Method for discriminating serum samples, citrated plasma samples and EDTA-treated plasma samples

Abstract

The present invention relates to a method for determining whether an unknown blood sample is a serum sample, a citrated plasma sample, or an EDTA-treated plasma sample. The invention also relates to a method for the diagnosis and / or monitoring of a patient suffering from a hemostasis disorder.

Description

[0001] Method for discriminating between serum, citrated plasma, and EDTA-treated plasma samples

[0002] The present invention relates to a method for determining whether an unknown blood sample is a serum sample obtained from a collection tube without anticoagulant, a plasma sample obtained from a tube containing citrate (also called "citrated plasma") or a plasma sample obtained from a tube containing EDTA.

[0003] Such a process makes it possible to discriminate between samples of serum, citrated plasma and EDTA-treated plasma, which is particularly useful for hemostasis testing.

[0004] Hemostasis encompasses all the physiological reactions that stop bleeding and prevent hemorrhages and thromboses. The hemostasis laboratory plays a crucial role in the diagnosis and monitoring of patients with hemostasis disorders (i.e., disorders of coagulation factors and / or coagulation inhibitors). It serves as the foundation for monitoring antithrombotic or hemophilia therapies. Its role is to implement the necessary control tools to ensure high-quality analytical results as quickly as possible. The quality of a result depends, in particular, on the quality of the sample and its subsequent processing.

[0005] Centralized analysis laboratories receive daily blood samples from patients which are either collected in primary tubes (i.e., collection tubes) containing an anticoagulant such as sodium citrate or EDTA. In this case, after centrifugation, plasma is obtained.

[0006] Plasma is the liquid portion of blood obtained after centrifuging blood collected in a tube containing an anticoagulant. After centrifugation, the plasma, which is light yellow, rises to the top, while the red blood cells and other blood cells form the sediment. Plasma can also be collected in test tubes without an anticoagulant (i.e., so-called dry tubes). Dry tubes contain nothing (no anticoagulant). Once centrifuged, the result is serum.

[0007] Serum is the liquid portion of blood obtained after collection in a dry tube. Upon contact with the tube, the blood initially coagulates, releasing an exudate (serum). During centrifugation, the clot settles to the bottom of the collection tube, leaving the serum above. Once the samples have been separated from their original tube, it is no longer possible to identify the anticoagulant used during collection, or even its presence (creating doubt as to the nature of the sample, serum or plasma).

[0008] However, it is necessary to know the specific sample before proceeding with the various tests, or at least to validate the results of hemostasis tests performed on this tube. Indeed, only collection tubes containing sodium citrate (3.2% or 3.8%) allow for the preservation of coagulation factors and the diagnosis of hemostasis disorders. Samples collected in EDTA (plasma) or dry tubes (serum) are not suitable for this diagnosis and must be discarded, or the results must be invalidated if applicable.

[0009] To date, there is no automated test that allows biologists to rule out results obtained from EDTA tubes or serum.

[0010] Therefore, there is a very strong need, particularly among hemostasis laboratories, for a reliable, sensitive, and specific test that can determine whether a given sample is serum, citrated plasma, or EDTA-treated plasma. Such a test must also be easy to perform and available on an automated system (especially for automated routine testing).

[0011] This test should allow discriminating between different blood samples, i.e. serum samples, citrated plasma and EDTA-treated plasma.

[0012] In addition, hemostasis laboratories need such a test to be very quick to interpret; indeed, its result allows the appropriate hemostasis test to be applied to the sample concerned, within a fairly short overall turnaround time for results (such as less than 24 to 48 hours excluding weekends).

[0013] The present invention makes it possible to meet these expectations.

[0014] The invention thus relates to a method for determining whether an unknown blood sample is a serum sample, citrated plasma or EDTA-treated plasma (“method of determination according to the invention”), comprising the following steps: a) mixing the unknown biological sample (unknown blood sample) with o-cresolphthalein-complexone, at a temperature between 30°C and 40°C, and without the addition of 8-hydroxyquinoline, b) a first measurement of the absorbance A1 of the mixture obtained in a) at a first time t1, and a second measurement of the absorbance A2 of the same mixture at a second time t2, with the conditions that t2>t1 and that the interval (t2-t1) is between 10 and 200 seconds,

[0015] A1 and A2 being measured at a wavelength between 520 and 580 nm, then c) the calculation of the absorbance difference (A2-A1), in which: if the absorbance difference (A2-A1) is zero, then the unknown sample is an EDTA-treated plasma sample, if the absorbance difference (A2-A1) is between [-0.150; -0.080], then the unknown sample is a citrated plasma sample, and if the absorbance difference (A2-A1) is between [-0.070;-0.040], then the unknown sample is a serum sample, and / or the process includes step a') measuring the initial absorbance of the mixture obtained in a) as soon as it is made at t0, at a wavelength between 520 and 580 nm, in which: if the initial absorbance is zero, then the unknown sample is an EDTA-treated plasma sample, and if the initial absorbance is strictly less than a threshold value, then the unknown sample is a citrated plasma sample, and if the initial absorbance is strictly greater than the threshold value, then the unknown sample is a serum sample.;

[0016] By "zero initial absorbance", we mean a significantly zero initial absorbance.

[0017] By "threshold value" we mean an absorbance value that allows us to discriminate between citrated plasma samples and serum samples.

[0018] The determination method according to the invention thus comprises:

[0019] - either steps a) to c). In this case, the process is based on the difference in absorbance (A2- A1) of the mixture;

[0020] - either steps a) to c) and a'). In this case, the process relies both on the difference in absorbance (A2-A1) of the mixture and on the initial absorbance of the mixture;

[0021] - either steps a) and a'). In this case, the process is based on the initial absorbance of the mixture.

[0022] According to one embodiment, preferably, the determination method according to the invention comprises steps a) to c), and does not include step a'). According to one embodiment, preferably, the determination method according to the invention comprises steps a) and a'), and the threshold value is equal to 0.8.In this case, preferably, the determination method according to the invention comprises: a) mixing the unknown blood sample with o-cresolphthalein-complexone, at a temperature between 30°C and 40°C, and without the addition of hydroxy-8-quinoline, and a') measuring the initial absorbance of the mixture obtained in a) as soon as it is made at t0, at a wavelength between 520 and 580 nm, in which: if the initial absorbance is zero, then the unknown sample is an EDTA-treated plasma sample, and if the initial absorbance is strictly less than 0.8, then the unknown sample is a citrated plasma sample, and if the initial absorbance is strictly greater than 0.8, then the unknown sample is a serum sample.

[0023] More preferably, if the initial absorbance is between 0.60 and 0.75, then the unknown sample is a citrated plasma sample, and if the initial absorbance is strictly between 0.85 and 1.00, then the unknown sample is a serum sample.

[0024] According to one embodiment, preferably, the determination method according to the invention comprises the steps a) to c) and a'): a) mixing the unknown biological sample (unknown blood sample) with o-cresolphthalein-complexone, at a temperature between 30°C and 40°C, and without the addition of 8-hydroxyquinoline, then a') measuring the initial absorbance of the mixture obtained in a) immediately after its preparation at t0, at a wavelength between 520 and 580 nm, preferably 540 nm, b) a first measurement of the absorbance A1 of the mixture obtained in a) at a first time t1, and a second measurement of the absorbance A2 of the same mixture at a second time t2, with the conditions that t2>t1 and that the interval (t2-t1) is between 10 and 200 seconds,

[0025] A1 and A2 being measured at the same wavelength as the initial absorbance, then c) the calculation of the absorbance difference (A2-A1), in which: if the absorbance difference (A2-A1) is zero or if the initial absorbance is zero, then the unknown sample is an EDTA-treated plasma sample, if the absorbance difference (A2-A1) is between [-0.150; -0.080] or if the initial absorbance is strictly less than a threshold value, preferably strictly less than 0.8, then the unknown sample is a citrated plasma sample, and if the absorbance difference (A2-A1) is between [-0.070; -0.040] or if the initial absorbance is strictly greater than the threshold value, preferably strictly greater than 0.8, then the unknown sample is a serum sample.

[0026] According to another embodiment, the method aims to determine whether an unknown blood sample is a serum sample, citrated plasma, or EDTA-treated plasma, and comprises the following steps: a) mixing the unknown blood sample with o-cresolphthalein-complexone at a temperature between 30°C and 40°C, without the addition of 8-hydroxyquinoline, then a') measuring the initial absorbance of the mixture obtained in a) immediately upon its preparation at t0, at a wavelength between 550 and 580 nm, b) a first measurement of the absorbance A1 of the mixture obtained in a) at a first time t1, and a second measurement of the absorbance A2 of the same mixture at a second time t2, with the conditions that t2 > t1 and that the interval (t2 - t1) is between 10 and 200 seconds.

[0027] A1 and A2 being measured at the same wavelength as the initial absorbance, then c) the calculation of the absorbance difference (A2-A1), in which: if the absorbance difference (A2-A1) per minute is between [0; 0.0036], or if the absorbance difference (A2-A1) is between [0; 0.010], or if the initial absorbance is zero, then the unknown sample is an EDTA-treated plasma sample, and if the initial absorbance is strictly less than 1000, then the unknown sample is a citrated plasma sample, and if the initial absorbance is strictly greater than 1000, then the unknown sample is a serum sample.

[0028] Thus, this other embodiment according to the invention comprises steps a) to c) and a').

[0029] The threshold value of this other embodiment according to the invention is equal to 1000.

[0030] The invention also relates to a method for diagnosing and / or monitoring a patient with a hemostasis disorder, comprising: identifying a sample of citrated plasma according to the method of determination of the invention, then performing a hemostasis test or measuring a coagulation parameter on said sample of citrated plasma.

[0031] The invention is now explained in detail as follows.

[0032] The determination method according to the invention comprises the following steps: a) mixing the unknown biological sample (unknown blood sample) with o-cresolphthalein-complexone, at a temperature between 30°C and 40°C, and without the addition of 8-hydroxyquinoline, b) a first measurement of the absorbance A1 of the mixture obtained in a) at a first time t1, and a second measurement of the absorbance A2 of the same mixture at a second time t2, with the conditions that t2>t1 and that the interval (t2-t1) is between 10 and 200 seconds,

[0033] A1 and A2 being measured at a wavelength between 520 and 580 nm, then c) the calculation of the absorbance difference (A2-A1).

[0034] One embodiment of the invention includes these same steps a) to c), with the sole difference that the absorbances are measured at a wavelength between 550 and 580 nm.

[0035] Step a) involves mixing the unknown biological sample (unknown blood sample) with o-cresolphthalein-complexone at a temperature between 30°C and 40°C. This mixture does not contain 8-hydroxyquinoline.

[0036] The principle underlying this step a) is the detection and quantification of Ca2+ ions in the unknown sample.

[0037] The principle of the assay is based on the formation of a colored complex between the calcium present in the unknown sample and ortho-cresolphthalein complexone (o-CPC) in an alkaline medium and in the presence of AMP (2-amido-2-methyl-1-propanol). The formation of this complex results in a violet color measured at 570 nm. The optimal absorbance of this complex is reached at a wavelength of 577 nm. The wavelength at which measurements are taken may vary around this value depending on the spectrophotometer and the wavelengths available on the diagnostic analyzer used. The intensity of the color obtained is directly proportional to the amount of Ca2+ present in the sample being analyzed.

[0038] This occurs when the unknown sample is a citrated plasma sample. Indeed, the acidification of the medium upon the addition of o-CPC (which is an acidic reagent) eliminates the interactions of calcium ions with proteins. This acidification of the citrated plasma allows the release of calcium ions that have been chelated by citrate ions.

[0039] The reaction is represented schematically as follows:

[0040] + AMP

[0041] 2-Amido-2-methyl-l-propanol

[0042] Citrated plasma + o-CPC - ► “Ca-o-CPC” complex (purple)

[0043] In an alkaline environment, pH ~ 10.7

[0044] Conversely, in the absence of Ca2+ ions, the solution remains colorless; this is the case when the unknown sample is a plasma sample treated with EDTA, because in the presence of EDTA, the Ca2+ ions remain chelated to EDTA under these assay conditions.

[0045] The hypothesis considered to explain the principle of the titration is as follows:

[0046] In an alkaline environment, the affinity of Ca2+ ions for EDTA is stronger than for o-CPC (competition). Therefore, the Ca2+ ions remain bound to EDTA, and the solution remains colorless. Conversely, the affinity of Ca2+ ions for o-CPC is greater than their affinity for citrate. Consequently, the Ca2+ ions bind to o-CPC and form violet Ca-o-CPC complexes, and the solution takes on a violet color whose intensity is directly proportional to the amount of Ca2+ present in the sample.

[0047] Finally, in serum, Ca2+ ions are not chelated: they therefore bind to o-CPC and form violet Ca-o-CPC complexes, and here again, the solution takes on a violet color of intensity directly proportional to the amount of Ca2+ present in the sample.

[0048] Conventionally, to ensure the specificity of the assay for Ca2+ ions, 8-hydroxyquinoline is added: this compound eliminates interactions with other metal ions, considered interfering, and in particular divalent cations such as Mg2+ or Fe2+. In such a case, only the Ca-o-CPC complex is measured by its absorbance at 570 nm. According to the invention, the mixture in step a) does not contain 8-hydroxyquinoline. This allows o-CPC to bind to metal ions other than Ca2+. The mixture in step a) is therefore not specific to calcium.

[0049] Indeed, in the case of a serum sample, several inorganic ions are present, notably Ca2+, Mg2+, Na+, and K+. These will therefore bind to the o-CPC, and the color intensity is higher than with a citrated plasma sample, where the citrate chelates the majority of free Ca2+ ions. However, given the presence of Mg2+ ions in a citrated plasma sample, these can bind to the reagent and contribute to maintaining a stronger residual color.

[0050] The mixing in step a) is carried out at a temperature between 30°C and 40°C. Preferably, the temperature is between 35°C and 40°C, and preferably around 37°C. Such a temperature destabilizes the Ca-o-CPC complex, but to varying degrees depending on the sampling tube used, as the complexation with metal ions differs in each tube.

[0051] In the case of a serum sample, there are more free Ca2+ ions (and metal ions) which give rise to more complexons, the equilibrium state between free Ca2+ ions and Ca2+ ions complexed with o-CPC is less disturbed than in the case of a citrated plasma sample, which has fewer residual Ca2+ ions capable of complexing with o-CPC.

[0052] Preferably, the mixture from step a) is incubated for 10 to 60 seconds, preferably 20 to 40 seconds.

[0053] Preferably, the mixture from step a) is incubated for 10 to 50 seconds, preferably for 10 to 40 seconds, preferably for 10 to 30 seconds.

[0054] According to another embodiment, preferably, the mixture from step a) is incubated for 20 to 50 seconds, preferably for 20 to 30 seconds.

[0055] According to another embodiment, preferably, the mixture from step a) is incubated for 30 to 50 seconds, preferably for 30 to 40 seconds.

[0056] Preferably, the mixture from step a) is incubated for 10 seconds.

[0057] Preferably, the mixing in step a) is carried out in the presence of a buffer with a pH between 9.0 and 11.0, preferably around 10.7. Preferably, the buffer also contains 2-amino-2-methyl-1-propanol (AMP). Preferably, the mixing in step a) is carried out with a weight ratio (sample unknown): (o-cresolphthalein-complexone solution): (buffer) of between 8-12:40-60:40-60.

[0058] Preferably, the mixing is carried out under agitation, especially with a ball.

[0059] Next, the determination method according to the invention includes a step b) in which the absorbance of the mixture obtained in a) is measured at a first time t1; this corresponds to a first measurement of absorbance A1.

[0060] Then a second absorbance measurement A2 of the same mixture is carried out, at a second time t2; t2 is later than t1 (in other words, t2>t1).

[0061] Furthermore, the interval (t2-t1) is between 10 and 200 seconds, preferably between 50 and 190 seconds, preferably between 100 and 180 seconds.

[0062] The times t1 and t2 correspond to the times elapsed since the end of step a).

[0063] Preferably, t1 is between 10 and 20 seconds. Preferably, t1 is between 10 and 20 seconds, and t2 is between 170 and 200 seconds. Typically, the first absorbance measurement A1 is performed approximately 12 seconds (t1) after mixing in step a). Typically, the second absorbance measurement A2 is performed approximately 180 seconds (t2) after mixing in step a).

[0064] Preferably, A1 and A2 are measured at 540 nm or 577 nm, preferably at 540 nm. Preferably, absorbances are measured at a wavelength between 550 and 580 nm, preferably at 575 nm.

[0065] Finally, the determination method according to the invention includes a step c) of calculating the difference in absorbance (A2-A1).

[0066] The difference in absorbance (A2-A1) obtained is negative in the case of a citrated plasma sample or a serum sample, i.e., it corresponds to a decrease in absorbance over time. It is zero in the case of a plasma sample treated with EDTA.

[0067] Based on the difference (A2-A1), the criteria for determining the process are as follows: - if the absorbance difference (A2-A1) is zero, then the unknown sample is an EDTA-treated plasma sample, - if the absorbance difference (A2-A1) is between [-0.150; -0.080], then the unknown sample is a citrated plasma sample, and

[0068] -if the difference in absorbance (A2-A1) is between [-0.070; -0.040], then the unknown sample is a serum sample.

[0069] By "zero absorbance difference (A2-A1)", we mean that (A2-A1) does not vary significantly. Typically, a zero absorbance difference (A2-A1) is between [-0.020; 0.020].

[0070] Preferably, the determination method according to the invention is carried out automatically, preferably on an automated machine.

[0071] Preferably, these measurements are performed kinetically on an automated diagnostic instrument, preferably a coagulation analyzer. More preferably, the absorbance decay (A2-A1) and / or the initial absorbance are measured on a Stago STA-R Max® type analyzer. It can also be measured on the Stago sthemO® platform, such as sthemO® 301, 201, or even 101; or on the CS 2500, CS 5100, CN 3000, and CN 6000 series of analyzers marketed by Siemens Sysmex, the ACL Top® series of analyzers marketed by Werfen, the Cobas t® series of analyzers marketed by Roche Diagnostics, or the Vidas® series of analyzers marketed by bioMérieux. Preferably, all the steps of the determination method according to the invention are carried out on such an analyzer. Without wanting to be linked to any particular theory, this decrease seems to be related to the dissociation of the Ca-o-CPC complex.The reaction appears to be reversible when the Ca2+ ions are not chelated.

[0072] Preferably, the decrease in absorbance (A2-A1) is measured and calculated by the automaton.

[0073] Thus, typically, EDTA-treated plasma samples will not be colored, because the Ca2+ ions are chelated by EDTA and therefore no Ca-o-CPC complex is formed, the absorbance difference (A2-A1) is close to 0.

[0074] In contrast, typically, in the case of citrated plasma or serum samples, a Ca-o-CPC complex forms (the color turns purple) and decreases slowly over time. The difference in absorbance (A2-A1) is measurable and non-zero, specifically negative (decreasing), which allows them to be distinguished from EDTA tubes. At the end of the determination process according to the invention, it is thus possible to differentiate between serum, citrated plasma, and EDTA-treated plasma samples. This demonstration is particularly useful for hemostasis tests, as it allows the test to begin with a sample that has been correctly collected and pretreated for a given test.

[0075] With regard to the decrease in absorbance according to an embodiment of the invention, starting from the difference (A2-A1), the criteria of the process are as follows:

[0076] -if the absorbance difference (A2-A1) per minute is between [0; 0.0036], or if the absorbance difference (A2-A1) is between [0; 0.010], then the unknown sample is an EDTA-treated plasma sample, and

[0077] -if the initial absorbance (absorbance at t0, or A0) is strictly less than 1000, then the unknown sample is a citrated plasma sample, and

[0078] -if the initial absorbance is strictly greater than 1000, then the unknown sample is a serum sample.

[0079] Typically, an embodiment according to the invention is carried out automatically, preferably on an automated machine.

[0080] Preferably, these measurements are performed kinetically on an automated diagnostic instrument, preferably a coagulation analyzer. For example, the absorbance difference (A2-A1) per minute is measured on a Siemens CS 2500® type analyzer. For example, all the steps according to the invention are implemented on such an instrument.

[0081] Preferably, the difference in absorbance (A2-A1) per minute, called DDO / min, is measured and calculated by the automaton for serum samples, samples taken on citrate and samples taken on EDTA.

[0082] Typically, the DDO / min does not change between t1 and t2 for samples collected in EDTA; it remains constant. Therefore, the DDO / min has a constant value regardless of the type of EDTA sample. Thus, based on the DDO / min, we can distinguish between samples collected in citrate or serum on the one hand, and samples collected in EDTA on the other.

[0083] Similarly, it is possible to obtain results in absorbance difference (A2-A1), called DDO, by applying a correction factor to the initial DDO / min result. This correction factor corresponds to the time lapse, in minutes, between t1 and t2. The DDO therefore also has a constant value, regardless of the samples collected on EDTA. Thus, based on the DDO obtained with the correction factor, it is possible to distinguish between samples collected on citrate or serum, on the one hand, and samples collected on EDTA, on the other. The presentation of results according to another embodiment of the invention, obtained in particular with an automated system, can thus be in two possible formats: 1 ers results in DDO / min, then 2 èmes results in DDO.

[0084] Thus, according to this other embodiment of the invention, if the difference in absorbance (A2- A1) per minute is between [0; 0.0036], or if the difference in absorbance (A2-A1) is between [0; 0.010], then the unknown sample is a plasma sample treated with EDTA.

[0085] However, at this stage of the analysis, this other embodiment according to the invention does not allow for distinguishing samples taken on citrate from serum samples.

[0086] With yet another embodiment according to the invention, the distinction between samples taken on citrate and serum samples is possible graphically on the basis of the OD at tO (initial absorbance or A0); this is step a'). Indeed, based on the graphical results generated by the CS 2500® analyzer, it is observed that: -for samples taken on citrate: A0 < 1000; and -for serum samples: A0 > 1000.

[0087] Thus, for this particular embodiment according to the invention:

[0088] -if A0 is strictly less than 1000, then the unknown sample is a citrated plasma sample, and

[0089] -if A0 is strictly greater than 1000, then the unknown sample is a serum sample.

[0090] Preferably, this alternative embodiment according to the invention makes it possible to determine whether an unknown blood sample is a serum sample, a citrated plasma sample, or an EDTA-treated plasma sample, and comprises the following steps: a) mixing the unknown blood sample with o-cresolphthalein-complexone, at a temperature between 30°C and 40°C, and without the addition of 8-hydroxyquinoline, then a') measuring the initial absorbance of the mixture obtained in a), called A0, as soon as it is prepared at t0, A0 being measured at a wavelength between 550 and 580 nm, b) a first measurement of the absorbance A1 of the mixture obtained in a) at a first time t1, and a second measurement of the absorbance A2 of the same mixture at a second time t2, with the conditions that t2>t1 and that the interval (t2-t1) is between 10 and 200 seconds,

[0091] A1 and A2 being measured at the same wavelength as A0, then c) the calculation of the absorbance difference (A2-A1), in which: if the absorbance difference (A2-A1) per minute, called DDO / min, is between [0; 0.0036], or if the absorbance difference (A2-A1) is between [0; 0.010], then the unknown sample is an EDTA-treated plasma sample, and if A0 is strictly less than 1000, then the unknown sample is a citrated plasma sample, and if A0 is strictly greater than 1000, then the unknown sample is a serum sample.

[0092] Preferably, AO, A1 and A2 were measured at a wavelength of 575 nm.

[0093] At the end of this alternative embodiment according to the invention, it is thus possible to differentiate between serum, citrated plasma, and EDTA-treated plasma samples. This differentiation is particularly useful for hemostasis tests, as it allows for the use of a properly collected and pre-treated sample for a given test.

[0094] The invention also relates to a method for diagnosing and / or monitoring a patient with a hemostasis disorder, comprising: identifying a citrated plasma sample according to the determination method according to the invention, then performing a hemostasis test or measuring a coagulation parameter on said citrated plasma sample.

[0095] At the end of the determination process according to the invention, at least one sample of citrated plasma can be identified.

[0096] This sample can then be subjected to at least one hemostasis test or measurement of a coagulation parameter; the results obtained will be reliable.

[0097] Hemostasis tests are generally routine and include the prothrombin time (expressed in seconds) or the prothrombin time (PT) expressed as INR (International Normalized Ratio, which is the ratio of the patient's PT to the control PT, multiplied by an international calibration index), the activated partial thromboplastin time (aPTT), the plasma fibrinogen level, and D-dimer testing. Tests measuring a coagulation parameter are typically chosen from those that measure factors of the endogenous or exogenous pathway, coagulation inhibitors, or primary hemostasis.

[0098] The invention is now illustrated by the following examples of implementation of the invention.

[0099] Example 1: Determination method according to the invention carried out with the Stago STA-R Max® automated system

[0100] 30 serum samples obtained from collection tubes without anticoagulant, 30 plasma samples obtained from tubes containing citrate and 30 plasma samples obtained from tubes containing EDTA were used.

[0101] Of the 30 plasma samples obtained from tubes containing citrate, 15 were obtained from tubes containing 3.2% citrate, and the other 15 were obtained from tubes containing 3.8% citrate.

[0102] A STA-R Max® automated system from the Stago group is used to carry out the process according to the invention.

[0103] - The o-CPC reagent is placed in position R1 of the STA-R Max® analyzer.

[0104] - The AMP reagent (AMP buffer) is placed in position R2 of the STA-R Max® analyzer.

[0105] Step a):

[0106] - The samples are placed in the automaton: a volume of 25pL of sample is mixed with 125 pL of o-CPC and 125 pL of AMP buffer for 30 seconds, at a temperature of approximately 37°C.

[0107] Step b):

[0108] A first measurement of the absorbance A1 of the mixture obtained in a) is carried out at a first time t1 = 12s, and a second measurement of the absorbance A2 of the same mixture at a second time t2 = 180s. In this case, the interval (t2-t1) is equal to 168s.

[0109] The wavelength is equal to 540 nm.

[0110] Step c): The calculation of the absorbance difference (A2-A1) of step c) of the process according to the invention is carried out by the Stago STA R-Max® automated system according to the following formula: [Math 1]

[0111] Difference DO (or DDO) = (DO(t2-2s) + DO(t2) + DO(t2-2s)) / 3 - (DO(ti) + DO(ti+2s)) / 2

[0112] The results are as follows (Table 1 and Figure 1):

[0113] Table of difference measurements of absorbances (here optical densities or OD) obtained on STA-R Max®

[0114] [Table 1]

[0115] Figure 1 represents the results of absorbance difference (here difference of DO or DDO or ADO) obtained for the different samples tested.

[0116] All these results show that when the difference in absorbance (A2-A1) is zero (value between [-0.02 ; 0.02]), then the unknown sample is a plasma sample treated with EDTA.

[0117] When the absorbance difference (A2-A1) is between [-0.150 ; -0.080], then the unknown sample is a citrated plasma sample.

[0118] When the absorbance difference (A2-A1) is between [-0.070 ; -0.040], then the unknown sample is a serum sample.

[0119] The results obtained show that the process according to the invention is reliable, sensitive and specific; it effectively discriminates the nature of the sample, i.e. serum sample, citrated plasma or EDTA-treated plasma.

[0120] Alternatively, step a') is carried out:

[0121] Step a'):

[0122] An initial absorbance measurement (A0) of the mixture obtained in a) is carried out immediately after its preparation at t0. The wavelength is equal to 540 nm.

[0123] The results obtained show that:

[0124] - AO measured for EDTA-treated plasma samples is zero;

[0125] - The measured AO for citrated plasma samples is around 0.7; and

[0126] - AO measured for serum samples is around 0.9.

[0127] All these results show that when the initial absorbance is zero, then the unknown sample is a plasma sample treated with EDTA.

[0128] When the initial absorbance is strictly less than 0.8 (threshold value), preferably around 0.7, then the unknown sample is a citrated plasma sample.

[0129] When the initial absorbance is strictly greater than 0.8 (threshold value), preferably around 0.9, then the unknown sample is a serum sample.

[0130] Example 2: Determination method according to the invention carried out with the CS automated system

[0131] Siemens 2500®

[0132] Five serum samples obtained from collection tubes without anticoagulant, 11 plasma samples obtained from tubes containing citrate (1 sample taken on 3.8% citrate, and 10 samples taken on 3.2% citrate) and 5 plasma samples obtained from tubes containing EDTA were used.

[0133] The samples are tested in duplicate.

[0134] A Siemens CS 2500® PLC is used to implement another embodiment according to the invention.

[0135] The absorbance measurement wavelength chosen here is 575 nm. Note that the 540 nm wavelength is not always available on diagnostic analyzers.

[0136] - The samples are placed in the automaton: a volume of 25pL of sample is mixed with 125 pL of o-CPC and 125 pL of AMP buffer for 10 seconds, at a temperature of approximately 37°C.

[0137] The initial absorbance A0 is measured immediately after mixing (t0, at 575 nm). Step b):

[0138] A first measurement of the absorbance A1 of the mixture obtained in a) is carried out at a first time t1 = 12s, and a second measurement of the absorbance A2 of the same mixture at a second time t2 = 180s. In this case, the interval (t2-t1) is equal to 168s.

[0139] The absorbance measurement wavelength is equal to 575 nm.

[0140] Step c):

[0141] The calculation of the absorbance difference (A2-A1) of step c) of the process according to the invention is carried out by the CS 2500® automated system.

[0142] The results are as follows (Table 2):

[0143] Although the DO observed graphically between t1 and t2 decrease on CS 2500® for the citrate and serum samples, the DDO / min deviations returned by the analyzer are positive, probably due to algorithmic signal processing by the analyzer.

[0144] On the other hand, the OD does not change for samples taken on EDTA; the deviation DDO / min is equal to 0.001.

[0145] Table of measurements of difference in absorbances per minute (here DDO / min) obtained on

[0146] CS 250CP

[0147] [Table 2]

[0148] Based on the deviation DDO / min (slope), it is possible to distinguish between samples taken on citrate or serum on the one hand, and samples taken on EDTA on the other.

[0149] However, at this stage of the analysis, it is not possible to distinguish samples collected in citrate from those collected in serum. It is nevertheless possible to obtain the results in DDO by applying a correction factor of

[0150] 2.8 to the initial result DDO / min (see Table 3). This correction factor of 2.8 corresponds to the time lapse in minutes between t1 and t2 (i.e. 180-12=168 seconds or 168 / 60=2.8 minutes).

[0151] The results from the CS 2500® analyzer can therefore be displayed in two possible formats: 1 ers results in DDO / min (Table 2), then 2 èmes results in DDO (Table 3).

[0152] Table of difference measurements of absorbances (here DDO) obtained on CS 2500® [Table 3]

[0153] Finally, the distinction between samples collected in citrate and those collected in serum can be graphically distinguished based on the OD at tO (initial absorbance AO, step a'). Indeed, based on the graphical results generated by the CS 2500® analyzer, we observe that:

[0154] -for samples taken on citrate: OD at tO (A0) < 1000; and

[0155] -for samples taken in serum: DO at tO (A0) > 1000.

[0156] Here, 1000 is the threshold value.

[0157] In conclusion, it is possible according to the invention: to distinguish samples taken on EDTA from samples taken on citrate on the basis of the DDO / min or the DDO of these samples, to distinguish samples taken on citrate from serum samples on the basis of the DO obtained at T0 (initial absorbances or A0), and to distinguish samples on citrate from serum samples by combining the DDO / min or DDO of these samples with the DO obtained at T0.

Claims

DEMANDS 1. A method for determining whether an unknown blood sample is a serum sample, citrated plasma, or EDTA-treated plasma, comprising the following steps: a) mixing the unknown blood sample with o-cresolphthalein-complexone at a temperature between 30°C and 40°C, without the addition of 8-hydroxyquinoline; b) a first measurement of the absorbance A1 of the mixture obtained in a) at a first time t1, and a second measurement of the absorbance A2 of the same mixture at a second time t2, with the conditions that t2 > t1 and that the interval (t2 - t1) is between 10 and 200 seconds. A1 and A2 being measured at a wavelength between 520 and 580 nm, then c) the calculation of the absorbance difference (A2-A1), in which: if the absorbance difference (A2-A1) is zero, then the unknown sample is an EDTA-treated plasma sample, if the absorbance difference (A2-A1) is between [-0.150; -0.080], then the unknown sample is a citrated plasma sample, and if the absorbance difference (A2-A1) is between [-0.070;-0.040], then the unknown sample is a serum sample, and / or the process includes step a') measuring the initial absorbance of the mixture obtained in a) as soon as it is made at t0, at a wavelength between 520 and 580 nm, in which: if the initial absorbance is zero, then the unknown sample is an EDTA-treated plasma sample, and if the initial absorbance is strictly less than a threshold value, then the unknown sample is a citrated plasma sample, and if the initial absorbance is strictly greater than the threshold value, then the unknown sample is a serum sample.; 2. A method according to claim 1, characterized in that a zero absorbance difference (A2-A1) is an absorbance difference (A2-A1) which does not vary significantly, typically the absorbance difference (A2-A1) is between [-0.020; 0.020], 3. A method according to one of claims 1 or 2, characterized in that A1 and A2 are measured at 540 nm or 577 nm, preferably at 540 nm.

4. A method according to any one of the preceding claims, characterized in that it comprises steps a) and a'), and the threshold value is equal to 0.

8.

5. A method according to any one of the preceding claims, characterized in that if the initial absorbance is between 0.60 and 0.75, then the unknown sample is a citrated plasma sample, and if the initial absorbance is strictly between 0.85 and 1.00, then the unknown sample is a serum sample.

6. A method according to any one of claims 1 to 3, characterized in that it comprises steps a) to c), and in that it does not comprise step a').

7. A method according to claim 1, comprising the following steps: a) mixing the unknown blood sample with o-cresolphthalein-complexone, at a temperature between 30°C and 40°C, and without the addition of 8-hydroxyquinoline, then a') measuring the initial absorbance of the mixture obtained in a) immediately upon its preparation at t0, at a wavelength between 550 and 580 nm, b) a first measurement of the absorbance A1 of the mixture obtained in a) at a first time t1, and a second measurement of the absorbance A2 of the same mixture at a second time t2, with the conditions that t2>t1 and that the interval (t2-t1) is between 10 and 200 seconds, A1 and A2 being measured at the same wavelength as the initial absorbance, then c) the calculation of the absorbance difference (A2-A1), in which: if the absorbance difference (A2-A1) per minute is between [0; 0.0036], or if the absorbance difference (A2-A1) is between [0; 0.010], or if the initial absorbance is zero, then the unknown sample is an EDTA-treated plasma sample, and if the initial absorbance is strictly less than 1000, then the unknown sample is a citrated plasma sample, and if the initial absorbance is strictly greater than 1000, then the unknown sample is a serum sample.

8. A method according to any one of the preceding claims, characterized in that it is carried out automatically, preferably on an automated machine.

9. A method according to any one of the preceding claims, characterized in that the mixture of step a) is incubated for 10 to 60 seconds, preferably 10 to 40 seconds, preferably 20 to 40 seconds.

10. A method according to any one of the preceding claims, characterized in that t1 is between 10 and 20 seconds, and t2 is between 170 and 200 seconds.

11. A method according to any one of claims 7 to 10, characterized in that the initial absorbances, A1 and A2 are measured at 575 nm.

12. A process according to any one of the preceding claims, characterized in that the mixing in step a) is carried out in the presence of a buffer with a pH between 9.0 and 11.0, preferably around 10.7, and comprising 2-amino-2-methyl-1-propanol.

13. Process according to claim 12, characterized in that the mixing of step a) is carried out with a weight ratio (unknown sample): (o-cresolphthalein-complexone solution): (buffer) of between 8-12: 40-60: 40-60.

14. Method for diagnosing and / or monitoring a patient with a hemostasis disorder, comprising: identifying a citrated plasma sample according to the method according to any one of claims 1 to 13, then performing a hemostasis test or measuring a coagulation parameter on said citrated plasma sample.

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