RU176274U1 - DEVICE FOR PREPARATION OF BLOOD FOR DETERMINING THE CONCENTRATION OF GLUCOSE IN BLOOD PLASMA - Google Patents

Abstract

The utility model relates to medicine, more specifically to clinical laboratory diagnostics and endocrinology, and may find application for the diagnosis of diabetes.

The device is a vacuum tube with a barcode and a gray protective cap assembly with a rubber stopper, on the bottom of which a separating inert gel is placed in an amount of 0.6-1.2 g and a mixture of potassium oxalate with sodium fluoride is applied to the walls of the tube.

The device provides increased accuracy in determining the concentration of glucose in blood plasma, reduces the time it takes to prepare the sample, reduces the risk of hemolysis during transportation and storage of blood, and reduces the risk of infection of laboratory personnel with blood contact infections.

Description

The utility model relates to medicine, more specifically to clinical laboratory diagnostics and endocrinology, and may find application for the diagnosis of diabetes.

The prevalence of diabetes mellitus, impaired glucose tolerance and especially gestational diabetes mellitus (GDM) throughout the world and our country has been steadily increasing in the last 10-20 years. HSD is a disease characterized by hyperglycemia, first detected during pregnancy, but not meeting the criteria for “manifest” (first detected) diabetes mellitus. The multicenter international study “Hyperglycemia and Unfavorable Outcomes of Pregnancy”, conducted in the 2000s, showed that the criteria for the diagnosis of GDM require revision. Thus, among the observed women, adverse pregnancy outcomes increased in direct proportion, starting from a significantly lower glycemia level than previously accepted as a criterion for the presence of GDM. In 2008, in the USA, the International Association of Diabetes and Pregnancy Study Groups IADPSG proposed for discussion new criteria for the diagnosis of GDM, based on the results of a study that included more than 23 thousand pregnant women who underwent a 75-gram oral glucose tolerance test (OGTT). The study showed that in women who exceeded at least one of the cut-off points of OGTT in terms of glucose in the plasma of venous blood, the frequency of birth of large children or preeclampsia was doubled. These pregnant women also showed a 45 percent increase in the preterm birth rate and the need for a cesarean section. During 2010-2012, a number of developed countries, including the USA, Japan, Germany, Israel, Finland, independently adopted new criteria for diagnosing GDM, guided by the results of this study. The experts of the Russian Association of Endocrinologists and the Russian Association of Obstetricians and Gynecologists as a result of repeated discussions of this issue also came to the conclusion that it is necessary to adopt new criteria for the diagnosis of GDH. Later, the Russian Consensus on the diagnosis and treatment of GDM was created. In 2013, clinical recommendations appeared (treatment protocol) "Gestational diabetes mellitus: diagnosis, treatment, postpartum monitoring", approved by the Ministry of Health of the Russian Federation. In these documents, the requirements for the correct diagnosis and detection of hyperglycemia (high blood glucose concentration) are most clearly formulated. There are no such clear recommendations for the non-pregnant population, therefore, in various medical institutions the concentration of glucose in blood plasma, in blood serum, in capillary blood and in whole blood is determined.

The standards for use in accredited clinical diagnostic laboratories in the EU and Russia recommend the determination of glucose in blood plasma using fluoride to prevent a decrease in glucose during storage of samples. Such recommendations are particularly relevant in connection with the centralization of laboratory research, i.e. analysis in large clinical diagnostic laboratories, where blood samples are delivered by transport from collection points, for example, from clinics or private clinics. For example, from a blood sampling point in Murmansk by air transport, INVITRO company delivers samples to a laboratory in Moscow, where glucose levels are determined. In this regard, the relevance of the correct and timely diagnosis of diabetes and impaired glucose tolerance is very high. First of all, this concerns the diagnosis of carbohydrate metabolism disorders in pregnant women.

In accordance with the Consensus and Recommendations, the diagnosis of carbohydrate metabolism disorders during pregnancy should be carried out in 2 stages. When a pregnant woman first visits an obstetrician-gynecologist or family doctor for a period of up to 24 weeks, all women must carry out the following tests without fail: determination of fasting glucose in venous blood plasma, or determination of venous plasma glucose at any time of the day, regardless of food intake, or determination of glycohemoglobin level (HbA1c) using a certified according to the National Glycohemoglobin Standartization Program (NGSP) and standardized according to the reference values adopted in DCCT (Diabetes Control and Com plications Study). In the event that the result of the study corresponds to the category of manifest diabetes mellitus (DM), its type is specified, and the patient should be immediately transferred for further management to the endocrinologist. If a randomly determined plasma glucose level is less than 11.1 mmol / L, fasting venous plasma glucose should be determined: according to Consensus, with a fasting venous plasma glucose level of more than 5.1 mmol / L, but less than 7.0 mmol / L, the diagnosis of GDS ; with a fasting plasma glucose level of less than 5.1 mmol / l, all pregnant women who have a high risk of developing GDM should immediately undergo an OGTT. In this case, it is advisable and correct to assign a fasting glucose determination in a specialized laboratory with blood sampling from a vein into a test tube with sodium fluoride (a test tube with a gray cap, designed to determine glucose concentration). In this case, analysis of capillary blood in laboratories is recommended to be carried out only in case of emergency due to the noticeably lower accuracy of the result. It is important to note that, according to experienced endocrinologists, the harm from an incorrect diagnosis of GDM on time is much higher than overdiagnosis, since the further algorithm for treating the disease involves a diet and careful monitoring of the glucose level and other laboratory parameters in a pregnant woman. At the second stage of GDM diagnosis, for patients without hyperglycemia detected at the first stage, a study using OGTT should be carried out at 24-28 weeks, namely, a test with 75 g of glucose, which is considered a safe load diagnostic test to detect carbohydrate metabolism disorders during pregnancy. A test is performed in the morning on an empty stomach after an 8-14 hour night fast. Venous plasma glucose testing should only be performed in a stationary laboratory using biochemical analyzers or stationary glucose analyzers. The use of portable self-monitoring tools (blood glucose meters) for testing in accordance with Consensus 2012 is prohibited.

It should be noted that in a recent study, when comparing the accuracy of 43 glucometers of various manufacturers certified by the European Union (EU), only 27 met the accuracy criteria (ISO 15197), and the bias of the results from the readings of reference stationary analyzers reached 14% [J Diabets Sci Technol. 2012 Sep 1; 6 (5); 75]. An accurate determination of glucose concentration is very important for the diagnosis of GDM and the subsequent management of such patients, since the variability of the results obtained depending on the time elapsed from blood sampling to its analysis is quite large. Specialists are well aware that blood red blood cells use plasma glucose to maintain their vital functions, and therefore plasma glucose levels after blood sampling are constantly reduced for each subsequent hour. This can lead to hypodiagnosis of diabetes. When performing an analysis in the laboratory later than 1 hour from blood sampling, sodium fluoride should be added to the blood sampling tube to inhibit glycolysis in red blood cells; after centrifuging the blood, it is recommended to transfer blood plasma into a separate tube.

This utility model relates to the improvement of a blood sample preparation apparatus (vacuum tubes) to accurately determine the glucose concentration in the blood of patients.

We conducted a search in the patent and scientific and technical literature on the production and use of tubes for blood sample preparation for determining the concentration of glucose in blood plasma.

Patents CN 105193427. 2015818 FUZHOU CHANGGENG MEDICAL DEVICES CO LTD; CN 104510481. 20130926 WENZHOU GAODE MEDICAL INSTR CO LTD; CN 203169191. 20121018 FU SHTMING, CN 202489959. 20120228, DAYONG XU, CN 202430210. 2011123. SHIMING, China. The patents describe blood collection tubes containing sodium fluoride and anticoagulants, and a method for applying them to the walls of the tube. EDTA (ethylenediaminetetraacetate), oxalate, heparin, and others are proposed as anticoagulants. Test tubes are used for routine blood tests.

Patents CN 202430219, 20111123, China. Gel tubes for the extraction, purification and activation of mononuclear cells and blood lymphocytes are described. As anticoagulants, EDTA or citrate, heparin, Hanks buffer solution are used. The extraction of mononuclear cells increases the accuracy of the examination of patients.

EP 0193279. JAN 29, 1985; 2548507, Dec. 28, 2010, Japan. Vacuum tubes and their manufacture are described for collecting blood with a coating of the inner walls of the tube with a hydrophilic substance that prevents blood cells from adhering to the walls of the tube, and with a separation gel to separate serum from blood cells for routine blood analysis.

WO 2015132774, Mag. 7, 2014. Pasteur Institute - a vacuum tube with an anticoagulant for collecting blood and a method for its manufacture.

WO 02097432, 20010601, Finland. - a tube with an anticoagulant and a method for performing a blood test from its sample. EDTA was used as an anticoagulant.

US patents 5320812, 5860937 Becton Dickinson, 5326535, 5246660 Becton Dickinson, Company (Franklin Lakes, NT) - patents 1975-1999 of the oldest American company "Becton Dickinson" (BD), founded in 1887, manufacturing and selling instruments, tools and reagents, including blood collection tubes. The company conducts business in 50 countries, has 30 thousand employees worldwide and subsidiaries in Ireland and the UK. Vakuteyner trademark for single plastic tubes with dosed vacuum, designed for sampling venous blood. Test tubes with a gray cap - for glucose analysis, contain sodium fluoride and oxalate, do not contain a separation gel.

These patents describe blood collection devices with an anticoagulant, some of which are silicone coated on their walls.

US 3920549. Nov. 18, 1975. Corning Glass Works (Corning NY) - a blood collection device containing a gel-like inert material, followed by analysis of blood plasma, without fluoride.

Search results for companies producing test tubes for glucose analysis:

Greiner Bio-one Preanalytics (Austria) - tubes for determination of glucose in venous blood.

Corway, Estonia - blood collection tubes.

RUSTECH, Germany - systems for vacuum blood sampling.

In Russia, test tubes for glucose analysis with fluoride (gray cap) are produced by the following companies:

Eiliton LLC, patent for PM 114277 - a microtube with a locking cap and anticoagulant for taking capillary blood.

Vaku Meyer LLC "Minimed", Bryansk region., Founded in 1992, TK 548792.

Crossmed.ru - produces tubes with EDTA, oxalate, heparin and sodium fluoride for the study of glucose in diabetology.

UNIVAC, TK 564110.

It is known to use a gel-like product together with EDTA (US Pat. No. 3,920,549 by applying a gel to the bottom of the tube before adding EDTA, which is sprayed onto the surface of the gel. Such a tube (with a lilac cap) is intended for plasma analysis, in particular for molecular diagnostics and determination of virus concentration not suitable for glucose determination.

Based on the analysis of patent and company documentation, we noted that to determine the concentration of glucose in blood plasma, test tubes with a mixture of sodium fluoride and an anticoagulant deposited on its walls are used, most of which use EDTA (ethylene diamine tetra acetate) - a test tube with a gray cap.

As a prototype for this utility model, we took a test tube from UNIVAC (CJSC A / O Unimed, Moscow) for testing glucose with sodium fluoride and EDTA (gray cap). The test tube is intended for the study of blood plasma in diabetology (glucose measurement). Venous blood is taken by a “closed” vacuum method. The tube is sterilized by radiation. The tube material is polyethylene terephthalate (PET). Test tube cap made of gray plastic (in accordance with the international standard ISO 6710). Bromobutyl rubber cap with recess for multiple punctures. Filler - sodium glycolysis inhibitor fluoride and anticoagulant (EDTA) to prevent blood coagulation.

Sample preparation of blood according to the prototype is described in the Consensus for the diagnosis of GDM and includes the following procedures: after taking the patient's venous blood into a primary vacuum tube containing an anticoagulant and fluoride, the tube is thoroughly mixed to dissolve these substances. Next, the primary tube is transported to a laboratory, for example, by road or air. In the laboratory, the specified tube is centrifuged under standard conditions, the lid is opened and the laboratory personnel selects the top layer (blood plasma) and transfers it to the secondary (empty) tube to place the plasma on board the biochemical analyzer (the plasma glucose level is stable for a long time) . Previously, the secondary tube is numbered or glued with a barcode to identify the patient.

In the prototype, as in other sources of information we found, in blood sample preparation for glucose analysis after centrifugation of blood due to the lack of a clear boundary between the plasma layers and blood cells, the possibility of mixing the layers during transportation, shaking, etc. plasma is transferred to another tube, followed by determination of glucose concentration in it. Our studies have shown that the results of such an analysis are not accurate enough and also inconvenient for practical work.

The disadvantages of the prototype can also include a high probability of destruction of red blood cells (hemolysis) during transportation of blood tubes due to shaking and the duration of storage of whole blood, especially in elderly and debilitated patients, as well as in patients with artificial heart valves. Plasma staining in red color occurs in these test tubes due to hemolysis and this is observed in 4-10% of cases. The appearance of hemolysis in a patient’s sample leads to a laboratory refusal to perform analyzes in such a sample (rejection of tubes), since the appearance of hemoglobin in the plasma leads to erroneous results during the work of biochemical analyzers. Typically, the laboratory interprets the presence of hemolysis as the presence of a mistake by medical personnel during blood sampling and transportation and requires taking blood from the patient again. The disadvantages of the prototype should include possible errors in blood transfusion to obtain plasma and laboratory personnel usually signing the patient’s name or number on a secondary tube placed in a biochemical analyzer (human factor), which does not guarantee the determination of glucose in the patient whose blood is delivered to the lab. When performing sample preparation according to the prototype, it is also possible that the personnel may be infected with blood contact infections (unscrewing the cap of the primary tube with the hands of personnel, transfusion of blood plasma into a secondary tube with a manual dispenser), and the laboratory staff’s time associated with transfusion of plasma from the primary tube into the secondary tube will increase, which leads to an increase in the cost of analysis. At the same time, it is necessary to take into account the laboratory costs for disposable tips of dispensers for blood transfusion and the cost of secondary tubes, which, after performing glucose analysis in the biochemical analyzer, must be further disinfected and disposed of.

The technical result of this utility model is to increase the accuracy of determining glucose concentration by taking blood into a test tube, on the bottom of which a separating inert gel is placed.

This result is achieved by the fact that in the known vacuum tube with a barcode, a gray protective cap assembly with a rubber stopper and a mixture of sodium fluoride and anticoagulant deposited on the tube walls, followed by sterilization of the tube by the radiation method, according to the claimed utility model, it additionally contains a separation inert gel in the amount of 0.6-1.2 g, located at the bottom of the tube, potassium oxalate as an anticoagulant.

The use of an inert gel separator (as a result of raising it from the bottom of the tube during centrifugation of blood) allows you to separate the top layer (blood plasma) from the formed elements, eliminating hemolysis and a decrease in glucose concentration due to the impossibility of catabolism ("eating") of plasma glucose by red blood cells - and thereby guarantees high accuracy in determining its concentration.

Due to the use of a separation gel, blood plasma is not required to be transferred to another tube after centrifugation, which allows the determination of glucose concentration in the primary tube, which reflects the true glucose levels in the patient's blood, increasing the accuracy of determining its concentration and decreasing the time of blood preparation and reducing the risk infection of personnel with blood contact infections.

The amount of separating gel added - 0.6-1.2 g - was found by us empirically and in the process of multiple determination of glucose concentration it was shown that this amount is necessary and sufficient for reliable separation of the formed elements from blood plasma during the standard procedure of centrifuging the tube for 10 minutes at a volume taken blood 3-4 ml.

In addition, the use of a separation gel eliminates the entry into the plasma of the lower blood layer containing clots and red blood cells, allowing you to determine the glucose concentration on the automatic analyzer immediately after centrifugation of the tube and eliminating the possibility of clogging the needle of the sample probe of the biochemical analyzer and its failure.

The necessity of using test tubes with a gel for blood sample preparation for glucose analysis was prompted by the results of our performed on the basis of the FSBI “Northwest Medical Research Center named after V.A. Almazova "of the Ministry of Health of Russia in 2016 - January 2017 of the research work" Study of the effect of the separation gel in vacuum systems for blood collection and storage on the correct determination of glucose in blood plasma ". We measured the concentration of glucose and the degree of hemolysis in the blood plasma of 30 patients.

Comparison of the results of the determination of glucose after blood sampling in the proposed device with a separation gel containing sodium fluoride and as an anticoagulant potassium oxalate, and tubes with sodium fluoride and EDTA (prototype) are shown in table. one.

1. EDTA fluoride gel

2. Oxalate fluoride gel

3. EDTA fluoride - a device without a separation gel (prototype)

Tab. 1. The level of hemoglobin in blood plasma (g / l) after blood sampling of donors using devices for sample preparation using gel and prototype (EDTA and potassium oxalate were added as anticoagulants.

As follows from Table 1, our test tubes containing potassium oxalate as an anticoagulant showed a statistically significant lower hemolysis than tubes containing EDTA.

In this regard, the use of potassium oxalate as an anticoagulant, we consider an essential sign of PM.

At a plasma concentration of free hemoglobin of more than 0.8 g / l, hemolysis was already determined visually, which in the usual way of working by laboratory personnel leads to the rejection of such blood samples, i.e. makes them unsuitable for analysis.

From this study it follows the appropriateness of centrifuging the blood directly at the place of its collection, before transportation, if this is due to its duration or the possibility of vibration or temperature differences in the path. In this case, the probability of hemolysis in the future is completely excluded.

Preparation of a device for taking blood from a patient and its further investigation is carried out as follows.

After taking the blood in a vacuum way, the blood in the device is immediately mixed to dissolve the potassium fluoride and potassium oxalate. Further, the blood is centrifuged for 1 hour from the moment of blood sampling at 3000 rpm for 10 minutes to separate the plasma, if it is planned to transport blood with the possibility of vibration, shaking or long-term storage at room temperature. The determination of glucose in blood plasma is carried out up to 24 hours of storage at room temperature. If the laboratory performing the blood glucose test is located near the blood sampling point, the centrifugation of the device can be performed directly in the laboratory, after which the device is placed in a biochemical analyzer for analysis. Determination of glucose is carried out on any stationary biochemical automatic analyzer, placing a bar-coded device on board the analyzer.

The proposed device in comparison with the known has several advantages.

Improves glucose detection accuracy.

It allows you to abandon the procedure for transfusion from a blood sample of the upper plasma layer into a separate (secondary) tube, which reduces the risks to the health of laboratory personnel and the possibility of errors due to incorrect labeling of secondary tubes.

The device can be placed in an automatic analyzer for determining glucose immediately after centrifugation of the blood without the risk of clots and red blood cells catching the sample probe with the needle.

In the case of centrifugation of blood directly at the site of blood sampling, the risk of hemolysis of the sample is excluded; the risk of hemolysis of the sample during storage and transportation

The device was developed and manufactured by the authors at the OV InterVak Technology vacuum systems plant in Narva and was tested at the Federal State Budgetary Institution North-West Federal Medical Research Center. V.A. Almazova Ministry of Health of Russia in 2016-2017 with a positive result.

Claims (1)

A device for the preparation of blood for determining the concentration of glucose in blood plasma, which is a vacuum tube with a barcode, a gray protective cap assembled with a rubber stopper and a mixture of sodium fluoride and an anticoagulant deposited on the walls of the tube, characterized in that it additionally contains an inert separator gel in an amount of 0.6-1.2 g, located at the bottom of the tube, and potassium oxalate as an anticoagulant.