RU2772466C2 - Blood sampler with heparin base without platelet activation - Google Patents

Abstract

FIELD: medicine; diagnostics.

SUBSTANCE: group of inventions relates to diagnostical analysis of a blood sample. A method is disclosed for the determination of an indicator(s) of blood gases, an indicator(s) of a basic metabolic panel (hereinafter – BMP) and a number of platelets in a single blood sample in vitro. The method includes stages of providing a blood sampler containing heparin and at least one antiplatelet substance selected from prostaglandin analogues, where the blood sampler does not contain any of MgSO₄, EDTA and citrate; supplying blood to the blood sampler to obtain a single blood sample; mixing the single blood sample in the blood sampler, and determining an indicator(s) of blood gases, an indicator(s) of a basic metabolic panel (hereinafter – BMP) and a number of platelets in a single blood sample. A blood sampler in the form of a syringe is also disclosed.

EFFECT: group of inventions provides for “3 in 1” analysis, namely the determination of an indicator(s) of blood gases, an indicator(s) of a basic metabolic panel (hereinafter – BMP) and a number of platelets in a single sample.

8 cl, 8 dwg, 6 tbl, 5 ex

Description

Technical field

The present invention relates to a diagnostic analysis of a blood sample.

Level of invention

Rapid collection of blood samples is fundamental to the diagnosis and treatment of acute illness. Determination of acid-base balance and degree of oxygenation is at the heart of modern algorithms for the treatment of emergency conditions, for which arterial blood gas (BG) analysis is used. In addition, emergency testing devices provide data on, for example, electrolytes, cardiac biomarkers, kidney function (creatinine), and inflammation (C-reactive protein).

The measurement of the basic metabolic panel (BMP) is used in humans to analyze renal electrolytes and kidney status and acid-base balance, as well as to determine blood glucose levels, all of which are associated with human metabolism. The basic metabolic panel can also be used to monitor patients in the hospital and in people with certain diagnosed conditions, such as hypertension and hypokalemia.

Additionally, in some diseases, the number of leukocytes in the blood (WBC) is an important biomarker. A differential WBC count can be performed and five different types of blood cells can be distinguished (5-diff analysis or partial 5-dif analysis), namely, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Alternatively, leukocytes can be subdivided into granulocytes (neutrophils, basophils, and eosinophils, defined as a single group), lymphocytes, and monocytes (3-diff or partial 3-diff analysis). The number of cells in each group is indicated as a percentage. A shift in percentage indicators may indicate a pathological condition.

Additionally, another indicator is the number of platelets (also called platelets). Platelets are a small group of cells essential for normal blood clotting. Platelet count data can be used to screen for or diagnose various diseases and conditions that can cause problems with blood clots. This indicator, among others, can be used, for example, in the treatment of pathological bleeding, disease of the bone marrow, or in pathologies such as excessive blood clotting.

This assay can be used as a monitoring tool for people who have these conditions or who are being treated with drugs that have a significant effect on platelets. This analysis can also be used to monitor the condition of patients during their treatment for platelet disorders, to determine the effectiveness of treatment.

However, in general, blood sampling for the purpose of diagnostic measurement of the above indicators must be carried out separately. For example, for BG and BMP analysis, heparin is the standard anticoagulant. Heparin prevents blood coagulation, but it does not prevent the activation and aggregation of platelets (platelets), which leads to the formation of platelet clots. Therefore, heparin is not currently used for the complete clinical blood test (CBC/CBC), which includes the determination of the number of leukocytes, platelets, 3-dif or 5-dif, red blood cell concentration (RBC), hematocrit, hemoglobin concentration, and descriptive parameters of red blood cells. Determination of the number of platelets in heparinized blood can be carried out, in particular, using known automated hematology analyzers, which are not able to distinguish single platelets from aggregated platelet clots. However, hematology analyzers may misclassify platelet clots as leukocytes, and therefore false data on high leukocyte counts can be obtained, which can lead to misdiagnosis or error messages and flagging, which makes the results of the study unacceptable.

Other standard anticoagulants commonly used in hematology are ethylenediaminetetraacetic acid (EDTA) and disodium, dipotassium, and tripotassium salts. The use of EDTA is generally accepted for the safe and reliable performance of complete blood count (CBC). Additionally, EDTA salts are compatible with standard blood smear staining protocols, i.e. do not interfere with staining. If there are problems with EDTA-dependent pseudothrombocytopenia, citrate is used as an alternative anticoagulant. However, EDTA or citric acid should not be used to analyze BG and BMP values because these anticoagulants seriously interfere with electrolyte measurements. For example, EDTA and citric acid complex with Ca ²⁺ and thus affect Ca ²⁺ levels, and these anticoagulants can even damage calcium sensors in automated analyzers.

Currently, blood tests, and specifically the complete blood count (CBC) are performed on blood samples with anticoagulants EDTA or citrate, and not on heparinized blood samples. Therefore, a comprehensive analysis of CBC, BG, and BMP parameters currently has to be performed on individual blood samples with differential anticoagulation, and on individual devices.

Schnuff-Wernet et al. (Br. J. Haematol. 162, 684, 2013) describe the possibility of using MgSO ₄ for blood analysis of patients with pseudothrombocytopenia as an anticoagulant, as an alternative to EDTA or citrate.

US 2010/0280412 discloses clotting factor Xa inhibitors and a method for anticoagulating human blood in which the calcium concentration in the blood remains constant, thrombin formation does not occur, and platelet function is not affected.

US 6,880,384 B2 describes an automated blood analyzer for measuring blood gases, metabolism and electrolytes, as well as a blood sampler containing a blood sample and a mixing element.

In light of the above, there is a need for a method for obtaining a single blood sample that can be used to determine BG, BMP, platelet and leukocyte count, and a method for determining all indicators using a single automated blood analyzer and a single blood sample. This will reduce the burden on the patient associated with the need to take several blood samples to determine the various blood parameters described above.

Thus, it is an object of the present invention to provide methods for preparing a blood sample for analysis that can be used for 3-in-1 analysis of BG, BMP and platelet count, and optionally white blood cell count (or complete blood count (CBC)).

A further object of the present invention is to provide suitable means for preparing said blood sample for analysis, which can be used for 3-in-1 analysis of BG, BMP and platelet count, and optionally white blood cell (or WBC) count.

The essence of the invention

The objects of the invention are achieved by the method of preparing a blood sample in vitro , wherein the blood is pooled:

a) with at least one anticoagulant for blood gas measurements and the main metabolic panel; and

b) at least one antiplatelet agent.

The objectives of the invention are also achieved by a method for preparing a sample for a blood test, and said method includes the step

a) drawing blood from the patient into a blood sampler; wherein the blood sampler contains at least one anticoagulant for determining blood gases and BMP and at least one antiplatelet agent.

Additionally, the objectives are achieved by a method for determining blood gas and BMP values and platelet count in an in vitro blood sample, and said method includes the step of

a) determination of blood gases, BMP and/or platelet count in the blood sample obtained as described above.

In addition, the present invention relates to the use of at least one anticoagulant for measuring blood gases and BMP and at least one antiplatelet agent for preparing a blood sample for analysis of BG and BMP and platelet count, preferably in a blood sampler.

In another aspect, the goals are achieved by using a blood sampler, and said device contains blood, at least one anticoagulant for determining BG and BMP values, and at least one antiplatelet agent for determining BG, BMP values and/or platelet count.

In addition, the objectives of the invention are achieved using a blood sampler containing

a) at least one anticoagulant for determining BG and BMP values; and

b) at least one antiplatelet agent.

Detailed description

The present invention relates to a method for preparing a blood sample in vitro , wherein the blood is combined: a) with at least one anticoagulant to determine blood gas and BMP values; and b) at least one antiplatelet agent. Surprisingly, it has been found that the blood sample prepared according to the method of the invention is suitable for the analysis of BG and BMP values, as well as for determining the number of platelets. Thus, one of the advantages of the method of the present invention is that it allows a 3-in-1 analysis to be performed on a single blood sample. In a preferred embodiment, the blood sample is further suitable for determining the number of leukocytes. Thus, the blood sample obtained by the method according to the invention is preferably suitable for the analysis of BG and BMP values, as well as for performing CBC.

In another aspect, the present invention relates to a method for preparing a sample for blood analysis, and said method includes the step of

a) drawing blood from the patient into a blood sampler;

wherein the blood sampler contains at least one anticoagulant for the analysis of blood gases and BMP, and at least one antiplatelet substance. The blood sample for analysis preferably has a relatively low (ie, small) volume of blood, for example, compared to a donation blood draw, which may be about 450 ml. In a preferred embodiment, the volume of blood for analysis drawn from the patient is from about 20 µl to about 10 ml, preferably from about 35 µl to about 10 ml, more preferably from 50 µl to about 5 ml, and even more preferably from about 0 .5 to approximately 2 ml.

Additionally, the present invention also relates to an in vitro method for determining blood gas and BMP and platelet count in a blood sample, and this method includes step a), during which BG, BMP and/or platelet count is determined in a blood sample obtained by the described above ways. One of the advantages of the method according to the invention is that the measurement of all the indicators described above can be carried out in a single blood sample. In a preferred embodiment, the method further comprises the step of determining the white blood cell count.

In a preferred embodiment, the determination of BG and BMP values, as well as platelet counts, is performed using an automated blood analyzer. Suitable blood analyzers are described in, for example, US Pat. No. 5,564,419 or US Pat. No. 6,880,384.

The blood sample is usually whole blood. Blood can be of venous or arterial origin. Preferably, the blood is arterial. However, it may be preferable to use venous blood if the method of the invention is applied to blood sampling in an emergency department setting.

In one embodiment, the blood may be capillary. This embodiment is particularly preferred in the case of obtaining a blood sample from newborns.

In a preferred embodiment, the blood in the sample is not a purified fraction of a specific blood cell type, such as a population of washed platelets.

In a preferred embodiment of the above methods of the invention, the at least one BG and BMP anticoagulant is selected from the group consisting of indirect factor Xa inhibitors or direct factor Xa inhibitors, or combinations thereof.

In a preferred embodiment, the anticoagulant is selected from the group of heparinates or heparinoids, or combinations thereof. In preferred embodiments, the heparinates are heparin, such as unfractionated high molecular weight heparin (HMWH), low molecular weight heparin (LMWH), including bemiparin, certoparin, dalteparin, enoxyparin, nadroparin, pamaparin, reviparin, tinzaparin; and oligosaccharides such as fondaparinax, idraparinax. In preferred embodiments, the heparinoids include danaparoid, dermatan sulfate, and sulodexide.

In preferred embodiments, direct factor Xa inhibitors include apixaban, bntrixaban, darexaban, edoxaban, otamixaban, and rivaroxaban.

In a preferred embodiment, at least one anticoagulant suitable for BG and BMP analysis comprises heparin. As described above, heparin is the standard anticoagulant for BG and BMP assays. Heparin is a naturally occurring polysaccharide that inhibits coagulation, that is, the mechanism leading to thrombosis. Natural heparin consists of molecular chains with variable lengths or molecular weights. It is also used as an anticoagulant drug (to thin the blood). Heparin binds to the enzyme inhibitor antithrombin III (AT), causing a conformational change that leads to its activation by increasing the flexibility of its reactive site loop. The activated AT then inactivates thrombin, factor Xa, and other proteases.

In a preferred embodiment, the anticoagulant is electrolyte balanced heparin, (also referred to as balanced heparin). Heparin is known to bind to positively charged electrolytes and this can interfere with electrolyte measurements. In a preferred embodiment, electrolyte balanced heparin compositions include heparin in the form of a lithium, zinc, sodium, potassium or ammonium salt. In a preferred embodiment, the electrolyte balanced heparin composition comprises lithium heparin and sodium heparin.

In another embodiment, the heparin anticoagulant is human heparin, porcine heparin, or synthetic heparin. In a preferred embodiment, the anticoagulant is porcine heparin.

In another preferred embodiment, the anticoagulant is unfractionated heparin.

In a preferred embodiment, the final concentration of the anticoagulant, eg, heparin, is from about 10 IU/mL to about 200 IU/mL, preferably from about 20 IU/mL to about 100 IU/mL. In a particularly preferred embodiment, the final concentration of the anticoagulant, eg heparin, is about 60 IU/mL.

In one embodiment, at least one BG and BMP anticoagulant used according to the present invention may be in liquid form, also referred to as "liquid heparin", or in dry form, for example, in the form of dry balanced heparin, when combined with blood. One example of a dry form of at least one anticoagulant is a lyophilized anticoagulant, such as lyophilized heparin, or lyophilized balanced heparin.

In one embodiment, at least one anticoagulant injected into the blood for determining BG and BMP values is in lyophilized form.

In another preferred embodiment, the at least one antiplatelet agent is selected from the group consisting of Ilb/IIIa glycoprotein inhibitors, ADP/P2Y ₁₂ receptor inhibitors, prostaglandin analogs, COX inhibitors, thromboxane inhibitors, phosphodiesterase inhibitors, cloricromene, ditazole, vorapaxar, or a combination thereof. .

The Ilb/IIIa glycoprotein, also known as the αIIbβ3 integrin, is an integrin complex located on platelets. It is a receptor for fibrinogen and von Willebrand factor and promotes platelet activation. Ilb/IIIa glycoprotein inhibitors can be used to prevent blood clots from forming in order to reduce the risk of heart attack or stroke. Examples of Ilb/IIIa glycoprotein inhibitors include, without limitation, abciximab, eptifibatide (also known as integrillin), orbofiban, lotrafiban, roxifiban, sibrafiban, and tirofiban (also known as aggrastat) or a salt thereof. A preferred Ilb/IIIa glycoprotein inhibitor is eptifibatide and tirofiban, or a salt thereof.

Adenosine diphosphate (ADP) / P2Y ₁₂ receptor inhibitors) belong to a class of antiplatelet agents used for the treatment of acute coronary syndrome or for prophylaxis in patients at risk of thromboembolism, myocardial infarction or stroke. Their mechanism of action is to antagonize the P2Y ₁₂ protein and thus prevent ADP from binding to the P2Y ₁₂ receptor. This leads to a decrease in platelet aggregation and prevents the formation of a thrombus. The P2Y ₁₂ receptor is an associated surface protein located on platelets. Such receptors are referred to as G-protein-associated purinergic receptors (GPCR) and are chemoreceptors for ADP. Examples of ADP/P2Y ₁₂ receptor inhibitors include, without limitation, thienopyridines such as clopidogrel, prasugrel, and ticlopidine, or a salt thereof; and nucleotide/nucleoside analogs/receptor antagonists such as cangrelor, elinogrel, ticagrelor, sodium suramin and 2-MeSAMP. The thienopyridines are less preferred ADP/P2Y ₁₂ receptor inhibitors of the present invention because they are prodrugs that do not have an in vitro ADP/P2Y ₁₂ receptor inhibitory action. Therefore, nucleotide/nucleoside analogs/receptor antagonists such as cangrelor, elinogrel, ticagrelor or their salt, sodium suramin and 2-MeSAMP are the preferred ADP/P2Y ₁₂ receptor inhibitors of the present invention.

Prostaglandins can induce or inhibit platelet aggregation and affect vasoconstriction to dilate blood vessels. Prostaglandin analogs are a class of drugs that bind to the prostaglandin receptor. Examples include, without limitation, beraprost, iloprost (also known as ZK36374), prostacyclin, epoprostenol, and treprostinil.

Cyclooxygenase (COX), officially called prostaglandin endoperoxide synthase (PTGS), is an enzyme responsible for the formation of prostanoids, including thromboxanes and prostaglandins. Examples of COX inhibitors include, without limitation, acetylsalicylic acid, aloxypyrine, calcium carbasalate, ibuprofen, triflusal, sulfinpyrazone, and nitroaspirin (NCX-4016).

Thromboxane is a member of a family of lipids called eicosanoids. The two main thromboxanes are thromboxane A2 and thromboxane B2. A distinctive feature of thromboxanes is a 6-membered ether-containing ring. Thromboxane is so named in connection with its importance in the formation of a blood clot. The enzyme thromboxane-A-synthetase, located in platelets, converts the arachidonic acid derivative prostaglandin H2 into thromboxane. Thromboxane inhibitors include thromboxane synthetase inhibitors such as dipyridamole, picotamide, terbogrel, daltroban, seratrodast, SQ-29548 and ramatroban; and thromboxane receptor antagonists such as terbogrel and terutroban.

Phosphodiesterase is an enzyme that breaks phosphodiester bonds. Enzymes of the phosphodiesterase group (PDE) are often targets for pharmacological inhibition due to their unique tissue distribution, structural and functional properties. Phosphodiesterase inhibitors are able to prolong or increase the effects of physiological mechanisms mediated by cAMP or cGMP by inhibiting their degradation by phosphodiesterases. PDE inhibitors have been identified as new potential drugs for the treatment of pathologies such as pulmonary arterial hypertension, coronary heart disease, dementia, depression, schizophrenia, asthma, chronic obstructive pulmonary disease (COPD), protozoal infections, including malaria. Additionally, cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) are two important intracellular second messengers with potent inhibitory effects on essential platelet functions. By catalyzing the hydrolysis of cAMP and cGMP, phosphodiesterases limit intracellular levels of cyclic nucleotides, thereby regulating platelet function. In this regard, PDE inhibition may exhibit a significant inhibitory effect on platelets (Gresele et al. Br. J. Clin. Pharmacol. 2011 Oct;72(4):634-46). Examples of PDEs include, without limitation, Cilostazol, Dipyridamole, Triflusal, Milrinone, Anagrelide, and Theophylline.

Antiplatelet drugs that do not belong to one of the above groups include, without limitation, cloricromene, ditazol, vorapaxar, and L-arginine, or salts thereof.

Cloricromen is a vasodilator antiplatelet drug used in the treatment of thromboembolic conditions.

Ditazol is a non-steroidal anti-inflammatory drug with analgesic and antipyretic effects, similar to phenylbutazone. Additionally, ditazol is an inhibitor of platelet aggregation and is sold in Spain and Portugal under the trade name Ageroplas®.

Vorapaxar, formerly known as SCH 530348, is a thrombin receptor (protease-activated PAR-1 receptor) antagonist based on the natural product gimbacin.

The oral form of L-arginine has been shown to inhibit platelet aggregation via the nitrate oxide pathway (Adams et al., J. Am. Coll. Cardiol. 1995 Oct;26(4):1054-61).

Preferably, the at least one antiplatelet agent is selected from the group consisting of Ilb/IIIa glycoprotein inhibitors, ADP/P2Y ₁₂ receptor inhibitors, prostaglandin analogs, cloricromene, ditazole, vorapaxar, or combinations thereof.

In another preferred embodiment, the at least one antiplatelet agent is selected from the group consisting of Ilb/IIIa glycoprotein inhibitors, prostaglandin analogs, cloricromene, ditazole, vorapaxar, or combinations thereof.

In another preferred embodiment, the at least one antiplatelet agent is selected from the group consisting of Ilb/IIIa glycoprotein inhibitors and prostaglandin analogues.

In a preferred embodiment, at least one antiplatelet agent comprises a prostaglandin analog.

In another preferred embodiment, the at least one antiplatelet agent is an Ilb/IIIa glycoprotein inhibitor selected from the group consisting of abciximab, eptifibatide, orbofiban, lotrafiban, roxifiban, sibrafiban and tirofiban, or a salt thereof. In a preferred embodiment, the at least one antiplatelet agent comprises eptifibatide and/or tirofiban or a salt thereof. In a preferred embodiment, eptifibatide is administered in the form of the acetate salt of eptifibatide. It is also preferred to use tirofiban in the form of tirofiban hydrochloride salt, and more preferably in the form of tirofiban hydrochloride monohydrate.

In yet another preferred embodiment, the at least one antiplatelet agent is a prostaglandin analogue selected from the group consisting of beraprost, iloprost, prostacyclone, epoprostenol, treprostinil, or a salt thereof.

In a particularly preferred embodiment, the at least one antiplatelet agent comprises eptifibatide, tirofiban, iloprost, a salt thereof, or a combination thereof.

It is particularly preferred if at least one antiplatelet agent contains iloprost. Iloprost is particularly preferred as an antiplatelet agent because not only is it suitable for preparing a blood sample for BG, BMP and platelet count analysis, but it has also surprisingly been found to inhibit leukocyte activation and thus significantly reduce leukocyte aggregation in a blood sample. .

In another embodiment, at least one antiplatelet agent comprises ADP/P2Y ₁₂ receptor inhibitors, wherein the ADP/P2Y ₁₂ receptor inhibitors are a nucleotide/nucleoside analog/receptor antagonist. Preferred ADP/P2Y ₁₂ receptor inhibitors are selected from the group consisting of cangrelor, elinogrel, ticagrelor or their salt, suramin sodium, 2-MeSAMP.

In another preferred embodiment, the method of the invention does not include the step of combining MgSO ₄ , EDTA or citric acid salt with blood in the blood sample. These anticoagulants are not suitable for preparing a blood sample for BG and BMP analysis.

In another preferred embodiment, the method of the invention further comprises the step of mixing the blood sample. This step has the advantage that without agitation, the blood sample coagulates or precipitates, or the blood can react with the air in the sample prior to analysis. Stirring can be carried out, for example, by agitation. Mixing can be done manually by repeatedly inverting the blood sample or rolling it in a horizontal plane. A stirring element can also be added to the blood sample. Also, the blood sample can be agitated using moveable elements, for example, as described in US Pat. No. 6,880,384 B2. Stirring the blood sample facilitates the dissolution of the anticoagulant, such as heparin, and thus prevents the formation of a precipitate. Insufficient dissolution of the anticoagulant may lead to the formation of microclots, which may interfere with the results and/or damage the analyzer. If a precipitate forms, the blood sample may become inhomogeneous, and the results of the analysis will mislead specialists.

In another aspect, the present invention also relates to a blood sampler, and said sampler comprises:

a) at least one anticoagulant for the analysis of indicators of BG and BMP, described above; and

b) at least one antiplatelet agent as described above.

A blood sampler may be used to carry out the method of the invention described above. The at least one BG and BMP anticoagulant may be present in the blood sampler in liquid form, or it may be present as a dry composition.

In one embodiment, the blood sampler contains an additional element comprising at least one anticoagulant for determining BG and BMP values and/or at least one antiplatelet agent. For example, the additional element may be a "package", for example, the so-called "heparin pack". In this context, the use of a "package" means that at least one anticoagulant is on a "cushion" of an inert filler material, while dissolving the "cushion" heparin is distributed throughout the sample with proper mixing, and such "cushions" can be injected into devices during manufacture to deliver a repeatable amount of heparin to each sample. One example of such a "cake", for example, is a piece of cellulose impregnated with at least one anticoagulant for determining BG and BMP values and/or at least one antiplatelet agent. However, other additional elements may also be used which, upon contact with blood, may release at least one BG and BMP anticoagulant and/or at least one antiplatelet agent, such as using a matrix covering the walls of a blood sampler that applied by spraying to the inner surface of this device.

In one embodiment, the blood sampler is made of plastic or glass.

In another preferred embodiment, the blood sampler has a cap. The sampler cap is a cap attached to the open end of a blood sampler, such as the tip of a syringe or the open end of a capillary tube or test tube. The cap on the blood sampler avoids gas exchange with the environment, which can affect the results of the blood gas analysis. Suitable sampler caps are described, for example, in WO 2004/000412.

In another preferred embodiment, the blood sampler includes a mixing element. The preferred mixing element is a spherical element or a cylindrical element with rounded ends. A particularly preferred mixing element has a spherical shape. The ball can be made of steel or plastic, for example.

In another embodiment, the mixing element is coated with an inert material. The inert material preferably does not or substantially does not affect the blood test. For example, the inert material may be selected from the group consisting of gold, platinum, palladium, or rhodium. In a preferred embodiment, the inert material is gold.

In a particularly preferred embodiment, the stirring element is a gold plated ball, preferably a gold plated steel ball.

In one embodiment, the mixing element is the mixing element described in US Pat. No. 6,880,384 B2.

The movement of the stirring element is also described in US Pat. No. 6,880,384 B2. Preferably, the movement of such a mixing element takes place with the help of driving devices, for example, mechanical devices. The driving devices can be an automated manipulator or a device for moving, for example, rocking or rotating, a manipulator and / or a stand for the analyzer, so that the movement of the mixing element located in any sample occurs by means of gravitational forces.

In a preferred embodiment, the blood sampler may be filled with blood from 20 µl to about 10 ml, preferably from about 35 µl to about 10 ml, more preferably from 50 µl to about 5 ml, and most preferably from about 0.5 to about 2 ml of blood.

In another aspect, the present invention relates to the use of at least one anticoagulant for blood gas and BMP measurements and at least one antiplatelet agent to prepare a blood sample for blood gas, BMP and platelet count measurements. At least one antiplatelet agent and at least one anticoagulant for determining BG and BMP values are as described above. In a preferred embodiment, the preparation of the blood sample is carried out in a blood sampler.

In still another aspect, the present invention relates to the use of a blood sampler comprising at least one anticoagulant for blood gas and BMP and at least one antiplatelet agent for blood gas, BMP and/or platelet count. The blood sampler, at least one antiplatelet agent, and at least one anticoagulant for determining BG and BMP values are as described above. In a preferred embodiment, the blood sampler further contains blood.

Used in the invention, the term "indicators" means any piece of clinical information about a blood sample.

As used herein, the term "blood gas", for example in the context of "blood gas readings", refers to the amount of gaseous substances in the blood and includes the amount of certain gases (e.g., oxygen and carbon dioxide) dissolved in the blood, usually arterial blood. Blood gas indicators include parameters such as pH, pCO ₂ , pO ₂ , oxygen saturation (sO2), total hemoglobin concentration (ctHb or tHb), oxyhemoglobin fraction (FO ₂ Hb or O ₂ Hb), carboxyhemoglobin fraction (FCOHb or COHb ), methemoglobin fraction (FMetHb or MetHb), deoxyhemoglobin fraction (FHHb or RHb), and fetal hemoglobin fraction (FHbF). The expression "blood sample suitable for blood gas analysis" means that this blood sample can be used to measure at least one blood gas, but preferably suitable for measuring all blood gases, such as pH, pCO ₂ , pO ₂ , ctHb, FO ₂ Hb, FCOHb, FMetHb, FHHb and FHbF. It may be preferable that the measurement capability refers to at least pH, tHb, FCOHb and FMetHb.

Used in the invention, the term "basic metabolic panel", for example, in the context of "analysis of indicators of the main metabolic panel", refers to the biochemical parameters of the blood, in particular to electrolytes, namely, the concentration of Na ⁺ , K ⁺ , Cl ^- , HCO ₃ ^- , urea, creatinine, glucose (glu), Ca ²⁺ , lactate (lac) and total bilirubin (tBil). Thus, a "blood sample suitable for BMP analysis" is a blood sample that can be used to measure at least one, but preferably all of the aforementioned BMP values. Preferably, the measurement capability refers to at least Na ⁺ , K ⁺ , Cl ^- , Ca ²⁺ , glu, lac and tBil.

Used in the invention, the terms "platelet count" or "platelet count" means a diagnostic study in which the number of platelets in the patient's blood is determined. Platelets, also called platelets, are small disc-shaped blood cells that form in the bone marrow and are involved in blood clotting. Typically, there are 150,000 to 450,000 platelets in each microliter of blood. A low platelet count or the presence of irregularly shaped platelets is associated with abnormal bleeding. A large number of platelets may indicate diseases of the bone marrow.

Used in the invention, the expression "at least one", for example, in the context of "at least one anticoagulant" or "at least one antiplatelet agent" means that only one type of substance or different substances can be present in the sample, for example, different anticoagulants . In a preferred embodiment, "at least one" means "one" type of substance, such as one type of anticoagulant, such as heparin.

Used in the invention, the term "anticoagulant" means a substance that prevents or reduces blood coagulation, that is, the coagulation cascade leading to the polymerization of fibrin and, accordingly, to the formation of a fibrin clot. Thus, anticoagulants increase clotting time by inhibiting the coagulation cascade via clotting factors after initial platelet aggregation.

The term "anticoagulant for determining BG and BMP values" as used herein means that a substance is suitable as an anticoagulant for a blood sample such that the blood sample can be used to analyze BG and BMP values. It is known that some anticoagulants, such as EDTA, are not suitable for determining BG and BMP values, for example, because they form a complex with Ca ²⁺ , thereby making it impossible to reliably determine the calcium concentration in a blood sample. Thus, EDTA is not an anticoagulant for determining BG and BMP values.

Used in the invention, the term "antiplatelet substance" means a substance that reduces platelet aggregation and/or inhibits the formation of a thrombus, that is, a substance that inhibits the initial aggregation of platelets in the blood coagulation mechanism. Thus, the antiplatelet agent interferes with the platelet activation cascade leading to activated platelet adhesion to fibrin fibers, other components of the extracellular matrix, or aggregation into platelet clots. It should be noted that the coagulation cascade and the platelet aggregation cascade are two separate mechanisms, even though some proteins, such as thrombin, may be involved in both cascades. Antiplatelet agents can reversibly or irreversibly inhibit the mechanism associated with platelet activation, which leads to a decrease in the tendency of platelets to adhere to each other and to damaged endothelium of blood vessels, or to surfaces of foreign materials, such as blood sampler material.

As used herein, "blood sample" or "blood sample" refers to a blood sample that is suitable for diagnostic or analysis purposes. Thus, the blood sample has a relatively low blood volume (20 µl to 10 ml of blood), i.e. not the volume required, for example, when donating blood (up to approximately 450 ml of blood). The term "blood sample suitable for analysis of BG and BMP values and platelet count" means that such a blood sample is suitable for use in the determination of BG, BMP values, and platelet count, while the anticoagulant and/or antiplatelet substance does not affect or at least essentially does not affect the definition of any of the indicators.

As used herein, the term "blood sampler" refers to a blood sampling device such as a syringe, capillary tube or test tube, e.g. blood sampling.

As used herein, the term "white blood cell count" refers to a diagnostic analysis of the white blood cell count in a patient's blood sample. The average normal range is 3500 to 10500 leukocytes per microliter of blood.

As used herein, the term "Complete Blood Count (CBC)" means a diagnostic test that includes a white blood cell count, a platelet count, and any of the differential counts: 3-dif (partial 3-dif) or 5-diff (partial 5-diff) ). The CBC does not include the number of red blood cells in a patient's blood sample.

As used herein, "substantially no effect" means that the maximum effect fraction is less than 5, preferably less than 2.5, and more preferably 1 or less.

Used in the invention, the term "approximately" in relation to numerical values in the text of this application, for example, "approximately 10 ml", means that the value indicated immediately after "approximately" includes minor deviations from the exact numerical value, for example, due to errors measurements, etc. In a preferred embodiment, "about" refers to a value in the range of 15% (±15%) of the value immediately following "about", including any numeric value within that range, a value equal to the upper limit (i.e., + 15%) and a value equal to the lower limit (that is, -15%) of this range. For example, "about 100" covers any numeric value between 85 and 115, including 85 and 115 (with the exception of "about 100%", which always has an upper limit of 100%). In one aspect, "about" means ±10%, more preferably ±5%, even more preferably ±1%, or less than ±1%.

In general, all salts of known reagents, for example, at least one antiplatelet agent or at least one anticoagulant for determining BG and BMP values, are preferably included in the present invention, provided that these salts do not or substantially do not affect the performance blood test. Examples of salts include inorganic and organic acid addition salts and basic salts. Salts include, without limitation, metal salts such as cesium salt, alkaline salts such as lithium salt, sodium salt, potassium salt, calcium salt or magnesium salt, organic amine salts such as triethylamine salt, picoline salt, pyridine salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N'dibenzylethylenediamine salt and the like; inorganic acid salts such as citrate, tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate, trifluoroacetate, dichloroacetate, trifluoroacetate, oxalate, formate and the like, sulfonates such as methanesulfonate, benzenesulfonate, p-toluenesulfonate and the like .; and amino acid salts such as arginate, glutamate, and the like. Acid addition salts include, without limitation, hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, phosphoric acid, oxalic acid, dichloroacetic acid and the like.

Brief description of the figures

Figure 1 :

Single platelet counts and platelet clot counts were performed on blood collected with a PICO70 sampler containing liquid heparin only (LiHep, no gold (Au) bead, gentle manual mixing). The PICO70 syringe was placed in a SAM mixer (PicoSAM); EDTA anticoagulant and liquid heparin or PicoSAM complete reagent was used in combination with antiplatelet drugs: eptifibatide, tirofiban or iloprost, at two different concentrations of these drugs. In samples with three antiplatelet drugs, single platelets were counted and the absence or a small number of platelet aggregates was detected, which is comparable to the standard method of adding EDTA anticoagulant to the blood under study.

Figure 2:

Single platelet counts and platelet clot counts were performed on blood collected with a PICO70 sampler containing only liquid heparin (LiHep, no gold bead, gentle manual mixing). The PICO70 syringe was placed in a SAM mixer (PicoSAM); EDTA anticoagulant and liquid heparin or PicoSAM complete reagent was used in combination with the antiplatelet agent MgSO ₄ at two different concentrations.

Figure 3:

Single platelet counts and platelet clot counts were performed on blood collected with a PICO70 sampler containing only liquid heparin (LiHep, no gold bead, gentle manual mixing). The PICO70 syringe was placed in a SAM mixer (PicoSAM); EDTA anticoagulant and liquid heparin or PicoSAM complete reagent was used in combination with the antiplatelet drug ticlopidine at two different concentrations.

Figure 4:

Single platelet counts and platelet clot counts were performed on blood collected with a PICO70 sampler containing only liquid heparin (LiHep, no gold bead, gentle manual mixing). The PICO70 syringe was placed in a SAM stirrer (PicoSAM), EDTA anticoagulant and liquid heparin or PicoSAM complete reagent was added in combination with the antiplatelet drug L-arginine at two different concentrations.

Figure 5:

Single platelet counts and platelet clot counts were performed on blood collected with a PICO70 sampler containing only liquid heparin (LiHep, no gold bead, gentle manual mixing). The PICO70 syringe was placed in a SAM stirrer (PicoSAM), EDTA anticoagulant and liquid heparin or PicoSAM complete reagent was added in combination with the antiplatelet drug dipyridamole at two different concentrations.

Figure 6: Results of microscopic examination of blood samples after the addition of anticoagulants (A) heparin and (B) EDTA. EDTA prevents platelet activation and promotes the retention of platelets in a single state in a blood sample, while heparin allows or even enhances platelet aggregation induced by other agonists or foreign materials. According to microscopy (C), this effect of heparin was not observed when 20 μM eptifibatide was added to heparin.

Figure 7: Blood sample mixed by hand with stain/hemolyze reagent. Stained leukocytes and platelets prepared as a wet preparation on a coverslip were examined under a Leica microscope using a 40x objective in brightfield mode.

(A) Example of leukocytes showing interaction with platelets. (B) Example of leukocyte clots.

Examples

Example 1 Platelet Aggregation Assay Using Heparin and Various Antiplatelet Agents

Each experiment was performed on a separate day with blood from a single volunteer donor, testing a single investigational antiplatelet drug. For each experiment, samplers in the form of PICO70 syringes (Radiometer Medical ApS) were prepared immediately before blood sampling. For tests under “LiHep” (liquid heparin) conditions, the gold bead and heparin stick were removed from the PICO70 sampler and 15 µl of aqueous liquid balanced heparin containing lithium heparin (Celsus Laboratories) and sodium heparin (Celsus Laboratories) was added (final concentration of heparin 60 IU/ ml of blood) and 15 µl of diluted test drug. For “PicoSAM” conditions, unmodified PICO70 sample devices (with gold bead and heparin pack) were used and 15 µl of dissolved test drug was added. For “LiHep xxx drug” conditions, the gold bead and heparin stick were removed from the PICO70 sampler as described for “LiHep” conditions, and 15 µl of liquid balanced heparin (final heparin concentration 60 IU/ml blood) and 15 µl of dissolved test drug were added. For the “PicoSAM xxx drug” condition, unmodified PICO70 sample devices (gold bead and heparin pack) were used and 15 µl of dissolved test drug was added. The following candidate antiplatelet drugs and their respective solvents were tested: eptifibatide acetate (Sigma, SML1042; diluted in saline, final concentration 5 and 20 μM), MgSO ₄ (Sigma, M7506; diluted in saline, final concentration 3 and 12 mM), tirofiban hydrochloride monohydrate (Sigma, SML0246; diluted 1:200 in DMSO in saline, final concentration 0.5 and 1 μM), iloprost (Sigma, SML1651; diluted in ethanol 1:1000 or 1:10000 in saline, final concentration 10 and 100 nM (later also 1 μM)), ticlopidine hydrochloride (diluted in saline, final concentration 60 and 600 μM), L-arginine (Sigma, A5006; diluted in saline, final concentration 600 μM and 6 mM), and dipyridamole (Sigma, D9766; diluted in DMSO 1:10 or 1:100 in saline, final concentration 10 and 100 μM). Into an EDTA tube (BD Vacutainer coated with K ₂ EDTA, 10 ml) and into PICO70 syringes, two samplers each using all the above conditions, venous blood was drawn through a butterfly needle closed with a ventilated cap (VTC), filling self-filling devices for PICO70 samples with venous whole blood in a volume of 1.5 ml. Immediately after blood collection, the devices were inverted 8 times to ensure proper anticoagulation of the samples. Blood samples were gently mixed manually (devices without gold ball) or with a SAM stirrer (Radiometer Medical ApS) within 15 minutes after sampling. After mixing, the samples were immediately fixed in 10% formalin solution (1:1 dilution of blood in formalin) for at least 10 minutes and then diluted 1:10 in a solution of diluted platelets to hemolyse red blood cells for an estimated manual platelet count using a hemocytometer. Manual counting of the number of single platelets (not aggregated), platelet clots and, if possible, determination of the size of clots (number of platelets in a clot) was performed twice for each sample, counting platelets in a hemocytometer using a Leica 750 microscope with phase contrast air lenses 10x and 20x. To calculate the concentration of single platelets, the number of single platelets was selected for dilution with liquid heparin and/or a solution of the diluted drug.

With the addition of eptifibatide, tirofiban and iloprost, the formation of platelet clots was reduced compared to the reference conditions "LiHep" and "PicoSAM" (see figure 1).

MgSO ₄ also reduced the formation of platelet clots (see figure 2).

The "ticlopidine+LiHep" condition showed a similar number of single platelets compared to the "EDTA" reference condition, and a slight decrease in the number of clots compared to the "LiHep" and "PicoSAM" control conditions (see Figure 3). With the addition of L-arginine, a decrease in the number of platelets and an increase in the number of platelet clots was found compared to the EDTA control, but at the same time, a higher number of platelets than in the control tests with LiHep and PicoSAM (see figure 4).

The dipyridamole, phosphodiesterase inhibitor, and thromboxane inhibitor tests showed slightly higher platelet counts compared to the control tests with LiHep and PicoSAM (see Figure 5).

Example 2 Platelet Aggregation Studies in Heparinized Blood and EDTA Blood Treatment

Venous blood sampling was performed, mixed and fixed with 10% formalin solution as described above in Example 1, then wet preparations were prepared on a glass slide and covered with a coverslip. After that, wet preparations of fixed blood cells of the samples were examined using a Leica 750 microscope with a 40x phase contrast air objective. In heparinized blood, aggregated platelets were found among many erythrocytes, in contrast to non-aggregated single platelets in blood with the anticoagulant EDTA and in heparinized blood with the addition of, for example, eptifibatide.

The results are shown in Figure 6. Platelet clots were found in heparinized blood, which cannot be found in blood samples treated with EDTA or in a blood sample treated with heparin and an antiplatelet drug such as eptifibatide.

Example 3 Microscopy of Wet Slides of Stained Blood Samples

Venous blood sampling and mixing was performed as described in Example 1 above and these samples were used to prepare stained wet slides for imaging. Blood samples were mixed with staining and hemolyzing agent (methylene blue and deoxycholic acid, respectively) and incubated at 47°C in a water bath for 30 seconds. Then, wet preparations were obtained from stained and hemolyzed blood samples on a glass slide and covered with a coverslip. Images of stained samples were examined using a Leica 750 microscope with a 40x air objective in bright field mode. Image processing software (FIJI, ImageJ) was used to select regions of interest (ROI) from representative stained leukocytes.

Samples under “PicoSAM” conditions (heparinized blood without antiplatelet drug) showed many aggregated platelets (small round cells).

In PicoSAM samples with the addition of 1 μM tirofiban or 20 μM eptifibatide, single platelets were detected, as well as the phenomenon of platelet satelliteism, that is, the adhesion of platelets to leukocytes. This is for example shown in figure 7A. Additionally, leukocyte aggregation was also detected, as shown in Figure 7B.

In PicoSAM samples supplemented with 100 nM iloprost (or in samples with balanced LiHep and 100 nM iloprost), single platelets were detected, and no leukocyte aggregation or platelet satelliteism was detected. The same results were obtained in the study of blood with the anticoagulant EDTA.

Example 4 Single Platelet Concentrations in Iloprost and Heparin Samples Compared to EDTA Reference Samples

Manual platelet count

Blood samples were prepared in the same way as in Example 1.

Platelet Counting in the ABX Analyzer

Blood samples after taking and mixing for 15 minutes as described above were analyzed using an automated ABX hematology analyzer (Horiba, ABX Pentra 60C +) for the purpose of a complete clinical blood test (CBC) with the determination of 5 dif, including the concentration of leukocytes, the concentration of platelets, mean platelet volume (MPV), concentrations and fractions of neutrophils, lymphocytes, monocytes, eosinophils and basophils, erythrocyte concentration, hematocrit, hemoglobin concentration, erythrocyte descriptive parameters (MCV - mean erythrocyte volume, MCH - mean erythrocyte hemoglobin content, MCHC - mean concentration hemoglobin in the erythrocyte mass, RDW - distribution width of erythrocytes). Samples were obtained from several donors. The measured platelet concentration was used for comparison with data obtained by manually counting the concentration of platelets in samples with iloprost and heparin (in PICO70 sample devices), and in samples with the anticoagulant EDTA. The ABX analyzer may underestimate the concentration of platelets when they are aggregating, although due to the shear force in the flow, some of the weakly connected platelets may be separated. For control, EDTA blood samples without platelet clots were used as a reference measurement for the ABX automated measured platelet concentration.

For one donor, manually counted platelet counts in PicoSAM samples with 100 nM iloprost compared to EDTA reference samples were 97%.

For eight donors, when manually counted, the mean platelet counts in PicoSAM samples with 1 μM iloprost compared to EDTA reference samples were 93%.

In contrast, when the corresponding sample was tested under PicoSAM conditions, i.e., without the addition of iloprost but with heparin, the performance was less than 40% compared to the reference samples with EDTA.

For seven donors, in ABX platelet counts, mean platelet counts in PicoSAM samples with 1 µM iloprost compared to EDTA reference samples were 97%.

Table 1 shows the results of platelet counts in donor samples, both types of platelet counts (manual count and ABX platelet count) were performed.

Table 1 Comparative Platelet Counts

Donor number Mean platelet count measured in ABX (PLT/nL) Mean platelet count, manually counted (PLT/nl) 4 PicoSAM
Iloprost 1 µM 204 198 EDTA 200 200 5 PicoSAM
Iloprost 1 µM 253 242 EDTA 259 242 6 PicoSAM
Iloprost 1 µM 173 154 EDTA 185 189 7 PicoSAM
Iloprost 1 µM 233 247 EDTA 240 249 eight PicoSAM
Iloprost 1 µM 191 203 EDTA 202 207 nine PicoSAM
Iloprost 1 µM 216 202 EDTA 236 255

Example 5. Analysis of the effect of an antiplatelet drug on the performance of the ABL90 analyzer

Each experiment was performed on a separate day with blood from a single voluntary donor. For each experiment, three PICO70 (Radiometer Medical ApS) syringe sample devices (unmodified, containing a gold bead and a heparin cake) were prepared. One PICO70 was used as an unmodified control (Control), one PICO70 was filled with a 15 µl diluted antiplatelet drug to achieve the indicated final concentration in the blood sample. The third PICO70 sample was filled with 15 µl of diluent (the specific diluent that is used to dilute the antiplatelet drug under test: a reference sample). Venous blood samples were taken into PICO70 sample syringes through a butterfly needle and sealed with a vented cap (VTC), while 1.5 ml whole blood was drawn into PICO70 self-priming samplers. Immediately after filling the syringes, the blood samples were inverted eight times to ensure proper heparinization of the blood. The blood samples were stirred using a SAM (Radiometer Medical ApS) stirrer, while in the blood sample, a gold-plated steel ball was moved to ensure homogeneous and repeatable mixing for 15 minutes after blood collection. After that, blood gas parameters were determined in the ABL90 analyzer (Radiometer Medical ApS). Measurements were also carried out in alternative samples (control, reference sample, drug sample) five times in each sample (5 repeated measurements), carefully turning the blood samples manually to keep the blood in a state of homogeneous mixing during the measurements. All measured values are shown in Table 3. After that, the blood samples were centrifuged to separate the plasma fraction. The plasma of all samples was analyzed three times in turn using the ABL90 analyzer to assess the concentration of free hemoglobin in plasma, which indicates a possible unwanted hemolysis of erythrocytes in whole blood samples. In hemolyzed samples, the effect is shown, for example, on the measured K ⁺ concentration.

The measured values are described in Table 2.

Table 2. Parameters measured in the ABL90 blood gas analyzer

Indicator Description pH pH level tHb (g/dL) total hemoglobin concentration COHb (%) carboxyhemoglobin (carboxylated hemoglobin) MetHb (%) methemoglobin (methylated hemoglobin) K ⁺ (mol/l) potassium concentration Na ⁺ (mol/l) sodium concentration Ca ²⁺ (mol/l) calcium ion concentration Cl ^- (mol/l) chloride concentration Glu (glucose, mol/l) glucose concentration Lac (lactate, mol/l) lactate concentration tBil (total bilirubin, µmol/l) bilirubin concentration

The measurement procedure performed is described in Table 3, and the description of the analyzed samples is given in Table 4.

Table 3. Measured values

Mean Average of 5 replicate measurements performed on a whole blood sample or of 3 replicate measurements of hemoglobin performed on a plasma sample (data not shown) SD Standard deviation calculated from 5 repeated measurements (data not shown) Difference Difference in measured values between specific samples (drug test sample - vehicle-only reference sample) (data not shown) Maximum Influence Share Difference divided by the maximum allowable influence for each parameter (a value >1 indicates an influence, while a value <1 is acceptable)

Table 4. Designations of samples

The control Control sample collected in an unmodified sampling device - syringe PICO70 saline solution Reference sample containing saline solution (saline NaCl/saline solution) DMSO/Saline (1:200) Reference sample containing dimethyl sulfoxide solvent in saline NaCl solution (dilution 1:200) EtOH / saline (1:1000) Reference sample containing solvent ethanol in saline (1:1000 dilution) EtOH / saline (1:100) Reference sample containing solvent ethanol in saline NaCl (dilution 1:100) Eptifibatide 20 µmol in saline Eptifibatide dissolved in saline NaCl to reach a final concentration of 20 µmol eptifibatide in a blood sample Tirofiban 1 µmol in DMSO/Saline Tirofiban diluted in DMSO/NaCl saline (1:200) to reach a final concentration of 1 µM tirofiban in the blood sample Iloprost 100 nM in EtOH/Saline Iloprost, diluted ethanol/saline NaCl (1:1000) to achieve a final concentration of 100 nM iloprost in a blood sample Iloprost 1 µM in EtOH/Saline Iloprost, diluted ethanol/saline NaCl (1:1000) to achieve a final concentration of 1 µM iloprost in a blood sample MgSO ₄ 12 mM in saline MgSO ₄ dissolved in saline NaCl to reach a final concentration of 12 mM MgSO ₄ in the blood sample

Valid reference ranges for adults and maximum influence in the examples are shown in Table 5.

The results of measurements (in the form of a share of the maximum influence) are shown in Table 6.

Table 5. Reference values for determining the maximum allowable influence

pH tHb (g/dL) COHb
(%) MetHb
(%) K ⁺ (mmol/l) Na ⁺ (mmol/l) Reference range in adults 7.32-7.43 11.4-17.5 2-3% (non-smokers)
7-9% (smokers) 1-2% 3.4-4.5 136-145 Maximum Permissible Influence <0.010 <0.5 <1% <1% <0.1 <1 Ca ²⁺ (mmol/l) Cl ^- (mmol/l) Glu (mmol/l) Lac (mmol/l) tBil (µmol/L) Reference range in adults 1.15-1.33 98-107 3.6-5.3 0.56-1.39 0-34 Maximum Permissible Influence 0.02 one 0.1 0.1 thirty

Table 6. Proportion of maximum effect for antiplatelet drugs

Eptifibatide Tirofiban Iloprost MgSO ₄ pH 0.18 0.18 0.32 5.56 tHb (g/dl) 0.04 0.12 0.12 0.08 COHb (%) 0.02 0.06 0.08 0.04 MetHb (%) 0.08 0.04 0.04 0.04 K ⁺ (mmol/l) 0 0 0.18 2.0 Na ⁺ (mmol/l) 0.4 0 0.14 5.6 Ca ²⁺ (mmol/l) 0.2 0.1 0.1 2.7 Cl ^- (mmol/l) 0 0 0.06 8.6 Glu (mmol/l) 0 0.8 0.86 0.2 Lac (mmol/l) 0.6 0 0.62 4.6 tBil (µmol/L) 0.013 0.000 0.013 0.140

Eptifibatide, tirofiban, and iloprost had no effect on the ABL analyzer values, while MgSO4 affected various parameters (pH, N+, K+, Ca ²⁺ , Cl ^- and Lac) (difference in the proportion of maximum effect >2).

Thus, eptifibatide, tirofiban and iloprost at the concentrations studied should be safe to add to heparinized blood used for blood gas measurements and the main metabolic panel.

Claims (12)

1. A method for determining the indicator(s) of blood gases, the indicator(s) of the main metabolic panel (BMP) and the number of platelets in a single in vitro blood sample, including the steps: a) providing a blood sampler containing heparin and at least one antiplatelet agent selected from prostaglandin analogs, where the blood sampler does not contain any of MgSO ₄ , EDTA and citrate; b) supplying blood to a blood sampler to obtain a single blood sample; c) mixing a single blood sample in a blood sampler; and d) determination of blood gas index(es), primary metabolic panel (BMP) index(es) and platelet count in a single blood sample. 2. The method of claim 1, wherein the one antiplatelet agent is a prostaglandin analog selected from the group consisting of beraprost, iloprost, prostacyclin, epoprostenol, treprostinil, or a salt thereof. 3. The method of claim 1 or 2, wherein the at least one antiplatelet agent comprises iloprost. 4. A blood sampler in the form of a syringe for a single blood sample from 20 μl to 10 ml, where the syringe contains (a) heparin and (b) at least one antiplatelet agent selected from prostaglandin analogs, where the blood sampler does not contain any of MgSO ₄ , EDTA and citrate. 5. The blood sampler of claim 4, wherein the at least one antiplatelet agent is a prostaglandin analog selected from the group consisting of beraprost, iloprost, prostacyclin, epoprostenol, treprostinil, or a salt thereof. 6. The blood sampler according to claim 4 or 5, wherein the at least one antiplatelet agent comprises iloprost. 7. A blood sampler according to any one of claims 4 to 6, wherein said blood sampler comprises a mixing element. 8. The blood sampler of claim 7, wherein the mixing element is a spherical element or a cylindrical element with rounded ends.

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