Concentrated lysis solution

Abstract

The invention relates to a lysis solution intended to be mixed with a diluent for hematology analyses, characterized in that it comprises a solvent, a cross-linking agent at a mass concentration relative to the solvent of between 10 g·L-1 and 100 g·L-1, and a lysis agent at a mass concentration relative to the solvent of between 1 g·L-1 and 60 g·L-1.

Description

Concentrated lysis solution

[0001] The field of the invention relates to a lysis solution for hematological analyses.

[0002] Lysis solutions (sometimes called lysis reagents) are used in hematology analyses. More specifically, lysis solutions are a set of reagents intended to be mixed with biological samples, such as human or animal blood, or any other biological fluid or preparation, to lyse red blood cells (erythrocytes) in order to preserve the remaining cells for measurement. This allows, in particular, for white blood cell counting and differentiation, and quantitative hemoglobin determination in a flow cytometry hematology analyzer.

[0003] Examples of lysis solutions available on the market include the products called Eosinofix®, Nucediff® and Whitediff® marketed by the Applicant.

[0004] Typically, lysis solutions are sold at a working concentration (denoted "X1"), which is the concentration at which they are introduced into hematology analyzers to be mixed with biological samples. This results in several constraints, including large volumes consumed and the labor required to handle these heavy volumes.

[0005] The X1 concentration also presents drawbacks related to throughput limitations. Indeed, the need to distribute a predetermined volume (often on the order of 1 to 2 mL) into hematology analyzers results in unavoidable handling times.

[0006] Furthermore, a stability and / or expiry issue arises with state-of-the-art solutions. Indeed, while a shelf life of approximately 12 months is expected for hermetically sealed solution bottles, only about 2 to 4 months is expected for opened bottles. The bottles must therefore be replaced regularly. Consequently, the solutions contained in the bottles are sometimes not fully used and / or are wasted. This leads to environmental drawbacks and / or increased costs.

[0007] Furthermore, each hematology analyzer is associated with a specific type of lysis solution. Although the formulations differ only slightly from one solution to another, this difference is nonetheless sufficient to impact the results of the hematology analysis. In other words, there is no flexibility, or alternative, regarding the choice of solution used for a given hematology analyzer. This limits, or even restricts, the applications of each lysis solution available on the market.

[0008] Ultimately, the mixture of a biological sample and a lysis solution must be in a predefined quantitative ratio (sample:solution). This ensures that a sufficient quantity of biological sample is available to perform hematological analyses. However, sample availability is generally limited.

[0009] The present invention improves the situation.

[0010] In this respect, the invention relates to a lytic solution for hematology analysis intended to be mixed with a diluent, characterized in that it comprises a solvent and a crosslinking agent at a mass concentration relative to the solvent of between 10 g·L -1 and 100 g·L -1 , a lysis agent at a mass concentration relative to the solvent between 1 g·L-1 and 60 g·L-1.

[0011] In one embodiment, the concentration of the crosslinking agent is between 15 g·L -1 and 25 g·L -1 and the concentration of the lysing agent is between 40 g·L -1 and 60 g·L -1 .

[0012] In another embodiment, the solution of the invention comprises a protective agent at a mass concentration relative to the solvent of between 20 g·L -1 and 200 g·L -1 and a cleaning agent at a mass concentration relative to the solvent of between 1 g·L -1and 10 g·L -1 In this embodiment, the concentration of the crosslinking agent can be between 70 g·L -1 and 80 g·L -1 the concentration of the lysing agent can be between 2 g·L -1 and 10 g·L -1 The concentration of the protective agent can be between 150 g·L -1 and 200 g·L -1 and the concentration of the cleaning agent can be between 2 g·L -1 and 10 g·L -1 .

[0013] In preferred embodiments:

[0014] - the crosslinking agent is chosen from the group consisting of glutaraldehyde, dimethylurea (DMU), or diazolidinyl urea (DZU);

[0015] - the lysis agent is chosen from the group consisting of a saponin and a quaternary ammonium compound (QAC), preferably from dodecyltrimethylammonium chloride (DTACl) and dodecyltrimethylammonium bromide (DTABr);

[0016] - the protective agent is chosen from the group consisting of Tween 20 and Tween 80;

[0017] - the cleaning agent is chosen from the group consisting of surfactants, preferably Triton X-100, and a quaternary ammonium compound (QAC), preferably dodecyltrimethylammonium bromide (DTABr);

[0018] - the solvent is chosen from the group consisting of ethylene glycol, propylene glycol, methanol, ethanol or a mixture thereof; and / or

[0019] - the diluent includes an anticoagulant, preferably EDTA, a pH buffer and a surfactant, and optionally one or more salts and / or a preservative.

[0020] In a particular embodiment, the lytic solution of the invention consists exclusively of the solvent, the crosslinking agent and the lysis agent.

[0021] In another particular embodiment, the lytic solution of the invention consists exclusively of the solvent, the crosslinking agent, the lysis agent, the protective agent and the cleaning agent.

[0022] Other features, details, and advantages will become apparent upon reading the detailed description below and analyzing the attached drawings, which illustrate this.

[0023] shows a comparative diagram between a prior art lysis solution and a concentrated lysis solution according to the invention;

[0024] shows a comparison between two hematological analysis matrices from blood samples respectively treated with a prior art lysis solution and a lysis solution according to the invention;

[0025] shows comparative hemograms of a blood sample between an analysis with a prior art solution and with a lysis solution of the invention; and

[0026] shows comparative hemograms of a control blood sample between an analysis with a prior art solution and with a lysis solution of the invention.

[0027] The figures, tables, and description that follow essentially contain elements of a certain nature. The figures and tables form an integral part of the description and may therefore not only serve to better understand the present invention but also contribute to its definition, if necessary.

[0028] In this description, and in particular with reference to the invention, the terms "lysis solution(s)" and "lytic solution(s)" shall be used interchangeably.

[0029] In general, the invention relates to a formulation and production process for a non-aqueous lysis reagent for in vitro diagnostics. The invention is particularly suitable for erythrocyte lysis (RBC), leukocyte enumeration and differentiation (WBC), and hemoglobin determination. The lysis solution of the invention is approximately 20 times more concentrated than a conventional lysis solution.

[0030] The principle of producing hematology reagents in a non-aqueous medium (ethylene glycol) is also applied to the preparation of staining reagents. For example, thiazole orange is used to detect nucleic acids in cells. However, it is not used to prepare lysis reagents because it is generally difficult to induce lysis in a non-aqueous medium. More generally, it is known to solubilize an organic molecule, such as a dye, in an organic solvent. The organic solvent stabilizes the organic molecule, resulting in a stable solution. Conversely, it is difficult, if not impossible, to solubilize molecules or substances with lytic properties in an organic solvent. Therefore, lytic molecules or substances in the prior art are solubilized in aqueous media.

[0031] The Applicant discovered, somewhat surprisingly, that an organic solvent could preserve the active agents, thereby increasing the stability of the reagent(s) present in the lysis solution. Indeed, and remarkably, the invention ensures the preservation of the active agents without the need for any preservatives. The concentrated lysis solution of the invention uses an organic solvent (ethylene glycol) instead of water. Consequently, the concentrated lysis solution differs essentially from the standard lysis reagent (at a working concentration) in that it contains only the active agents necessary for erythrocyte lysis and leukocyte differentiation, and at a concentration 20 times higher than standard lysis reagents.

[0032] A conventional lysis solution contains, in addition to the lysis agent(s), substances such as EDTA, salts, and a pH buffer. In the present invention, the lysis solution, in its basic form, is free of these substances. More specifically, in the context of the present invention, these substances are introduced via a diluent (which is separate from the lysis solution).

[0033] This illustrates the principle of the invention. The prior art lysis solution "Diff Lysis" (left) is composed of EDTA, salts, pH buffer, and preservative, as well as glutaraldehyde (crosslinking agent), saponin (lytic agent), Tween20 (protective agent), and QAC (lytic or cleaning agent) dissolved in water. The concentrated lysis solution of the invention "CDL" consists of glutaraldehyde (crosslinking agent), saponin (lytic agent), Tween20 (protective agent), and QAC (lytic or cleaning agent) in ethylene glycol (solvent). The "CDL" lysis solution is formulated to be diluted in a specific diluent, "Diluent," which consists of EDTA, salts, pH buffer, a surfactant, and a preservative, diluted in osmotic water. As an example, the standard lysis solution "Diff Lysis" can be reconstituted by mixing one part of the concentrated lysis solution of the invention "CDL" with 19 parts of diluent.The Applicant has surprisingly identified a concentrated lytic solution consisting only of specifically selected ingredients, which, after being mixed with a diluent, are suitable for forming a solution usable in a hematology analyzer.

[0034] Overall, the advantages of the invention are increased stability of the lysis solution and / or of the reagent(s), a reduction in environmental impact and water consumption, as well as a decrease in production, transport and storage costs.

[0035] Furthermore, lysis performed with the concentrated solution of the invention allows for great flexibility of use, as the single concentrated solution can be adapted to various types of cycles and devices. In particular, the concentrated solution can be used independently for leukocyte differentiation into 3 subpopulations (called "3Diff") or 5 subpopulations (called "5Diff"), for total nucleated cell counting (TNC), and for basophil count (BASO count). Moreover, the concentrated solution can be used independently in human or veterinary applications. A primary application is the use of the concentrated solution in flow cytometry techniques.

[0036] Another advantage of the invention is its ease of use. Indeed, to reconstitute the lysis solution (or lysis reagent) to the working concentration, it is sufficient to mix the appropriate amount of concentrated lysis solution with the necessary amount of diluent specific to the application of interest. Examples include diluents such as "ABX Diluent" or "MiniDIL," available from the Applicant, or more conventional diluents such as "PBS+Tween20," i.e., a mixture of phosphate saline buffer with polysorbate 20.

[0037] More specifically, the concentrated lytic solution of the invention drastically reduces the volumes used. This results in a reduction of the labor required to handle them. The invention therefore makes it possible to increase the work rate, particularly in laboratories, the pharmaceutical industry, and / or diagnostics.

[0038] Furthermore, reducing the volume also addresses issues of stability and / or expiry of lysis solutions. Bottles are generally used entirely and / or not wasted. This offers environmental benefits and / or reduces costs.

[0039] Furthermore, the lytic solution of the invention can be mixed with various diluents in a range of quantitative ratios (solution:diluent). This allows for its use in numerous applications. The lytic solution can thus be used in different types of hematology analyzers, resulting in significant flexibility.

[0040] Furthermore, the solution of the invention allows for working with cascading dilution ranges. This reduces the amount of solution used, and consequently the amount of biological sample (particularly blood) required for hematological analysis (sample:solution). Therefore, a reduced availability of biological samples is less of an issue.

[0041] In summary, the invention offers several advantages:

[0042] - for the user, including in particular: increased reagent stability, increased analysis rate because less reagent volume is distributed, storage space savings, reduced weight and / or reagent replacement, and increased autonomy and / or working time savings;

[0043] – on the environment, including in particular: a reduction of plastic waste and a reduction of the carbon footprint; and

[0044] - for the manufacturer, including in particular: a reduction in water consumption and waste and a reduction in production, logistics and / or transport costs.

[0045] The present invention proposes for the 1 ère once a universal concentrated lysis solution for hematology analyzers. It should be noted that, to the inventors' knowledge, a concentrated lysis solution called IOTest® 3 exists (available from Beckman Coulter). This is a solution concentrated tenfold (noted "10X") which, once reconstituted, contains all the lysis components at the 1X working concentration, including the pH buffer, concentrated in an aqueous solvent. However, this solution is intended for specific immunophenotyping applications. Furthermore, it requires storage at a temperature between 2°C and 8°C.

[0046] EXAMPLES OF PROJECTS

[0047] The solution of the invention can be used in various applications. In particular, the solution can be used in the differentiation of leukocytes into 3 subpopulations (called "3Diff") or into 5 subpopulations (called "5Diff").

[0048] Table 1 shows reagent ranges for preparing a concentrated lysis solution of the invention according to its use for a "3Diff" and "5Diff" application. In this Table 1, the symbol "[c]pref." means "preferred concentrations".

[0049] Function Product Concentration (g / L) Concentration % (w / w) 3 DIFF[c]pref. % (w / w) 5 DIFF[c]pref. % (w / w) Crosslinking agent (protects leukocyte cell membranes) Glutaraldehyde, dimethylurea (DMU), or diazolidinyl urea (DZU or diazolidinyl urea) 10-100 1-10% 1.5-2.5% 7-8% Lytic agent Saponin, QAC (DTACl, DTABr) 1-500 0.1-5% 4-6% 0.2-1% Protective agent Tween 20, Tween 80 20-200 2-20% na 15-20% Cleansing agent Surfactants (Triton X-100), QAC (DTABr) 1-100 0.1-1% na 0.2-1%

[0050] As shown in Table 1, the main difference between the "3Diff" and "5Diff" versions is that the lysis solution does not contain a protective agent or cleaning agents in the "3Diff" version. Another difference relates to the preferred concentrations of the crosslinking and lytic agents.

[0051] EXAMPLE: 3DIFF LYSIS SOLUTION

[0052] A specific example for use in "3Diff" is given by a concentrated lysis solution (3DIFF version) containing 20.0 g / L of dimethylurea (DMU) and 50.0 g / L of dodecyltrimethylammonium chloride (DTACl). Preferably, the concentrated lysis solution (3DIFF version) consists of 20.0 g / L of DMU and 50.0 g / L of DTACl.

[0053] EXAMPLE: 5DIFF LYSIS SOLUTION

[0054] A specific example for use in "5Diff" is given by a concentrated lysis solution (5DIFF version) containing 74.2 g / L glutaraldehyde, 8.0 g / L dodecyltrimethylammonium bromide (DTABr), 176.8 g / L Tween 20, and 8.0 g / L saponin. Preferably, the concentrated lysis solution (5DIFF version) consists of 74.2 g / L glutaraldehyde, 8.0 g / L DTABr, 176.8 g / L Tween 20, and 8.0 g / L saponin.

[0055] Table 2 shows examples of preparation according to different applications.

[0056] ApplicationTNCBASODIFFFLOW CYTOMETRYDilution rate (1 / x)25020012110Diluent (µL)*200020002400110CDL (µL) [5DIFF]1401407010Dye (TOC) (µL)27Incubation time (s)12122590Temperature (°C)35453735

[0057] *The diluent used can be a generic diluent for hematological applications. Typically, such a diluent contains at least: an anticoagulant, a pH buffer, and a surfactant. In particular, the diluents mentioned above are suitable (ABX Diluent or MiniDIL, available from HORIBA ABX SAS, or PBS+Tween20). Depending on the application, the CDL concentration is adjusted with the diluent. For example, for leukocyte differentiation applications, the TOC staining reagent is used.

[0058] EXAMPLE: HEMATOLOGICAL APPLICATION 5DIFF

[0059] This example concerns 5DIFF type hematological cycles.

[0060] A comparative test is performed between a prior art lysis solution marketed under the name "Whitediff" available from HORIBA ABX SAS and a lysis solution according to the invention, "CDL". For this purpose, a hematological analysis of 26 separate samples is performed using either "Whitediff" or "CDL" as the lysis solution.

[0061] In a Yumizen H550 hematological analyzer available from HORIBA ABX SAS, the standard operating procedure uses Whitediff solution as the lysis reagent for leukocyte counting and differentiation (WBC) and for hemoglobin parameter measurement (RBC). To perform this test, Whitediff solution was used as a reference, then replaced with a concentrated lysis solution (CDL) diluted 20 times in ABX Diluent.

[0062] The mixture produced in the analyzer is as follows:

[0063] - 20 µL of blood sample;

[0064] - 1 mL of "ABX Diluent" thinner; and

[0065] – 1.4 mL of lysis reagent ("Whitediff" or "CDL" diluted X20 in "ABX Diluent"). When using CDL, solutions conforming to Table 1 are suitable.

[0066] The mixture is homogenized and incubated in a tray for 25 seconds at 37°C, then transferred to the optical tank for analysis.

[0067] Lamontre compares the two hematological analysis matrices from the 26 blood samples respectively treated with the "Whitediff" or "CDL" lysis solution.

[0068] Comparison of the matrix from human blood samples with the "Whitediff" lysis solution (top) and a reagent of the invention "CDL" (bottom) shows similar results. Therefore, there is no alteration in performance when using a lysis solution according to the invention.

[0069] Lamontre demonstrates the correlation in human blood between the commercial reagent "Whitediff" and the reagent according to the invention, CDL, with regard to the parameters of the leukocyte differential. A correlation is observed in human blood between the commercial lysis solution "Whitediff" and the "CDL" solution of the invention. The correlation is shown for the following parameters of the leukocyte differential: nbWBC (R2 = 0.84), Hb (R2 = 0.98), pLym (R2 = 0.96), pMon (R2 = 0.74), pNeu (R2 = 0.95), pEos (R2 = 0.92), pIG (R2 = 0.64).

[0070] Lamontre presents a linearity test on three levels of control blood, marketed under the name "ABX Difftrol," with three different blood cell concentration levels. The "ABX Difftrol" test is a three-level, multiparametric control for in vitro diagnostics, designed to verify the accuracy and precision of HORIBA Medical blood cell counters. Here, a correlation is observed on the control blood between the "Whitediff" reagent and the reagent according to the invention, "CDL," on the parameters of the leukocyte differential: nbWBC (R2 = 1.00), Hb (R2 = 1.00), pLym (R2 = 0.92), pMon (R2 = 0.95), pNeu (R2 = 0.96), pEos (R2 = 0.85), pBASO (R2 = 0.63).

Claims

1. A lytic solution for hematology analysis intended to be mixed with a diluent, characterized in that it comprises a solvent and a crosslinking agent at a mass concentration relative to the solvent of between 10 g·L -1 and 100 g·L -1 , a lysis agent at a mass concentration relative to the solvent of between 1 g·L -1 and 60 g·L -1 .

2. A lytic solution according to claim 1, wherein said concentration of the crosslinking agent is between 15 g·L -1 and 25 g·L -1 and in which said concentration of the lysing agent is between 40 g·L -1 and 60 g·L -1 .

3. A lytic solution according to claim 1, further comprising a protective agent at a mass concentration relative to the solvent of between 20 g·L -1 and 200 g·L -1 and a cleaning agent at a mass concentration relative to the solvent of between 1 g·L -1and 10 g·L -1 .

4. A lytic solution according to claim 3, wherein said concentration of the crosslinking agent is between 70 g·L -1 and 80 g·L -1 said concentration of the lysing agent is between 2 g·L -1 and 10 g·L -1 the said concentration of the protective agent is between 150 g·L -1 and 200 g·L -1 and the said concentration of the cleaning agent is between 2 g·L -1 and 10 g·L -1 .

5. A lytic solution according to any one of the preceding claims, wherein the crosslinking agent is selected from the group consisting of glutaraldehyde, dimethylurea (DMU), or diazolidinyl urea (DZU).

6. A lytic solution according to any one of the preceding claims, wherein the lysing agent is selected from the group consisting of a saponin and a quaternary ammonium compound (QAC), preferably from dodecyltrimethylammonium chloride (DTACl) and dodecyltrimethylammonium bromide (DTABr).

7. A lytic solution according to any one of the preceding claims, wherein the protecting agent is selected from the group consisting of Tween 20 and Tween 80.

8. A lytic solution according to any one of the preceding claims, wherein the cleaning agent is selected from the group consisting of surfactants, preferably Triton X-100, and a quaternary ammonium compound (QAC), preferably dodecyltrimethylammonium bromide (DTABr).

9. A lytic solution according to any one of the preceding claims, wherein the solvent is selected from the group consisting of ethylene glycol, propylene glycol, methanol, ethanol or a mixture thereof.

10. A lytic solution according to any one of the preceding claims, wherein the diluent comprises an anticoagulant, preferably EDTA, a pH buffer and a surfactant, and optionally one or more salts and / or a preservative. 11.Lytic solution according to claim 1, consisting of the solvent, the crosslinking agent and the lysis agent. 12.Lytic solution according to any one of claims 3 to 10, consisting of the solvent, the crosslinking agent, the lysis agent, the protective agent and the cleaning agent.

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