WO2017100882A1 - Composição corante de células de base aquosa, respectivo processo de preparação e uso da mesma, métodos de preparação de amostra e contagem de células - Google Patents
Composição corante de células de base aquosa, respectivo processo de preparação e uso da mesma, métodos de preparação de amostra e contagem de células Download PDFInfo
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- WO2017100882A1 WO2017100882A1 PCT/BR2016/000163 BR2016000163W WO2017100882A1 WO 2017100882 A1 WO2017100882 A1 WO 2017100882A1 BR 2016000163 W BR2016000163 W BR 2016000163W WO 2017100882 A1 WO2017100882 A1 WO 2017100882A1
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
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- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C—CHEMISTRY; METALLURGY
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
Definitions
- Blood cells can be classified into three groups: red cells (erythrocytes), white blood cells (leukocytes) and platelets (thromfoocytes). As blood permeates the entire animal body, these cells are transferred to other parts of it, in particular to body fluids.
- red cells erythrocytes
- white blood cells leukocytes
- platelets thromfoocytes
- Leukocytes represent an important group of cells that are involved in the body's defense against infectious agents and foreign substances. Therefore, they are present in various body fluids, in addition to blood, urine, cerebrospinal fluid, cavitary fluids such as ascites, pleural, peritoneal and synovial, alimentary fluids such as milk.
- cavitary fluids such as ascites, pleural, peritoneal and synovial, alimentary fluids such as milk.
- - Excessive leukocytes in animal milk may represent a major public health problem or financial loss as it indicates the presence of different infections that may contaminate the entire batch of milk collected due to a sick animal Py ⁇ r ⁇ lâ ⁇ 2003 ⁇ ; Normative Instruction No. 51 of 2011 of the Ministry of Agriculture, Ordinance Anv ⁇ sa,
- O Normal human blood contains from 4000 to 10000 leukocytes per cubic millimeter of blood. Under normal health conditions, mammals have five types of leukocytes: neutrophils, basophils, eosinophils, lymphocytes and monocytes (MIRCIC, 2006; RAWAT, 2015).
- the Leukocytes that contain granules are called granulocytes, represented by neutrophils, eosinophils and basophils.
- Cells without granules are called agranulocytes, represented by lymphocytes and monocytes (MARIEB, 2011).
- White cell distribution typically occurs at the following intervals: neutrophils occur between 40% and 60%; lymphocytes between 20% and 40%; monocytes between 2% and 8%; eosinophils between 1% and 4%; and basophils 0.1% and 1%, as described by MATHUR et al f 2013. Variations in the total amount of leukocytes and / or proportions of each type are indicative of the presence of a large number of diseases.
- Neutrophils are by far the most abundant leukocytes in the human immune system (CARRERAS, 1994). They are rounded cells, ranging in size from 12 to 14 ⁇ m in diameter (RAWAT et al, 2015). It has a lobulated nucleus, which usually contains two to five lobes, connected by a thin strand of chromin. The mature neutrophil is defined by round lobes showing chromatin. condensed, and sometimes the strand of croraatin may not be visible as they are overlapped by the nuclear lobes. Croatine is coarse, grouped into portions. The cytoplasm is clear and contains several small granules evenly distributed throughout the cell, but may not be visible when they are over the nucleus. These granules are peroxidase-positive and peroxidase-negative in a two to one ratio (BEUTLER et al, 20.01).
- Lymphocytes represent a heterogeneous group of cells that can be easily differentiated from other leukocytes by their characteristic nuclear and cytoplasmic morphology. However, the general morphology is shared by its three main types: T cells, B cells and natural killer NK cells "(BEUTLER et al, 2001).
- Classic blood studies show lymphocytes with spherical or ovoid cells with ranging from 5 to 17 m (R ⁇ WAT et al, 2015) Small cells are considered to be 6 to 9 m in diameter, while large lymphocytes are 9 to 17 m in diameter.
- Small lymphocytes when stained by Romanowsky derivatives have an oval or brawny nucleus, which stains purple, and occupies about 90% of the cell area.
- the small cytoplasmic border is stained light blue. nucleoli, which are rarely seen on blood strain slides when they are stained with Giemsa (BEUTLER et al, 2001).
- ⁇ Monocytes are the largest normal blood cells whose diameter ranges from 14 ⁇ m to 24 pm (RAWAT et al, 2015).
- the nucleus is of various shapes, from round, kidney-shaped, oval or yolk, and may appear bent. Chromatin is organized into thin strands with well defined margins.
- the cytoplasm When stained with Romano-wsky derivatives the cytoplasm is light blue or gray and often vacuolated / especially in slides made from blood collected with the EDTA anticoagulant.
- the gray color of the cytoplasm (as opposed to blue) is due to fine graining containing RNA (blue coloration) and helps distinguish between monocytes and reactive lymphocytes.
- Laonocyte nuclear chromatin contains a thin structure, unlike the coarser clusters of the lymphoid chromatin (BEUT.LER et al f 2001).
- Eosinophils were first described in 1845 by Jones (McEWEN, 1992), and since then their presence is associated with parasitic infections, as well as in a number of other diseases, including dermatoses, allergies, polyarteritis, and some neoplastic diseases (BASTEN et al. , 1970).
- BE LA ⁇ . ⁇ ' ⁇ describes eosinophils as cells slightly larger than neutrophils. Its diameter is between 12 and 15 um (Frawat et al, 2015). Usually the nucleus has only two wolves. The chromatin pattern. It is the same found in neutrophils, but the nucleus tends to be more, slightly stained.
- the main distinguishing feature of these cells is the presence of many refringent granules, which Roanowsky derivatives stain reddish orange and are evenly distributed throughout the cell (BEUTLER et al, 2001).
- the granules contain mainly the major basic protein (PBM), the cationic eosinophilic protein (PCE) and the eosinophilic peroxidase (POE) (SPRY, 1985; McEWEN, 1992; SPRY, 1992; STEVENS, LO and 1993; TIZARD, 1998).
- Basophils represent a population of granulocytic cells: morphologically distinct in the blood and are the main mediators of allergic inflammation (KURIMOTO, 1989; FALCONE, 2000). Basophils are less numerous.
- Sorting and counting blood cells and other body fluids is important for assessing the individual's health status under numerous conditions.
- Examples of criteria used to classify leukocytes in imaging examinations include differences in texture, affinity with different dyes, size, content and morphology of the nucleus and cytoplasm of each cell type.
- Counting and characterization of blood cells, or blood count is the most requested laboratory test for monitoring and screening the patient's health status. Morphological counting and evaluation of blood cells has been an important tool not only for disease diagnosis but also as an "attestation" of the patient's overall health on periodic examinations and checkups. (ROSENFELD, 2012).
- leukocyte differential counting has a long tradition as a diagnostic tool in medicine, helping the clinician to make decisions (DUCREST, 2005). Therefore, over the years, information derived from differential leukocyte counts has become fundamental in the patient's diagnostic assessment, and is also routinely used for screening and monitoring hematological and non-hematological diseases.
- leukocytes in the various body fluids such as blood, urine, milk, cerebrospinal fluid, cavity fluids such as ascites, pleural, peritoneal, synovial, require different types of tests. All of these fluids are predominantly aqueous media; however, all dyes that are used to differentiate leukocytes are available only in alcoholic solutions. As the differential dyes are in an alcoholic medium, it is necessary that the body fluid to be stained is distended on a slide, dried and fixed, before proceeding to the differential staining of the leukocytes.
- Some of the currently available dye compositions may perform differential staining in alcoholic liquid medium.
- coagulation or agglutination of solids forming lumps occurs as shown in the integral solution in Figure 1, or as shown in detail in the Neubauer chamber image shown in Figure 2.
- the results of these stains are of no practical use because particle agglutination makes cell counting and trumpet classification unfeasible. No : It is thus possible to separate the cells contained in the agglutinate for counting.
- leukocyte staining in non-alcoholic liquid medium is mainly made with Methylene Blue or its derivatives, forming, for example, the M liquid Tur.3 ".
- this type of staining does not: allows the differentiation of leukocytes into This type of dyes only allows detecting the presence of leukocytes in the sample, but does not allow them to be differentiated in order to make a qualitative analysis of them.
- the dyes used for blood distention were developed by the Russian physician Dimitri L. Romanowsky (1861-1921) in the late 19th century. After it emerged several derivatives of the same dye, which act on the same principles and with which similar results are obtained. They are the dyes known as: Wright, Giemsa, Lesh ⁇ man, May-Grunwaid and Rosenfeld among others. These dyes are so old that there is no patent pending or in force for them. In addition to being derived from Romanowsky, they all have another feature in common: they act only on fixed strains. None of these or any other dye known to date is capable of staining leukocytes in liquid medium so as to allow them to be differentiated and counted. expert visual evaluation or image processing.
- Dyes based on Romanowsky's formulation and the manual method used to differentiate leukocytes are widely used in hematology.
- coloration for visual differentiation has not undergone methodological developments for several decades. Therefore, there is a need in the art for staining compositions that stain, differentially form cells and do not form cell clumps, allowing them to be counted.
- red blood cells Another important fact is that none of these dyes or the method used for manual differential counting eliminates the presence of red blood cells, which are also stained.
- the presence of red blood cells hinders the work of specialists, who need to search for scattered leukocytes amid a multitude of red blood cells. (The normal ratio is 1000 red cells for each leukocyte).
- the presence of red blood cells has little effect on the work of experienced specialists who have learned to ignore them. However, it drastically affects leukocyte segmentation and recognition algorithms in image processing programs. For this reason, there is one. a great need in the art for a dye that eliminates or does not red blood cells while preserving differently stained leukocytes.
- Such dyes contain among their essential components, " a mixture in which a basic cationic component such as azur-B imparts the blue or blue-violet color to nucleic acids, DNA and RNA. This is because they attach to phosphate groups. found in acids, nucleoproteins, basophil granules and, to a lesser extent, eosinophil granules.An anionic acid dye, also present in the mixture, gives the hemoglobin, eosinophil granules an orange color and also helps in the coloration of the nucleus.
- Eosin is a dye that meets this demand and is usually combined with azur-B (WITTEKIND, 1979) or methylene blue to produce one of the satisfactory dyes derived from Romanowsky [MARSHALL et (1975, 1975) .
- the stains produced with these mixtures are pH dependent, including that of the slide wash water. before use, otherwise they form particulate deposits on strains, which can lead to misreading, such as the visualization of corpuscles in erythrocytes.
- These combinations of dyes use methanol as a solvent and diluent, making them unsuitable for staining blood cells in liquid because they coagulate the blood and do not allow visualization of the stained cells.
- the two main dyes in the solutions that make up the Romano isk derivatives are methylene blue and Eosin Y, whose characterizations are presented.
- methylene blue whose chemical structure is presented below is a basic dye belonging to the phenothiazine class. It has the following characteristics: it is organic, aromatic, - heterocyclic, soluble in water or alcohol (LIMA et al., 2007).
- Basic dyes are soluble in. water and produce colored cations in solution. Thus, they are generally referred to as cationic dyes (POGGERE et al, 2011). It has maximum absorption at 661 nm in aqueous solution and 656 nm when in methanol solution (H.OROBIN, 2008).
- Eosin Y is an anionic hydroxixanthene acid dye with maximum absorption at 516 nm in alkaline or neutral aqueous solution (HOROBIN, 2008)stiIn alcoholic solution it absorbs at 524 nm. Its chemical structure is presented in the structure below. It is widely used to stain cytoplasm and cytoplasmic structures such as eosinophilic granules &.
- Another class of known dyes which are mainly used for staining histological sections, but which also stains blood cells, are those which have deoxyribbonucleic acids (DNA) and nucleic acid (RNA), such as Methyl Green (VM) and Pyronine (P).
- DNA deoxyribbonucleic acids
- RNA nucleic acid
- VM Methyl Green
- P Pyronine
- VM has a strong cationic character, which leads it to be electrostatically attracted to phosphate groups, which are located on the outside of the DNA strand. This results in green-blue stained leukocyte nuclei.
- Pyronine is slightly cationic, which gives it greater affinity for flat and less polymerized structures, having high affinity for ribonucleic acid molecules. Thus, it stains the nucleoli and areas containing RNA in. pink tones ⁇ PERRY et al, 1956; KIERNAN, 1999; HOROBIN, 2008).
- Another dye that may be part of this group is fluorescein, which stains cytoplasmic granules and other structures that have basic protein content because of its anionic character. Thus, eosinophilic granules turn an orange-yellow color.
- This grouping of dyes produces three color groups, the combination of which allows the differentiation of leukocytes in blood strains.
- the three dyes described above have desirable characteristics for leukocyte differentiation in manual examinations as it provides another route of recognition that goes beyond.
- morphological analysis which is the main characteristic for differentiation, in staining with Romanowsky derivatives.
- the only descriptions of these dyes in blood distention were presented by Ornste ⁇ en et al, (19 * 74 ⁇ , TYC O et al. ( 1976. According to Tycho et al.
- This staining has the potential to differentiate leukocytes by image processing). However, it did not prosper because the acting time in distension blood is long (greater than 30 minutes) and the resulting staining is less contrasting than those obtained with Romanowsky derivatives for visual analysis. In these works no detail has been presented on how these dyes are prepared or how they work.
- the dye composition of the present invention although it may comprise concentrations and proportions similar to known compositions, differs from all of its priorities.
- teriti a method of preparation which stabilizes the composition in aqueous medium and therefore modifies all results obtained in staining cells in liquid medium
- the current dye solutions are prepared in alcoholic media because the alcohol performs cell membrane permeabilization, which facilitates dye entry into the cell.
- the present invention relates to compositions which perform membrane permeabilization for dye penetration even in predominantly aqueous medium.
- the dyes penetrate the cells, react with the cell structures with which they have affinity, as described above, and allow the differentiation of leukocytes in an appropriate manner.
- the present invention provides, in one embodiment, a dye composition capable of staining cells in liquid medium based on water, particularly leukocytes, differentiated between the respective types, without forming particle agglutinates and, with the possibility of simultaneously carrying out the red cell lysis if necessary.
- This new form of staining provides that this invention also contemplates a new method of leukocyte counting in a liquid medium, which does not require the preparation of blood distension, performing the procedure of global and differential cell counting simultaneously in a Neubauer chamber or a similar device.
- the aim of this invention is to perform the global and differential leukocyte counting in one step, making the preparation of blood and differential staining of cells in liquid medium, so that they are counted directly under the microscope and photographed for verification or future evaluation of results, without the need for repetition of readings.
- a dye composition which penetrates and differentially stains leukocytes has been developed.
- the composition disclosed herein comprises combinations of the Methyl Green ( " VM), Pyronine (P) Fluorescein (F) dyes which may or may not be associated with a lysing agent, forming a dye / lysing agent complex which acts in a liquid medium stage.
- the dye composition of the present invention allows leukocyte differentiation without the need to perform cell morphological analysis, which represents a major advance for image processing recognition, so cells can be classified at lower magnifications at 100x without the need for use. of immersion oil, which further simplifies the counting process.
- the method uses simple information obtained from histograms and a simple classification logic, making it possible to obtain an automated classification that is equivalent to a flow cytometer.
- Methyl Green acts as a triaryl ethane cationic dye that has a maximum absorption at 635 nm and a subsidiary peak at 410 nm when diluted in water (KOROBIN, 2008).
- Pyronine acts as a mildly cationic dye that has good affinity for RNA. It has a maximum absorption peak at 546 nm when diluted in water, whereas when in 50% aqueous ethanol solution, the maximum absorption occurs at 549 nm (HOROBIN, 2008).
- Fluorescein is an anionic dye that has affinity for cytoplasm proteins such as eosinophilic granules. Sla has a maximum absorption of 490 nm when diluted in water. Since pH is important in defining the absorption range where it acts, it is possible to find the absorption peak at other wavelengths if the pH is different. (HOROBIN, 2008; BANCROFT, 2013 ⁇ . Its chemical structure is presented below.
- CSC Load selectivity
- DNA demonstration can be performed by different techniques such as those showing deoxyribose, or by staining with VM and P in which the phosphate clusters combine with the base of the ' VM dye at acid pH or can be displayed by methods. fluorescent, using dyes such as acridine orange.
- the demonstration of RNA can be performed by staining with acridine orange, or other forms that require complex extraction processes.
- the method of choice for DNA and RNA disclosure is the MV and P staining technique as described by Bancroft (2013).
- both dyes used in combination are cationic with the VM preferentially and specifically binding to DNA, allowing P to bind to RNA.
- the specific reactivity of VM is attributed to the spatial alignment of its NH3 ⁇ 4 groups with phosphate radicals on the double strand of DNA. P staining, on the other hand, does not show this spatial affinity, so other negatively charged constituents stain red.
- these dyes are soluble in water or aqueous acetate solution, they also do not clot the blood when used in medium. have useful characteristics when staining in this medium is desired.
- the present invention aims to provide aqueous compositions comprising cell dyes or salts and complexes thereof.
- Such compositions may further comprise one or more cell isolating agent (s) and an alcohol concentration of less than or equal to 35% by volume.
- the present invention relates to the process of preparing the composition comprising mixing dyes and optionally one or more isolating agents (s).
- lysing agents may be cyanide free.
- the present invention aims to provide a method of counting cells derived from body fluids stained with the cell dye composition described above.
- the present invention provides a cell counting method comprising (i) obtaining an image of the cells suspended in the dye composition; (ii) overall cell type counting and different cell subtype counting in a single step, where such counts can be done by traditional / microscope methods, or by processing the image obtained in step (i).
- the present invention relates to the use of the dye compositions for preparing a cell sample for cell analysis.
- compositions, methods and uses of the present invention consist of the evolution of the current technique, manual method by collecting micro samples of body fluids that are diluted in dye solution and lysing agents (where appropriate), which allows for the preparation and availability of cells for reading in one step.
- the global and differential counts can be made automatically, for image processing, or the health professional directly microscópio- Digital images of the cells can be stored for possible future evaluations without the need for new collection and examination.
- the present invention relates in a first aspect to cell dye compositions comprising a mixture of dyes in predominantly aqueous medium.
- a liquid medium can be understood as a solution comprising less than 50% alcohol.
- a predominantly aqueous medium should be understood as a medium comprising less than 40%, less than 35% or less than 30%, less than 20% or less than 10% alcohol.
- compositions of the present invention stain cell types and subtypes differentially.
- compositions of the present invention comprise the Methyl Green, Pyronea and Fluorescein dyes, or salts and complexes thereof, in. kinda predominantly aqueous.
- dyes with equivalent physicochemical properties for example dyes of the Triarylmethane (or triphenylmethane) chemical group, where dye green is found.
- compositions of the present invention may optionally comprise a cell lysing agent which, according to the laws and safety standards in force in various countries, must be free of cyanide.
- a suitable cell lysing agent for the present invention may be selected from quaternary agents, such as Quaternary Ammonium, which are capable of disrupting the membranes of red blood cells.
- compositions of the present invention comprise from 0.3 to 8 mM Methyl Green. or equivalent dye, preferably from 4.0 mH to 7.0 mM, 0.5 mM to 7 mM, from 5 mM to 8.0 mM or from 1.0 mM to 6 mM, or for example 2 0.0 mM to 5 mM, optionally from 3.0 mM to 4 mM.
- Prionine may be present in a concentration of 0.3mM to 8mM, preferably 0.5mM to 7mM, or 1.0mM to 6mM, or for example from 2.0mM to 5mM. mM, optionally from 3.0 mM to 4 mM.
- Fluorescein concentration may range within the range from 0.1 M to 3 mM, optionally from 0.3 mM to 2.5 mM or, for example, from 0.5 mM: to 2 mM or 1.0 mM to 1.5 mM. Finally, a.
- the concentration of cell lysing agent, such as Quaternary Ammonium, if present in the composition may range from 25 g / l to 80 g / l diluted in aqueous medium.
- compositions of the present invention have a.
- the particularity is that the dyes, for example Methyl Green, Pyroxyin, and Fluorescein, and optionally one. lysing agent, form a complex in a predominantly aqueous medium, or at an alcohol concentration of 35% or less.
- the present invention discloses the method of obtaining the dye compositions. Such a method involves three basic steps, namely:
- Methyl Green and Pyronine are mixed at concentrations between 0.3mM and 8mH each, preferably from 0.5mM to 7mM, or 1.0mM to 6mM, or, for example, from 2mM to 6mM. 0.5 mM to 5 mM, optionally from 3.0 mH to 4 mM having the VMP solution a pH of 3.8 to 5.5.
- Fluorescein is added in one. ratio of one part to two parts PMV fluorescein at a concentration of 0, IM 3 .mm, optionally, 0.3mM to 5mM and f 2, for example, 0.5 mM or 2 mM L 0mm al 5mM , and two and a half parts of VMP to one part of Fluorescein, thus obtaining the VMPF solution with. pH between 4.8 and 6.4.
- the lysing agent may be added to the composition of the present invention in a ratio of one part of lysing agent (in the ratio of 25 g / l to 80 g / l associated in the ratio of 2 g / l to 9 g / l, both previously diluted in aqueous medium for two parts VMPF solution to two parts lysant for one part VMPF solution, where the final VMPF dye solution (H ⁇ should remain at a pH between 5.5 and 6.6,
- the present invention further relates to a method of cell sample preparation comprising cell lysis and differential staining of cell types in single step aqueous medium.
- a method of cell sample preparation comprising cell lysis and differential staining of cell types in single step aqueous medium.
- Such a method comprises the step of mixing a cell sample with a dye or dye / lysate composition as described above. This step allows permeaphilization of cell membranes in predominantly aqueous medium.
- a cell sample is mixed with a dye or dye / lysate composition in the ratio of one part body fluid to 5 to 200 parts of the dye composition, depending on the type of body fluid to be analyzed.
- a part body fluid for 10 to 150 parts dye composition, or one part body fluid for 20 to 130 parts dye composition / one part body fluid for 40 to 100 parts dye composition or, for example, one part 60 to 90 parts of the dye composition is mixed with a dye or dye / lysate composition in the ratio of one part body fluid to 5 to 200 parts of the dye composition, depending on the type of body fluid to be analyzed.
- a part body fluid for 10 to 150 parts dye composition, or one part body fluid for 20 to 130 parts dye composition / one part body fluid for 40 to 100 parts dye composition or, for example, one part 60 to 90 parts of the dye composition is optionallys a part body fluid for 10 to 150 parts dye composition, or one part body fluid for 20 to 130 parts dye composition / one part body fluid for 40 to 100 parts dye composition or, for example, one part 60
- the present invention relates to a cell counting method comprising the steps of mixing a cell sample with a dye or dye / lysant composition, placing the cell sample mixture with the dye or dye / lysant composition on a equipment for cell counting and, finally, to count the total value of cell units, identifying determination of the proportions of each cell subtype in the body fluid sample in a single step.
- the cell counting method of the present invention is characteri ed in that it comprises ⁇ i) obtaining images of cells suspended in the coloring composition; (ir) the global cell type count and cell count, differential subtypes in one step,
- the counting of the cell counting method of the present invention is done by image processing, or by a microscope-aided individual, so such method may further comprise the step of recording images of stained and lysed cells and archiving such. images for future sample evaluations.
- cell samples used in any of the methods of the invention consist of or are obtained from animal or human body fluids and may comprise erythrocytes, leukocytes and trorobocytes, wherein there are selected cellular subtypes of neutrophils, basophils, eosinophils. , lymphocytes (Type B e) and monocytes, as well as the immature forms of these cells.
- the present invention relates to the use of a composition as defined above for the preparation of a cell sample for cell analysis.
- the sample subjected to the analysis methods may be a sample obtained from any body fluid, secretions, blood and other fabrics.
- Cells contained in amestra- / turn may be cells of any animal.
- compositions obtained by different processes have been developed resulting in satisfactory methods of sample preparation and cell counting.
- Some examples of compositions and methods that fall within the scope of the present invention are provided in the present specification / without, however, have the intention of limiting the scope of protection- / since the present invention and its concepts allow the compositions design comprising different combinations of dyes and cell lysants.
- Figure 1 Agglutination of particles of blood stained by dyes prepared in alcoholic medium, indicating (1) the lumps of blood stained in alcoholic solution and (2) whole blood with EDTA in Guiemsa standard solution.
- Figure 2 formation of lumps of diluted and Leishman-stained blood in an alcoholic medium, visualized in a Neubauer chamber.
- Figure 3 Absorbance spectra of the individual components of the proposed dye solution and the final solution.
- Figure 4 Absorbance spectra of the final VMPF (H) solution read on 9/19/2104, six months after and fourteen months after which is when pyronein degradation begins.
- Figure 5 Eosinophils in VMPF-stained blood distension. (a) with an increase of 400 thirteen; (b) with 1000x magnification.
- Figure 6 Basophil in VMPF-stained blood distension at 1000-fold magnification.
- Figure 7 Samples of liquid-stained neutrophils, stained in liquid medium with VMPF (H) and VMPF, obtained from whole blood and synovial fluids. Images taken at 1000 times magnification.
- Figure 8 Lymphocyte samples stained in. liquid medium with VHPF (H) and VMPF obtained from whole blood and synovial fluids. Images taken at 1000 times magnification.
- Figure 9 Samples of monocytes, stained in liquid, stained in liquid with VMPF ⁇ (H) and VMPF, obtained from whole blood and synovial fluids. Images taken at 1000 times magnification.
- FIG 10 Samples of eosinophils, stained in liquid, stained in liquid with VMPF (H) and VMPF, obtained from whole blood and synovial fluids. Images taken at 1000 times magnification.
- FIG 11 Basophil sample, stained in liquid medium with VMPF (H), obtained from whole blood. Image taken at 1000 times magnification.
- Figure 14 Bland-Alt an analysis comparing Neutrophil differential counts performed by automatic LAC and manual VMPF (H) equipment.
- Figure 15 Bland-Altman analysis comparing Neutrophil differential counts in liquid medium stained with. VMPF (H) and in Leisiirtian stained blood
- Figure 16 BIand-Altman analysis comparing lymphocyte differential counts performed by automatic LAC and manual VMPF (H) equipment.
- Figure 17 Bland-Altman analysis comparing differential lymphocyte counts in VMPF (H) -stained liquid medium and Leishman-stained blood distention.
- Figure 18 Bland-Altman analysis comparing Monocyte differential counts performed by automatic LAC and manual equipment with VMPF (H).
- Figure 19 Bland-Altman analysis comparing Monocyte differential counts in VMPF (H) stained liquid medium and Leishman stained blood distention.
- Figure 20 Bland-Altman analysis comparing Eosinophil differential counts performed by automatic LAC and manual VMPF (H) equipment.
- Figure 21 Bland-Altman analysis comparing Eosinophil differential counts in VMPF (H) stained liquid medium: and in Leishman stained blood distention.
- Figure 22 - RG3 histograms obtained from neutrophil images.
- (A) are the R channel histograms;
- (B) are the histograms of channel (G);
- (C) are the histograms of channel B.
- Figure 23 RGB histograms obtained from lymphocyte images.
- A are the R channel histograms;
- B are the histograms of channel (G);
- C are the B channel histograms.
- Figure 24 RGB histograms obtained from monocyte images. Where (A) are the R channel histograms; (B) are the histograms of channel (G); (C) are the histograms of channel B. Figure 25 - RGB histograms obtained from eosinophil images. Where (A) are the R channel histograms; (B) are the channel histograms (G) (C) are the channel B histograms.
- Figure 26 RGB histogram obtained from basophil image.
- (A) are the R channel histograms;
- (B) are the histograms of channel (G);
- (C) are the B channel histograms.
- stepwise preparation of the preparation is to achieve a balanced staining solution that meets three requirements for obtaining acceptable differential staining for diagnosis; 1) not produce coagulation or agglutination of particles; 2) maintain leukocyte integrity long enough for characterization and counting; 3) that in the case of. staining of leukocytes from whole blood, allows hemolysis without the presence of important cellular debris that may disrupt leukocyte recognition and counting.
- differential liquid staining protocol is the differential liquid staining protocol:
- Step 1 VM Preparation and Purification:
- a partially isotonic buffer solution is prepared so that it has buffering power in the pH range of not less than 3.5 and not more than 5.5 to obtain dye performance methyl green.
- the buffer solution should be adjusted for each body fluid type so that staining and counting requirements are met.
- methyl green is prepared in the partially isotonic aqueous buffer solution at a concentration of not less than 0.3 M and not more than 8.0 mM, depending on the type of stain desired and the type of body fluid in which it will be applied.
- methyl green is prepared in the partially isotonic aqueous buffer solution at a concentration of not less than 0.3 M and not more than 8.0 mM, depending on the type of stain desired and the type of body fluid in which it will be applied.
- methyl green is necessary to purify it.
- purification protocol may vary according to. the product quality that is provided by each manufacturer.
- Step 2 Adding Pyronine
- the Pyronin dye is added to the already purified VK solution at a concentration of not less than 0.3mM and not greater than 7.0mM. After homogenization, the solution should be filtered to obtain a solution (VMP) with pK not less than 3,8 and not more than 5,5.
- the preparation of Fluorescein is made in a hot alcoholic medium, in a concentration no lower than C ) , 1mM and not greater than 3,. After filtration a solution with a pH of not less than 9 and not more than 10 is obtained.
- the VMPF solution is obtained by mixing the VMP solution with. the fluorescein solution in proportion not less than a part of VMP stops. two parts of fluorescein and not more than two and a half parts of VMP for one part of fluorescein, thus obtaining the VMPF solution with a pH of not less than 4.8 and not more than 6.4.
- the mixture of the VMP solution and the fluorescein solution should be equilibrated to safely prevent coagulation or particle agglutination in the medium.
- the balance of the solution should be optimized for the body fluid in which it will be used. This solution is used for staining cells in blood distention or in liquid medium, but without. he heyse.
- the dye solution is already viable, as it is possible to stain the leukocytes present in most body fluids that do not have too many red blood cells, such as urine, cerebrospinal fluid (CSF), cavitation fluids such as ascites., the pleural, the peritoneal and the bell ial; food fluids such as milk.
- red blood cells such as urine, cerebrospinal fluid (CSF), cavitation fluids such as ascites., the pleural, the peritoneal and the bell ial; food fluids such as milk.
- Step 5 Preparation of VMPF (H) Solution with Addition of Lysing Agent
- the addition of the lysing agent is an alternative to. leukocyte staining in blood, whole.
- the lysing agent may be quaternary ammonium compound in a ratio of 5 g / L to 80 g / L associated with a buffer solution and diluted in aqueous medium. This solution is incorporated into the VMPF solution, producing the VMFF (H) solution.
- the ratio of VMPF to lysing agent may be carried out in the range of one part lysing agent to two parts dye solution to the ratio two parts lysing agent to one part dye solution. The ratio should ensure heolysis without damage to the leukocytes and: allowing their staining.
- the final VMPF (H) dye solution should remain at a pH no lower than 5, 5 and no higher than 6,6.
- Example 2 Method of preparing & reading cells.
- a whole blood sample is diluted in the dye solution in the proportion of one part blood to 20 parts of the dye solution. Placing this solution in a Neubauer chamber counts the differently stained cells. Thus, the count occurs for both the overall value (total leukocytes per cubic millimeter of blood) and the proportions of each cell type.
- the complete protocol can be performed in less than two minutes, collecting an accurate volume of blood by digital puncture and presenting the same results as the counts performed on. automated counter and manual methodology, which takes up to 60 minutes to obtain the result of each exam.
- the same dye solution without the addition of the lysing agent, (VMPF;) also stains the blood distention in one step, but with action time occurs in the interval of 15 to 30 minutes.
- the time of action depends on the melting conditions, temperature and fixation technique used.
- Example 3 Characterization and stability evaluation of coloring solutions
- the dye solutions that gave rise to the invention which is the final solution called VMPF (H)
- VMPF final solution
- the first evaluation took place the day after its initial preparation on 19 / 09/2014.
- the same solution was evaluated again / under the same conditions, six months, nine months, twelve months and fifteen months after its preparation.
- the chemical stability of the solutions, as well as their action on cell staining, remained unchanged until the twelfth month.
- Verification of the chemical stability of the dye solutions was carried out by two-step absorption spectrophotometry. At. First, all individual solutions of each dye were read in the spectrophotometer to assess the individual composition of each component. The absorbances of the intermediate dye solutions and the final solution in two versions VMPF (without lysing agent) and VMPF (H) (with lysing agent) were also read. noirTo know if the individual solutions react with each other and form other substances. In the second stage the temporal chemical stability was evaluated, ie, if there is degradation of the final dye solution after long periods of time. The graph of figure 3 shows relative values of the absorption of each solution and its combinations as a function of wavelength (between 350nm: and 750nm).
- the relative values of the absorptions vary, depending on the dilutions, that were necessary for the spectrophotometer to operate within its working range. Therefore, the absorptions presented are in relative values. Analyzing the absorption curves presented in figure 3, we have that the VM in. Acetate buffer solution has an absorption band between 590rim and 680 nm, peaking at 64 °. Pyronine in solution buffer absorbs in the 80nm to 570nm band, peaking at 505n. Fluorescein was an alcoholic medium absorbing in the range of 440nm to 520nm, with a peak of 490nm. However, it is noteworthy that to obtain this spectrum both VM and Pyronine were diluted in.
- VMPF (H) line is the overlap of 25x diluted VM lines with 25.x diluted Pironine (P).
- F Fluorescein
- the absorption spectra of the first stage were obtained on 1.9 / 09/2014.
- the same spectrophotometer reading was performed under the same temperature and relative humidity conditions on 03/06/2015 of the final solution.
- the spectra comparing the final solution read 19/03/2014 with the same solution read 06/03/2015 are shown in figure 4.
- Example 5 Evaluation of cells and celilar structures in images obtained in VMPF-stained blood strains
- Sosinophils were stained with VMPF dye solution, as observed in figures 5 (a) and 5 (b).
- the slides containing the blood strains were previously fixed in methanol, which facilitates the entry of Fluorescein dye. in cells and the coloring of the characteristic granules of this cell type.
- peripheral blood leukocytes were identified, classified and counted into five types.
- morphological neutrophils, lymphocytes, monocytes, eosinophils and basophils.
- leukocytes present in the other body fluids urine, iiquor (cerebrospinal fluid), cavitaria-Sy liquids as aseitic, pleural, peritoneal and s-inovial were also characterized and categorized by of your images.
- iiquor cerebrospinal fluid
- cavitaria-Sy liquids as aseitic, pleural, peritoneal and s-inovial were also characterized and categorized by of your images.
- classification and counting the images of the cells contained in these liquids have the same characteristics and information as the images of the cells contained in the blood.
- the cells were visualized in. its characteristic size and spherical shape, showing no rupture of cell membranes.
- the images shown in figures 7 to 11 had their relative sizes preserved, ie, considering that all images had the same magnification (1000X), the cropped areas around the cell was always the same (500x500 pixels). Therefore, the cell area is proportional to the size of the section window.
- the cytoplasm was clear, clear and without granulation.
- the nucleus appeared in its form segmentation feature. Both rods and polymorphonuel cells were classified under the name of neutrophils. Nuclei with 2 to 4 segmentations could be observed, as can be seen in the examples shown in Figure 7. Due to the affinity of the VM component with DNA, the nuclear segments stained in bluish-green.
- nucleoli presented a reddish color that detached from the nucleus, which can be related to the affinity of pyronein for the RNA present in such a structure.
- Cytoplasmic strips stained with different intensities of pink (p ⁇ nk) confirmed the affinity of the Pironin component of the VMPF (H) or VMPF solution for RNA, characterizing differences in cellular activity, and no cytoplasmic granulations were observed.
- Example 7 Evaluation of the dye solution by comparing cell counts with established protocols and the new dye.
- VMPF (H) dye solution As a leukocyte differential disclosure instrument, with a view to counting the blood count, it is necessary to determine if the counts performed are compatible with the counts performed by the established methodologies. Then, the global count and the differential count were evaluated by ANOV analysis of variance and Blend-Altman agreement analysis.
- leukocyte differential counts were performed by three techniques, one automated by the LAC, the second done by the inventors manually using VMPF (H) and the third by the inventors manually, in Leishman-stained blood distention ..
- the ANQVA test results were: p - 0.51, with critical F greater than 2.6, and the obtained F was 0.73.
- the p value much greater than 0.05 and F less than the critical F indicate that the counts do not present statistical differences, ie they can be considered equivalent.
- Table 4 ⁇ Result of differential monocyte count by three techniques: Automatic, performed by an independent Clinical Analysis Laboratory. Blood distention stained with Leishman. Manual stained with VMPF (H). Amounts expressed in quantity: total cells / mean 3 .
- monocyte count shows a high variability, largely due to an expected variation, but mainly due to differences between counting techniques, as normal or reactive lymphocytes can be misidentified. as monocytes, making monocyte counts sometimes an unsatisfactory measurement.
- Another source of interference in the statistical analysis is the results presented by LAC, which occur in multiples of 100. Assuming that a reading error of about 50 cells may occur and since this error represents a Significant percentage of total cells statistical results tend to differ from expected values.
- FIGS. 20 and 21 show Bland-Altman analyzes for LAC x VMPF (H) and Leishman x VMPF (H). However, due to the low number of cells available per patient and the fact that the LAC maintains the count in multiples of 100 it is not possible to perform an appropriate statistical analysis. Then, the counts data are presented based on the characterization and visual recognition of cells by experienced hematologists.
- Basophils are rare cells, and their count is common, resulting in null values in most blood counts performed.
- the IAC maintains the count in multiples of 100, the result for most patients was null for absolute values, and non-null values were presented in the relative results (as a percentage of total leukocytes), as shown in table 6.
- the Statistical analysis or 31and-Altman agreement analysis are meaningless. Then, the counts data are presented based on the characterization and visual recognition of the cells by experienced hematologists.
- Example 8 Evaluation of the effectiveness of the method of classifying and counting cells by image processing.
- Figure 20 depicts the uniformity of behavior of neutrophil histograms, which is different from the uniformity of histograms obtained from lymphocyte images (figure 21) from monocyte images (figure 22) from eosinophil images ( figure 23) & basophil images (figure 24).
- CARRERAS Maria Cecilia et al. Kinetics of nitric oxide and hydrogen peroxide production and formation. of peroxynitrite during the respiratory burst of human neutrophils.
- DUCREST S. et al. Flowcytometry analysis of basophil counts in human biood and inaccuracy of hematology analyzers. Allergy, v. 60, no. 11, p. 1445-1450, 2005.
- MAHAJAN S. GQL ⁇ TI, S. S .; Meshes, A .; JICHLKAN, N. Revie: Detection of Types of Acut.e Leukeraia. International Journal of Computer Science and Mobile Computing, v.3, n. 3, p. 104-111, March 2014.
- PATERNITI Irene et al. PDE 7 inhibitors; new potential drugs for spinal cord therapy in ury. PLoS One, v. 6, n, 1, p. El5937, 2011.
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EP16874165.0A EP3392343A4 (en) | 2015-12-17 | 2016-12-19 | COLORING COMPOSITION OF AQUEOUS-BASED CELLS, CORRESPONDING PROCESS FOR PREPARING AND USING THE SAME, METHODS FOR SAMPLE PREPARATION AND CELL COUNTING |
BR112018011173A BR112018011173A2 (pt) | 2015-12-17 | 2016-12-19 | composição corante de células de base aquosa, respectivo processo de preparação e uso da mesma, métodos de preparação de amostra e contagem de células |
US16/063,276 US20180370929A1 (en) | 2015-12-17 | 2016-12-19 | Water-based cell staining composition, method for preparing same and use thereof, cell sample preparation and cell counting methods |
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PCT/BR2016/000163 WO2017100882A1 (pt) | 2015-12-17 | 2016-12-19 | Composição corante de células de base aquosa, respectivo processo de preparação e uso da mesma, métodos de preparação de amostra e contagem de células |
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Non-Patent Citations (6)
Title |
---|
KONDO, T.: "Mechanisms of hemolysis by surface active agents", ADVANCES IN COLLOID AND INTERFACE SCIENCE, vol. 6, no. 2, 1976, pages 139 - 172, XP 009002619, DOI: doi:10.1016/0001-8686(76)85002-6 * |
NASCIMENTO, V. A. O. ET AL.: "Colorado de leucocitos em meio liquido para aquisição e diferenciação por processamento de imagens. In: XXIV Congresso Brasileiro de Engenharia Biomedica", XXIV CONGRESSO BRASILEIRO DE ENGENHARIA BIOMEDICA, vol. 1, October 2014 (2014-10-01), pages 1816 - 1819, XP 055391408 * |
ORNSTEIN, L. ET AL.: "Spectral matching of classical cytochemistry to automated cytology", J HISTOCHEM CYTOCHEM, vol. 22, no. 7, 1974, pages 453 - 469, XP 055391415 * |
POLLACK, A ET AL.: "Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green", CYTOMETRY, vol. 3, no. 1, 1982, pages 28 - 35, XP 055391417, DOI: doi:10.1002/cyto.990030108 * |
See also references of EP3392343A4 * |
TYCKO, D. H. ET AL.: "Automatic leukocyte classification using cytochemically stained smears", J HISTOCHEM CYTOCHEM, vol. 24, no. 1, 1976, pages 178 - 194, XP 055391412, DOI: doi:10.1177/24.1.56388 * |
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WO2017100869A1 (pt) | 2017-06-22 |
US20180370929A1 (en) | 2018-12-27 |
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