RU2427838C1 - Method of quantitative analysis of red blood cell deformability in differential diagnostics of obstructive jaundice - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: pressure equal to existing capillary pressure required to pass the entire red cell suspension by micro-sieving through a calibrated filter is generated in a simulated medium. It involves filtration time recording of the analysed blood by a stop watch. Passage time of the analysed red cell suspension shows a red cell deformability index - RCDI which is calculated by formulae:

and

where X_i is an average filtration time, n is number of observations.

EFFECT: use of the method allows quick and precise quantitative analysis of red cell deformability.

1 tbl, 3 ex

Description

The invention relates to medicine and can be used in laboratory research methods to identify the causative factor of obstructive jaundice in patients.

There is a method for assessing the deformability of red blood cells, in which physiological saline and a 60% suspension of red blood cells are applied to an anesthetized filter, a visual assessment is made of the staining intensity of a spot of a 60% suspension of red blood cells, and in the case of signs of uneven deformability of its red blood cells, namely, in the center of the spot there is a rounded zone of the highest staining intensity, surrounded by a lighter peripheral ring and a clearly distinguished boundary between the lateral ring and the central zone erythrocyte suspension, determine the width of the peripheral ring of the erythrocyte suspension, represented by a small amount of the most deformed red blood cells. Next, calculate the indicator of deformability of red blood cells according to the formula (see patent No. 2173461, IPC G01N 33/48, Bull. No. 25, 09/10/2001).

A disadvantage of the known method for assessing the deformability of red blood cells is that it allows only a semi-quantitative assessment of the deformability of the studied population of red blood cells.

There is a known method of filtering red blood cells, which is based on the separation of red blood cells into populations according to their ability to pass through the pores of a filter paper for a certain time, consisting of a network of crimped tubules of various diameters, which ensure the separation of the red blood cell suspension into several fluid flows and subject each of them to viscometry. The ability of red blood cells to filter or filterability is, first of all, the result of the individual elasticity of each cell. The study is carried out using filter paper firms "Sinpor" and "Millipore", with microchannels from 3 to 6 microns. The low shear stress (less than 10 dyne / cm) and the low shear rate (less than 0.5 s ^-1 ) at which filtering occurs, help to detect the slightest changes in the deformability of red blood cells (see Chernukh AM, Aleksandrov PN, O.V. Alekseev, Microcirculation, M., 1984, p. 432).

However, this method of assessing the deformability of red blood cells has a significant drawback - the ability to filter red blood cells not only through the bottom of the filter (where hydrostatic pressure creates the most stable shear rate), but also through its sides. This significantly reduces the accuracy of the method.

The closest in technical essence and the achieved result to the present invention is a method for determining the deformability of red blood cells, in which the flow time of the first 200-300 μl of a suspension of red blood cells through a membrane filter is recorded, registration is carried out using a sensitive sensor and an electronic registration unit. A erythrocyte suspension is prepared from whole blood or red blood cells washed with physiological saline. The deformability of red blood cells is estimated by the rigidity index, which is calculated by the formula (see patent No. 2052194, IPC G01N 33/48, publ. 01.10.1996).

However, the known method performs an incomplete assessment of the deformability of red blood cells, that is, using this method, you can only make a semi-quantitative assessment. Also, the known method is laborious and time consuming.

The technical task of the invention is to develop a method for the quantitative assessment of the state of deformability of red blood cells in the differential diagnosis of obstructive jaundice, which will distinguish patients with obstructive jaundice from tumor and gallstone, thereby helping to establish the correct diagnosis and will affect the timeliness of treatment, its successful outcome.

The technical result of the invention is to reduce the examination time, the speed of determining the nature of obstructive jaundice in a patient, the ease of implementation and the availability of a method for determining the deformability of red blood cells.

The technical result of the invention is achieved by the fact that in a method for quantifying the state of deformability of red blood cells, comprising passing a suspension of red blood cells through a filter, recording the filtration time of a suspension of red blood cells, determining an index of deformability of red blood cells, according to the invention, to determine the state of deformability of red blood cells creates an artificial pressure equal to the existing one intracapillary pressure required to pass the entire suspension of erythrocytes by microsifting through a calibrated filter, at the same time the stopwatch is turned on and the filtering time of the test blood is recorded, which is used as a suspension of red blood cells; the time taken for the suspension of red blood cells to be studied is used to determine the index of erythrocyte deformability index - IDE, which is calculated by the formulas

where X _i is the average value of the filtration time, n is the number of observations.

Distinctive features of the present invention are its higher sensitivity compared to the known methods for determining the erythrocyte deformability, a fundamentally different mechanism for determining the degree of erythrocyte deformability is a complete rather than semi-quantitative assessment of the condition of the patient erythrocyte blood membranes, the exclusion of the influence of the external temperature factor on the result of the study, the creation of a “natural "Pressure (4.2 kPa) in an artificial environment, necessary for the passage of the entire suspension of red blood cells in the blood through a calibrated filter equal vnutrikappilyarnomu existing pressure that allows to achieve the technical result consisting in the reduction of inspection time, a rapid determination of the nature of jaundice in a patient, the simplicity and performance of the method for determining the availability of the deformability of erythrocytes index - EDI.

From known sources in the scientific and technical literature and patent documentation, the authors have not identified analogues with the proposed combination of distinctive features of the claims.

The invention is illustrated by drawings, where FIG. 1a shows a device for determining the quantitative assessment of the state of deformability of red blood cells, FIG. 1b shows a calibrated filter from above, and FIG. 2 shows a graph of the filtration time of a suspension of red blood cells depending on the cause of jaundice in patients and healthy patients. .

The inventive method for the quantitative assessment of the state of deformability of red blood cells in the differential diagnosis of obstructive jaundice is as follows.

To study the suspension of red blood cells in the differential diagnosis of obstructive jaundice, a device is used to determine the quantitative assessment of the state of deformability of red blood cells (see figa, b), containing a glass graduated cylinder 1, enclosed in a transparent plastic casing 2, which has an upper 3 and a lower 4 silicone tubes equipped with plugs 5 and 6, respectively. Placing the glass graduated cylinder 1 completely inside the transparent plastic casing 2 provides thermostating and eliminates the influence of the external temperature factor on the result of determining the degree of deformability of red blood cells. The lower tube 4 is intended for supplying water pre-heated to + 36 ° C to the cavity of the plastic casing 2. The upper tube 3 is designed to allow air to escape from the plastic casing 2. At the bottom of the graduated glass cylinder 1, a plastic grate 7 is installed; a calibrated filter 9 is mounted on it with a metal ring 8, which is made of a polymer film with a pore diameter of 5 μm so that the minimum pore dispersion (spread) of 2-5%, a smooth mirror surface (less than the conditions for microaggregation), does not emit low molecular weight impurities in the filtrate (there is no side effect on the result). A pore diameter of 5 μm for the calibrated filter 9 corresponds to the main diameter of the capillary bed. The glass graduated cylinder 1 has an upper inlet 10 and a lower outlet 11 of the hole. The lower outlet 11 is provided with a device 12 for the implementation of the provisions of "open" or "closed". Inside the glass graduated cylinder 1 there is a piston 13 with a rod 14. Two different diameter channels 15 are made in the piston 13, at the junction of which there is a metal ball 16, which performs the purpose of the exhaust valve for air. The pressure generated by the piston 13 with the rod 14 in the device during measurement is 4.2 kPa, which generally equals the natural intracapillary pressure, which averages 4.3 kPa. The device is equipped with an electronic stopwatch (not shown in FIG. 1a) for recording the transit time of the studied suspension of red blood cells through a calibrated filter 9.

To conduct a study of a suspension of red blood cells, a plug 6 is opened and water is preliminarily heated to + 36 ° C through a lower silicone tube 4 through a lower plastic tube 4. When the lower outlet opening 11 is closed, a suspension of red blood cells of a sick patient in an amount of 2 ml diluted with physiological saline in a ratio of 1: 2 is poured from a test tube through a graduated glass cylinder 1 through an upper inlet 10. A piston 13 is introduced into the glass graduated cylinder 1 with the rod 14, not fully screwed in. The piston 13 is advanced to the liquid level in the glass graduated cylinder 1, while air from the cylinder 1 is released through the channels 15 in the piston 13 using a metal ball 16 that performs the purpose of the exhaust valve. Then the rod 14 is screwed to the stop in the piston 13 and clamp the metal ball 16 (exhaust valve is closed). Next, the device 12 of the lower outlet 11 of the glass graduated cylinder 1 is opened, under the pressure of the piston 13 and the rod 14 in the cylinder 1, the whole suspension of red blood cells is passed through a calibrated filter 9 and at the same time an electronic stopwatch is turned on, by which the transit time of the entire studied suspension of red blood cells is recorded through calibrated filter 9 in seconds.

An increase in the time of passage of a suspension of red blood cells through a calibrated filter 9 characterizes a decrease in the deformability of red blood cells, and, on the contrary, a decrease in time characterizes an increase in the deformability of red blood cells. In figure 2, this graphically manifests itself in the form of a shift in the curve of the passage of suspension of red blood cells through a calibrated filter 9. A shift to the right occurs when the deformability of red blood cells decreases, a left shift occurs when the deformability of red blood cells increases.

After recording the transit time of the studied suspension of red blood cells through a calibrated filter 9 of the device, the authors determine the index of deformability of red blood cells - IDE, which they propose to calculate as the decimal logarithm of the filtering time of the suspension of red blood cells using the following formulas:

where X _i is the average value of the filtration time, n is the number of observations.

As a result, the authors calculated the average values of the time and the filtration index of the suspension of red blood cells in healthy and sick patients, and using further the formulas

the authors also calculated the standards of these quantities. The obtained results of IDE statistics in healthy and sick patients are shown in the table.

Table Red blood cell deformability indices in healthy and sick patients Group view The number of examined

The average filtering time of red blood cells, s

Filtration time standard, c S ₁ The average index of deformability of red blood cells, s (log x) Filtration Index Standard, with S ₂ Healthy 276 3.53 ± 0.05 (p <0.03) 0.40 0.55 ± 0.01 (p <0.03) 0.05 Cancer patients with obstructive jaundice 91 8.41 ± 0.08 (p <0.05) 0.38 0.93 ± 0.01 (p <0.05) 0.02 Patients with cholelithiasis complicated by jaundice 52 4.25 ± 0.10 (p <0.05) 0.37 0.63 ± 0.01 (p <0.05) 0.04

фильтрации эритроцитов конкретного обследуемого пациента лежит в доверительном интервале 3,53±0,05, то можно с уверенностью 97% считать, что он здоров. Если же среднее время фильтрации лежит в интервале 8,41±0,08 или 4,25±0,10, то можно с уверенностью 95% признать, что он болен раком или желчнокаменной болезнью соответственно. Аналогично для ИДЭ (lg x) - обследуемый пациент здоров с достоверностью 97%, если его ИДЭ входит в интервал 0,55±0,01, или с достоверностью 95% болен соответственно раком или желчнокаменной болезнью, если его ИДЭ находится в интервале 0,93±0,01 или 0,63±0,01.The table shows if the average time

erythrocyte filtration of a particular patient under examination lies in a confidence interval of 3.53 ± 0.05, then we can safely assume that 97% is healthy. If the average filtration time lies in the range of 8.41 ± 0.08 or 4.25 ± 0.10, then we can confidently 95% admit that he is ill with cancer or gallstone disease, respectively. Similarly for IDE (log x) - the examined patient is healthy with a confidence of 97% if his IDE is in the range of 0.55 ± 0.01, or with a confidence of 95% he is ill with cancer or gallstone disease, respectively, if his IDE is in the range of 0, 93 ± 0.01 or 0.63 ± 0.01.

Examples confirming the implementation of the method for the quantitative assessment of the state of deformability of red blood cells in the differential diagnosis of obstructive jaundice

Example 1

Patient A., 35 years old, was admitted to the II surgical department of the Republican Oncology Center on 03/26/09 by transfer from the city infectious diseases hospital, where he had been undergoing infectious hepatitis since the end of January this year. The appearance of jaundice was preceded by a pain attack in the right hypochondrium, chills. In the infectious diseases hospital in the clinical picture of the disease, persistent jaundice and an increase in the level of transaminases came to the fore. However, at the end of the first quarter, the diagnosis of hepatitis was withdrawn. Upon admission to the department, the condition is relatively satisfactory. Yellowness of sclera and integument, subfebrile temperature, pain in the right hypochondrium were observed.

Laboratory and biochemical studies: HB - 131 g / l, L - 9.4 · 10 / l, ESR - 18 mm / h, mercuric chloride test - 1.19 ml, thymol test - 10 units, ACT - 0.69 mmol / l, ALT - 1.39 mmol / l, total bilirubin - 105.3 mmol / l, blood sugar - 4.5 mmol / l, potassium - 3.6 mmol / l, blood sodium - 140.3 mmol / L, blood Ca - 2.39 mmol / L, blood cholesterol - 5.16 mmol / L.

As a result of studies and calculations in a patient, the following indicators: filtration index - IDE - 0.63, filtration time - 4.31 s.

Patient A. 03/28/09 after admission taken for surgery. After laparotomy, a congestive liver was found. A large calculus fixed in the common bile duct is determined by palpation, which was confirmed by the data of intraoperative cholangiography. The gall bladder is scarred and wrinkled, it contains 2 stones. The operation was performed: cholecystectomy, choledochotomy. The postoperative course is smooth. Recovery.

In this example, with a duration of obstructive jaundice of 49 days, the value of the IDE indicator even before the operation indirectly indicated the calculous nature of the disease, which was confirmed by the data of intraoperative diagnosis.

Example 2

Patient D., 73 years old, was admitted to the II surgical department of the Republican Oncological Dispensary on 05.21.09 with a direct diagnosis: obstructive jaundice. Jaundice appeared in early May of this year. The patient was disturbed by skin itching, pain in the right hypochondrium and general weakness. Upon admission, a state of moderate severity, marked yellowness of sclera and skin was noted. Symptom Courvoisier was not determined.

Laboratory research data: HB - 120 g / l, L - 5.0 · 10 / l, ESR - 38 mm / h, total blood bilirubin - 217.0 mmol / l, ACT - 1.1 mmol / l, ALT - 2.23 mmol / l, sublimate test - 1.5 ml, thymol test - 30 units, blood sugar - 7.2 mmol / l, potassium - 4.00 mmol / l, blood sodium - 133.6 mmol / l , Ca - 2.15 mmol / L, blood cholesterol - 12.0 mmol / L.

The preoperative diagnosis in the patient according to ultrasound was formulated - cholelithiasis, obstructive jaundice. Studies, measurements and calculations of the IDE indicator in patient D. — 0.94, filtration time — 8.8 s cast doubt on the correctness of the diagnosis.

With a laparotomy on May 29, 2009, a tumor of extrahepatic bile ducts was detected. An operation draining the biliary tract has been performed. Postoperative wound healing by primary intention. Patient D. was discharged 06/14/09 in a relatively satisfactory condition.

Determination of the IDE indicator in the patient suggested the tumor genesis of obstructive jaundice, which was fully confirmed during surgery.

Example 3

Patient K., 37 years old, was transferred to the II surgical department of the Republican Oncology Center from the city infectious diseases hospital with a diagnosis of Pancreatic head cancer, obstructive jaundice. From the anamnesis it is known that at the end of January 2009, patient K. had complaints of lethargy and decreased appetite. In mid-March 2009, jaundice joined, which was rapidly increasing. Upon receipt, her condition was relatively satisfactory. Intense yellowness of sclera and skin was noted. Palpation of the right hypochondrium is painless. It was not possible to determine the bottom of the gallbladder. The 3 cm liver protruded from under the edge of the costal arch.

Laboratory research results: HB - 140 g / l, L - 6.9 · 10 / l, ESR - 25 mm / h, total blood bilirubin - 179.3 mmol / l, ACT - 1.70 mmol / l, ALT - 2.72 mmol / l, sublimate test - 1.8 ml, thymol test - 20 units, blood sugar - 8.7 mmol / l, blood potassium - 3.5 mmol / l, blood sodium - 137.8 mmol / l, blood calcium - 2.26 mmol / l, blood cholesterol - 10 mmol / l.

The studies and IDE calculations confirmed the clinical idea of patient K. - IDE indicator - 0.93, filtration time - 8.6 s. A repeated study of this indicator before surgery, performed 6 days after the first, was identical to the previous one - IDE indicator - 0.94, filtering time - 8.8 s.

04/25/09 patient K. was performed laparotomy. The results of an intraoperative audit confirmed the diagnosis: a malignant tumor of the pancreatic head, the third stage. For obstructive jaundice, an operation was performed - cholecystoenterostomy with intestinal anastomosis. The postoperative period without complications. At discharge, the IDE indicator had the following value - 0.89, filtering time - 7.80 s.

Such dynamics of the IDE indicator was characteristic of a favorable course of the postoperative period.

The present invention "A method for quantifying the state of deformability of red blood cells in the differential diagnosis of obstructive jaundice" compared with the prototype (see patent No. 2052194, IPC G01N 33/48, publ. 01.10.1996) has the following advantages:

- reflection of the deviation of the IDE indicator, depending on the nature of jaundice,

- the detected dynamics of changes in IDE makes it directly dependent on the degree of development of cholemia and necrobiotic changes in the liver parenchyma,

- the IDE indicator reliably determines the nature of jaundice, which allows you to promptly establish the correct diagnosis and conduct a full treatment of diseases that have this complication,

erythrocyte deformability index - IDE, helping to detect jaundice caused by a malignant tumor of the pancreatic head and extrahepatic bile ducts, can be used as a diagnostic test in the differential diagnosis of obstructive jaundice,

- the simplicity of the method, its availability and speed make it possible to use it at the prehospital stage for early detection of the nature of obstructive jaundice and timely hospitalization in appropriate specialized medical institutions.

The claimed invention can be used to examine patients and make a timely diagnosis in the laboratories of polyclinics of the city, countryside, surgical departments of hospitals.

Claims (1)

A method for the quantitative assessment of the state of red blood cell deformability, comprising passing a suspension of red blood cells through a filter, recording the filtration time of a suspension of red blood cells, determining an indicator of red blood cell deformability, characterized in that to determine the state of deformability of red blood cells, an artificial pressure is created equal to the existing intracapillary pressure the whole suspension of red blood cells by microsifting through a calibrated filter, corresponding yuschy core diameter of the capillary bed, at the same time start the stopwatch and record the time study blood filtration, which takes as erythrocyte suspension at the time of passage of red blood cells suspended investigated through a calibrated filter index parameter determines the EDI deformability of erythrocytes by the following formulas:

and

where X _i is the average value of the filtration time, n is the number of observations.