FR2840992A1 - Counting leucocytes, useful e.g. for quality control of plasma products, by permeabilization, labeling, retention on filter and determining number of retained cells - Google Patents
Counting leucocytes, useful e.g. for quality control of plasma products, by permeabilization, labeling, retention on filter and determining number of retained cells Download PDFInfo
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Abstract
Description
joint soude (1) a controler.seal (1) to be checked.
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PROCEDE DE DENOMBREMENT ET/OU D' IDENTIFICATION METHOD OF INVOLVING AND / OR IDENTIFYING
MORPHOLOGIQUE DE LEUCOCYTES EN PHASE SOLIDE. MORPHOLOGY OF SOLID PHASE LEUKOCYTES.
La presente invention se rapporte a un procede de detection et/ou de comptage et/ou d/identification The present invention relates to a method for detecting and / or counting and / or identifying
morphologique de leucocytes en phase solide. morphology of solid phase leukocytes.
La reduction des leucocytes residuels dans les produits sanguine, notamment dans des produits sanguine labiles (PSL) comprenant notamment, le plasma et les cellules sanguines et leurs derives, ci-apres designee leucoreduction, est une des priorites dans le monde de la transfusion sanguine et elle est devenue une technologie bien etablie, pratiquee par les centres de transfusion The reduction of residual leukocytes in blood products, in particular in labile blood products (PSL) including in particular plasma and blood cells and their derivatives, hereinafter referred leucoreduction, is one of the priorities in the world of blood transfusion and it has become a well-established technology, practiced by transfusion centers
sanguine du monde entier.worldwide.
On entend par produit sanguin, toute preparation issue du fractionnement du sang, comprenant ou non des composants cellulaires, a titre d'exemple, on entend par produit sanguin les concentres des globules rouges ou de plaquettes, mais aussi les preparations The term "blood product" means any preparation resulting from the fractionation of the blood, whether or not comprising cellular components. By way of example, the term "blood product" means the concentrates of the red blood cells or platelets, but also the preparations.
plasmatiques ou seriques.plasma or seric.
Dans le domaine de la transfusion sanguine, il a ete montre que l'utilisation de derives sanguine ayant un taux de leucocytes reduit permet de diminuer la frequence de complications severes liees a la transfusion sanguine, incluant l 'apparition de reactions febriles, la formation d'alloanticorps, la transmission de virus ou encore la In the field of blood transfusion, it has been shown that the use of blood derivatives with a reduced leukocyte content makes it possible to reduce the frequency of severe complications associated with blood transfusion, including the appearance of febrile reactions, the formation of alloantibody, the transmission of viruses or the
maladie de Creutzfeldt-Jacob.Creutzfeldt-Jacob disease.
On constate dans les modeles experimentaux d'encephalite spongiforme transmissible que l'infectiosite sanguine est essentiellement associee (90% des cas) aux leucocytes. (Rapport de Fevrier 2002 de l'Agence Francaise Experimental models of transmissible spongiform encephalitis indicate that blood infectivity is mainly associated (90% of cases) with leukocytes. (February 2002 report of Agence Francaise
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De Securite Sanitaire Des Produits De Sante (AFSSAPS): << Analyse du risque de transmission de la variante de la maladie de Creutzfeldt-Jakob par les medicaments d'origine humaine et les produits sanguine labiles >>) et que de ce fait la leucoreduction contribue a diminuer les risques Health Products Safety (AFSSAPS): << Analysis of the risk of transmission of variant Creutzfeldt-Jakob disease by drugs of human origin and labile blood products >>) and that therefore leukoreduction helps reduce risks
potentials lies a l' agent causal de la BSE. potentials related to the causal agent of BSE.
Ainsi, dans le but de securiser le sang les organismes charges de la securite des produits sanguine recommandent une reduction significative des concentrations de ces cellules dans les plasmas, en utilisant, par Thus, for the purpose of securing the blood the bodies responsible for the safety of the blood products recommend a significant reduction of the concentrations of these cells in the plasma, using, for example,
exemple, des filtres ecran.for example, screen filters.
Les concentrations acceptables de leucocytes dans le plasma vent d'environ 106 cellules par litre, ce seuil sera certainement abaisse a environ 104 cellules par The acceptable concentrations of leukocytes in the plasma wind of about 106 cells per liter, this threshold will certainly be lowered to about 104 cells per
litre, soit environ 10 cellules/ml.liter, about 10 cells / ml.
Les normes europeennes recommandent un controle de qualite sur 1% des produits sanguine avec un minimum de controles/mois/produit sanguin. Ces normes vent satisfaites si 90% des unites d'echantillonnage se trouvent dans les fourchettes de valeurs indiquees. (Guide pour la preparation, l'utilisation et l' assurance qualite des composants sanguine -7eme edition-2001-Editions du Conseil European standards recommend quality control on 1% of blood products with a minimum of controls / month / blood product. These standards are satisfied if 90% of the sample units are within the indicated ranges of values. (Guide to the Preparation, Use and Quality Assurance of Blood Components-7th Edition-2001-Council Editions
de ['Europe).from Europe.
I1 apparalt que la mise a disposition des etablissements charges de controler la securite des produits sanguine, de procedes gables et sensibles de detection et comptage de leucocytes residuels dans lesdits It appears that the provision of facilities to control the safety of blood products, gable and sensitive processes of detection and counting of residual leucocytes in said
produits sanguine, presente un interet certain. blood products, has a certain interest.
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A ce jour, deux techniques vent utilisees couramment pour le denombrement de leucocytes residuals, toutes les deux vent des techniques en phase liquide, il s'agit du comptage par cytometric de flux (CF) et du comptage avec un hematocytometre comme par exemple la To date, two commonly used techniques for the enumeration of leucocyte residuals, both of which are liquid phase techniques, are flow cytometric (CF) counting and hematocytometer counting such as
Chambre de Nageotte.House of Nageotte.
Des etudes visant a comparer ces deux techniques, et a mettre en lumiere leurs avantages et inconvenients font etat d'une meilleure fiabilite pour les procedes de denombrement mettant en uvre la CF, car elle Studies to compare these two techniques, and to highlight their advantages and disadvantages, indicate a better reliability for the enumeration procedures implementing FC, as it
presente une variabilite moindre.has less variability.
Parmi les publications qui mentionnent l'interet de la leucoreduction, on peut citer l' article du Pr. Maurice Masse (Transfus Clin Biol 2001 Jun; (3):297-302 << Universal leuLoreduction of cellular and plasma components: process control and performance of the leukoreduction process. >>) Les auteurs font reference au besoin d'optimisation des methodes de reduction des concentrations leucocytaires, non senlement dans les globules rouges, ou les plaquettes, mais aussi dans les plasmas pour Among the publications which mention the interest of leucoreduction, one can quote the article of Pr. Maurice Masse (Transfus Clin Biol 2001 Jun; (3): 297-302 << Universal leuLoreduction of cellular and plasma components: process control performance of the leukoreduction process. >>) The authors refer to the need for optimization of the methods of reducing leukocyte concentrations, not senescent in red blood cells, or platelets, but also in plasma for
['amelioration de la securite transfusionnelle. [Improvement of transfusion safety.
Aussi les auteurs font clairement allusion au besoin du developpement de techniques elaborees de detection et comptage des leucocytes residuals. Le but etant d'atteindre des sensibilites elevees, generalement The authors therefore clearly refer to the need for the development of elaborate techniques for detecting and counting leucocyte residues. The goal is to achieve high sensitivities, usually
par concentration prealable de ltechantillon. by pre-concentration of the sample.
Cette concentration prealable permet d'atteindre des sensibilites elevees avec une limite de detection de l'ordre de 10 cellules leucocytes/ml pour les globules rouges (GR) ou les plaquettes et de 0,5 cellules This preliminary concentration makes it possible to reach high sensitivities with a detection limit of the order of 10 leukocyte cells / ml for red blood cells (RBCs) or platelets and 0.5 cells.
leucocytes /ml pour le plasma.leukocytes / ml for plasma.
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L' article de Rebulla P. et al. (Transfusion 1993 Feb;33(2):128-33 << White cell-reduced red cells prepared by filtration: a critical evaluation of current filters and methods for counting residual white cells >>) evalue la reduction de globules blancs a partir de preparations de leucocytes, par filtration sur differents The article by Rebulla P. et al. (Transfusion 1993 Feb; 33 (2): 128-33 "White cell-reduced red cells prepared by filtration: a critical evaluation of current filters and methods for counting residual white cells") assesses the reduction of white blood cells from leukocyte preparations, by filtration on different
filtres commerciaux.commercial filters.
En meme temps, les auteurs de cette publication comparent trots procedes de denombrement de leucocytes residuels: soit dans une Chambre de Burke (CB), soit dans une chambre de Nageotte (CN), soit par cytometric de flux (CF). Alors qu'aucun leucocyte n'est detecte avec la CB, les courbes d'etalonnage obtenues avec la CN et par CF avec des echantillons contenant des concentrations connues en globules blancs, allant de 1 a 1000 cellules par microlitre, montrent que les deux derniers procedes detectent en moyenne 67% des globules blancs presents dans les echantillons Le procede utilisant la CF montre une faible variabilite, (rendement de 61-70%) pour les differents niveaux de globules blancs alors que la variabilite avec le procede utilisant la CN est plus importante (rendement de 39-91%). Cette variabilite plus importante empeche la correction des denombrements en CN par un facteur simple et illustre les difficultes d'une standardisation de la technique. Il apparalt cependant que les besoins en securite dans ce domaine necessitent des procedes de denombrement plus gables, presentent moins de variabilite, At the same time, the authors of this publication compare three residual leucocyte enumeration methods: either in a Burke chamber (CB), or in a Nageotte chamber (CN), or by flow cytometry (CF). While no leucocyte is detected with CB, the calibration curves obtained with CN and CF with samples containing known white blood cell concentrations, ranging from 1 to 1000 cells per microliter, show that the last two On average, 67% of the white blood cells in the samples are detected in the samples. The method using CF shows a low variability, (61-70% yield) for the different levels of white blood cells, while the variability with the process using the CN is higher. significant (yield of 39-91%). This greater variability prevents the correction of CN counts by a single factor and illustrates the difficulties of standardizing the technique. It appears, however, that the security needs in this area require more manageable enumeration methods, have less variability,
et plus sensibles.and more sensitive.
Afin de resoudre ce probleme de manque de sensibilite des procedes de denombrement de leucocytes In order to solve this problem of lack of sensitivity of leukocyte enumeration methods
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precedemment cites, la demanderesse a mis en muvre un procede de denombrement et/ou d' identification de leucocytes residuels dans les fluides biologiques presentant une sensibilite accrue et realisable dans des delais plus courts. Dans le cadre de la presente invention, l entend par fluide biologique tout fluide issu d'un organisme vivant, comme par exemple: le plasma, le fait, la salive, les larmes, la sueur, l' urine ou le liquide cephalorachidien, mais aussi les produits issus du fractionnement du sang, comprenant ou non des composants cellulaires, ainsi que les preparations plasmatiques ou . previously cited, the Applicant has implemented a method of enumeration and / or identification of residual leukocytes in biological fluids having increased sensitivity and achievable within shorter time. In the context of the present invention, the term biological fluid means any fluid from a living organism, such as for example: plasma, the fact, saliva, tears, sweat, urine or cephalorachidian fluid, but also the products of the fractionation of the blood, including or not cellular components, as well as the plasma preparations or.
s erlques.s erlques.
Ce procede est particulierement bien adapte au This process is particularly well adapted to
denombrement des leucocytes dans le plasma. enumeration of leukocytes in the plasma.
Le procede de denombrement de leucocytes de l 'invention comprend une detection en phase solide des leucocytes residuels sur une membrane de filtration et ne necessite pas d'etape de concentration prealable des cellules. De plus, l' identification secondaire des sous populations leucocyLaires (polynucleaires, lymphocytes, monocytes) sur une base morphologique est realisable par observation au microscope a epifluorescence de la membrane The method for counting leukocytes of the invention comprises solid phase detection of residual leukocytes on a filtration membrane and does not require a prior cell concentration step. In addition, the secondary identification of leukocyte subpopulations (polynuclear, lymphocytes, monocytes) on a morphological basis is feasible by observation with epifluorescence microscopy of the membrane.
de filtration.filtration.
Plus precisement, l' invention a pour objet un procede de denombrement de leucocytes eventuellement presents dans un fluide biologique caracterise en ce qu'il comprend les etapes suivantes: More precisely, the subject of the invention is a process for the enumeration of leukocytes possibly present in a biological fluid characterized in that it comprises the following stages:
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a) l' incubation d'un echantillon audit fluide biologique avec un agent permeabilisant des membranes cellulaires, b) l' incubation de l'echantillon obtenu a l'etape (a) avec un marqueur des leucocytes, c) la filtration de l'echantillon obtenu a l'etape (b) sur une membrane non autofluorescente, susceptible de retenir les leucocytes, pour obtenir une preparation en phase solide des leucocytes marques, eventuellement presents dans ledit echantillon, d) l'analyse de la preparation en phase solide de ladite membrane non autofluorescente de ltetape (c) pour denombrer les leucocytes marques eventuellement presents (a) incubating a sample with said biological fluid with a cell membrane permeabilizer, (b) incubating the sample obtained in step (a) with a leukocyte marker, (c) filtering the sample obtained in step (b) on a non-autofluorescent membrane, capable of retaining leukocytes, to obtain a solid phase preparation of the marked leukocytes, possibly present in said sample, d) the analysis of the solid phase preparation of said non-autofluorescent membrane of step (c) for counting leukocytes possibly present marks
sur celle-ci.on this one.
L' invention a egalement pour objet un procede d'identification morphologique des leucocytes eventuellement presents dans un produit sanguin caracterise en ce qu'il comprend les etapes (a) a (c) du procede precedemment decrit suivies d'une etape (e) d'observation visuelle au microscope des preparations obtenues a l'etape ( c) Cette observation visuelle de la phase solide permet l' identification morphologique de polynucleaires, lymphocytes et/ou monocytes eventuellement presents dans le The invention also relates to a process for the morphological identification of leukocytes possibly present in a blood product characterized in that it comprises the steps (a) to (c) of the process previously described followed by a step (e) of visual observation under the microscope of the preparations obtained in step (c) This visual observation of the solid phase allows the morphological identification of polynuclear, lymphocytes and / or monocytes possibly present in the
fluide biologique.biological fluid.
Par agent permeabilisant des membranes, on entend tout compose susceptible de modifier la structure des membranes cellulaires et de laisser ainsi passer vers l'interieur de la cellule des composes tels que des marqueurs, comme les marqueurs fluorescents ou des molecules couplees a un fluorochrome, de maniere a ce que Membrane permeabilizer is understood to mean any compound capable of modifying the structure of cell membranes and thus allowing compounds such as markers, such as fluorescent markers or fluorochrome-coupled molecules, to pass into the interior of the cell. way that
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ceux-ci puissent atteindre le cytoplasme et/ou le noyau these can reach the cytoplasm and / or the nucleus
cellulaire et se fixer a des elements intracellulaires. cell and attach to intracellular elements.
Parmi les agents permeabilisants des membranes cellulaires, on peut citer des produits detergents et des fixateurs. Par membrane nonautofluorescente on entend toute membrane qui ntemet pas de la fluorescence, ni par elle-meme, ni en presence de fluorochromes, ni sous lteffet des excitations lumineuses de l'analyseur, de telle maniere qu'elle n' interfere pas avec les mesures de fluorescence Among the permeabilizing agents of cell membranes, mention may be made of detergent products and fixatives. By non-autofluorescent membrane is meant any membrane which does not fluoresce, either by itself, or in the presence of fluorochromes, or under the effect of the luminous excitations of the analyzer, in such a way that it does not interfere with the measurements. fluorescence
effectuees par l'analyseur.performed by the analyzer.
Lors de differents travaux menes par la demanderesse pour la mise en muvre de ['invention, il a ete constate qu'une detection optimale est obtenue avec des concentrations allant de 1 a 50000 leucocytes par In various works conducted by the applicant for the implementation of the invention, it has been found that optimal detection is obtained with concentrations ranging from 1 to 50,000 leucocytes per
preparation en phase solide.solid phase preparation.
Ainsi, lorsque la concentration en leucocytes est trop elevee, l'echantillon de ltetape (a) est prealablement dilue avec un tampon osmotiquement compatible avec les leucocytes, avant son incubation avec le Thus, when the leucocyte concentration is too high, the sample of step (a) is previously diluted with an osmotically compatible buffer with leukocytes, prior to incubation with the leukocyte.
detergent.detergent.
Ce tampon de dilution osmotiquement compatible avec les leucocytes est choisi parmi le groupe comprenant: du PBS a pH 7,4, du TRIS a pH 7,4, du tampon Tyrode a pH This osmotically compatible dilution buffer with leukocytes is chosen from the group comprising: PBS at pH 7.4, TRIS at pH 7.4, Tyrode buffer at pH
7,4 ou du serum physiologique (NaCl a 0,9%). 7.4 or saline (0.9% NaCl).
Parmi les detergents que lton peut utiliser, seuls ou en melange, lors de ltetape (a) on peut mentionner ceux du groupe comprenant: Triton X-100, saponine, n Among the detergents that can be used alone or in a mixture, in step (a), mention may be made of the group comprising: Triton X-100, saponin, n
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ocLyl-glucopyranoside, Tween 20, Tween 80, sodium dodecyl sulfate. Le detergent, ou le melange de detergents, incube avec l'echantillon a l'etape (a) vent utilises a une concentration finale comprise entre 0,05% et 5%, de preference entre 0,01% et 2% et tout preferentiellement a une concentration de 0,1% pour le Triton X-100, de 1% pour le n-ocLyl glucopyranoside, le Tween 20, le Tween 80 et le α-L-glucopyranoside, Tween 20, Tween 80, sodium dodecyl sulfate. The detergent, or mixture of detergents, is incubated with the sample in step (a) vent used at a final concentration of between 0.05% and 5%, preferably between 0.01% and 2% and most preferably at a concentration of 0.1% for Triton X-100, 1% for n-octyl glucopyranoside, Tween 20, Tween 80 and
sodium dodecyl sulfate, de 0,2% pour la saponine. sodium dodecyl sulfate, 0.2% for saponin.
La duree dtincubation de l'echantillon avec le detergent a ltetape (a) est comprise entre 5 minutes et 45 minutes, de preference entre 10 et 20 minutes et tout The duration of incubation of the sample with the detergent at step (a) is between 5 minutes and 45 minutes, preferably between 10 and 20 minutes and all
preferentiellement elle est de 15 minutes. preferentially it is 15 minutes.
Selon de modes preferes de mise en xuvre du procede de ['invention, le marqueur des leucocytes utilise a l'etape (b) est choisi parmi le groupe comprenant: un According to preferred embodiments of the method of the invention, the leukocyte marker used in step (b) is selected from the group consisting of:
colorant, un fluorochrome, un marqueur d'acides nucleiques. dye, a fluorochrome, a nucleic acid marker.
De preference, le marqueur de leucocytes utilise a l'etape (b) est un marqueur d'acides nucleiques choisi parmi le groupe comprenant: orange d'acridine, Picogreen@, cyanine asymetrique (SYTO), cyanine monomere Preferably, the leukocyte marker used in step (b) is a nucleic acid marker selected from the group consisting of: acridine orange, Picogreen®, asymmetric cyanine (SYTO), cyanine monomer
(YOPRO), cyanine dimere, SYBRO et Iodure depropidium. (YOPRO), dimeric cyanine, SYBRO and propidium iodide.
La duree dtincubation de l'echantillon avec le marqueur a l'etape (b) peut etre comprise entre 5 et 45 minutes, de preference entre 10 et 20 minutes et tout The duration of incubation of the sample with the marker in step (b) can be between 5 and 45 minutes, preferably between 10 and 20 minutes and all
preferentiellement elle est de 15 minutes. preferentially it is 15 minutes.
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Selon un mode particulier de mise en uvre du procede de l' invention, la membrane non autofluorescente mise en uvre According to a particular mode of implementation of the method of the invention, the non-autofluorescent membrane implemented
a l'etape (c) est une membrane noire. in step (c) is a black membrane.
De preference ladite membrane noire est une membrane en en polyester ou polycarbonates ou polyether sulfone de Preferably said black membrane is a membrane made of polyester or polycarbonates or polyether sulfone
diametre 25 mm.diameter 25 mm.
La taille des pores de la membrane mise en muvre a l'etape (c) peut-etre comprise entre 0,05,um et 2,um, de preference elle est comprise entre 0,1, um et 1,um The pore size of the membrane implemented in step (c) may be between 0.05 μm and 2 μm, preferably between 0.1 μm and 1 μm.
et preferentiellement elle est de 0,4 um. and preferentially it is 0.4 μm.
Eventuellement, le procede de l' invention peut comporter une etape supplementaire de ravage, apres la filtration de l'etape (c) par filtration d'un volume de tampon. Ce ravage permet d'eliminer un eventuel bruit de Optionally, the method of the invention may comprise an additional step of ravage, after the filtration of step (c) by filtration of a volume of buffer. This havoc eliminates a possible noise of
fond sur la membrane provoque par des traces du marqueur. background on the membrane causes traces of the marker.
L'analyse de la membrane a l'etape (d) du procede peut etre effectuee au moyen d'un analyseur capable de detecter des evenements marques et en particulier au moyen d'un analyseur laser a balayage et/ou d'un microscope The analysis of the membrane at step (d) of the process can be carried out by means of an analyzer capable of detecting marked events and in particular by means of a scanning laser scanner and / or a microscope
a epifluorescence.epifluorescence.
Le procede de l' invention est particulierement utile pour le controle de qualite des preparations de plasmas et derives. Il peut egalement etre utilise pour le controle de qualite des filtres de leucocytes et en controle qualite dans des barques de sang pour le controle The process of the invention is particularly useful for the quality control of plasma and derivative preparations. It can also be used for quality control of leukocyte filters and quality control in blood vessels for control
des procedes de leucoreduction.leucoreduction methods.
D'autres avantages et caracteristiques de l 'invention apparaltront a la lecture des exemples qui suivent et des figures en annexe dans lesquelles - La figure 1 est un schema illustrant les etapes pour la preparation des leucocytes a analyser, selon un modele experimental pour obtenir une gamme de concentrations de leucocytes dans du plasma et etablir la Other advantages and characteristics of the invention will become apparent on reading the following examples and the appended figures in which - Figure 1 is a diagram illustrating the steps for the preparation of the leucocytes to be analyzed, according to an experimental model to obtain a concentration range of leukocytes in plasma and establish the
limite de detection et la sensibilite du procede. detection limit and the sensitivity of the process.
- la figure 2 montre un schema du procede de denombrement de leucocytes de l' invention en cytometric en phase solide et de la validation des resultats obtenus par FIG. 2 shows a diagram of the leukocyte enumeration method of the invention in solid phase cytometry and the validation of the results obtained by FIG.
lecture au microscope des phases solides analysees. microscopic reading of the analyzed solid phases.
La membrane filtre non autofluorescente (noire) (1) introduite dans l'analyseur (2) est balayee ligne par ligne avec un laser Argon (3) et ['emission de fluorescence verse est traduite en un nombre d'evenements fluorescents dont la position sur le filtre est visualisee sur ltecran de l'ordinateur(4). L'analyseur a balayage laser est connecte avec un microscope a epifluorescence qui permet la confirmation des resultats obtenus et l' identification The non-autofluorescent filter membrane (black) (1) introduced into the analyzer (2) is scanned line by line with an Argon laser (3) and the fluorescence emission is translated into a number of fluorescent events whose position on the filter is displayed on the computer screen (4). The laser scanning analyzer is connected with an epifluorescence microscope which allows the confirmation of the obtained results and the identification
morphologique des soul-populations leucocyLaires. morphologic leucocytic leukocyte populations.
- la figure 3 illustre la linearite de la methode. Wile illustre le denombrement de leucocytes effectue avec le procede de l' invention pour des dilutions en serie de leucocytes dans du plasma filtre sur un filtre - Figure 3 illustrates the linearity of the method. Wile illustrates the enumeration of leukocytes performed with the process of the invention for serial dilutions of leukocytes in plasma filter on a filter
de porosite 0,22 um.porosity 0.22 μm.
- la figure 4 illustre le denombrement de leucocytes effectue avec un compteur de cellules pour des dilutions en serie de leucocytes dans du plasma filtre avec FIG. 4 illustrates the count of leukocytes carried out with a cell counter for serial dilutions of leucocytes in plasma with
un filtre de porosite 0,22 um.a 0.22 μm porosity filter.
- la figure 5 illustre l' etude comparative entre le denombrement de leucocytes obtenu avec le procede de l' invention et celui obtenu avec un compteur de cellules. - la figure 6 illustre le denombrement de leucocytes avec le procede de ['invention, en duplicate, sur deux echantillons differents A et B. avant leucoreduction du plasma frais congele a usage therapeutique. - la figure 7 illustre le denombrement de leucocytes avec le procede de ['invention, en duplicate, sur deux echantillons differents A et B. apres leucoreduction du plasma frais congele a usage - Figure 5 illustrates the comparative study between the count of leukocytes obtained with the process of the invention and that obtained with a cell counter. FIG. 6 illustrates the counting of leukocytes with the process of the invention, in duplicate, on two different samples A and B. before leucoreduction of fresh frozen plasma for therapeutic use. FIG. 7 illustrates the enumeration of leucocytes with the process of the invention, in duplicate, on two different samples A and B. after leucoreduction of the fresh frozen plasma for use
therapeutique.therapeutic.
PARTIE EXPERIMENTALE- EXEMPLESEXPERIMENTAL PART - EXAMPLES
I - DETERMINATION DU SEUIL DE DETECTION DES I - DETERMINATION OF THE DETECTION THRESHOLD
LEUCOCYTESLEUCOCYTES
L'objet de cette etude est de determiner la capacite du procede de denombrement de leucocytes en phase solide pour detecter de faibles nombres de leucocytes dans The purpose of this study is to determine the ability of the solid phase leukocyte enumeration method to detect low numbers of leukocytes in
du plasma.plasma.
Pour effectuer cette etude, un modele experimental a ete defini: une serie de dilutions en cascade de leucocytes issus de couche leucoplaquettaire a ete realisee dans du plasma, filtre sur un filtre de To carry out this study, an experimental model was defined: a series of cascading dilutions of leukocytes from the buffy coat was carried out in plasma, filtered on a filter of
porosite 0,22,um, exempt de cellules. 0.22 micron porosite, cell free.
I.1 Materiel À Produits sanguine: À Plasma frais congele, prepare selon les normes en place a l'EFS, (<< Guide pour la preparation, l'utilisation et l' assurance de qualite des composants sanguine >>, recommendation n R (95) 15, 7eme edition, Janvier 2001, Editions du conseil de l' Europe) I.1 Blood Materials: Fresh frozen plasma, prepared according to the standards in place at EFS, ("Guide to the Preparation, Use and Quality Assurance of Blood Components", Recommendation n R (95) 15, 7th edition, January 2001, Council of Europe Publishing)
À Sang total preleve sur anticoagulant CPD (citrate- Whole blood taken from anticoagulant CPD (citrate-
phosphate-dextrose) À Tampon phosphate (PBS pH 7,4) phosphate-dextrose) with phosphate buffer (PBS pH 7.4)
12 284099212 2840992
À Unites de filtration de 0.22 um.At filtration units of 0.22 μm.
À Membranes filtres noires en polyester de Black Polyester Filter Membrane
diametre 25 mm et de porosite 0,4,um. diameter 25 mm and porosity 0.4 μm.
À Triton X-100 5.To Triton X-100 5.
À Sous membrane noire en ester de cellulose Under Black Cellulose Ester Membrane
ou polyether sulfone de diametre 25 mm. or sulfonated polyether of diameter 25 mm.
À Acridine orange, solution mere a 5 mg/ml Acridine orange, 5 mg / ml stock solution
dans du DMSO.in DMSO.
À Analyseur en phase solide a balayeur laser Solid Phase Laser Scanner
et microscope a epifluorescence.and epifluorescence microscope.
À Appareil dthematologie MICRO 60-OT (Abx) de numeration /formule des cellules sanguines À Hemocytometre de Malassez I.2 Methode 1.2.1 Obtention des leucocytes En premier lieu, on procede a la preparation, d'une part, de plasma depourvu de leucocytes contaminants et d'autre part d'une couche leucoplaquettaire, egalement designee << suffy Coat >> ou BC, constituee par la couche mince jaunatre de leucocytes recouvrant les cellules rouges To MICRO 60-OT (Abx) Methodology of Blood Cell Count / Formulation to Malassez Hemocytometer I.2 Method 1.2.1 Obtaining Leukocytes Firstly, the preparation of plasma without contaminating leucocytes and secondly of a leucoplaquettaire layer, also designated "suffy Coat" or BC, constituted by the thin yellowish layer of leucocytes covering the red cells
compactees apres avoir centrifuge le sang. compacted after centrifuging the blood.
Preparation de la couche leucoplaquettaire: Dix millilitres de sang total vent centrifuges Preparation of the buffy coat: Ten milliliters of centrifugal whole blood
durant 15 minutes a 4000 tours par minute. for 15 minutes at 4000 rpm.
Le surnageant (plasma) est elimine.The supernatant (plasma) is eliminated.
La couche leucoplaquettaire est transferee dans The buffy coat is transferred to
un autre tube et le culot de globules rouges est elimine. another tube and the red blood cell pellet is eliminated.
1.2.2 Preparation du plasma filtre À On decongele le plasma frais congele au 1.2.2 Preparation of the Plasma Filter The frozen fresh plasma is thawed at
Bain Marie a 37 C.Bain Marie has 37 C.
13 284099213 2840992
À On aspire le plasma dans une seringue de 5 ml. À On positionne une unite de filtration de 0,22 um sur ltembout de la seringue À On fait passer le plasma sur l 'unite de filtration en recueillant le filtrat dans un tube (Plasma filtre). 1.2.3 Preparation de l'acridine orange On pipette dans un tube, 15 ul d'acridine orange a 5 mg/mL et on ajoute 1, 485 ml de tampon PBS pour Plasma is aspirated into a 5 ml syringe. A 0.22 μm filter unit was positioned on the tip of the syringe. The plasma was passed to the filter unit by collecting the filtrate in a tube (filter plasma). 1.2.3 Preparation of acridine orange 15 μl of 5 mg / ml acridine orange were pipetted into a tube and 1.485 ml of PBS buffer
obtenir une solution de travail de 50 ug/ml. obtain a working solution of 50 μg / ml.
I.2.4. Preparation des series de dilution des leucocytes Cent microlitres de la couche leucoplaquettaire (BC) vent dilues avec 900 ul de plasma filtre (dilution 10 1) Cent microlitres de la dilution 10-1 vent dilues avec 900 ul de plasma filtre (dilution 10-2), et ainsi de I.2.4. Preparation of leukocyte dilution series One hundred microliters of the buffy coat (BC) diluted with 900 μl of plasma filter (10 1 dilution) One hundred microliters of the 10-1 dilution diluted with 900 μl of plasma filter (dilution 10-2 ) and so
suite jusqu'a une dilution 10-6.continued until dilution 10-6.
1.2.5 Denombrement des leucocytes a) en phase solide avec l'analyseur a balayage laser. - On preleve 220 ul de l'echantillon contenant 1.2.5 Enumeration of leukocytes a) in solid phase with the laser scanning analyzer. - Take 220 μl of the sample containing
eventuellement les leucocytes.possibly leucocytes.
- On dilue ledit echantillon par addition de - said sample is diluted by addition of
220 ul de tampon PBS.220 μl of PBS buffer.
- On additionne 9 ul de Triton-X 100 5% pour obtenir une concentration de 0.1% final en Triton-X 100 9 μl of 5% Triton-X 100 are added to obtain a final 0.1% concentration of Triton-X 100
dans l'echantillon dilue.in the diluted sample.
- On incube le melange echantillon-detergent - Incubate the sample-detergent mixture
durant 15 minutes a temperature ambiante. for 15 minutes at room temperature.
14 284099214 2840992
- On marque les leucocytes eventuellement presents dans l'echantillon par addition de 45,ul de la - The leukocytes possibly present in the sample are marked by the addition of 45 μl of the
solution de travail d' Orange d'acridine. Orange acridine working solution.
- On incube l'echantillon dilue contenant les leucocytes et le marqueur durant 15 minutes a temperature ambiante. - On filtre 500 ul de l'echantillon comprenant les leucocytes marques sur une membrane filtre noire en polyester de 0.4um de porosite non autofluorescente, - On filtre ensuite 500,ul de tampon PBS pour The diluted sample containing the leukocytes and the marker are incubated for 15 minutes at room temperature. 500 μl of the sample comprising the marked leukocytes are filtered on a 0.4 μm polyester black filter membrane of non-autofluorescent porosite. 500 μl of PBS buffer are then filtered.
laver la membrane.wash the membrane.
- En tent que temoin, on utilise 220 ul de plasma filtre sur un filtre de porosite 0,22 um a la place In the test, 220 μl of plasma filter is used on a 0.22 μm porosite filter instead.
des 220,ul de l'echantillon initial. 220, ul of the initial sample.
b) avec un compteur de cellules Selon le protocole decrit dans le manuel de b) with a cell counter According to the protocol described in the manual of
procedures de l'appareil Micro 60 de la societe Abx. procedures of the Micro 60 device of the company Abx.
Le principe de la mesure repose sur la variation d'impedance engendree par le passage des cellules The principle of the measurement is based on the impedance variation generated by the passage of the cells
a travers un micro-orifice calibre.through a micro-orifice caliber.
L' etude de la distribution volumetrique des Globules blancs (Gs) permet de denombrer les trots sous populations leucocyLaires suivantes: lymphocytes, The study of the volumetric distribution of white blood cells (Gs) makes it possible to count trots under leucocyte populations as follows: lymphocytes,
monocytes et granulocytes.monocytes and granulocytes.
Le principe de l'analyse repose sur l' etude volumetrique des leucocytes apres ['action d'un reactif lytique. À On presente chaque tube correspondent a une dilution de la serie au niveau de la sonde The principle of the analysis is based on the volumetric study of leucocytes after the action of a lytic reagent. Each tube corresponds to a dilution of the series at the probe
d' aspiration.suction.
À L'appareil preleve 10,ul de suspension cellulaire. À Le resultat est obtenu dans un delai d' environ une minute et la zone de linearite est comprise To the apparatus collected 10 μl of cell suspension. The result is obtained within about a minute and the linearite zone is included
entre 0,5 et 80 x 106 leucocytes/ml. between 0.5 and 80 x 106 leukocytes / ml.
c) avec un hemocytometre: À On effectue une dilution 1/10 de la couche c) with a hemocytometer: A 1/10 dilution of the layer is carried out
leucoplaquettaire (BC) dans du plasma filtre. leukoplaquettaire (BC) in plasma filter.
À On distribue 20ul dans l'hemocytometre de Malassez. 20ul is distributed in the Malassez hemocytometer.
À On denombre les cellules au microscope. The cells are counted under a microscope.
I.3 Resultats Les resultats des differents denombrements I.3 Results The results of the different counts
effectues vent indiques dans les tableaux 1, 2 ci-dessous. carried out in tables 1, 2 below.
Le tableau 1 indique les resultats du denombrement en phase solide effectue selon le procede de Table 1 shows the results of the solid phase enumeration carried out according to the method of
['invention, a partir des 220 pl d'echantillon de depart. ['invention, from the 220 pl sample departure.
Tableau 1Table 1
Denombrement de leucocytes en phase solide Resultats apres Resultats validation (*) (leucocytes x 103/ mL) (leucocytes/membrane) Controle Negatif: O O Plasma Filtre O O Dilution de 13272 60 leucocytes: 10- 3 15930 72 Dilution de 1305 5,9 leucocytes: 10-9 1101 4,9 Dilution de 150 0,68 leucocytes: 1O-s 90 0,4 Dilution de 4 0,02 leucocytes: 10- 6 O O (*) Les resultats apres validation se referent a la validation effectuee au microscope sur un nombre de champs representatifs (> 50 champs) pour definir le nombre de leucocytes par membrane c'est-a-dire Enumeration of leukocytes in solid phase Results after Validation Results (*) (leukocytes x 103 / mL) (leukocytes / membrane) Negative Control: OO Plasma Filter OO Dilution of 13272 60 leukocytes: 10- 3 15930 72 Thinning of 1305 5.9 leukocytes : 10-9 1101 4.9 Dilution of 150 0.68 leukocytes: 10-s 90 0.4 Dilution of 4 0.02 leukocytes: 10-6 OO (*) The results after validation refer to the validation carried out under the microscope on a number of representative fields (> 50 fields) to define the number of leucocytes per membrane, that is to say
dans 220 ul de preparation.in 220 μl of preparation.
Il apparalt que les concentrations en leucocytes vent bien analysees par le marquage avec l' grange d'acridine et que l'on peut detecter moins de 10 leucocytes a partir de 220 It appears that the leucocyte concentrations are well analyzed by labeling with the acridine barn and that less than 10 leucocytes can be detected from 220
microlitres de plasma.microliters of plasma.
Tableau 2Table 2
Denombrement de leucocytes avec un compteur de cellules ("Micros 60") PREPARATION Leucocytes Controle Negatif 0 Plasma filtre Couche leucoplaquettaire (Buffy coat= BC) Saturation non diluee Dilution BC dilution 11 500 -1 Dilution BC dilution 1 200 -2 Dilution BC dilution 200 -3 Dilution BC dilution 100 -4 Controle Positif 400 Plasma non filtre La gamme de detection de leucocytes avec un compteur de cellules est comprise entre 5 x 1Os cellules/ml et 1Os cellules/ml. Le tableau 3 ci- dessous montre les resultats de cette etude comparative entre les trots procedes de denombrement de leucocytes pour mesurer le nombre de cellules dans la couche leucoplaquettaire: en phase solide, au moyen d'un compteur de particules (Micros 60) et Enumeration of leukocytes with a cell counter ("Micros 60") PREPARATION Leukocytes Negative control 0 Plasma filter Buffy coat (BC) Undiluted saturation BC dilution dilution 11 500 -1 BC dilution dilution 1 200 -2 BC dilution dilution 200 BC Dilution Dilution 100 -4 Control Positive 400 Non-Filter Plasma The detection range of leukocytes with a cell counter is between 5 x 10 cells / ml and 10 cells / ml. Table 3 below shows the results of this comparative study between the three leucocyte enumeration methods for measuring the number of cells in the buffy coat: in the solid phase, using a particle counter (Micros 60) and
au moyen d'un hemocytometre (Cellule de Malassez). by means of a hemocytometer (Cell of Malassez).
17 284099217 2840992
Tableau 3Table 3
Concentration estimee de la couche leucoplaquettaire (BC) Phase solide Compteur de Hemocytometre Procede de particules ['invention 4.8 107 cellules /ml 1.1 108 cellules /ml 1 108 cellules /ml Le procede de denombrement en phase solide est particulierement adapte et presente une meilleure sensibilite par rapport aux deux autres methodes pour les Estimated Concentration of the Buffy Coat (BC) Solid Phase Hemocytometer Counter Particle Process [invention 4.8 107 cells / ml 1.1 108 cells / ml 1 108 cells / ml The solid phase enumeration method is particularly suitable and has better sensitivity compared to the other two methods for
falbles nombres de leucocytes.falble numbers of leucocytes.
II - VALIDATION DE LA METHODE DE DENOMBREMENT II - VALIDATION OF THE METHOD OF ENUMERATION
DE LEUCOCYTES EN PHASE SOLIDE.LEUKOCYTES IN SOLID PHASE.
De maniere a valider le procede de denombrement de leucocytes de ['invention, avec des echantillons de plasma a usage therapeutique, des analyses desdits plasmas ont ete effectuees a partir de tubes scelles avant et apres In order to validate the leukocyte enumeration method of the invention, with plasma samples for therapeutic use, analyzes of said plasmas were made from sealed tubes before and after
avoir effectue une leucoreduction.have a leucoreduction.
II.1 Materiel - Echantillon de plasma (A) sous forme de tubulure prelevee a partir de la poche de plasma avant II.1 Material - Plasma sample (A) in the form of tubing taken from the forward plasma bag
leucoreduction et apres leucoreduction. leucoreduction and after leucoreduction.
- Echantillon de plasma (B) sous forme de tubulure prelevee a partir de la poche de plasma avant - Plasma sample (B) in the form of tubing taken from the front plasma bag
leucoreduction et apres leucoreduction. leucoreduction and after leucoreduction.
Le volume des echantillons est d' environ 2 ml The sample volume is about 2 ml
(30 cm de tubulure).(30 cm of tubing).
- Orange d'Acridine, prepare a une concentration de 5 mg/ml dans du DMSO Tampon salin phosphate (PBS) a une concentration de 10 mM et pH 7.4 prepare dans de l'eau distillee - Des membranes filtres noires non autofluorescentes en polyester de diametre 25 mm et de - Acridine orange, prepared at a concentration of 5 mg / ml in DMSO phosphate buffered saline (PBS) at a concentration of 10 mM and pH 7.4 prepared in distilled water - Black autofluorescent polyester filter membranes of diameter 25 mm and
porosite 0,4 um.porosity 0.4 μm.
- Sous membrane noire en ester de cellulose ou - Under black membrane made of cellulose ester or
polyether sulfone de diametre 25 mm. sulfonated polyether of diameter 25 mm.
- Du detergent Triton X100 Solution a une - Triton X100 Solution detergent has a
concentration de 5% dans du PBS.concentration of 5% in PBS.
- Unites de filtration de porosite 0.22 um - Analyseur en phase solide a balayeur laser - 0.22 μm porosite filtration units - Laser scanned solid phase analyzer
et microscope a epifluorescence.and epifluorescence microscope.
II.2 Methode - Preparation du controle negatif: Du plasma extrait du tube et filtre a travers un filtre de porosite 0.22um est utilise comme controle negatif, comme indique au II.2 Method - Preparation of negative control: Plasma extracted from the tube and filtered through a 0.22um porosity filter is used as a negative control, as indicated in
paragraphe 1.2.2 ci-dessus.paragraph 1.2.2 above.
- Preparation de la solution de travail - Preparation of the working solution
d' grange d'acridine.of acridine barn.
On prepare une solution d' Orange d'acridine a une concentration finale de 5Oug/ml dans du PBS, par dilution 1/100 de la solution mere d' Orange d'acridine (5 A solution of Acridine Orange at a final concentration of 50 μg / ml in PBS is prepared by diluting 1/100 of the acridine orange stock solution (5 μg / ml).
mg/ml dans du Pss).mg / ml in Pss).
Procedure de marquage et analyse de l'echantillon. On preleve 100 ul d'echantillon (plasma filtre ou echantillon frais) On ajoute 100,ul de PBS On ajoute 4 ul de Triton X100 a une concentration de 5% (concentration finale: 0.1%) Tagging procedure and sample analysis. 100 μl of sample (filter plasma or fresh sample) 100 ul of PBS 4 μl of Triton X100 at a concentration of 5% (final concentration: 0.1%) are added
19 284099219 2840992
On incube le melange 15 minutes a temperature ambiante. On ajoute 20,ul de la solution de travail The mixture is incubated for 15 minutes at room temperature. 20 μl of the working solution is added
d' Orange d'Acridine.of Acridine Orange.
On incube durant le melange echantillon-marqueur durant 15 minutes a temperature ambiante. On filtre la preparation ainsi obtenue sur une Incubate during the sample-label mixture for 15 minutes at room temperature. The resulting preparation is filtered on a
membrane filtre non autofluorescente (noire). Non autofluorescent filter membrane (black).
On analyse la membrane avec un analyseur laser a balayage II. 3 Resultats Les resultats du denombrement des leucocytes a la suite de cette procedure de marquage et analyse vent presentes dans le tableau 4 ci- dessus pour chacun des deux The membrane is analyzed with a scanning laser analyzer II. 3 Results The results of counting leukocytes following this marking procedure and analysis are shown in Table 4 above for each of the two
echantillon A et B. avant et apres leucoreduction. sample A and B. before and after leucoreduction.
Tableau 4Table 4
Denombrement de leucocytes en phase solide (a partir de 100,ul d'echantillon) Resultats apres Resultats (*) validation (**) (leucocytes/membrane) (leucocytes/mL) Echantillon avant leucoreduction A Plasma filtre sur 0.22 um 0 0 avant leucoreduction 58 580 Plasma nonfiltre 50 530 apres leucoreduction O O Plasma filtre sur 0.22 pm 0 0 apres leucoreduction 1 10 Plasma non-filtre 1 10 Echantillon avant leucoreduction B Plasma filtre sur 0.22 um 0 0 avant leucoreduction 76 760 Plasma non-filtre 81 810 apres leucoreduction O O Plasma filtre sur 0.22 um 0 0 apres leucoreduction O O Plasma non- filtre O O 2 o 2840992 La concentration en leucocytes avant la leucoreduction est d' environ, 5X102 a 8X102 cellules/ml et elle est inferieure a 10 cellules /ml apres la leucoreduction. Ainsi, il apparalt que ce procede de denombrement des leucocytes en phase solide, apres leur marquage avec un colorant fluorescent, tel que l' grange d'acridine, permet leur detection dans le plasma de maniere gable. Les differents travaux experimentaux conduits par la demanderesse ont permis de verifier que la limite de detection du procede est inferieure a 10 cellules/ml. Wile est d' environ 1 leucocyte/ml de plasma (200 Enumeration of leukocytes in solid phase (from 100, μl of sample) Results after Results (*) validation (**) (leukocytes / membrane) (leukocytes / mL) Sample before leukoreduction A Plasma filter on 0.22 μm 0 0 before leucoreduction 58 580 Plasma non-filter 50 530 after leucoreduction OO Plasma filter on 0.22 pm 0 0 after leucoreduction 1 10 Plasma non-filter 1 10 Sample before leucoreduction B Plasma filter on 0.22 um 0 0 before leucoreduction 76 760 Plasma non-filter 81 810 after leucoreduction OO Plasma filter on 0.22 μm 0 0 after leucoreduction OO Plasma non-filter OO 2 o 2840992 The leucocyte concentration before leucoreduction is about 5X102 to 8X102 cells / ml and it is less than 10 cells / ml after leucoreduction. Thus, it appears that this method of solid phase leukocyte enumeration after labeling with a fluorescent dye, such as the acridine barn, allows their detection in plasma in a manageable manner. The various experimental works conducted by the applicant have made it possible to verify that the detection limit of the process is less than 10 cells / ml. Wile is about 1 leucocyte / ml of plasma (200
leucocytes/poche) avec environ 5 ml d'echantillon. leukocytes / pouch) with about 5 ml of sample.
Ce procede assure une detection de 100% des leucocytes residuels dans un echantillon de plasma, lorsqu'il est compare aux denombrements obtenus avec un This method provides 100% detection of residual leukocytes in a plasma sample when compared to counts obtained with a
hemocytometre tel qu'une cellule de Malassez. hemocytometer such as a cell of Malassez.
D' autres experiences ont ete conduites dans du plasma prepare pour des usages therapeutiques. La concentration en leucocytes avant leucoreduction etait d'environ 5X102 cellules/ml (superieur a 1O: cellules/poche), alors qu'apres leucoreduction cette concentration est inferieure a 10 cellules/ml (inferieur a Other experiments have been conducted in plasma prepared for therapeutic use. The concentration of leukocytes before leukoreduction was about 5X102 cells / ml (greater than 10 cells / bag), whereas after leukoreduction this concentration was less than 10 cells / ml (below
2 103 cellules/poche).2,103 cells / pocket).
Le procede de denombrement de leucocytes de l' invention est sur et les denombrements obtenus vent coherents avec ccux decrits dans la litterature.( Masse M., Transfus Clin Biol 2001 Jun;8(3):297- 302 << Universal The leukocyte enumeration method of the invention is on and the counts obtained are consistent with those described in the literature (Mass M., Transfus Clin Biol 2001 Jun; 8 (3): 297-302 << Universal
2 1 28409922 1 2840992
leuLoreduction of cellular and plasma components: process control and performance of the leukoreduction process.>>) Cette etude montre que le procede de denombrement de leucocytes de ['invention permet la detection de leucocytes residuels dans le plasma et, compare aux procedes conventionnels de denombrement de leucocytes, il offre une plus grande exactitude. This study shows that the leukocyte enumeration method of the invention allows the detection of residual leukocytes in the plasma and, compared to conventional enumeration methods, the leukocyte enumeration method of the invention allows the detection of residual leucocytes in the plasma and, compared with conventional enumeration methods. leukocytes, it offers greater accuracy.
De plus, la sensibilite du procede de l 'invention peut etre optimisee aisement en augmentant le volume de l'echantillon. Un volume d'echantillon plus important conduira a une meilleure recuperation des leucocytes qui In addition, the sensitivity of the method of the invention can be optimized easily by increasing the volume of the sample. A larger sample volume will lead to better recovery of leukocytes
seront plus representatifs de l'echantillon. will be more representative of the sample.
22 284099222 2840992
Claims (15)
Priority Applications (3)
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FR0207492A FR2840992B1 (en) | 2002-06-18 | 2002-06-18 | METHOD FOR ENUMERATION AND / OR MORPHOLOGICAL IDENTIFICATION OF LEUKOCYTES IN SOLID PHASE |
PCT/FR2003/001865 WO2003107005A1 (en) | 2002-06-18 | 2003-06-18 | Method for counting and/or morphological identification of leukocytes in the solid phase |
AU2003260603A AU2003260603A1 (en) | 2002-06-18 | 2003-06-18 | Method for counting and/or morphological identification of leukocytes in the solid phase |
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FR0207492A FR2840992B1 (en) | 2002-06-18 | 2002-06-18 | METHOD FOR ENUMERATION AND / OR MORPHOLOGICAL IDENTIFICATION OF LEUKOCYTES IN SOLID PHASE |
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CN112020384A (en) * | 2018-05-02 | 2020-12-01 | 里珍纳龙药品有限公司 | Method for evaluating the suitability of a biochemical filter |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0525398A2 (en) * | 1991-07-29 | 1993-02-03 | Toa Medical Electronics Co., Ltd. | Method of preparing specimen for classifying and counting leukocytes |
DE4309328A1 (en) * | 1993-03-18 | 1994-09-22 | Volker Ost | Method for distinguishing between erythrocytes and leucocytes in whole blood by the methods of scattered-light measurement in a through-flow cytometer for cell counting and cell sorting |
WO1997002482A1 (en) * | 1995-06-30 | 1997-01-23 | Biometric Imaging, Inc. | Volumetric cell quantification method and system |
-
2002
- 2002-06-18 FR FR0207492A patent/FR2840992B1/en not_active Expired - Fee Related
-
2003
- 2003-06-18 AU AU2003260603A patent/AU2003260603A1/en not_active Abandoned
- 2003-06-18 WO PCT/FR2003/001865 patent/WO2003107005A1/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0525398A2 (en) * | 1991-07-29 | 1993-02-03 | Toa Medical Electronics Co., Ltd. | Method of preparing specimen for classifying and counting leukocytes |
DE4309328A1 (en) * | 1993-03-18 | 1994-09-22 | Volker Ost | Method for distinguishing between erythrocytes and leucocytes in whole blood by the methods of scattered-light measurement in a through-flow cytometer for cell counting and cell sorting |
WO1997002482A1 (en) * | 1995-06-30 | 1997-01-23 | Biometric Imaging, Inc. | Volumetric cell quantification method and system |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112020384A (en) * | 2018-05-02 | 2020-12-01 | 里珍纳龙药品有限公司 | Method for evaluating the suitability of a biochemical filter |
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