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

Abstract

Method for discriminating between serum, citrated plasma, and EDTA-treated plasma samples. The present invention relates to a method for determining whether an unknown blood sample is a serum, citrated plasma, or EDTA-treated plasma sample. It also relates to a method for diagnosing and / or monitoring a patient with a hemostasis disorder. Figure for abstract: none

Description

Title of the invention: Method for discriminating between samples of serum, citrated plasma and EDTA-treated plasma

[0001] 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.

[0002] Such a process makes it possible to discriminate between samples of serum, citrated plasma and EDTA-treated plasma, which is particularly useful for hemostasis tests.

[0003] Hemostasis is the set of physiological reactions that stop bleeding and prevent hemorrhages and thromboses. The hemostasis laboratory plays an important 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 basis for monitoring antithrombotic therapies. Its role is to implement the necessary control tools to ensure, as quickly as possible, a high-quality analytical result. The quality of a result depends in particular on the quality of the sample and its subsequent processing.

[0004] Centralized analysis laboratories receive daily blood samples from patients who 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.

[0005] Indeed, plasma is the liquid part of blood obtained after centrifugation of blood collected in a tube containing an anticoagulant. After centrifugation of the tube, the plasma, which has a light yellow color, appears at the top of the tube, while the red blood cells and other blood cells form the pellet; - either collected in test tubes without anticoagulant (i.e., so-called dry tubes). Dry tubes contain nothing (no anticoagulant). Once centrifuged, serum is obtained.

[0006] Serum is the liquid portion of blood obtained after collection in a dry tube. Upon contact with the tube, the blood initially coagulates, leaving an exudate (serum). During centrifugation, the clot moves to the bottom of the collection tube, leaving the serum at the top.

[0007] Once the samples have been decanted from their primary tube, it is no longer possible to identify the anticoagulant used during the sampling, nor even its presence (doubt about the nature of the sample, serum or plasma).

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

[0009] To date, there is no automated test allowing biologists to discard results obtained on EDTA tubes or on serum.

[0010] There is therefore 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 (in particular for automated routine testing).

[0011] This test must make it possible to discriminate 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 time frame for delivering results (such as, for example, 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, a citrated plasma sample or an EDTA-treated plasma sample ("determination method according to the invention"), comprising the following steps:

[0015] a) mixing the unknown biological sample with o-cresolphthalein-complexone, at a temperature between 30°C and 40°C, and without the addition of 8-hydroxyquinoline,

[0016] b) a first measurement of the absorbance Al 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>tl and that the interval (t2-tl) is between 10 and 200 seconds,

[0017] A1 and A2 being measured at a wavelength between 520 and 580 nm, then

[0018] c) the calculation of the absorbance difference (A2-A1),

[0019] in which:

[0020] if the absorbance difference (A2-A1) is zero, then the unknown sample is an EDTA-treated plasma sample,

[0021] if the absorbance difference (A2-A1) is between [-0.150; -0.080], then the unknown sample is a citrated plasma sample, and

[0022] if the difference in absorbance (A2-A1) is between [-0.070 ; -0.040], then the unknown sample is a serum sample.

[0023] The invention also relates to a method for diagnosing and / or monitoring a patient with a hemostasis disorder, comprising: - the identification of a citrated plasma sample according to the determination method according to the invention, then - the performance of a hemostasis test or the measurement of a coagulation parameter on said citrated plasma sample.

[0024] The invention is now explained in detail as follows.

[0025] The determination method according to the invention comprises the following steps:

[0026] a) mixing the unknown biological sample with o-cresolphthalein-complexone, at a temperature between 30°C and 40°C, and without the addition of 8-hydroxyquinoline,

[0027] b) a first measurement of the absorbance Al 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>tl and that the interval (t2-tl) is between 10 and 200 seconds,

[0028] Al and A2 being measured at a wavelength between 520 and 580 nm, then

[0029] c) the calculation of the absorbance difference (A2-A1).

[0030] Step a) includes mixing the unknown biological sample with o-cresolphthalein-complexone at a temperature between 30°C and 40°C. This mixture does not contain 8-hydroxyquinoline.

[0031] The principle underlying this step a) is the detection and quantification of Ca2+ ions in the unknown sample.

[0032] 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-l-propanol). The formation of this complex results in a violet color measured at 570 nm. The intensity of the color obtained is directly proportional to the amount of Ca2+ present in the sample to be assayed.

[0033] This is the case 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 Citrated plasma allows the release of calcium ions that have been chelated by citrate ions.

[0034] The reaction is schematically represented as follows: Dried plasma + o-CPC -------------► Ca-a-CPC complex (purple) (acidic environment) pH 10.7

[0035] 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.

[0036] The hypothesis considered to explain the principle of the titration is as follows:

[0037] In acidic conditions, the affinity of Ca2+ ions for EDTA is greater 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. The Ca2+ ions thus bind to o-CPC and form violet Ca-o-CPC complexes, and the solution takes on a violet color of intensity directly proportional to the amount of Ca2+ present in the sample.

[0038] 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.

[0039] Conventionally, to ensure the specificity of the assay with respect to Ca2+ ions, 8-Thydoxyquinoline is added: this compound eliminates interactions with other metal ions, considered as interfering factors, 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.

[0040] 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 non-specific for calcium.

[0041] Indeed, in the case of a serum sample, several inorganic ions are present, notably Ca2+, Mg2+, Na-, K+, and Fe2+. These will therefore bind to o-CPC, and the intensity of the staining is greater than with a citrated plasma sample, where the citrate chelates the majority of free Ca2+ ions. However, given the presence of Mg2+ and Fe2+ ions in a citrated plasma sample, significant staining is retained.

[0042] 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, preferably around 37°C. Such a temperature destabilizes the Ca-o-CPC complex, but in different proportions depending on the sampling tube used, because the complexation with metal ions is different in each:

[0043] 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.

[0044] Preferably, the mixture from step a) is incubated for 10 to 60 seconds, preferably 20 to 40 seconds.

[0045] 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 includes 2-amino-2-methyl-l-propanol (AMP).

[0046] Preferably, the mixing in step a) is carried out with a weight ratio (unknown sample): (o-cresolphthalein-complexone solution): (buffer) of between 8-12: 40-60: 40-60.

[0047] Preferably, the mixing is carried out under agitation, in particular with a ball.

[0048] 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 tl; this corresponds to a first measurement of absorbance Al.

[0049] Then a second absorbance measurement A2 of the same mixture is carried out, at a second time t2; t2 is later than tl (in other words, t2>tl).

[0050] Furthermore, the interval (t2-tl) is between 10 and 200 seconds, preferably between 50 and 190 seconds, preferably between 100 and 180 seconds.

[0051] The times t1 and t2 correspond to the times elapsed from the end of step a).

[0052] Preferably, t1 is between 10 and 20 seconds, and t2 is between 170 and 200 seconds. Typically, the first absorbance measurement Al is carried out at approximately 12 seconds (t1) after the mixing in step a).

[0053] Typically the second absorbance measurement A2 is carried out at approximately 180 seconds (t2) after the mixing of step a).

[0054] Preferably, Al and A2 are measured at 540 nm or at 577 nm, preferably at 540 nm.

[0055] Finally, the determination method according to the invention includes a step c) of calculating the difference in absorbance (A2-A1).

[0056] 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.

[0057] Starting from the difference (A2-A1), the criteria for determining the process are as follows:

[0058] -if the absorbance difference (A2-A1) is zero, then the unknown sample is a plasma sample treated with EDTA,

[0059] -if the absorbance difference (A2-A1) is between [-0.150 ; -0.080], then the unknown sample is a citrated plasma sample, and

[0060] -if the difference in absorbance (A2-A1) is between [-0.070; -0.040], then the unknown sample is a serum sample.

[0061] By "zero absorbance difference (A2-A1)", it is meant that (A2-A1) does not vary significantly. Typically, a zero absorbance difference (A2-A1) is between [-0.020 ; 0.020].

[0062] Preferably, the determination method according to the invention is carried out automatically, preferably on an automated machine.

[0063] Preferably, these measurements are performed kinetically on an automated diagnostic instrument, preferably a coagulation analyzer. More preferably, the decrease in absorbance (A2-A1) is 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. Preferably, all the steps of the determination method according to the invention are carried out on such an instrument. Without wishing to be bound by any particular theory, this decrease appears to be linked to the dissociation of the Ca-o-CPC complex. The reaction appears to be reversible when the Ca2+ ions are not chelated.

[0064] Preferably, the decrease in absorbance (A2-A1) is measured and calculated by the automaton.

[0065] 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.

[0066] 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 (decrease), which allows them to be distinguished from EDTA tubes.

[0067] At the end of the determination process according to the invention, it is thus possible to discriminate between serum, citrated plasma, and EDTA-treated plasma samples. This differentiation is particularly useful for hemostasis tests, as it allows one to start with a sample that has been correctly collected and pretreated for a given test.

[0068] The invention also relates to a method for diagnosing and / or monitoring a patient with a hemostasis disorder, comprising: - the identification of a citrated plasma sample according to the determination method according to the invention, then - the performance of a hemostasis test or the measurement of a coagulation parameter on said citrated plasma sample.

[0069] At the end of the determination process according to the invention, at least one sample of citrated plasma can be identified.

[0070] This sample can then be subjected to at least one hemostasis test or assay of a coagulation parameter; the results obtained will be reliable.

[0071] Hemostasis tests are generally routine tests, which include the Quick time (expressed in seconds) or the Prothrombin Time (PT) expressed in INR (International Normalized Ratio, which is the ratio of the PT - prothrombin rate - of the patient to the PT of the control, multiplied by an international calibration index), the activated partial thromboplastin time (aPTT), the plasma level of fibrinogen or the D-dimer assay.

[0072] Tests for measuring a coagulation parameter are chosen in particular from tests for measuring factors of the endogenous or exogenous pathway, or inhibitors of coagulation, or even of primary hemostasis.

[0073] The invention is now illustrated by the following example of implementation of the invention. Example#:

[0074] 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.

[0075] Among 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.

[0076] A STA-R Max® automated system from the Stago group is used to carry out the process according to the invention.

[0077] - The o-CPC reagent is placed in position RI of the STA-R Max® analyzer.

[0078] - The AMP reagent (AMP buffer) is placed in position R2 of the STA-R analyzer Max®. Step a)#:

[0079] - The samples are placed in the automated analyzer: 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. Step b)#:

[0080] A first measurement of the absorbance Al of the mixture obtained in a) is carried out at a first time tl = 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-tl) is equal to 168s.

[0081] The wavelength is equal to 540 nm. Step c)#:

[0082] 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:

[0083] [Math 1]

[0084] Difference DO (or DDO) = (DO(t2 2s) + DO(t2) + DO(t2 2s)) / 3 - (DO(tl) + DO(tl+2s)) / 2

[0085] The results are as follows (Table 1 and [Fig. 1]):

[0086] Table of difference absorbance measurements (here optical densities or OD) obtained on STA-R Max®

[0087] [Tables 1] Statistics - D DO Citrate 3.2% Citrate 3.8% EDTA Serum Number of samples 15 15 30 30 Mean -0.087933 -0.097467 -4.03E-03 -0.058633 Standard deviation 0.0099173 0.011594 0.0020254 0.0091557 Minimum -0.099 -0.116 -0.011 -0.072 Maximum -0.063 -0.081 -0.001 -0.038

[0088] [Fig.1] Fig.1 represents the results of difference in absorbance (here difference in DO or DDO or ADO) obtained for the different samples tested.

[0089] 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.

[0090] When the absorbance difference (A2-A1) is between [-0.150 ; -0.080], then the unknown sample is a citrated plasma sample.

[0091] When the difference in absorbance (A2-A1) is between [-0.070 ; -0.040], then the unknown sample is a serum sample.

[0092] 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.

Claims

Demands

1. A method for determining whether an unknown blood sample is a serum sample, citrated plasma, or EDTA-treated plasma sample, comprising the following steps: a) mixing the unknown biological 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; and then c) calculating the absorbance difference (A2 - A1), wherein: 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.;

2. A method according to claim 1, characterized in that a zero absorbance difference (A2-A1) is an absorbance difference (A2-A1) that does not vary significantly, typically the absorbance difference (A2-A1) is between [-0.020 ; 0.020].

3. A method according to claim 1 or 2, characterized in that it is carried out automatically, preferably on an automated machine.

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

5. 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.

6. A method according to any one of the preceding claims, characterized in that Al and A2 are measured at 540 nm or 577 nm, preferably at 540 nm.

7. 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-l-propanol.

8. The process according to claim 7, characterized in that the mixing in step a) is carried out with a weight ratio (unknown sample): (o-cresolphthalein-complexone solution): (buffer) of between 8-12: 40-60: 40-60.

9. A 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 8, then - performing a hemostasis test or measuring a coagulation parameter on said citrated plasma sample.

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