcohol, also hemolyzed, but its main function is to reduce the volume of leukocytes to be differentiated into three populations: lymphocytes (LY), monocytes (MO) and granulocytes (GR).

Glycerin.- It is a colorless substance, with a sweet taste to alcohol, soluble in water, avoids the separation of the quaternary ammonium salts and slows the crystallization of these low temperatures (4 -8 degrees Celsius).

Figure 4 shows the leukocyte histogram obtained in the Coulter Counter JT., By mixing the hemolysate with anticoagulated whole blood. The components of the hemolyzer, lyse the erythrocytes, leaving the leukocytes in suspension, to decrease the volume and be differentiated in their three main populations, being as follows: Lymphocytes (LY) 45-105 femptolithoids, Monocytes (MO) 105-180 femptolítros and Granulocytes (GR) 180-450 femptolítros.

These components are present in the hemolyzing solution in the following proportions: HEMOLYZING FORMULA Concentration Preferred concentration range - Anhydrous sodium citrate (99%) 0.2% P / V 0.1 to 0.5% P / V - Cetyl dimethyl ethyl ammonium bromide (98%) 0.4% P? / 0.2 to 0.8% P / V - Dodecyltrimethylammonium Chloride (50%) 3.0% V / V 1.5 to 7.0% VA / (Isopropyl Alcohol 40%, Water 10%) - Glycerin 0.3% V V 0.1 to 0.5% V / V PROCESSING OF ELABORATION The procedure consists of 3 stages: the first one is to mix the Cetyl dimethyl ethyl ammonium bromide with the sodium citrate in water, stirring until homogenizing at room temperature; the second is to add the Dodecyltrimethylammonium Chloride with constant agitation at room temperature, until its complete homogenization and the third is to add the glycerin with stirring at room temperature.

Next, an example of the elaboration of the solution is presented. 1. Preparation of the hemolyzing solution, used in the following example: a) In a flask with a capacity of 2000 ml, add 25% V V of distilled water. b) With constant stirring and at room temperature, add Cetyl dimethyl ethyl ammonium bromide 0.4% P? / and sodium citrate 0.2% P / V. c) With constant agitation and at room temperature, add the 3.0% VA / Dodecyltrimethylammonium Chloride to the previous mixture until completely dissolved. d) To the previous mixture and with constant agitation, add Gliceerina 0.3% V / V e) Add to all the previous solution another 75% VV of distilled water, stirring constantly at room temperature.

RESULTS Application examples of the haemolysing solution of example 1.

Blood samples (90) were run in a Coulter Counter JT instrument, comparing the cyanide-free hemolyzer of the present invention with cyanide-containing hemolysate (Lyse Ill. De Coulter Counter). As you can see they were similar.

Hemoglobin concentration (g / dl) Hemoglobin concentration (g / dl) (hemolyzer without cyanide) (hemolyzer with Lyse cyanide lll) 13. 1 13.2 12.7 12.7 14.8 14.7 15.1 15.1 10.4 10.5 9.2 9.2 16.4 16.3 15.0 15.1 13.6 13.6 17.7 17.9 14.2 14.1 13.9 14.0 11.5 11.6 11.9 11.9 7.6 7.7 15.4 15.5 13.4 13.6 16.8 16.7 15.0 15.0 13.1 13.0 12.7 12.6 9.8 9.8 11.0 10.9 12.0 12.0 15.1 15.2 17.9 17.8 18.7 18.6 18.1 18.1 8.8 8.8 6.9 7.0 13.0 13.0 18.1 18.0 17.9 17.9 16.6 16.5 16.6 16.7 14.0 14.1 17.2 17.4 7.1 7.1 16.9 17.0 15.3 15.3 14.9 15.0 15.1 15.3 16.9 17.2 15.9 16.0 9.3 9.2 10.8 10.7 11.0 1 1.0 14.2 14.3 16.5 16.4 18.0 18.1 12.0 12.2 13.0 13.1 CN O r- O ooo co 00 o r- "f 00 O h- CD N- CO 00 co v- CD LO CN OO LO co ^ CD O 00 1- ^ oo oo o 00 LO "< t co o ~ CN 00 * 5T r- ~ CD CD I? 00" Ü "O O - 00 CN CO-CN O O 00 CD T- or CN 0) O O CD CD CN h- 00 CD ~ 00 CD - CN co r- T- CD * ÑT CO CD -! • «- co o CD O CN f- oo co CD 00 LO "= r 00 or T- CN 00 T h- CD? LO 00 -f O T T- co T- CO rc co CN o We can conclude with these results, that the concentrations of hemoglobin obtained with the hemolyser of the present invention are very similar to those commercially sold today. The advantage of the hemolyser of the invention with respect to those commercially available, is that to measure the concentration of blood hemoglobin does not require cyanide salts, solving the problem of not throwing this dangerous poison to drain.

Claims (10)

REINVIDICATIONS Having described the invention, the content of the following clauses is claimed as my property:

1) A procedure for the preparation of a hemolysate, which consists, in adding to a cetrimide of sodium citrate, together with the Dodecyltrimethylammonium Chloride and glycerin; in this same order, with constant agitation and at room temperature. A) Add to a solution of 0.2% P? / To 0.8% P / V of cetrimide the citrate of dio to complete a concentration of this, from 0.1% P? / To 0.5% P? /. B) To solution A), add Dodecyltrimethylammonium Chloride until achieving a concentration of this, between 1.5% V? / To 7.0% V? /; in constant agitation and at room temperature. C) Add glycerin at a concentration of 0.1% V? to 0.6% V? /, maintaining the agitation and at room temperature.

2) The procedure of claim 1), wherein the cetrimide is Cetyldimethylethylammonium Bromide and the concentration in the final solution is preferably 0.4% P? /.

3) The procedure of the reinvidation 1) where the Dodecyltrimtrimilamonium Chloride is in a concentration of 3.0% V V in the final solution.

4) The method of claim 1) wherein the sodium citrate is in a concentration of 0.2% P? / In the final solution.

5) The method of claim 1) wherein the final concentration of the glycerin is 0.3% V? /.

6) A blood hemolysate comprising: a) Cetrimide Bromide Cethyldimethylethylammonium 0.2% to 0.8% b) Sodium Citrate 0.1% at 0.5%. c) Dodecyltrimethylammonium Chloride 1.5% at 7.0% d) Glycerin 0.1% at 0.6% as solvent, distilled or deionized water.

7) The hemolyser of claim 6), wherein the cetrimide is Cetyl dimethyl ethylammonium bromide and is at an ideal concentration of 0.4% P? .

8) The hemolyser of claim 6) wherein the sodium citrate is in a concentration of 0.2% P? /.

9) The hemolyser of claim 6), wherein the Dodecyltrimethylammonium Chloride is at a concentration of 3.0% P? /.

10) The hemolyser of claim 6) wherein the glycerin is in a 0.3% V? / concentration.