FORMULATION FOR COUNTING AND MORPHOLOGICAL-BASED INFERENCING OF ERYTHROCYTES AND PLATELETS AND METHOD OF MAKING AND USING SAME

Abstract

The invention relates to a formulation for counting and morphological-based inference of erythrocytes and platelets to for use in clinical analyses, in particular hemograms, with the purpose of counting both erythrocytes and platelets in a liquid medium, and performing qualitative inferences of erythrocyte and platelet morphology, through image processing in Point-of-Care devices or through an optical microscope. This formulation preserves the natural morphological characteristics of erythrocytes and platelets, and uses methylparaben, propylparaben, sodium chloride, sodium bicarbonate, bovine serum albumin (BSA), sodium citrate, potassium citrate, K.sub.2EDTA and deionized water, which is advantageous in terms of operational simplicity and low cost.

Claims

1. A formulation for counting and morphological-based inferencing of erythrocytes and platelets for hematological analyses of partially isotonic characteristic that preserves the morphology of erythrocytes and platelets, comprising deionized water in a range of 84.4701% to 93.4174% by mass, wherein the formulation additionally comprises the following compounds in the following mass percentages: TABLE-US-00012 Compound Min (%) Max (%) methyl paraben 0.0009 0.0090 Propylparaben 0.0002 0.0025 sodium chloride 0.2324 11.4266 Sodium bicarbonate 0.0433 3.4020 BSA 0.0000 10.6198 sodium citrate 0.0000 4.1690 potassium citrate 0.0000 0.5296 K.sub.2EDTA 0.0871 10.7809

2. The formulation for counting and morphological-based inferencing of erythrocytes and platelets according to claim 1, comprising 88.9815% by mass of deionized water, 1.7297% by mass of K.sub.2EDTA and comprising the following compounds, in the following mass percentages: TABLE-US-00013 Compound % by mass methylparaben 0.0045 Propylparaben 0.0012 sodium chloride 5.0163 Sodium bicarbonate 1.4415 BSA 0.8649 sodium citrate 1.4415 potassium citrate 0.5189

3. A method for preparation of a formulation for counting and morphological-based inferencing of erythrocytes and platelets, comprising the following steps: 1. preparing a preservative formulation, by weighing methylparaben and propylparaben, followed by dilution in deionized water; the homogenization of the formulation carried out on a thermal magnetic plate heated to 100° C., or an equivalent procedure, until complete dilution of the aforementioned compounds; the preservative formulation can be replaced by the addition of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (3% by mass) dispersed in modified glycol (93-95% by mass) and alkyl carboxylate (2-3% by mass); 2. preparing an isotonic formulation, by weighing sodium bicarbonate, sodium chloride, bovine serum albumin (BSA), sodium citrate, potassium citrate and K.sub.2EDTA, followed by dilution in deionized water; and 3. preparing a final composition by mixing the preservative formulation with the isotonic formulation, followed by filtration through No. 1 filter paper, and buffering to obtain a pH measurement in the range between 7.5 and 9.5.

4. A method of counting erythrocytes and platelets in an animal blood sample comprising the steps of diluting and homogenizing the blood sample in the formulation according to claim 1 to obtain a mixture and inserting the mixture into a hemocytometer for evaluation under an optical microscope.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

[0020] The preparation of the formulation for counting and morphological inference of erythrocytes and platelets (final composition) consists of three steps, described below:

[0021] 1) Preparation of the preservative formulation, by weighing methylparaben and propylparaben, followed by dilution in deionized water. The homogenization of the formulation is carried out on a thermal magnetic plate heated to 100° C., or an equivalent procedure, until complete dilution of the aforementioned compounds. This preservative formulation can be replaced by the addition of a commercially available product such as Proain 300® [5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (3% by mass) dispersed in modified glycol (93-95% by mass) and alkyl carboxylate (2-3% by mass)], but resulting in lower effectiveness;

[0022] 2) Preparation of the isotonic formulation, by weighing sodium bicarbonate, sodium chloride, bovine serum albumin (BSA), sodium citrate, potassium citrate and K.sub.2EDTA, followed by dilution in deionized water; and

[0023] 3) Preparation of the final composition, by mixing the preservative formulation with the isotonic formulation, followed by optional filtration through No. 1 filter paper for better analysis results, and subsequent pH measurement, which must be in the range between 7.5 and 9.5.

[0024] The main characteristics and technological functions of the components used in the final composition are described below:

[0025] a) Methylparaben, used with a high degree of purity (analytical grade), molar mass of 152.15 g/mol, CAS number 99-76-3, commercially known as Nipagin®, has a preservative action, being one of the responsible for keeping the formulation free from contamination by microorganisms as it is biocidal and biostatic. When added in insufficient amounts, the final composition is colonized by invading microorganisms, and when added in excess, it promotes cell death;

[0026] b) Propylparaben, used with a high degree of purity (analytical grade), molar mass of 180.20 g/mol, CAS number 94-13-3, commercially known as Nipazol®, also has a preservative action and keeps the final composition free of contamination by microorganisms as it is biocidal and biostatic. When added in insufficient amounts, the final composition is colonized by invading microorganisms, and when added in large amounts, it promotes cell death. Practical tests have shown that the joint use of methylparaben and propylparaben has a synergistic action, and when used alone, unsatisfactory results such as cell death or solution contamination are obtained;

[0027] c) Sodium bicarbonate, used with a high degree of purity (analytical grade), molar mass of 84.01 g/mol and CAS number 144-55-8, is a source of bicarbonate and sodium electrolytes, which are among the main blood electrolytes. If added in small amounts, the final composition is no longer partially isotonic and cell lysis occurs, and if added in excess, there will be changes in erythrocyte morphology and cell lysis;

[0028] d) Sodium chloride, used with a high degree of purity (analytical grade), molar mass of 58.44 g/mol and CAS number 7647-14-5, is a source of chloride and sodium electrolytes, which are among the main blood electrolytes. If less than the minimum is added, the final composition is no longer partially isotonic and promotes cell lysis, and if added in an amount greater than the maximum, there will be changes in erythrocyte morphology and cell lysis;

[0029] e) Bovine serum albumin (BSA), used with a high degree of purity (analytical grade), molar mass of 66.4 g/mol and CAS number 9048-46-8, is used as a protein source for the formulation. During research and tests, it was verified that its use is not mandatory for the correct functioning of the final composition, but when used, it improves the morphological characteristics of the erythrocytes and increases the stability of the formulation. When added in excess, it promotes changes in erythrocyte morphology and cell lysis;

[0030] f) Sodium citrate, used with a high degree of purity (analytical grade), molar mass of 294.10 g/mol and CAS number 68-04-2, is used as a source of sodium. During research and tests it was verified that its addition is not mandatory for the functioning of the final composition, but when added it improves the morphological characteristics of the erythrocytes. If added beyond the stipulated maximum, it causes changes in erythrocyte morphology and cell lysis;

[0031] g) Potassium citrate, used with a high degree of purity (analysis standard), molar mass of 306.39 g/mol and CAS number 6100-056, is used as a source of potassium. During research and testing, it was verified that its addition is not mandatory for the final composition to work. It has the function of improving the morphological characteristics of erythrocytes, but when added in excess, it causes changes in erythrocyte morphology and cell lysis;

[0032] h) K.sub.2EDTA, used with a high degree of purity (analytical grade), molar mass of 404.45 g/mol and CAS number 25102-12-9 prevents clotting of the blood sample. When added in small amounts, the formation of platelet aggregates occurs, which causes errors in the platelet count, and when added in excess, it promotes changes in erythrocyte morphology and cell lysis; and

[0033] i) Deionized water is the solvent for the formulation, and when added in small amounts or in excess, the final composition is no longer partially isotonic, which leads to morphological changes and cell death.

[0034] Exhaustive research and practical tests were carried out to determine the maximum and minimum amounts of each component used in the formulation for counting and morphological inference of erythrocytes and platelets. The tests that were performed to determine the minimum and maximum amount of each component of the composition for counting and morphological inference of erythrocytes and platelets (final composition) are shown below. All tests were performed following the same methodology.

[0035] Test methodology to determine the component ranges was as follows: a 1 μl sample of fresh capillary blood was diluted and homogenized in 199 μl of the final composition and, subsequently, 10 μl of this mixture was inserted into a hemocytometer for evaluation under an optical microscope. The evaluated parameters were: formulation stability, morphological characteristics of cells in contact with the final composition, cell lysis, and colonization by invading microorganisms. Tests 1 to 34 were performed to determine the minimum and maximum amount, as well as the preferred formulation.

[0036] Tests 1 to 4 determined the minimum and maximum amount of methylparaben are listed in table 1.

TABLE-US-00001 TABLE 1 Tests 1 to 4 for determination of amounts in % by mass of methylparaben. Compound 1 2 3 4 methylparaben 0.0008 0.0009 0.0090 0.0091 Propylparaben 0.0012 0.0012 0.0012 0.0012 sodium chloride 5.0163 5.0163 5.0163 5.0163 Sodium bicarbonate 1.4415 1.4415 1.4415 1.4415 BSA 0.8649 0.8649 0.8649 0.8649 sodium citrate 1.4415 1.4415 1.4415 1.4415 potassium citrate 0.5189 0.5189 0.5189 0.5189 K.sub.2EDTA 1.7297 1.7297 1.7297 1.7297 Deionized water 88.9852 88.9851 88.9770 88.9769

[0037] Test 1: Determination of the minimum methylparaben value of 0.0008%. The amount added was not enough to avoid colonization of the composition by invading agents. Therefore, this composition was disapproved.

[0038] Test 2: Determination of the minimum value of 0.0009% methylparaben. The amount of methylparaben added was sufficient to prevent colonization of the composition by invading agents. Therefore, this composition was approved.

[0039] Test 3: Determination of the maximum value of methylparaben of 0.0090%. This maximum amount of methylparaben prevented the colonization of the composition by invading agents and did not cause cell death. Therefore, this composition was approved.

[0040] Test 4: Determination of the maximum value of methylparaben of 0.0091%. The amount that was added prevented the colonization of the composition by invading agents but caused cell death. Therefore, this composition was disapproved.

[0041] Tests 5 to 8 determined the minimum and maximum amount of propylparaben are listed in table 2.

TABLE-US-00002 TABLE 2 Tests 5 to 8 for determination of amounts in % by mass of propylparaben. Compound 5 6 7 8 methylparaben 0.0045 0.0045 0.0045 0.0045 Propylparaben 0.0001 0.0002 0.0025 0.0026 sodium chloride 5.0163 5.0163 5.0163 5.0163 Sodium bicarbonate 1.4415 1.4415 1.4415 1.4415 BSA 0.8649 0.8649 0.8649 0.8649 sodium citrate 1.4415 1.4415 1.4415 1.4415 potassium citrate 0.5189 0.5189 0.5189 0.5189 K.sub.2EDTA 1.7297 1.7297 1.7297 1.7297 Deionized water 88.9826 88.9825 88.9802 88.9801

[0042] Test 5: Determination of the minimum value of 0.0001% propylparaben. The amount added was not enough to avoid colonization of the composition by invading agents. Therefore, this composition was disapproved.

[0043] Test 6: Determination of the minimum value of 0.0002% propylparaben. The amount of propylparaben added was sufficient to prevent colonization of the composition by invading agents. Therefore, this composition was approved.

[0044] Test 7: Determination of the maximum value of 0.0025% propylparaben. This maximum amount of propylparaben prevented the colonization of the composition by invading agents and did not cause cell death. Therefore, this composition was approved.

[0045] Test 8: Determination of the maximum value of 0.0026% propylparaben. The amount that was added caused cell death. Therefore, this composition was disapproved.

[0046] Tests 9 to 12 determined the minimum and maximum amount of sodium chloride are listed in table 3.

TABLE-US-00003 TABLE 3 Tests 9 to 12 for determination of amounts in % by mass of sodium chloride. Compound 9 10 11 12 methylparaben 0.0045 0.0045 0.0045 0.0045 Propylparaben 0.0012 0.0012 0.0012 0.0012 sodium chloride 0.2323 0.2324 11.4266 11.4267 Sodium bicarbonate 1.4415 1.4415 1.2300 1.2300 BSA 5.4000 5.4000 0.4000 0.4000 sodium citrate 1.4415 1.4415 0.7500 0.7500 potassium citrate 0.5189 0.5189 0.3000 0.3000 K.sub.2EDTA 1.7297 1.7297 1.2000 1.2000 Deionized water 89.2304 89.2303 84.6877 84.6876

[0047] Test 9: Determination of the minimum amount of sodium chloride of 0.2323%. This amount of sodium chloride made the composition no longer partially isotonic and promoted cell lysis. Therefore, this composition was disapproved.

[0048] Test 10: Determination of the minimum amount of sodium chloride of 0.2324%. This added amount kept the final composition partially isotonic and cell lysis did not occur. Therefore, this composition was approved.

[0049] Test 11: Determination of the maximum amount of sodium chloride of 11.4266%. The added amount kept the final composition partially isotonic, as well as preserving the erythrocyte morphology. Therefore, this composition was approved.

[0050] Test 12: Determination of the maximum amount of sodium chloride of 11.4267%. The high concentration of sodium chloride, remained partially isotonic, but promoted changes in erythrocyte morphology and cell lysis. Therefore, this composition was disapproved.

[0051] Tests 13 to 16 determined the minimum and maximum amount of sodium bicarbonate are listed in table 4.

TABLE-US-00004 TABLE 4 Tests 13 to 16 for determination of amounts in % by mass of sodium bicarbonate. Compound 13 14 15 16 methylparaben 0.0045 0.0045 0.0045 0.0045 Propylparaben 0.0012 0.0012 0.0012 0.0012 sodium chloride 5.0163 5.0163 5.0163 5.0163 Sodium bicarbonate 0.0432 0.0433 3.4020 3.4021 BSA 0.8649 0.8649 0.8649 0.8649 sodium citrate 1.4415 1.4415 1.4415 1.4415 potassium citrate 0.5189 0.5189 0.5189 0.5189 K.sub.2EDTA 1.7297 1.7297 1.7297 1.7297 Deionized water 90.3798 90.3797 87.0210 87.0209

[0052] Test 13: Determination of the minimum amount of sodium bicarbonate of 0.0432%. This amount of sodium bicarbonate caused the composition to stop being partially isotonic and promoted cell lysis. Therefore, this composition was disapproved.

[0053] Test 14: Determination of the minimum amount of sodium bicarbonate of 0.0433%. This added amount kept the final composition partially isotonic and cell lysis did not occur. Therefore, this composition was approved.

[0054] Test 15: Determination of the maximum amount of sodium bicarbonate of 3.4020%. The amount added maintained the final composition partially isotonic, as well as preserving the erythrocyte morphology. Therefore, this composition was approved.

[0055] Test 16: Determination of the maximum amount of sodium bicarbonate of 3.4021%. The high concentration of sodium bicarbonate promoted changes in erythrocyte morphology and cell lysis. Therefore, this composition was disapproved.

[0056] Tests 17 to 19 determined the minimum and maximum amount of bovine serum albumin are listed in table 5.

TABLE-US-00005 TABLE 5 Tests 17 to 19 to determine the amounts in % mass of bovine serum albumin Compound 17 18 19 methylparaben 0.0045 0.0045 0.0045 Propylparaben 0.0012 0.0012 0.0012 sodium chloride 5.0163 1.0000 1.0000 Sodium bicarbonate 1.4415 1.2300 1.2300 BSA 0.0000 10.6198 10.6199 sodium citrate 1.4415 0.7500 0.7500 potassium citrate 0.5189 0.3000 0.3000 K.sub.2EDTA 1.7297 1.2000 1.2000 Deionized water 89.8464 84.8945 84.8944

[0057] Test 17: Determination of the minimum amount of 0% bovine serum albumin. The final composition works satisfactorily in the absence of BSA. Therefore, this composition was approved.

[0058] Test 18: Determination of the maximum amount of bovine serum albumin of 10.6198%. The added amount caused the final composition to maintain the morphological characteristics of the erythrocytes and increased the stability of the final composition. Therefore, this composition was approved.

[0059] Test 19: Determination of the maximum amount of bovine serum albumin of 10.6199%. The increase in bovine serum albumin concentration promoted changes in erythrocyte morphology and cell lysis. Therefore, this composition was disapproved.

[0060] Tests 20 to 22 determined the minimum and maximum amount of sodium citrate are listed in table 6.

TABLE-US-00006 TABLE 6 Tests 20 to 22 to determine the amounts in % by mass of sodium citrate Compound 20 21 22 methylparaben 0.0045 0.0045 0.0045 Propylparaben 0.0012 0.0012 0.0012 sodium chloride 5.0163 5.0163 5.0163 Sodium bicarbonate 1.4415 1.4415 1.4415 BSA 0.8649 0.8649 0.8649 sodium citrate 0.0000 4.1690 4.1691 potassium citrate 0.5189 0.5189 0.5189 K.sub.2EDTA 1.7297 1.7297 1.7297 Deionized water 90.4230 86.2540 86.2539

[0061] Test 20: Determination of the minimum amount of 0% sodium citrate. The final composition works satisfactorily in the absence of sodium citrate. Therefore, this composition was approved.

[0062] Test 21: Determination of the maximum amount of sodium citrate of 4.1690%. The added amount caused the final composition to maintain the morphological characteristics of the erythrocytes. Therefore, this composition was approved.

[0063] Test 22: Determination of the maximum amount of sodium citrate of 4.1691%. The high concentration of sodium citrate promoted changes in erythrocyte morphology and cell lysis. Therefore, this composition was disapproved.

[0064] Tests 23 to 25 determined the minimum and maximum amount of potassium citrate are listed in table 7.

TABLE-US-00007 TABLE 7 Tests 23 to 25 to determine the amounts in % by mass of potassium citrate Compound 23 24 25 methylparaben 0.0045 0.0045 0.0045 Propylparaben 0.0012 0.0012 0.0012 sodium chloride 5.0163 5.0163 5.0163 Sodium bicarbonate 1.4415 1.4415 1.4415 BSA 0.8649 0.8649 0.8649 sodium citrate 1.4415 1.4415 1.4415 potassium citrate 0.0000 0.5296 0.5297 K.sub.2EDTA 1.7297 1.7297 1.7297 Deionized water 89.5004 88.9708 88.9707

[0065] Test 23: Determination of the minimum amount of 0% potassium citrate. The final composition works satisfactorily in the absence of potassium citrate. Therefore, this composition was approved.

[0066] Test 24: Determination of the maximum amount of potassium citrate of 0.5296%. The added amount caused the final composition to maintain the morphological characteristics of the erythrocytes. Therefore, this composition was approved.

[0067] Test 25: Determination of the maximum amount of potassium citrate of 0.5297%. The high concentration of potassium citrate promoted changes in erythrocyte morphology and cell lysis. Therefore, this composition was disapproved.

[0068] Tests 26 to 29 determined the minimum and maximum amount of K.sub.2EDTA are listed in table 8.

TABLE-US-00008 TABLE 8 Tests 26 to 29 to determine the amounts in % by mass of K 2 EDTA Compound 26 27 28 29 methylparaben 0.0045 0.0045 0.0045 0.0045 Propylparaben 0.0012 0.0012 0.0012 0.0012 sodium chloride 5.0163 5.0163 1.1000 1.1000 Sodium bicarbonate 1.4415 1.4415 0.5000 0.5000 BSA 0.8649 0.8649 0.8649 0.8649 sodium citrate 1.4415 1.4415 0.5200 0.5200 potassium citrate 0.5189 0.5189 0.5189 0.5189 K.sub.2EDTA 0.0870 0.0871 10.7809 10.7810 Deionized water 90.6242 90.6241 85.7096 85.7095

[0069] Test 26: Determination of the minimum amount of K.sub.2EDTA of 0.0870%. This amount of K.sub.2EDTA promoted platelet aggregation and platelet count errors. Therefore, this composition was disapproved.

[0070] Test 27: Determination of the minimum amount of K.sub.2EDTA of 0.0871%. This added amount kept the platelets without aggregating and maintained the cell morphology. Therefore, this composition was approved.

[0071] Test 28: Determination of the maximum amount of K.sub.2EDTA of 10.7809%. This added amount kept the cell morphology preserved and the platelets without aggregation, and cell lysis did not occur. Therefore, this composition was approved.

[0072] Test 29: Determination of the maximum amount of K.sub.2EDTA of 10.7810%. The high concentration of K.sub.2EDTA promoted changes in erythrocyte morphology and cell lysis. Therefore, this composition was disapproved.

[0073] Tests 30 to 33 determined the minimum and maximum amount of deionized water are listed in table 9.

TABLE-US-00009 TABLE 9 Tests 30 to 33 to determine the amounts in % by mass of deionized water Compound 30 31 32 33 methylparaben 0.0045 0.0045 0.0045 0.0045 Propylparaben 0.0012 0.0012 0.0012 0.0012 sodium chloride 5.0163 5.0163 0.5450 0.5449 Sodium bicarbonate 1.4415 1.4415 1.4415 1.4415 BSA 0.8649 0.8649 0.9000 0.9000 sodium citrate 3.2000 3.2000 1.4415 1.4415 potassium citrate 0.5189 0.5189 0.5189 0.5189 K.sub.2EDTA 4.4827 4.4826 1.7300 1.7300 Deionized water 84.4700 84.4701 93.4174 93.4175

[0074] Test 30: Determination of the minimum amount of deionized water of 84.4700%. The final composition was no longer partially isotonic with this amount of deionized water, there were morphological changes and cell death. Therefore, this composition was disapproved.

[0075] Test 31: Determination of the minimum amount of deionized water of 84.4701%. This added amount kept the composition partially isotonic, there were no significant morphological changes, nor cell death. Therefore, this composition was approved.

[0076] Test 32: Determination of the maximum amount of deionized water of 93.4174%. This added amount kept the composition partially isotonic, there were no significant morphological changes, nor cell death. Therefore, this composition was approved.

[0077] Test 33: Determination of the maximum amount of deionized water of 93.4175%. The final composition was no longer partially isotonic with this amount of deionized water, there were morphological changes and cell death. Therefore, this composition was disapproved.

[0078] In summary, the maximum and minimum amounts of the mass percentages of the components for the satisfactory functioning of the formulation for counting and morphological inference of erythrocytes and platelets are listed in table 10 below:

TABLE-US-00010 TABLE 10 Minimum and maximum amounts of components of the final composition in % by mass. Compound Min (%) Max (%) methylparaben 0.0009 0.0090 Propylparaben 0.0002 0.0025 sodium chloride 0.2324 11.4266 Sodium bicarbonate 0.0433 3.4020 BSA 0.0000 10.6198 sodium citrate 0.0000 4.1690 potassium citrate 0.0000 0.5296 K.sub.2EDTA 0.0871 10.7809 Deionized water 84.4701 93.4174

[0079] Test 34 determined the preferential amount of the components of the composition is listed in table 11. In this test, the final composition showed the best results in terms of cell morphology and stability, avoided contamination by invading microorganisms and the formation of cell aggregates, preserving the erythrocytes.

TABLE-US-00011 TABLE 11 Test 34 with preferred composition, amount in % by mass. Compound % by mass methylparaben 0.0045 Propylparaben 0.0012 sodium chloride 5.0163 Sodium bicarbonate 1.4415 BSA 0.8649 sodium citrate 1.4415 potassium citrate 0.5189 K.sub.2EDTA 1.7297 Deionized water 88.9815

[0080] The formulation for counting and morphological inference of erythrocytes and platelets can be used in venous blood with EDTA or in fresh capillary in the proportion of 1:100 to 1:200 parts by volume. After diluting the collected blood, the blood mixture and final composition must be homogenized and inserted into a hemocytometer for reading under an optical microscope, or in a Point-of-Care device, for analysis with a 10× or 40× magnifying objective lens. The formulation for counting and morphological inference of erythrocytes and platelets was evaluated with blood from rats, dogs, cats and horses, showing good results, and can also be used for veterinary purposes. The final composition can be stored for long periods of up to 4 months, provided it is protected from light.