WO2001011362A1 - Procede de numeration de cellules et trousse a cet effet - Google Patents

Procede de numeration de cellules et trousse a cet effet Download PDF

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Publication number
WO2001011362A1
WO2001011362A1 PCT/GB2000/002084 GB0002084W WO0111362A1 WO 2001011362 A1 WO2001011362 A1 WO 2001011362A1 GB 0002084 W GB0002084 W GB 0002084W WO 0111362 A1 WO0111362 A1 WO 0111362A1
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WO
WIPO (PCT)
Prior art keywords
sample
target cells
cells
binding
substrate
Prior art date
Application number
PCT/GB2000/002084
Other languages
English (en)
Inventor
Janet Rosemary Rider
Ruth Gilbert
Richard Turton
Original Assignee
The National Blood Authority
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The National Blood Authority filed Critical The National Blood Authority
Priority to AU50908/00A priority Critical patent/AU5090800A/en
Publication of WO2001011362A1 publication Critical patent/WO2001011362A1/fr

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Classifications

    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • This invention relates to a method for quantifying the number of target cells in a sample, and a kit for quantifying the number of target cells in a sample using such a method.
  • the leucocyte concentration in the blood product is commonly below 20 cells ⁇ LT 1 ,
  • the transplacental passage of fetal Rhesus D positive (D+) red blood cells (RRCs) into a Rhesus D negative (D-) woman before or after delivery results in the formation of anti-D antibodies.
  • the anti-D IgG is then transported back across the placenta into the fetus, resulting in haemolysis of fetal RBCs .
  • Anti-D mediated haemolytic disease can affect both the current pregnancy and subsequent pregnancies with a D+ fetus .
  • the production of maternal anti-D may be prevented by administration of prophylactic anti-D within 72 hours of the fetomaternal haemorrhage (FMH) .
  • Prophylactic anti-D is given to all D- women after delivery of a D+ neonate, or after any other event likely to cause a FMH.
  • the minimum dose 500-1500 IU of IgG is sufficient to clear 4-12mL of D+ cells, calculated at a ratio of 125 IU (25 ⁇ g) to lmL of cells.
  • the volume of the FMH must be determined to calculate whether additional anti-D is required. It is currently quantitated using either of the Kleihauer-Betke acid elution technique or by flow cytometry.
  • a method for quantifying the number of target cells in a sample which comprises;
  • binding specifically is used to indicate that the binding moiety has the capacity to recognise and interact specifically with a particular molecular site or "marker" which is characteristic of the target cell.
  • the sample is a fluid sample, such as a sample of a bodily fluid or derived from a bodily fluid.
  • the sample is, or is derived from blood.
  • the fluid sample may be whole blood, concentrated red cells, platelet concentrates or plasma.
  • the fluid sample may be a leucodepleted blood component .
  • the target cell may be any eukaryotic or prokaryotic cell. It is presently preferred that the target cell is a eukaryotic cell, for example a eukaryotic cell which may be found in a bodily fluid.
  • the target cell is a cell which can be found in blood, for example monocytes, macrophages, neutrophils, eosinophils, basophils, B lymphocytes, T lymphocytes, natural killer cells, dendritic ceils, erythrocytes or platelets .
  • the target cell is a leucocyte .
  • the substrate is defined so that it is capable of discretely accommodating multiple samples.
  • the solid substrate may be a microtitre plate, for example a 6-well, 12-well, 24-well, 48-well or a 96- well microtitre plate. Most preferably the solid substrate is a 96-well microtitre plate.
  • the target cells bind specifically to the substrate such that the remainder of the sample can be removed, leaving the target cells bound (indirectly or directly) to the substrate.
  • the substrate and the captured target cells are then washed to remove any extraneous non-specifically bound matter.
  • a surface of the substrate is provided with a plurality of binding moieties, each of which is capable of binding specifically to the target cells.
  • the binding moiety is preferably an antibody to a cell-surface molecule or a ligand for a cell surface molecule such as an adhesion molecule.
  • the binding moiety which interacts may be bound to the substrate directly, for example by adsorbtion of the molecule comprising the binding moiety on to the substrate, or indirectly, for example by way of a linker molecule.
  • a linker molecule is streptavidin or similar molecule (such as ExtrAvidin which is commercially available from Sigma Chemicals) .
  • the target cell is a leucocyte and the substrate is capable of binding to a leucocyte cell-surface antigen.
  • the solid substrate may have bound to its surface antibodies against one or more of the following leucocyte cell surface antigens: CD3 , CD5, CD13, CD14, CD15, CD16, CD19, CD20, CD34 and/or CD45.
  • Other CD targets also exist.
  • the solid substrate has bound to its surface antibodies against CD45 and CD15. More preferably, the solid substrate has bound to its surface antibodies against both CD45 and CD15.
  • leucocyte cell surface antigens There are a number of other leucocyte cell surface antigens in addition to those listed above. The choice of cell surface antigen will depend on the target cell.
  • the sample is a maternal blood sample of a rhesus- negative mother which is to be tested to quantitate the concentration of fetal, rhesus-positive red-blood cells in the maternal circulation.
  • the solid substrate may have bound to its surface a monoclonal antibody specific for D+ red blood cells, such as BRAD-3.
  • the cells are quantified by analysing a cellular component of the captured target cell.
  • This component may be an intracellular component or a cell surface component.
  • the cellular component of the target cells may be any molecule, the expression of which (in or on the target cell) is substantially uniform among the target cell population. In other words, the expression of intracellular or cell surface compor.ent of the target cells must be substantially proportional to the number of cells in the fluid sample.
  • the cellular component may be nucleic acid (DNA, rRNA, tRNA, mRNA) , or an intracellular or cell surface protein, glycoprotein, carbohydrate or lipid.
  • the intracellular component of the target cell is nucleic acid (DNA and RNA) .
  • the cellular component quantified is haemoglobin.
  • the cell membranes of the retained cells are disrupted or permeabilised prior to the quantification step.
  • the cells may be lysed by conventional techniques such as incubation in a cell lysis solution, freeze-thaw, or sonication.
  • Commonly used cell lysis solutions include solutions comprising a cell lysis agent such as a detergent or an enzyme, and hypotonic solutions such as distilled water.
  • a cell lysis agent such as a detergent or an enzyme
  • hypotonic solutions such as distilled water.
  • differential cell lysis solutions for example, Optilyse C and DNA Prep LPR from Beckman Coulter
  • the choice of lysis means will depend on the cell population targeted.
  • the amount of the cellular component of the target cells may be analysed using a probe.
  • such a probe may be capable of binding specifically to the cellular component of the target cells and capable of being detected when it is so bound.
  • the probe may be protein such as an antibody or a ligand for a cell-surface or intracellular protein.
  • Sucn a protein may be labelled, for example with a radiolabel, a fluorescent label or an enzyme label.
  • the probe may be capable of binding directly or indirectly to a cellular component of the target cells and being detected specifically when it is so bound.
  • the probe may be a dye.
  • the probe is a dye which is detectable when bound to nucleic acids .
  • the probe is a dye which exhibits fluorescent enhancement when bound to cellular nucleic acids.
  • the probe is CyQUANT GR dye (Molecular Probes) or PicoGreen dye (Molecular Probes) .
  • the bound probe may be detectable by any one of a number of known techniques such as fluorimetry, spectrophotometry, autoradiography or radioactive counting and chemiluminescence methods.
  • the bound probe is detectable by fluorimetry, for example using a fluorescence microplate reader.
  • a fluorescence microplate reader When the probe is CyQUANT GR dye or PicoGreen dye, a fluorescent microplate reader is commonly used with filters appropriate for fluorescein (for example, about 480nm excitation and about 520nm emission maxima) .
  • the cellular component may be analysed or detected directly without the use of a probe.
  • non-invasive techniques are known in the art by which an intracellular molecular component may be detected in situ in intact or lysed cells.
  • Haemoglobin in erythrocytes may be detected by 3 wavelength spectrophotometry in a manner known per se .
  • This method may be used in the aspect of the invention where the sample is a blood sample of a rhesus-negative mother which is to be tested to quantitate fetal rhesus-positive red-blood cells in the sample. More particularly, in the 3 wavelength spectrophotometry method mentioned above, after the lysis step, sample absorbance at wavelengths of 562nm, 578nm and 598nm is recorded. The concentration of haemoglobin is calculated as follows:
  • the concentration of haemoglobin remaining can be used to calculate the proportion of fetal cells in the maternal blood sample, and consequently the volume of FMH.
  • kit for quantifying the number of target cells in a sample using the method of the first " aspect of the invention which kit comprises: (i) a substrate, the surface of which is provided with a plurality of binding moieties each capable of binding specifically to the target cell, or the means for making such a substrate .
  • the kit of the second aspect of the invention also comprises:
  • the kit may comprise a substrate which is ready- provided with binding moieties capable of binding specifically to the target cell.
  • the kit may comprise the means for making such a substrate.
  • the kit may comprise a substrate and a solution of molecules which comprise target-cell specific binding moieties in a form suitable for application to the substrate.
  • the target cell-specific molecules may, for example, be absorbed on to the solid substrate by incubating the solution of the molecule on the solid substrate.
  • the kit may also comprise a cell lysis means, for example a cell lysis solution.
  • the kit may also comprise a means for detecting the probe.
  • many of the detection means which may be used with the method of the first aspect of the invention are unsuitable for incorporation into kit form.
  • the present invention discloses a method for quantifying cells which can be carried out in a microplate and which lends itself to simultaneous automated cell counting for multiple samples.
  • a technique for counting leucocytes in solution which involves capture of the leucocytes on a substrate, and staining the nucleic acid with a quantitative fluorescent stain.
  • a method for quantitating fetal rhesus -positive red-blood cells in a blood sample of a rhesus -negative mother which involves capture of the foetal rhesus -positive red- blood cells on a substrate, and measuring the amount of haemoglobin in the captured cells from which a measure of the proportion of fetal cells in the maternal blood sample can be determined, and hence the volume of FMH.
  • the method is capable of consistently detecting leucocytes at concentrations down to concentrations of the order of 1 leucocyte ⁇ L "1 in whole blood, concentrated red cells, platelet concentrates and plasma. Detection is generally linear throughout the range 0.1-100 leucocytes ⁇ L "1 .
  • the method can be used for many cell quantification applications. It is generally applicable to any circumstance when it is necessary to quantitate specific cell types which are present in low numbers in mixed populations (for example, cell types which are present in low numbers in body fluids) . Examples of such circumstances include detection of CD34+ haemopoietic stem cells, analysis of fetal rhesus-positive red-blood cells in rhesus-negative mothers, analysis of cancer cells in leukaemia and detection of cells in cerebrospinal fluid (CSF) .
  • CSF cerebrospinal fluid
  • Figure 2 Fluorescent intensity against leucocyte concentration in SAG-M red cells.
  • Figure 3 Fluorescent intensity against leucocyte concentration in whole blood.
  • Figure 4 Fluorescent intensity against leucocyte concentration in leucodepleted platelet concentrate.
  • Figure 5 Fluorescent intensity against leucocyte concentration in unfixed and fixed whole blood samples.
  • M red cells, plasma and platelets were leucodepleted by in process filtration, with an in-line ATSBC-1 Autostop (Pall) filter.
  • Plasma was leucodepleted using the Gelman filter from a Pathlnact Methylene Elue kit (Baxter) .
  • SAG-M red cells were leucodepleted using a BP4 (Pall) filter.
  • Whole blood was leucodepleted using an integral RS2000 filter (Asahi) .
  • the leucocyte content of all prefiltration samples, except for plasma, was determined using a Sysmex haematology analyser. All filtered products, and unfiltered plasma, were analysed by flow cytometry as described previously (Rider et al . , 1996 as above).
  • ExtrAvidin (Sigma) was prepared to 20 ⁇ g mL "1 in bicarbonate buffer (pH 9.6) (3g of NaHC0 3 , 1.5g of
  • Calibrators were prepared by dilution of component with PBS or ⁇ "ith the leucodepleted equivalent component to give a series of known leucocyte concentrations. To test the agreement of the assay with current routine tests, blood product samples already analysed by flow cytometry in routine Product Testing were used. In all nine apheresis platelet samples, one haemonetics platelet sample and ten citrate-phosphate-dextrose leucodepleted red blood cell (CPD-LD-R3C) samples were tested 1 . Plates, loaded with sample, were incubated with agitation for 2 hours at 37°C, before being either just blotted or blotted and gently washed with PBS. Plates were frozen at -70°C for at least one hour. After defrosting, "blank plate” readings without the addition of nucleic acid stain were taken, using a fluorescent plate reader with excitation at 485nm and emission at
  • CyQUANT (Molecular Probes Europe, Leiden, Netherlands) working reagent containing lysis buffer and DNA stain was prepared according to the manufacturer's instructions and 200 ⁇ L added to each well. The plate was incubated in the dark for 5 minutes before being analysed on the plate reader as above. Longer incubation periods (for example of about 1 hour) may alternatively be used. All fluorescence readings were corrected by subtraction of fluorescence blanks. An alternative approach is to use a well containing 200 ⁇ L of CyQUANT as the blank and then the value obtained is used to correct all other fluorescence readings.
  • Leucodepleted platelet concentrate was spiked with leucocytes isolated from a whole blood sample (7ml EDTA) using histopaque . A range of leucocyte concentrations from 6 to 200 leucocytes ⁇ L "1 were tested. Following a 2 hour incubation and freeze-thaw step, plates were s t ained with CyQUANT and read at excitation 485/20, emission 530/25 and sensitivity 64. This assay showed that all leucocyte concentrations down to 6 leucocytes ⁇ L "1 gave above background readings.
  • a 96-well Immulon4 plate was prepared using the standard protocol. Two whole blood samples (7ml EDTA) were obtained. 70 ⁇ l of fixative solution (Transfix-NEQAS) was added to one sample. The samples were diluted with PBS to give a range of leucocyte concentrations from 3 to 4000 leucocytes ⁇ L "1 . Following a 2 -hour incubation and freeze-thaw step, plates were stained with CyQUANT and " read at excitation 485/20, emission 530/25 and sensitivity 60. This assay showed that leucocyte concentrations down to 3 leucocytes ⁇ L "1 in samples, both with and without fixative, gave above background readings .
  • Example 2 A biotinylated monoclonal antibody specific for fetal D+ RBCs, such as ERAD-3, is adhered to the wells of a 96 well plate using ExtrAvidin. Duplicate lOO ⁇ L samples of maternal blood are added to the microplate. The plate is incubated, with gentle shaking, at room temperature for 2 hours to facilitate binding of the antibody to any fetal D+ cells present. Following the incubation period, the samples are washed to remove any unbound RBCs . The captured fetal RBCs are lysed and sample absorbance at wavelengths of at 562nm, 578nm and 598nm is recorded. The concentration of haemoglobin is calculated as follows:
  • the concentration of haemoglobin remaining can be used to calculate the proportion of fetal cells in the maternal blood sample, and consequently the volume of FMH.
  • the microplate assay represents a simple, high throughput alternative to the Kleihauer- Betke acid elution technique or to flow cytometry, for quantitating FMH.
  • the cells may be quantified by analysing a cellular component of the captured target cell which is a cell surface component, the expression of which (on the target cell) is substantially uniform among the target cell population, in other words that the expression of the cell surface component of the target cells is substantially proportional to the number of cells in the fluid sample.

Abstract

L'invention porte sur un procédé de numération des cellules cibles présentes dans un prélèvement comportant les étapes suivantes: a) application du prélèvement sur un substrat pourvu de plusieurs fragments de fixation se fixant chacun spécifiquement aux cellules cibles de manière à maintenir lesdites cellules fixées à la surface du substrat, le nombre des fragments étant suffisant pour retenir sensiblement toutes les cellules cibles du prélèvement; b) lavage du substrat et des cellules cibles fixées pour éliminer toute matière étrangère non spécifiquement fixée; c) lise des cellules cibles fixées; et d) numération d'au moins l'un des composants desdites cellules pour obtenir une indication du nombre total de cellules retenues.
PCT/GB2000/002084 1999-05-28 2000-05-30 Procede de numeration de cellules et trousse a cet effet WO2001011362A1 (fr)

Priority Applications (1)

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AU50908/00A AU5090800A (en) 1999-05-28 2000-05-30 Cell counting method and kit

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GBGB9912631.0A GB9912631D0 (en) 1999-05-28 1999-05-28 Cell counter
GB9912631.0 1999-05-28

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005090984A1 (fr) * 2004-03-16 2005-09-29 Ambion, Inc. Procede et reactifs pour l'extraction d'arn a partir de leucocytes du sang fractionnes
US7141369B2 (en) 2002-04-25 2006-11-28 Semibio Technology, Inc. Measuring cellular metabolism of immobilized cells

Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0311492A2 (fr) * 1987-09-30 1989-04-12 Elf Sanofi Trousse et méthode de dosage immunométrique applicables à des cellules entières
JPH05215750A (ja) * 1992-02-06 1993-08-24 Idemitsu Petrochem Co Ltd 免疫学的分析方法
JPH11258231A (ja) * 1998-03-13 1999-09-24 Kdk Corp 白血球計数方法および白血球計数装置

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0311492A2 (fr) * 1987-09-30 1989-04-12 Elf Sanofi Trousse et méthode de dosage immunométrique applicables à des cellules entières
JPH05215750A (ja) * 1992-02-06 1993-08-24 Idemitsu Petrochem Co Ltd 免疫学的分析方法
JPH11258231A (ja) * 1998-03-13 1999-09-24 Kdk Corp 白血球計数方法および白血球計数装置

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Title
DATABASE WPI Week 38, Derwent World Patents Index; AN 1993-300220, XP002151026, "Immunoassay method-by fixing antibody to surface antigen of analyte cell on baseplate, contacting sample containing analyte cell , etc" *
DATABASE WPI Week 47, Derwent World Patents Index; AN 1999-561701, XP002151027, "Immunological method for counting Leucocytes with a miniaturizable counter, at low cost, with accuracy, without interfrence from other nucleated cells, precipitated fibrin or the skill of operator" *
MURTHY V V ET AL: "A SIMPLE SPECTROPHOTOMETRIC ASSAY FOR URINARY LEUKOCYTE ESTERASE ACTIVITY", BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY,, vol. 40, 1988, pages 260 - 268, XP000918201 *
PATENT ABSTRACTS OF JAPAN vol. 017, no. 654 (P - 1653) 3 December 1993 (1993-12-03) *
PATENT ABSTRACTS OF JAPAN vol. 1999, no. 14 22 December 1999 (1999-12-22) *
PICARD F ET AL: "PRELIMINARY EVALUATION OF THE NEW HEMATOLOGY ANALYZER COULTER GEN.S IN A UNIVERSITY HOSPITAL", CLINICAL CHEMISTRY AND LABORATORY MEDICINE,WALTER DE GRUYTER UND CO,DE, vol. 37, no. 6, 1999, pages 681 - 686, XP000918440, ISSN: 1434-6621 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7141369B2 (en) 2002-04-25 2006-11-28 Semibio Technology, Inc. Measuring cellular metabolism of immobilized cells
US7855068B2 (en) 2002-04-25 2010-12-21 Semibio Holdings Limited Methods and kits for detecting a target cell
WO2005090984A1 (fr) * 2004-03-16 2005-09-29 Ambion, Inc. Procede et reactifs pour l'extraction d'arn a partir de leucocytes du sang fractionnes

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Publication number Publication date
GB9912631D0 (en) 1999-07-28
AU5090800A (en) 2001-03-05

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