CS274409B2 - Agent for simultaneous determination of thrombocytes and leucocytes number - Google Patents

Description

The invention relates to an agent for the simultaneous determination of platelet and leukocyte counts. j

The present invention provides an agent comprising acetone and formaldehyde or glutaraldehyde, mineral salts and dyes which is suitable for the simultaneous determination of platelet and leukocyte counts from whole blood by normal microscopic means.

The determination of platelet counts and leukocyte counts is well within the scope of standard clinical laboratory methods. Platelet counting can be performed by a variety of methods:

1. Electronic particle counting using automata developed for platelet counting. Although the method is quick and simple, it is necessary to provide very expensive instruments. This method is not always suitable to replace microscopic determination. The reproducibility of this procedure is also worse than the reproducibility of microscopic counting.

2. Platelet counting using a phase contrast microscope using a solution containing cocaine or novocaine or any other platelet counting solution. A phase contrast microscope is indispensable for carrying out this procedure. A further significant disadvantage is that the method is tedious, laborious and harmful to the eyes of the conducting experimenters, especially with a high number of experiments.

3. Determination of platelet count by staining method. This procedure is the simplest and can be performed using a microscope. According to the methods used today, crystal violet (gentian violet; chemical name: hexamethyl-p-rosaniline hydrochloride) is used as a dye.

It is rightly argued against these methods that they distort the results compared to the phase contrast assay, because not all platelets stain well in a visible way.

In hematological diagnostics, leukocyte counting is the most widely used method. The Turk solution, which is described in the Hungarian Pharmacopoeia (Ph. Hg. VI) and which is well preserved, is generally suitable for this purpose, but is not suitable for the determination of platelet counts. This is a considerable disadvantage, because very often both joint extended laboratory experiments are required. A dye solution is required to determine platelet counts and whole blood leukocyte counts using a normal light microscope.

(a) induces complete haemolysis of erythrocytes without reducing the platelet count and leukocyte count;

(b) fixes both platelets and leukocytes; and

c) it contains a dye which is bound to the above particles with high affinity and in this way is fixed under normal light.

Although a solution which meets the first two of the above requirements is known (cf.

Scand. 0. Clin. Invest. 33, 121 (1974)), however platelet counting can only be performed using a phase contrast microscope.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a dye solution which can be used to determine platelet counts and leukocyte counts from whole blood using a normal light microscope and to overcome the above-mentioned disadvantages of known agents.

The invention is based on the surprising discovery that by combining a hemolysis and fixative solution with a basic high affinity dye, a reagent solution is obtained which is suitable for co-determining platelet count and leukocyte count using a normal light microscope. The above conditions can be fulfilled if the hemolyzing and fixing solution is not incompatible with the dye, i. that it does not form any clot.

Microscopic clots should be avoided; in fact, such clots may have the character of platelets and thus distort the result of the assay.

The cresol violet dye solutions used hitherto only partially meet the above requirements. The dye components of cresol violet in the solution to be counted are of a slightly basic character. As a result, platelets stain very little and leukocytes do not appear unambiguous.

It has now been found according to the invention that the so-called thiazine is excellent for this purpose

CS 274 409 B2 dyes. Thiazine-type dyes are characterized by the presence of two chromophore groups. Thiazine-type compounds have already been used in medical practice in various fields (e.g., for staining bacteria, in histochemistry), but there is no indication in the scientific and patent literature of the invention. The most important representatives of this group of compounds are toluidine blue of formula I

thionine of formula II

(II)

Cl and methylene blue of formula

III (CH ₃ ) ₂ f

(III).

Cl

The present invention provides an agent for the simultaneous determination of platelet count and leukocyte count by a conventional microscopic method, characterized in that it contains 0.1 to 5 parts by weight of acetone, 0.05 to 2.0 parts by weight of formaldehyde and / or glutaraldehyde, 0.001 to 5 parts by weight. 0.1 parts by weight of a thiazine dye, preferably toluidine blue, 0.1 to 2.0 parts by weight of a mineral salt, preferably sodium chloride, and up to 100 parts by weight of water.

According to a preferred embodiment of the invention, the reagent comprises 0.1 to 5.0 parts by weight of acetone, 0.05 to 2.0 parts by weight of formaldehyde, 0.001 to 0.1 parts by weight of toluidine blue and 0.1 to 2 parts by weight of sodium chloride .

A particular advantage of the dyes used according to the invention is that these dyes also have a very high affinity for the acid macromolecules of platelets and leukocytes, which accordingly results in very intense and striking visibility.

The most important advantage of the agents of the invention is that the sensitivity of the agents is substantially higher compared to the known agents. This can be demonstrated by the following

CS 274 409 82 experiments.

Results from samples with unknown platelet and leukocyte counts are obtained over twenty consecutive platelet counts and leukocyte counts. The accuracy of the cell counting method is satisfactory if the standard deviation is less than 10%. At a platelet count of 218.8 G / l, the standard deviation (SO) is 5.0 and using these values the coefficient of variation (as standard deviation, expressed as%) is 2.28 (cf. Figure 1). It should also be noted that the G / 1 value is the platelet count and leukocyte count reported in the SI system [G / 1 = giga (cell count) / liter = cell count x 10J, see comparison with FIG.

1.

Platelet and leukocyte counting is performed using the reagent solution of Example 1 as described in Example 2. The same venous blood solution is used for measurements and the measurements are repeated 20 times. The cell count values obtained in each measurement, expressed in G / 1 (G / 1 = giga / 10 / cell count / liter) were plotted in Figure 1. As can be read from this Figure, the measured values vary between 210 G / 1 and 230 G / L; from these data, the calculated mean value (x) is 218.8 G / l. The standard deviation (SD) of the measurement is 5,0 5.0; coefficient of variation (CV), expressed in% of standard deviation, is 2.28. The numerical data on the ordinate in Fig. 1 represent the number of cells in G / 1; the ordinate shows deviations that correspond to twice the standard deviation value (+2 SD or -2 SD) and three times the standard deviation value (+3 SD or -3 SD).

The optimal leukocyte counting error is a mean value of 5.08 G / l, SD = -0.042, which corresponds to a coefficient of variation of 0.8%, see comparison with Figure 3.

The values obtained with 20 parallel leukocyte counts are shown in Fig. 3. The interpretation of the x, SD, CV symbols and numerical values on the ordinate of Fig. 3 is the same as in Fig. 1.

For comparison, in the case of platelet counting, the reagent solution of the invention is compared

P with a preparation known under the trade name THROMBOFIX intended for the counting of thrombocyte R cells and, in the case of the counting of leukocytes, it is compared with Turke solution. THROMBOFIX is a product of Gódecke, Germany and its chemical composition is unknown. However, it is commonly found on the market under this name. Comparative experiments were performed using the blood of 41 patients, in which the platelet count and leukocyte counts were determined in parallel.

The results are reported in the Descartes coordinate system, see comparison with Figures 2 and 4. The correlations between the two methods and the standard parameters (a, b) of the function y = ax + b describing the correlations are determined using a computer (Hewlett-Packard HP-90). ).

In platelet counting, the correlation (r) = 0.927; linear functional dependence between the two methods can be expressed by the equation y = 1.05 x - 0.94; y = the result obtained with the platelet counting solution of the invention; x = result obtained with THR0MB0FIX ^R , see Figure 2.

The platelet count obtained with the reagent solution of the invention is found to be about 5% higher than the platelet count found from the same sample using THR0MB0FIX ^R.

Due to the nature of the determination, the measurement of higher values is out of the question. The difference is due to the fact that the dye component of the reagent solution of the invention has a significantly higher affinity for the acid groups of the platelet macromolecules, and thus the image has more contrasts and can be perceived better. By using a solution of the agent according to the invention, it is also possible to eliminate the lower values systematically obtained using known dye solutions.

p

It should be noted that when using THROMBOFIX, even when using a phase contrast microscope, similar results are obtained compared to the actual results.

When counting leukocytes, the method of the invention shows a correlation of r = 0.959 with the Turke solution. The relation can be expressed by linear function y = 0.97 x + 0.23. This shows that

EN 274 409 B2 that there is no statistically significant difference between the two procedures.

Further details of manufacture and use of the reagent solution of the invention are provided in the following examples. However, the examples given do not limit the scope of the invention in any way.

The activity of the agent of the invention is illustrated by the drawings, wherein in Fig. 1 the numerical data on the ordinate indicate the number of cells in G / 1; the ordinate shows deviations that correspond to twice the standard deviation value (+2 SD or -2 SD) and three times the standard deviation value (+3 SD or -3 SD); platelet count (G / L) detected with the counting solution of the invention; the abscissa axis shows the platelet count (G / 1) detected by the known comparator THROMBOFIX®, Fig. 3 shows values obtained from 20 parallel leukocyte counts, with numerical data on the ordinate indicating the number of cells in G / 1; CV is the coefficient of variation, SD is the standard deviation, and x is the mean value, in FIG. 4 the ordinate shows the number of leukocytes (G / 1) detected by the counting solution according to the invention; on the abscissa axis is the number of leukocytes (G / 1) detected by Tiirke solution.

Example 1

Counting solution

200 ml of 0.9% sodium chloride solution prepared using distilled water are mixed with 5 ml of 35% formaldehyde and 770 ml of deionized distilled water. After vigorous stirring, 100 mg of toluidine blue is dissolved in the solution as a dye. The resulting solution was filtered through a G-4 glass filter. After adding 25 ml of acetone, the solution must remain clear and free of particulate matter.

Example 2

Determination of platelet count and leukocyte count in venous blood treated with anticoagulant

The blood is expediently treated with ethylenediaminetetraacetic acid as an anticoagulant, since in the presence of ethylenediaminetetraacetic acid there is no minimal excretion of thrombocytes. ml. Blood clotting can also be avoided by using a 3.3% solution of trisodium citrate in distilled water. When taking blood in a plastic cannula, the ratio is 9 parts blood to 1 part citrate.

To carry out the procedure:

To 25 µl of venous blood treated with a clotting agent, 475 µl of the counting solution was added in a plastic tube, and the mixture was allowed to stand at room temperature for 15 minutes without stirring. Platelet and leukocyte counts must be performed within 6 hours. Before counting, the solution is again mixed, dripped into the Burker chamber and allowed to settle in the humid chamber for 10 minutes.

a) Platelet counting

The sum of platelets counted in the 10 rectangles of the Burker chamber is multiplied by 2 (using ethylene diamine tetraacetic acid to prevent clotting) or 2.2 (using citrate to prevent clotting). The platelet count is obtained in G / L.

b) Leukocyte counting

The number of leukocytes found in four, three line delineated squares is divided by 18 (or multiplied by 0.055) when using citrate to prevent precipitation, or by 20 (or multiplied by 0.05) by using ethylenediaminetetraacetic acid as a composition

CS 274 409 B2 to prevent shrinkage. The leukocyte count is obtained in G / L.

Example 3

Determination of platelet count and leukocyte count from capillary blood

The disinfected finger tip or heel is injected, the first drop of blood is wiped and then 25 µl of blood is drawn into the pipette using a plastic syringe (type: Finnpipette, Gilson or Eppendorf, etc.). This amount of blood is measured in a plastic tube containing 25 µl of a 2 mg / ml solution of ethylenediaminetetraacetic acid. It is then well mixed with the anticoagulant. After addition of 450 µl of counting solution and thorough shaking, the solution is allowed to stand at room temperature for at least 15 minutes and, after re-mixing, is dropped into the Burker chamber and left to sediment in the wet chamber for 10 minutes.

Platelet counts and leukocyte counts are determined in a similar manner to blood treated with a clotting agent. When determining the platelet count, the factor by which it is multiplied is 2. When counting leukocytes, the value obtained must be divided by 20.

Example 4

The procedure is as in Example 1 except that 80 g of 25¾ glutaraldehyde is used instead of formaldehyde.

Claims (1)

SUBJECT OF THE INVENTION Reagent for the simultaneous determination of platelet and leukocyte counts by normal microscopy using a light microscope, characterized in that it contains 0.1 to 5 parts by weight. acetone, 0.05 to 2.0 parts by weight of acetone; % of formaldehyde and / or glutaraldehyde; 0.1 to 2.0 parts by weight of a thiazine dye, for example toluidine blue; mineral salts, for example sodium chloride, and up to 100 parts by weight of the composition; water.