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

Abstract

Method for counting leucocytes in a biological fluid by incubating the sample with an agent (I) that renders cell membranes permeable, incubation with a leucocyte marker (II), filtering through a non-autofluorescent membrane to retain any labeled leucocytes and analysis of the membrane to determine the number of labeled cells.

Description

seal (1) to be checked.

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  METHOD OF INVOLVING AND / OR IDENTIFYING

  MORPHOLOGY OF SOLID PHASE LEUKOCYTES.

  The present invention relates to a method for detecting and / or counting and / or identifying

  morphology of solid phase leukocytes.

  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

worldwide.

  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.

plasma or seric.

  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

Creutzfeldt-Jacob disease.

  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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  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 related to the causal agent of BSE.

  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,

for example, screen filters.

  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

liter, about 10 cells / ml.

  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

from Europe.

  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

  blood products, has a certain interest.

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  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

House of Nageotte.

  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

has less variability.

  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

  [Improvement of transfusion safety.

  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

  by pre-concentration of the sample.

  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.

leukocytes / ml for plasma.

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  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

commercial filters.

  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,

and more sensitive.

  In order to solve this problem of lack of sensitivity of leukocyte enumeration methods

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  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.

  This process is particularly well adapted to

  enumeration of leukocytes in the plasma.

  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.

filtration.

  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) 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

on this one.

  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

biological fluid.

  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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  these can reach the cytoplasm and / or the nucleus

  cell and attach to intracellular elements.

  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

performed by the analyzer.

  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

solid phase preparation.

  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.

  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 or saline (0.9% NaCl).

  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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  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, 0.2% for saponin.

  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

  preferentially it is 15 minutes.

  According to preferred embodiments of the method of the invention, the leukocyte marker used in step (b) is selected from the group consisting of:

  dye, a fluorochrome, a nucleic acid marker.

  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), dimeric cyanine, SYBRO and propidium iodide.

  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

  preferentially it is 15 minutes.

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  According to a particular mode of implementation of the method of the invention, the non-autofluorescent membrane implemented

  in step (c) is a black membrane.

  Preferably said black membrane is a membrane made of polyester or polycarbonates or polyether sulfone

diameter 25 mm.

  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.

  and preferentially it is 0.4 μm.

  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

  background on the membrane causes traces of the marker.

  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

epifluorescence.

  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

leucoreduction methods.

  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

  detection limit and the sensitivity of the process.

  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.

  microscopic reading of the analyzed solid phases.

  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

  morphologic leucocytic leukocyte populations.

  - 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

porosity 0.22 μm.

  FIG. 4 illustrates the count of leukocytes carried out with a cell counter for serial dilutions of leucocytes in plasma with

a 0.22 μm porosity filter.

  - 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

therapeutic.

EXPERIMENTAL PART - EXAMPLES

  I - DETERMINATION OF THE DETECTION THRESHOLD

LEUCOCYTES

  The purpose of this study is to determine the ability of the solid phase leukocyte enumeration method to detect low numbers of leukocytes in

plasma.

  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

  0.22 micron porosite, cell free.

  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)

  Whole blood taken from anticoagulant CPD (citrate-

  phosphate-dextrose) with phosphate buffer (PBS pH 7.4)

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At filtration units of 0.22 μm.

  Black Polyester Filter Membrane

  diameter 25 mm and porosity 0.4 μm.

To Triton X-100 5.

  Under Black Cellulose Ester Membrane

  or sulfonated polyether of diameter 25 mm.

  Acridine orange, 5 mg / ml stock solution

in DMSO.

  Solid Phase Laser Scanner

and epifluorescence microscope.

  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

  compacted after centrifuging the blood.

  Preparation of the buffy coat: Ten milliliters of centrifugal whole blood

  for 15 minutes at 4000 rpm.

The supernatant (plasma) is eliminated.

  The buffy coat is transferred to

  another tube and the red blood cell pellet is eliminated.

  1.2.2 Preparation of the Plasma Filter The frozen fresh plasma is thawed at

Bain Marie has 37 C.

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  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

  obtain a working solution of 50 μg / ml.

  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

continued until dilution 10-6.

  1.2.5 Enumeration of leukocytes a) in solid phase with the laser scanning analyzer. - Take 220 μl of the sample containing

possibly leucocytes.

  - said sample is diluted by addition of

220 μl of PBS buffer.

  9 μl of 5% Triton-X 100 are added to obtain a final 0.1% concentration of Triton-X 100

in the diluted sample.

  - Incubate the sample-detergent mixture

  for 15 minutes at room temperature.

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  - The leukocytes possibly present in the sample are marked by the addition of 45 μl of the

  Orange acridine working solution.

  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.

wash the membrane.

  In the test, 220 μl of plasma filter is used on a 0.22 μm porosite filter instead.

  220, ul of the initial sample.

  b) with a cell counter According to the protocol described in the manual of

  procedures of the Micro 60 device of the company Abx.

  The principle of the measurement is based on the impedance variation generated by the passage of the cells

through a micro-orifice caliber.

  The study of the volumetric distribution of white blood cells (Gs) makes it possible to count trots under leucocyte populations as follows: lymphocytes,

monocytes and granulocytes.

  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

suction.

  To the apparatus collected 10 μl of cell suspension. The result is obtained within about a minute and the linearite zone is included

  between 0.5 and 80 x 106 leukocytes / ml.

  c) with a hemocytometer: A 1/10 dilution of the layer is carried out

  leukoplaquettaire (BC) in plasma filter.

  20ul is distributed in the Malassez hemocytometer.

  The cells are counted under a microscope.

  I.3 Results The results of the different counts

  carried out in tables 1, 2 below.

  Table 1 shows the results of the solid phase enumeration carried out according to the method of

  ['invention, from the 220 pl sample departure.

Table 1

  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

in 220 μl of preparation.

  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

microliters of plasma.

Table 2

  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

  by means of a hemocytometer (Cell of Malassez).

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Table 3

  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

falble numbers of leucocytes.

  II - VALIDATION OF THE METHOD OF ENUMERATION

LEUKOCYTES IN SOLID PHASE.

  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

have a leucoreduction.

  II.1 Material - Plasma sample (A) in the form of tubing taken from the forward plasma bag

  leucoreduction and after leucoreduction.

  - Plasma sample (B) in the form of tubing taken from the front plasma bag

  leucoreduction and after leucoreduction.

  The sample volume is about 2 ml

(30 cm of tubing).

  - 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

porosity 0.4 μm.

  - Under black membrane made of cellulose ester or

  sulfonated polyether of diameter 25 mm.

  - Triton X100 Solution detergent has a

concentration of 5% in PBS.

  - 0.22 μm porosite filtration units - Laser scanned solid phase analyzer

and epifluorescence microscope.

  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

paragraph 1.2.2 above.

  - Preparation of the working solution

of acridine barn.

  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 in Pss).

  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

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  The mixture is incubated for 15 minutes at room temperature. 20 μl of the working solution is added

of Acridine Orange.

  Incubate during the sample-label mixture for 15 minutes at room temperature. The resulting preparation is filtered on a

  Non autofluorescent filter membrane (black).

  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

  sample A and B. before and after leucoreduction.

Table 4

  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

  leukocytes / pouch) with about 5 ml of sample.

  This method provides 100% detection of residual leukocytes in a plasma sample when compared to counts obtained with a

  hemocytometer such as a cell of Malassez.

  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 cells / pocket).

  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

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  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.

  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

  will be more representative of the sample.

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Claims (15)

CLAIM I CAT I ONS   1) Method for enumeration of leukocytes possibly present in a biological fluid, characterized in that it comprises the following steps: a) the incubation of a sample with said biological fluid with a permeabilizing agent of the cell membranes, b) the incubation of the sample obtained in step (a) with a leukocyte marker, c) filtration of the sample obtained in step (b) on a non-autofluorescent membrane, capable of retaining the leucocytes, to obtain a preparation in the solid phase of the marked leukocytes, possibly present in said sample, d) the analysis of the solid phase preparation of step (c), to count the leukocytes   possibly present on it.   2) Method according to claim 1, characterized in that the sample of step (a) is previously diluted with an osmotically compatible buffer with leukocytes, before incubation with the agent. permeabilizing membranes.   3) Method according to claim 2, characterized in that the dilution buffer is chosen from the group comprising: PBS, TRIS, buffer of Tyrode or saline.   4) Method according to any one of   preceding claims, characterized in that the agent   permeabilizer of the cell membranes is selected from   a detergent, and a fixing agent.   Process according to claim 4, characterized in that the detergent used in step (a) is selected from the group consisting of detergents: Triton X-100, saponin, n-octyl glucopyranoside, Tween 20, Tween 80, sodium dodecyl sulfate.   6) Method according to any one of   Claims 4 and 5, characterized in that the detergent   used in step (a) is used at a final concentration of between 0.05% and 5%, preferably between 0.01% and 2%.   7) Method according to any one of   Claims 4 to 6, characterized in that the detergent   used in step (a) is used at a final concentration of 0.1% for Triton X-100, 1% for n-octyl glucopyranoside, Tween 20, Tween 80 and sodium   dodecyl sulfate, 0.2% for saponin.   8) Method according to any one of   preceding claims is characterized in that the duration   The incubation period between the sample and the cell membrane permeabilizer in step (a) is from 5 to 45 minutes, preferably from 10 to 20 minutes.   all preferentially it is 15 minutes.   9) Method according to any one of   preceding claims, characterized in that the   leukocyte marker used in step (b) is selected from the group consisting of: a dye, a fluorochrome, a marker of nucleic acids.   Method according to claim 9, characterized in that the leukocyte marker used in step (b) is a nucleic acid marker selected from the group consisting of: acridine orange PicogreenO, asymmetric cyanine, cyanine monomer, dimeric cyanine , SYBRO 11) Method according to any one of   preceding claims, characterized in that the duration   The incubation period between the sample and the marker in step (b) is between 5 and 45 minutes, preferably between 20 and 20 minutes and most preferably it is 15 minutes.   12) Method according to one of the claims   precedence, characterized in that the non-autofluorescent membrane used in step (c) is a black membrane.   13) Method according to any one of   preceding claims, characterized in that the size   the pores of the membrane used in step (c) are between 0.05 μm and 2 μm, most preferably between 0.1 μm and 1 μm and preferably it is 0.4 μm. 14) Method according to any one of   preceding claims, characterized in that the   membrane is a polyester membrane, polyether sulfone or polycarbonate.   ) Method according to any one of   preceding claims, characterized in that 2840992   filtration of the step (c) a ravage of the   membrane by filtration of a buffer volume.   16) Method according to any one of   preceding claims, characterized in that the analysis   solid phase preparation at step (d) is   performed by means of a laser scanning analyzer.   17) Method for the morphological identification of leukocytes possibly present in a blood product, characterized in that it comprises the steps (a) to (c) of a   process according to one of claims 1 to 15,   followed by a step (e) of visual observation with an epifluorescence microscope of the preparations obtained a step (c).   18) Use of a method of enumeration of   Leukocytes according to any one of Claims 1 to 16   or a method for the morphological identification of leukocytes according to claim 17 for the control of quality of plasma preparations.   19) Use of a method of enumeration of   Leukocytes according to any one of Claims 1 to 16   or a process for the morphological identification of leukocytes according to claim 17 for the quality control of leukocyte filters) Use of a method for the enumeration of   Leukocytes according to any one of Claims 1 to 16   or a process for the morphological identification of leukocytes according to claim 17, for enumeration   leukocytes in biological fluids.   21) Use of a method of enumeration of   Leukocytes according to any one of Claims 1 to 16   or a process for the morphological identification of leukocytes according to Revindication 17 for the morphological identification of the different populations of leucocytes in biological fluids 22) Use of a method for the enumeration of   Leukocytes according to any one of Claims 1 to 16   or a method for the morphological identification of leukocytes according to claim 17 for the control of