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
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • 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
  • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/22—Haematology
  • G01N2800/222—Platelet disorders

Definitions

• the present invention relates to a method for the analysis of platelet activation induced by antibodies directed against the platelet factor 4 (FP4) modified by a polyanion, preferably directed against the FP4/heparin complex, as well as a method for the in in vitro diagnosis of heparin-induced thrombocytopenia or a diagnosis of exclusion of heparin-induced thrombocytopenia in a patient, comprising specific steps.
• FP4 platelet factor 4
• Heparin-Induced Thrombocytopenia is a severe complication of heparin therapy. It is the consequence of an atypical immune response characterized by the synthesis of IgG class antibodies specifically directed against platelet factor 4 (FP4) modified by a polyanion, for example heparin (H). These polyanion-modified anti-FP4 antibodies activate platelets via the Fc ⁇ RIlA receptor.
• FP4 platelet factor 4
• H heparin
• Heparin is widely used in hemodialysis, heart surgery including extracorporeal circulation (ECC), extracorporeal membrane oxygenation (ECMO) procedures and intensive care.
• ECC extracorporeal circulation
• ECMO extracorporeal membrane oxygenation
• UHF unfractionated heparin
• LMWH low molecular weight heparin
• HIT is in fact considered a clinical-biological syndrome, the complete diagnosis of which is based on:
• heparin-dependent activation of platelets by these antibodies demonstrate heparin-dependent activation of platelets by these antibodies.
• platelet activation is demonstrated in the presence of therapeutic concentrations of heparin (i.e. between 0.1 and 1 IU/ml of heparin), whereas it is completely inhibited by a high concentration of heparin (i.e. between 10 and 100 IU/ml).
• the most effective tests are the radiolabeled serotonin release test (SRA or Serotonin Release Assay) and the HIPA test (Heparin Induced Platelet Agregation Assay), both carried out with fresh washed platelets obtained from one or more healthy donors. .
• SRA and HIPA are greater than 95%.
• Other functional tests that also use platelets from healthy subjects are also available but show poorer performance.
• two commercial tests use the flow cytometry technique: Emo-test HIT Confirm® (5-Diagnostics, Basel, Switzerland) and HITAIertTM KIT (IQ Products, DL Groningen, Netherlands).
• Emo-test HIT Confirm® 5-Diagnostics, Basel, Switzerland
• HITAIertTM KIT IQ Products, DL Groningen, Netherlands.
• the performances of the functional tests available today are variable, with a heterogeneity of the results due to the use of different methodologies, the variability of platelets washed from healthy donors, but also different positivity criteria to
• HIT results from multicellular activation induced by pathogenic anti-FP4/H antibodies.
• monocytes and neutrophils are also stimulated by these antibodies and can potentiate platelet activation.
• HIMEA Heparin-lnduced Multiple Electrode Aggregometry
• the present invention makes it possible to meet these expectations.
• the present invention thus relates to a method for analyzing platelet activation in a patient suspected of having thrombocytopenia induced by anti-FP4 antibodies. modified with a polyanion, in particular antibodies directed against the heparin-modified factor FP4.
• the present invention also relates to a method for the in vitro diagnosis of a Tl H or for confirming the diagnosis of a Tl H in a patient suspected of having a Tl H.
• It also relates to a method for an in vitro diagnosis of exclusion of a Tl H in a patient suspected of having a HIT.
• the subject of the present invention is a method for analyzing heparin-dependent platelet activation in a patient suspected of having thrombocytopenia induced by anti-FP4 antibodies modified by a polyanion, in particular antibodies directed against heparin-modified factor FP4, comprising the following steps:
• Validation/Standardization (VS) series said Validation/Standardization series comprising:
• Test Patient series comprising:
• mixture 2b at least one mixture of a whole blood sample from said patient, diluted in an acceptable buffer, with FP4 and heparin in a concentration equal to that used for mixture 1b, in order to obtain mixture 2b;
• the method according to the invention is an in vitro method.
• step 4) of analyzing all the mixtures obtained in step 3) comprises measuring the specific labeling intensity of the activated platelets in the platelet population of each mixture of the VS series (1 a to 1c ) and TP series (Ago, Basal, and 2a to 2c).
• the method of the invention is a method of analysis by flow cytometry.
• the method of the invention comprises steps 1) to 4) set out above, as well as the following additional step 5), following step 4):
• the VS series is considered to be validated if one observes:
• step 5 consists of validating the series VS by calculating the activation ratio of this series and calculating the inhibition ratio of this same series.
• heparin-dependent platelet activation means a platelet activation induced by anti-FP4 antibodies (or antibody fragments) modified by a polyanion, or antibodies (or antibody fragments) mimicking polyanion-modified anti-FP4 antibody, which is observed in the absence of heparin and/or in the presence of low concentrations of heparin, and which is inhibited in the presence of high concentrations of heparin.
• low concentration of heparin we mean a concentration typically between 0.01 and 1 ⁇ l/ml.
• “High concentration of heparin” means a concentration typically between 10 and 200 IU/ml.
• the method of the invention preferably comprises a step 5′) of calculating the antibody-dependent platelet activation rate (called APA) of the Ago, 2a, 2b and 2c mixtures of the TP series .
• APA antibody-dependent platelet activation rate
• the method of the invention is implemented for the exclusion diagnosis of HIT, or the confirmation of a HIT diagnosis in a patient suspected of having the disease.
• the method of the invention comprises steps 1) to 5′) set out above, as well as a step for interpreting the results obtained on the TP series for establishing a diagnosis of HIT or an exclusion from TIH.
• the method of the invention comprises steps 1) to 5′) stated above, as well as a step 6), in which step 6) is the following:
• the method according to the invention uses whole blood samples from a patient.
• whole blood is meant a sample of blood comprising plasma and its formed elements (red blood cells, white blood cells and platelets and derived microparticles).
• the use of the patient's whole blood eliminates the variability of response to HIT antibodies in platelets from healthy donors.
• the real activating character of the patient's antibodies is directly evaluated on his own platelets.
• each sample is obtained from blood samples, the collection of which is carried out in the presence of a compound preventing the blood from clotting.
• the compound usually used is sodium citrate, but other compounds such as hirudin or citric acid-trisodium citrate-glucose (ACD) can also be used for this purpose.
• ACD citric acid-trisodium citrate-glucose
• the total patient whole blood sample volume is between 500 pL and 2 ml.
• the whole blood sample can be diluted in any physiologically acceptable buffer.
• Physiological saline is preferably used (ie aqueous solution of NaCl 9 g/l), but other buffers such as for example PBS buffer with different compositions (for example NaN 3 10X or Citrate NaN 3 10X) can also be used. for the dilution of the blood sample.
• All the samples of whole blood from the patient used in the method according to the invention are used undiluted or diluted up to 1/10th, preferably diluted in the proportion 1/2 to 1/ 10th , and preferably to a quarter, in physiological saline.
• a dilution makes it possible to use thrombocytopenic blood (ie in which the quantity of platelets less than 150 Giga/L).
• the VS series includes at least 3 mixtures:
• - mixture 1a which corresponds to the mixture of a sample of whole blood from said patient, diluted in an acceptable buffer, with exogenous FP4 and a monoclonal antibody directed specifically against FP4 modified by a polyanion, or one of its fragments ;
• mixture 1b which corresponds to the mixture of a sample of whole blood from said patient, diluted in an acceptable buffer, with exogenous FP4, a monoclonal antibody directed specifically against the FP4 modified by a polyanion, or one of its fragments, and heparin in a concentration of between 0.01 and 1 IU/ml;
• - mixture 1c which corresponds to the mixture of a sample of whole blood from said patient, diluted in an acceptable buffer, with exogenous FP4, a monoclonal antibody directed specifically against FP4 modified by a polyanion, or one of its fragments , and heparin in a concentration of between 10 and 200 IU/ml.
• each of the mixtures 1a to 1c has a volume of between 20 and 200 pl.
• the aforementioned antibody perfectly mimics the effects of Tl H antibodies developed by patients. It is used as a positive internal quality control and thus allows the technical validation of the test, as well as a standardized expression of the results, which contributes to the innovative character of the invention.
• the preparation of the VS series includes the preparation of at least 4 mixtures:
• two mixtures 1b are carried out on the one hand with a heparin concentration of between 0.01 and 0.1 IU/ml for the first mixture 1b', and on the other hand of between 0.2 and 1 IU/ml for the second mix 1 b”. More preferentially, the two mixtures 1b are carried out with a heparin concentration of between 0.02 and 0.08 IU/ml, preferably about 0.05 IU/ml, for the first mixture 1b'; and on the other hand with a heparin concentration between 0.3 and 0.8 IU/ml, preferably around 0.5 IU/ml, for the second mixture 1 b”.
• Mixture 1c is more preferably obtained with a heparin concentration of between 50 and 150 IU/ml, preferably between 80 and 120 IU/ml, preferably approximately 100 IU/ml.
• Mixes 1b (1 b’ and 1 b”) mimic the effects of low concentrations of heparin, while mixture 1c mimics the effects of high concentrations of heparin.
• polyanion-modified FP4 By “monoclonal antibody directed specifically against polyanion-modified FP4, or one of its fragments”, is meant an antibody or one of its active fragments (i) which binds to polyanion-modified FP4, and which does not bind, or binds insignificantly, to FP4 alone, and (ii) which induces platelet activation and aggregation in the absence and/or presence of low concentrations of heparin, and whose action is inhibited in the presence of high concentrations of heparin.
• such an antibody or one of its active fragments (i) binds to the FP4/heparin complex ("FP4/H complex"), and does not bind, or binds insignificantly, to FP4 alone , and (ii) induces platelet activation and aggregation in the absence and/or presence of low concentrations of heparin, and whose action is inhibited in the presence of high concentrations of heparin.
• FP4/H complex the FP4/heparin complex
• the low concentrations of heparin in the presence of which such an antibody activates the platelets are generally between 0.01 and 1 IU/ml.
• the high concentrations present which inhibition of the activating effect of said antibody is observed are generally between 10 and 200 IU/ml, typically of the order of 100 IU/ml.
• this antibody is a whole antibody.
• this antibody is an IgG, preferably an IgG1.
• This antibody can be chimeric, humanized, or human.
• Preferably it comprises a human Fc.
• antibody we mean a tetramer made up of two heavy chains of 50-70 kDa each (called the H chains for Heavy) and two light chains of approximately 25 kDa each (called the L chains for Light), linked by intra- and inter-catenary disulphide bridges which are identical to each other.
• This tetramer comprises at least two variable regions at the N-terminal end of each chain (called VL for the light chains and VH for the heavy chains) and a constant region at the C-terminal end, consisting of a single domain called CL for the light chain and three or four domains for the heavy chain called CH1, CH2, CH3 and possibly CH4.
• variable parts VH and VL The region which determines the specificity of the antibody for the antigen is carried by the variable parts VH and VL, it is these parts which are responsible for the recognition of the antigen.
• VH and VL variable region three loops are brought together to form an antigen binding site.
• Each of the loops is called a Complementarity Determining Region (or CDR).
• the base of the Y corresponds to the Fc constant region, or Fc fragment: it is recognized by complement proteins and Fc receptors in order to mediate the effector functions of the antibody, and
• the ends of the arms of the Y correspond to the respective assembly of the variable region VL of a light chain and of the variable region VH of a heavy chain, said ends constituting the Fab region and determining the specificity of the antibody for the antigen.
• the Fab fragment has the same affinity for the antigen as the whole antibody.
• the Fab fragment is formed from the entire light chain (VL+CL) and part of the heavy chain (VH+CH1). It is monovalent.
• the monoclonal antibody (or one of its fragments) directed specifically against the FP4 modified by a polyanion, and preferably against the FP4/H complex comprises the VH and VL sequences encoded by the following nucleic acid sequences: Sequence nucleic acid encoding the variable region VL (VJ): GAAATTGTGCTCACCCAGTCTCCAACCATGGCTGCATCTCCCGGGGAGAAGATC ACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTGGTATCAGC AGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATCCAATCTGGCTTCTGG AGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGG CACCATGGAGGCTGAAGATGTTGCCACTT ACTACTGCCAGCAGGGTAGTAGTATACC GCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG (SEQ ID NO:1)
• VDJ VH variable region
• the monoclonal antibody (or one of its fragments) directed specifically against the FP4 modified by a polyanion, and preferably against the FP4/H complex according to the invention comprises:
• variable region VL the amino acid sequence encoded by SEQ ID NO: 1.
• Such an antibody is in particular the 5B9 antibody, described in particular in Kizlik-Masson et al (Journal of Thrombosis and Haemostasis, 15: 2065-2075, 2017, 5B9, a monoclonal antiplatelet factor 4/heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies).
• such an antibody is used in the method according to the invention as a positive internal quality control, which allows standardized expression of the results taking into account the platelet activation measured with this antibody.
• the Test Patient (TP) series includes 5 mixtures, 2 of which are additional to the Validation/Standardization series: the Ago mixture and the Basal mixture.
• Mixtures 2a, 2b (possibly 2b' and 2b”) and 2c are similar to mixtures 1a, 1b (possibly 1 b' and 1 b”) and 1c respectively (ie same quantities, same ingredients and same volumes).
• the 5 mixtures of the Test Patient (TP) series do not include not the addition of monoclonal antibody directed specifically against the polyanion-modified FP4, or one of its fragments.
• the Test Patient series is used to assess the ability of polyanion-modified anti-FP4 antibodies present in the patient's blood to induce (i) platelet activation in the absence of heparin and/or in the presence of low concentrations of heparin, and (ii) their inactivation in the presence of high concentrations of heparin.
• the presence of a platelet activator or strong agonist in the Ago mixture is intended to activate the entire platelet population, and constitutes a non-specific platelet activation control making it possible to define the positivity threshold of the marker of platelet activation.
• the strong agonist is known in the state of the art.
• TRAPs thrombin receptor activator peptides, with 6 or 14 amino acids
• calcium ionophore A23187 (chemical formula below)
• arachidonic acid adenosine diphosphate (ADP)
• ADP adenosine diphosphate
• two types of strong agonists are used according to the final labeling of platelet activation: preferably, the strong agonist is chosen from TRAPs (thrombin receptor activator peptides, with 6 or 14 amino acids) or calcium ionophore A23187, whose chemical formula is given below:
• the Basal mixture is a control carried out in order to have the basal level of platelet activation.
• the buffer of the Basal mixture is a buffer known from the prior art.
• the buffer of the Basal mixture is a saline buffer such as a PBS buffer, a 1% PBS-BSA buffer, a 1% HEPES-BSA Tyrodes buffer, a 10X PBS-BSA-NaN 3 buffer, a PBS- 10X citrate-NaN 3 or 10X PBS-NaN 3 buffer.
• the Test Patient series preparation includes:
• mixture 2b at least one mixture of a whole blood sample from said patient, diluted in an acceptable buffer, with FP4 and heparin in a concentration equal to that used for mixture 1b, in order to obtain mixture 2b.
• mixtures 2b at least two mixtures of a sample of whole blood from said patient, diluted in an acceptable buffer, with FP4 and heparin are made, in order to obtain at least two mixtures 2b (2b' and 2b”).
• the heparin concentration of each mixture 2b being equal to that of each mixture 1b;
• steps 1) and 2) of the method according to the invention are carried out after 4 hours, preferably after 12 hours, preferably after 24 hours after obtaining the whole blood samples from the patient.
• steps 1) and 2) of the method according to the invention are carried out no later than 72 hours, preferably no later than 48 hours after obtaining the whole blood samples from the patient.
• the method according to the invention thus makes it possible to generate a heparin-dependent platelet activation, directly evaluated in the patient's whole blood, on his own platelets.
• Incubation is typically carried out for a period of 10 minutes to 4 hours at a temperature between 18°C and 37°C.
• the labeling of the platelets is carried out using at least one marker specific for the platelet population and at least one marker specific for the state of activation of the platelets.
• Labeling of platelets can be performed using at least one marker specific for the platelet population and possibly two markers specific for the state of platelet activation.
• This labeling with a specific marker of the activation state of the platelets consists in revealing and quantifying the presence of any compound or molecule whose expression, synthesis or activation is induced when the platelets are in an activated state and whose the level of expression of synthesis or activation can make it possible to evaluate the level of platelet activation.
• the compound is chosen from molecules or antigens expressed on the surface of the platelets, such as P-selectin (or CD62P), phosphatidylserine (PS), the LAMP-3 protein (or CD63), ADP, l ATP, phosphorylated proteins or combinations thereof.
• the compounds used for labeling are P-selectin (or CD62P) and phosphatidylserine (PS).
• CD62P (or P-selectin) is a relevant candidate to assess platelet activation.
• CD62P is indeed a protein contained in the alpha granules of platelets, which is expressed on the surface of platelets during platelet activation.
• an anti-CD62P antibody is therefore preferably used to mark the expression of CD62P.
• the protein Annexin V (or AnxV) can also make it possible to demonstrate the activation of the platelets. It has a strong affinity for phosphatidylserine (PS), which, initially located in the cytoplasmic layer of the resting cell, is exposed on the surface of activated platelets.
• PS phosphatidylserine
• Annexin V When Annexin V is used as a marker, all the mixtures used in the process according to the invention (steps 1 and 2) are carried out by adding a thrombin inhibitor.
• a thrombin inhibitor is preferably a direct thrombin inhibitor, preferably hirudin or argatroban. The addition of this inhibitor prevents the formation of thrombin, and secondarily of fibrin.
• all the mixtures used in the process according to the invention (steps 1 and 2) are carried out by adding a calcium buffer. Indeed, the addition of a calcium buffer is essential for the exposure of phosphatidylserines and therefore for the labeling by Annexin V.
• a specific labeling of the platelet population is used, targeting a compound expressed on the surface by all the platelets.
• a compound is for example the CD41 marker, the CD61 marker or else the combination of these CD41/CD61 markers.
• an anti-CD41 antibody is used for specific labeling of platelets in the sample.
• the markers used are coupled with fluorochromes, for example fluorescent proteins.
• fluorochromes are well known in the state of the art, and are for example allophycocyanin (APC), phycoerythrin (PE), phycoerythrin (PE)-Cy5 or Cy7, PerCP, PerCP-Cy5.5 , fluorescein (FITC) or Alexa Fluor dyes.
• a large volume of acceptable buffer typically between 100 ⁇ l and 5 ml, is added to each mixture. This last step makes it possible to slow down the activation of the platelets induced during steps 1 and 2, before the analysis.
• Buffer is preferably the same as that used specifically for labeling P-selectin or phosphatidylserines with Annexin V.
• the platelets Once the platelets have been labeled, they are analyzed, preferably by flow cytometry.
• the flow cytometry technique is a particularly effective tool for evaluating platelet activation in the presence of a reduced number of cellular elements. This is of major interest in this case, since the blood of patients with suspected HIT is most often thrombocytopenic.
• step 4) of analyzing all the mixtures obtained in step 3) comprises measuring the level of expression of CD62P and/or exposing phosphatidylserine to the surface platelets (via Annexin V labeling) of each mixture.
• the method further comprises a step 5') for calculating the level of antibody-dependent platelet activation (APA) in each of these mixtures.
• APA antibody-dependent platelet activation
• the analysis of the platelets of the mixtures of each of the 2 Validation/Standardization and Patient Test series (step 4) as well as the calculation of the antibody-dependent platelet activation rate (APA) of the Ago mixtures, 2a , 2b and 2c of the series TP (step 5′) are carried out according to a windowing strategy presented in the example, the description of which is given in more detail below.
• the proposed windowing strategy involves the following steps:
• a first sorting is carried out from the measurement of the intensity of labeling of a specific compound of the platelet population to select only the platelets among the other elements of the blood of each mixture, on the peak of a first histogram;
• the median labeling intensity of a compound expressed by the activated platelets is measured for all the mixtures of each series on the peak of a second histogram;
• a threshold is determined making it possible to distinguish the non-activated platelets from the activated platelets on a third histogram, placed at the intersection between the peak obtained with the Ago mixture (i.e. state of maximum activation of the platelets) in the previous step b) and the peak obtained with the Basal mixture (i.e. basal state of platelet activation);
• the percentage of doubly labeled activated platelets is calculated for each of the mixtures of each of the 2 series.
• the median total labeling intensity of the compound expressed by the activated platelets i.e. specific marker of the activation state of the platelets
• the labeling being preferably carried out with fluorescent markers, the median fluorescence intensity (MFI) is therefore measured.
• the results are expressed by multiplying the percentage (%) of cells doubly positive for the marker of the platelet population and for the specific marker of the state of platelet activation (i.e. for example CD41 +/CD62P+), by the total MFI of the specific marker of the state of platelet activation in the platelet population (i.e. MFI of the total labeling CD62P/CD41 +) (%xMFI).
• %xMFI total MFI of the specific marker of the state of platelet activation in the platelet population
• the method of the invention comprises steps 1) to 5′) stated above.
• step 5 consists of validating the series VS by calculating the activation ratio of this series and the inhibition ratio of this same series.
• two criteria are taken into account to technically validate the Validation/Standardization series:
• the activation ratio of the VS series is calculated between the Basal mixture in the absence of heparin and FP4, and the maximum platelet activation obtained in the presence of low concentrations of heparin tested, indicated mixtures 1b.
• This activation ratio of the VS series is calculated with the following formula: [Math 1 ]
• the heparin dependence of the platelet activation induced by the monoclonal antibody directed specifically against the FP4 modified by a polyanion, or one of its fragments, preferably the 5B9 antibody, is evaluated in calculating the inhibition ratio in the presence of the high concentration of heparin tested.
• This inhibition ratio of the VS series is calculated with the following formula:
• the positivity threshold of the VS series activation ratio is at least 5 for the CD62P protocol and at least 4 for the Annexin V protocol.
• the validated results of the Validation/Standardization series make it possible to calculate the antibody-dependent platelet activation rate (called APA) for the Basal, 2a, 2b and 2c mixtures of the Test Patient series (step 5').
• This rate expressed here as a percentage, is calculated from the raw results (% x MFI) expressed in AU obtained on the two series using the following formula:
• This calculation eliminates the variable reactivity of the platelets from one patient to another and from one manipulation to another by standardizing the expression of the results obtained on the Test Patient series.
• the test is considered doubtful if the APA is greater than or equal to the threshold of positivity in the presence of low concentrations of heparin (mixture 2b), but the percentage of inhibition at the high concentration of heparin (mixture 2c) is in the gray zone, i.e. between the negativity threshold and the positivity threshold. In this case, the test should be repeated, preferably on a new sample.
• the APA positivity threshold can also be defined by the mean and the standard deviation of the APA values measured in a population of patients with suspected HIT with a negative ELISA test and a negative SRA test.
• the test is considered doubtful if the APA is greater than or equal to the threshold defined in the presence of low concentrations of heparin (mixture 2b) but the percentage of inhibition at the high concentration of heparin (mixture 2c) is included between 30 and 50%. In this case, the test should be repeated, preferably on a new sample.
• the test is considered positive if the APA is greater than or equal to the threshold defined in the presence of low concentrations of heparin (mixture 2b) and the percentage of inhibition in the presence of the high concentration of heparin (mixture 2c) is greater than or equal to 50%.
• a threshold is set to distinguish platelets (CD41+) from other blood cells that do not express this marker (CD41-):
• the threshold for distinguishing CD41+/CD62P- cells (non-activated platelets) from CD41+/CD62P+ double positive cells (activated platelets) is positioned at the intersection of the two peaks between the condition activated with the TRAP (tube 5) and the resting condition (tube 6) on the third CD62P-PE versus Count histogram. After repositioning this threshold on the second histogram, the percentage of CD41+/CD62P+ double positive cells is calculated for each condition tested:
• PA A 100 x _ - _
• the maximum level of antibody-dependent platelet activation (APA) calculated in the presence of low concentrations of heparin (0.05 and/or 0.5 IU/ml, mixtures 2b ) (according to the formula described Math 3 above) is equal to 28% (positivity threshold > 20%), with 94% inhibition (threshold > 50%) in the presence of the high concentration of heparin tested (100 IU/ml, mix 2c).
• APA antibody-dependent platelet activation
• the percentage of antibody-dependent platelet activation calculated is equal to 215% (positivity threshold > 27%), with an inhibition of 96 % (threshold > 50%) in the presence of the high concentration of heparin tested (100 IU/ml, mixture 2c).
• the TP series is positive. The result of this functional test is in favor of the diagnosis of HIT.
• the SRA is a functional test for the diagnosis of HIT. This test is based on measuring the release, during platelet activation, of radiolabeled serotonin ( 14 C- serotonin), previously incorporated into the dense granules.
• the platelet washing steps allow the elimination of the plasma matrix.
• the addition of different concentrations of heparin allows the formation of FP4/H complexes on the surface of platelets from healthy donors.
• the pathogenic antibodies recognize, by their Fab fragment, the macromolecular complexes FP4/H and activate the platelets via their Fc fragment which binds to the FcyllA receptor. The content of the granules is released into the reaction medium and the radioactivity measured in the supernatant is directly proportional to platelet activation.

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