WO2011123070A1 - Procédé pour la surveillance du développement de parasites dans le sang - Google Patents

Procédé pour la surveillance du développement de parasites dans le sang Download PDF

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WO2011123070A1
WO2011123070A1 PCT/SG2011/000135 SG2011000135W WO2011123070A1 WO 2011123070 A1 WO2011123070 A1 WO 2011123070A1 SG 2011000135 W SG2011000135 W SG 2011000135W WO 2011123070 A1 WO2011123070 A1 WO 2011123070A1
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reacts
nucleic acid
dna
acid dye
blood
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Laurent RÉNIA
Benoît MALLERET
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Agency For Science, Technology And Research
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    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa

Definitions

  • the invention relates generally to methods and kits for determining parasitemia and blood quantification of a blood sample.
  • Haematozoa is a general term that includes blood parasites, mainly protozoans. Well known examples include the protozoan parasites causing malaria, Leishmaniasis and trypanosomasis. Intra-erythrocytic haematozoa results from the multiplication of parasites within erythrocytes (red blood cells ' ). Often the parasites have complex life cycles that include vertebrate hosts and invertebrate vectors. These diseases include malaria, filariasis, Chagas disease, leishmaniasis and African sleeping sickness. Some of these diseases cause severe anaemia or blood loss and may require the use of blood products or transfusions in order to save lives. Diagnosis and treatment reduces the need for blood transfusions.
  • the two diagnostic methods recommended by WHO are: optic microscopy to quantify malaria parasites via microscopic examination of Giemsa-stained thin blood smear; or rapid diagnostic tests (RDT) based on lateral flow immunochromatography.
  • the major limit of the optic microscopy technique is that the counts obtained vary depending on the expertise of the observer. Moreover, the process is tiring and time consuming. Implementation of RDT is not widespread due to poor product performance, inadequate methods to determine the quality of products and poor storage for such tests in tropical climates.
  • the cell composition of mammalian blood predominantly comprises erythrocytes, thrombocytes and leukocytes.
  • Mammalian erythrocytes are non-nucleated cells in their mature form. Because of the lack of nuclei and organelles, mature red blood cells do not contain DNA and cannot synthesize any RNA and consequently cannot divide and have limited repair capabilities.
  • Reticulocytes are immature erythrocytes (red blood cells). Reticulocytes develop and mature in the bone marrow and then circulate for about a day in the blood stream before developing into mature red blood cells. Like mature red blood cells, reticulocytes do not have a nucleus. They are characterized by a reticular (mesh-like) network of ribosomal RNA. The normal range of values for reticulocytes in the blood is 0.5% to 1.5%. However, if a person has anemia, their reticulocyte percentage is usually higher than normal. A very high number of reticulocytes in the blood can be described as reticulocytosis and this can happen during diverse pathologies such "as hemochromatosis
  • Mammalian thrombocytes are small, regularly-shaped clear cells also with no nuclei. Thrombocytes also called platelets are involved in blood clotting.
  • Leukocytes are found in blood. Leukocytes comprise granulocytes, lymphocytes and monocytes. The number of leukocytes in the blood is often an indicator of disease. There are normally between 4x l0 9 and l .l x lO 10 white blood cells in a litre of blood, making up approximately 1% of blood in a healthy adult. An increase in the number of leukocytes is called leukocytosis, and a decrease is called leukopenia.
  • One aspect of the invention provides a method for determining parasitemia and blood quantification of a blood sample comprising the steps of:
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the cells in the blood sample may refer to all the cells in the blood including erythrocytes, thrombocytes and leukocytes or the cells in the blood sample may refer to erythrocyte cells.
  • the method may further comprise calculating the number of infected cells and/or leukocytes and/or reticulocytes as a percentage of the total number of the cells in the blood sample.
  • kits to detect parasitemia and blood quantification of a blood sample comprising, a nucleic acid dye that reacts with deoxyribonucleic acid; a nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid and a antibody coupled to a flurorophore capable of selectively binding a marker present on all leukocytes.
  • Figure 1 Flow cytometry analysis of the parasitemia in a C57BL/6 mouse infected with the rodent malaria parasite Plasmodium berghei ANKA.
  • Figure 4 Flow cytometry analysis of the parasitemia in a patient infected with Plasmodium vivax Figure 5.
  • FIG. 6 Validation of the technique for drug testing. Plasmodium falciparum (strain 3D7) culture was treated in triplicate during 24 hours with 19 ng/mL of Artesunate (AS) or 512 ng/mL of Chloroquine (CQ). The percentage of inhibition on schizonts development is 83.7% and 79.0% respectively, with these two high doses.
  • AS Artesunate
  • CQ Chloroquine
  • Figure 7 Flow cytometry analysis of the parasitemia in a whole blood samples from two patients infected with Plasmodium vivax detected with ultra violet light (A) or a laser in the violet spectrum (B).
  • FIG. 8 (A) Representative Dot plot of a synchronized culture of P. falciparum (clone 3D7) with the Ethidium/Hoechst double staining before and after 24 hours of culture. The developmental blood stages were sorted by Flow Cytometry and (B) stained with Giemsa. Representatives images of (1) a single ring, (2) a double ring, (3) a trophozoite, (4) an early schizont and (5) a late schizont.
  • Figure 9 Flow cytometry analysis of the parasitemia in whole blood blood from two patients infected with P. falciparum.
  • Whole blood samples from two patients infected with P. falciparum were stored frozen in nitrogen. On the day of the experiment, blood samples were thawed and analyzed with the tri-color Flow Cytometry method.
  • FIG 10. Flow cytometry analysis of the parasitemia in a whole blood samples from one patient infected with P. vivax and identification of the different developmental blood stages in the different gates.
  • Whole blood samples from a P. vivax-infected patient was stored frozen in nitrogen. On the day of the experiment, it was thawed and analyzed by the tri-color Flow Cytometry (A).
  • the developmental blood stages were sorted by flow cytometry and stained with Giemsa.
  • B The pictures represent the different life cycle stage, (1) rings and (2) late cycle stages (trophozoite, schizont), present in the DNA gate after cell sorting and Giemsa staining.
  • Our tricolor method is based on the use of a flow cytometer having 3 lasers and in particular a UV laser for excitation of the Hoechst dye.
  • the method was adapted to be used for Flow cytometer having only two lasers (488nm and 635nm).
  • the Hoechst dye was replaced by the SYBR green dye.
  • Validation of the Tricolor method wit two laser Flow cytometers. by Comparison of the two techniques for measurement of parasitemia in whole blood of mice infected by P. berghei .
  • the technique includes a a method for determining parasitemia and blood quantification of a blood sample comprising the steps of: e) adding to the sample a nucleic acid dye that reacts with deoxyribonucleic acid (DNA); a nucleic acid dye that reacts with DNA and ribonucleic acid (RNA) and an antibody coupled to a fluorophore capable of selectively binding a marker present on a leukocyte;
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the cells in the blood sample may refer to all the cells in the blood including erythrocytes, thrombocytes and leukocytes or the cells in the blood sample may refer to erythrocyte cells.
  • the method may further comprise calculating the number of infected cells and/or leukocytes and/or reticulocytes as a percentage of the total number of the cells in the blood sample.
  • the number of cells containing a parasite and/or leukocytes and/or reticulocytes in relation to the total number of the cells in the blood samples may be measured as a ratio or a fraction.
  • the fraction may have a denominator of 10 or may have a denominator of 100 or may have a denominator of 1000.
  • Preferably the fraction is measured as a percentage of the total number of the cells in the blood sample.
  • the method has the advantage of being able to Screen a large number of samples in a short time. As the incubation time is short and no washing steps are required. In terms of accuracy, the counting has the added advantage when it is performed by a Flow cytometer of reducing any variability to a minimum and having high reproducibility.
  • the method may include analysing the light emission pattern, being a reflection of the events detected in a Flow Cytometer, in the following way:
  • the first proportion is calculated as a first percentage of the total number of the cells in the blood sample, the first percentage indicates a number of leukocytes cells as a percentage of the total number of the cells in the blood sample whereby greater than 1% in combination with a positive number for the forth proportion is further indication of parasitemia.
  • the third proportion is calculated as a third percentage of the total number of the cells in the blood sample, the third percentage indicates a number of reticulocyte cells as a percentage of the total number of the cells in the blood sample whereby when the third percentage is greater than 1.5% in combination with a positive number for the forth proportion is further indication of parasitemia.
  • the method is performed using a Flow Cytometer and the proportion of cells of interest in relation to the cells in the blood sample is determined. The calculations are preferably done in terms of percentage
  • the method can be adapted to monitor a range or parasites.
  • parasitemia is caused by haematozoans such as Plasmodia and Babesia
  • mycoplasma such as hemobartonella or other parasites with a blood phase such as Trypanosomas, Wuchereria Bancrofti, Loa loa, Brugia, Mansonella and Dirofilaria.
  • the parasite is an intra-erythrocytic parasite such as Plasmodia; babesia and bartonella.
  • the parasite is P. falciparum or other human Plasmodia such as P. vivax, P. ovale, P. malariae , rodent malaria parasites such as P_. berghei, P. yoelii, P. vinckei, P. chabaudi., or primate malaria such as P. knowlesi or P. cynomolgi.
  • the Haematozoa parasites are blood parasites having an extra-erythrocytic blood phase such as Trypanosoma, Wuchereria bancrofti, Loa loa, Brugia malayi/timori, Mansonella, Dirofilaria.
  • the technique can quantify a wide range of protozoans and haematozoan parasites known in the art.
  • Blood samples may be taken from any subject including a vertebrate or a mammal either living or dead. Preferable the blood sample is taken from a human subject. The sample may be a fresh sample drawn from a subject and tested immediately or it may be a stored sample. Samples are preferably stored at 4°C in sterile conditions for later detection.
  • the method uses blood containing intact cells referred to as an intact blood sample. This allows the calculation of the proportion of erythrocytes actually containing parasites in relation to the cells in the sample. As the technique does not require hemolysis, or the lysis of the blood cells, it reduces the processing steps and thus time of sample processing. Further, only the proportion of erythrocytes actually containing parasites is determined rather than the propotion of the total number of parasites in relation to the volume of the sample, which may result in more cells overestimating parasitemia.
  • a nucleic acid dye that reacts with deoxyribonucleic acid may include any DNA fluorochromes such as Hoeschst 33342, Ethidium Bromide Hoeschst 33258, DAPI, SYTOX Blue, Chromomycin A3, SYTOX Green, mithramycin, 7-AAD, TO-PRO-3, propidium iodide, SYBR green 1, thiazole orange (TO) and its derivatives, and propidium iodide (PI) or any other dyes known to those skilled in the art to react with DNA. Many of these dyes are commercially available. Effectively, such dyes can be used detect parasites and leukocytes.
  • DNA fluorochromes such as Hoeschst 33342, Ethidium Bromide Hoeschst 33258, DAPI, SYTOX Blue, Chromomycin A3, SYTOX Green, mithramycin, 7-AAD, TO-PRO-3, propidium io
  • nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid
  • a nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid may include dihydro ethidium, acridine orange, pyronin Y (PY), oxazine 1, LDS 751, 7-AAD, SYTOX Orange, or any other dyes known to those skilled in the art to react with both DNA and RNA. Effectively, such dyes can be used detect parasites leukocytes and reticulocytes. This allows quantification of reticulocytes.
  • a an antibody coupled to a fiuorophore capable of selectively binding a leukocyte cell marker present on all leukocyte populations may be coupled with any flurorophore known in the art to be capable of binding to an antibody and produce a signature emission pattern under a light stream.
  • the fluorescently labeled antibody is exposed to light of a proper wave length, its presence can then be detected due to fluorescence of the fluorophore.
  • fiuorophores fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin, phycocyanin, allophycocyanin (APC), o-phthaldehyde, sulforhodamine 101 acid chloride (Texas Red), fluorescamine or fluorescence-emitting metals such as 152 Eu or other lanthanides.
  • FITC fluorescein isothiocyanate
  • APC allophycocyanin
  • o-phthaldehyde o-phthaldehyde
  • sulforhodamine 101 acid chloride Texas Red
  • fluorescamine or fluorescence-emitting metals such as 152 Eu or other lanthanides.
  • metals are attached to antibodies using metal chelators.
  • the fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a fiuorometer or a photosensor such as CCD camera equipped with appropriate emission filters. Effective
  • the labels generally are light emitting fluorescent tags such as phycoerythrin, fluorescein (green light) or rhodamine (red light).
  • the fluorescent label often is conjugated to a binding agent, such as an antibody, capable of binding to a component of the cell surface.
  • Cell fluorescence is indicative of the presence of the binding partner, such as an antigen, of the binding agent on the cell surface.
  • the intensity of the fluorescence is a function of the number of fluorescent labels bound per cell, and is thus related to the number of binding partners available on the surface of the cell.
  • a leukocyte cell marker should be a cell marker that is found on all leukocyte cells and not found on red blood cells or reticulocyte cells. Commonly known leukocyte cell marker present on all leukocyte populations is CD45. Hence, the leukocyte cell marker is CD45.
  • the protein sequences of leukocyte cell marker CD45 maybe quickly obtained by a person skilled in the art at the swissprot/uniprot database at accession number Q64224. Based on the sequence obtained the protein can be manufactured using techniques well known in the art such as being synthesized chemically using known techniques such as liquid phase synthesis or solid phase synthesis such as t-BOC or Fmoc, or BOP SPPS or any chemical synthesis method known to those in the art.
  • the peptide may be made biologically within a cell or vector designed to use the machinery of translation and/or transcription for the leukocyte cell marker synthesis.
  • the cell surface marker recognizing all leukocytes can be bought commercially for use in making fluorophore-coupled antibodies capable of selectively binding to the specific cell surface marker required.
  • the present invention also provides polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, which are capable of specifically binding to the leukocyte cell polypeptides and fragments thereof.
  • Antibodies against the leukocyte cell marker, CD45 may be bought commercially from any of the known suppliers such as Abnova, Sigma, Biorad or other such companies or they may be made according to any of the methods known in the art. Exemplary methods include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, Fab expression library, humanized, bispecific, and heteroconjugate antibodies. According to the invention, a polypeptide of a leukocyte cell marker produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as an immunogen to generate antibodies that recognize the leukocyte cell marker polypeptide.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, as well as antigen binding portions of antibodies, including Fab, F(ab') 2 and F(v) (including single chain antibodies).
  • the phrase "antibody molecule” in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule containing the antibody combining site.
  • An “antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • the sample may be excited with light such as laser light and there may be a variety of wavelengths used.
  • the light may be in a UV wavelength where ethidium bromide is used as the nucleic acid dye that reacts with deoxyribonucleic acid (DNA).
  • An argon laser (488 nm) may be employed as the light source.
  • the light is emitted from a flow cytometer. In one embodiment two lasers cytometer are used at488-nm and 633 nm to detect Ethidium bromide and sybr green respectively.
  • the light emission pattern is detected and measured in a flow cytometer.
  • Flow cytometry is an analytical method that allows both the rapid measurement of scattered light for particle size determination and measurement of fluorescence emission produced by suitably illuminated particles. The particles are suspended in liquid and produce signals when they pass through a beam of light individually. Because measurements of each particle are made separately, the results are a correlated set of each individual particle's characteristics.
  • An important analytical feature of flow cytometry is its ability to measure multiple particle parameters such as scattered light and fluorescence emission. Scattered light collected in the same direction as the incident light (FSL) reflects cell size and granularity where as the fluorescence is dependent upon the presence of fluorochromes on particles. Thus, a combination of light scattering and fluorescence is a powerful approach to detect multiple targets in one sample without the need for a separation step.
  • the method is carried out using flow cytometry.
  • flow cytometry and “flow cytometric” and “cell sorter” refer to the instruments and procedures whereby a population of cells in a liquid suspension is directed through a fine liquid stream passing by the cytometer's laser into a device capable of measuring the physical and/or chemical characteristics of cells based on the light quanta emitted. The light passing through each cell is measured individually and can represent physical characteristics of cells that are unlabeled and also cells that have the various labels/dyes of the invention attached.
  • the cell sorter exposes the cells moving through the liquid stream to light, usually a specific wavelength, known as the excitation wavelength that corresponds to the fluorescent label used. In response to excitation wavelengths, the fluorescent label fluoresces and emits light.
  • the cell sorter detects and records the emission of light, both the number of discrete occurrences and the intensity of the light emitted.
  • the cell sorter can momentarily divert the cell stream so as to separate fluorescing cells from non-fluorescing cells, or separate cells having different wavelength fluorescence, or those having fluorescence exceeding a certain predetermined minimum intensity from the rest of the cells in the population.
  • the light quanta emitted is measured for each cell individually and presented as such in data representations.
  • a population of cells can be characterized by parameters such as percentage of fluorescing cells, percentage of non- fluorescing cells, and population profiles of cells having various fluorescence intensities (See Figures 1).
  • Multiparameter flow cytometry allows one to estimate, with high accuracy, relative quantities of a variety of cells simultaneously.
  • the measurements are recorded in a list mode, it is possible to attribute each of the several measured features to a particular cell and thus to obtain correlated measurements of these features on a cell by cell basis.
  • Cellular heterogeneity can thus be estimated and subpopulations with distinct characteristics can be discriminated.
  • multiparameter flow cytometry offers improved opportunities to describe the complex relationships in a sample.
  • the expression profile may include at least one component of the forward scatter, the side scatter and the fluorescence signature.
  • the expression profile includes all three components.
  • Parasitemia analysis is done on the basis of determining the percentage (%) of infected red blood cells per total red blood cells. It is calculated as the percentage of cells positive for the nucleic acid dye that reacts with deoxyribonucleic acid and the nucleic acid dye that reacts ribonucleic acid (containing both infected red blood cells and leukocytes) minus the percentage of cells positive for both the nucleic acid dye that reacts deoxyribonucleic acid and the antibody selectively binding the leukocyte cell marker (corresponding to the leukocytes).
  • the technology can differentiate the reticulocytes from the leukocytes or other nucleated cells because the reticulocytes are positive for the nucleic acid dye that reacts with ribonucleic acid and is negative for the nucleic acid dye that reacts with deoxyribonucleic acid and the antibody selectively binding the leukocyte cell marker while the leukocytes are positive for all three of the dyes i.e., the nucleic acid dye that reacts with ribonucleic acid; and the nucleic acid dye that reacts deoxyribonucleic acid; and the antibody selectively binding the leukocyte marker recognizing all leukocyte populations.
  • the method may also allow determining parasite developmental stages. Because the different developmental stages possess different quantity of DNA and RNA, they can be identified by this method.
  • a cell sorting experiment may be used to confirm that the parasite stages detected by the methods of the invention are indeed the real one (ascertained by Giemsa-stained blood smears).
  • Cell sorting is done by the step of enriching the blood sample with magnetic sorting.
  • To enriched the sample it may be magnetically sorted using the MACS Miltenyi technology or other magnetic sorting techniques such as dyna beads by dynal or any other method that sorts heme containing cells magnetically.
  • Late blood stage parasites synthesize high quantity of the pigment hemozoin (Hz), which is rich in the iron metal and thus can be retained by the magnet on the purification column more easily, particularly of late stage infection. This allows the technique the ability to detect the different stages of the parasites. The early and late developing stages of the parasites can be identified.
  • a kit to detect parasitemia and blood quantification of a blood sample comprising, a nucleic acid dye that reacts with deoxyribonucleic acid; a nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid and a fluorophore- coupled antibody capable of selectively binding a marker recognizing all leukocyte populations. Exemplary dyes and markers are listed above.
  • the kit is for use in with flow cytometry as described above.
  • the kit to specifically detect parasitemia and blood quantification of a blood sample comprising, a nucleic acid dye that reacts with deoxyribonucleic acid (DNA); a nucleic acid dye that reacts with deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and a fluorophore-coupled capable of selectively binding a marker recognizing all leukocyte populations.
  • the nucleic acid dye that reacts with DNA of the kit comprises Hoeschst 33342, Ethidium Bromide Hoeschst 33258, DAPI, SYTOX Blue, Chromomycin A3, SYTOX Green, mithramycin, 7-AAD, TO-PRO-3, propidium iodide SYBR green 1, thiazole orange (TO) and its derivatives, or propidium iodide (PI).
  • the kit may comprises Hoeschst 33342 as the nucleic acid dye that reacts with DNA.
  • the kit may comprises SYBR green as the nucleic acid dye that reacts with DNA.
  • the nucleic acid dye that reacts with DNA and RNA of the kit may comprise dihydroethidium, acridine orange, pyronin Y (PY), oxazine 1, LDS 751, 7-AAD, or SYTOX Orange.
  • the kit may comprises
  • the dyed antibody capable of selectively binding a leukocyte cell marker of the kit comprises a detectable label capable of producing a signature emission as described above.
  • the antibody of the kit may comprise antibodies capable of selectively binding a leukocyte cell marker described above such as CD45 .
  • the kit may further comprising magnetic beads suitable for cell sorting.
  • the parasites are stained using two dyes, dihydroethidium (Sigma) and Hoeschst 33342 (Sigma), which react with parasite DNA. Since red blood cells have no DNA, only parasite DNA is stained. We also detect leukocytes in the same sample using an antibody against the CD45 molecules coupled to allophycocyanine (APC) (Miltenyi). This marker is expresses on all leukocytes but not on red blood cells. The whole procedure requires 20 minutes to be performed at room temperature and no washing step. Acquisition of the data is performed using a flow cytometer with Blue and U.V lasers (See Fig 1).
  • APC allophycocyanine
  • Dot plots represent: (left) forward-scatter/side-scatter (FSC-A/SSC-A) scatter gram of representative whole blood sample Events (representing blood cells) are acquired and their size (FSC-A) and granularity (SSC-A) recorded. 2. A secondary analysis allows excluding duplets (FSC-A/SSC-W).
  • Duplets represents aggregated cells and may bias the analysis.
  • the parasitemia (% of infected red blood cells/total red blood cells) is calculated as the percentage of cell positive for Hoechst and Ethidium (containing both infected red blood cells and leukocytes) minus the percentage of cells positive for Hoechst and anti-CD45 (corresponding to the leukocytes).
  • the commercial application is the development of a kit for the parasitemia quantification in mammalian blood such as in human blood.
  • the Hoechst can be excited also with a violet laser or a U.V. laser.
  • the UV laser is preferable.
  • the excitation can be done with a green laser.
  • the technology can differentiate the reticulocytes from the leukocytes because the reticulocytes are positive for the Dihydroethidum staining and negative for the Hoechst and CD45 staining while the leukocytes are positive for the Dihydroethidum, Hoechst and CD45 staining.
  • the kit can be extended to monitor the Haematozoa (see the list below) parasites, grouped in two different subsets. The first one is the intra-erythrocytic Haematozoa, such as Plasmodium falciparum, and the second one is the extra- erythrocytic blood Haematozoans or mycoplasma. The technique can be easily adapted to quantify these parasites. [079]. List of Haematozoa infecting mammals:
  • Intra-erythrocytic Haematozoa protozoa
  • Figure 8 shows Representative Dot plot of a synchronized culture of P. falciparum (clone 3D7) with the Ethidium/Hoechst double staining before and after 24 hours of culture.
  • the developmental blood stages were sorted by flow cytometry and stained with Giemsa (1) single ring, (2) double ring, (3) trophozoite, (4) early schizont and (5) late schizont.
  • CD45/Hoechst staining and Ethidium/Hoechst staining was used in this experiment.
  • the invention described herein may include one or more range of values (eg size, concentration etc).
  • a range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

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Abstract

L'invention concerne un procédé et une trousse pour détecter une parasitémie et pour la quantification sanguine d'un échantillon sanguin avec plusieurs colorants dans un cytomètre de flux, ce qui permet la quantification d'une proportion de cellules infectées dans l'échantillon contenant un parasite par rapport à un nombre total d'érythrocytes dans l'échantillon. Ce procédé permet également la quantification de la proportion d'érythrocytes, de leucocytes ou de réticulocytes par rapport au nombre total de cellules présentes dans les échantillons.
PCT/SG2011/000135 2010-03-31 2011-03-31 Procédé pour la surveillance du développement de parasites dans le sang WO2011123070A1 (fr)

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