RU2849275C1 - Method for predicting multiple organ failure in critically ill patients - Google Patents

Abstract

FIELD: medicine, namely clinical laboratory diagnostics.

SUBSTANCE: invention can be used to predict multiple organ failure in critically ill patients. Blood samples are taken from patients. From the moment the patient in critical condition is transferred to the intensive care unit, the number of circulating endothelial cells (CEC) in the blood is determined using flow cytometry, in which the results are normalised to 3⋅10⁵ leukocytes. An initial measurement of CEC is taken when the patient is admitted to the intensive care unit, followed by a second measurement 24 hours later and a third measurement 48 hours after the start of treatment for the critical condition. If the initial CEC count is at least 4 cells per 3⋅10⁵ leukocytes, and its level remains the same or increases in subsequent measurements after 24 and 48 hours, the development of multiple organ failure is predicted. A decrease in CEC levels by more than 50% after 24 hours or more after the start of treatment indicates a reduced risk of multiple organ failure and the effectiveness of the therapy.

EFFECT: this method enables the timely detection of the risk of multiple organ failure and the rapid monitoring of the effectiveness of therapy through the continuous monitoring of CEC levels, which reflect the state of microcirculation and endothelial damage.

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Description

The invention pertains to medicine, specifically clinical laboratory diagnostics and intensive care for critical conditions. The proposed method is designed to predict the development of multiple organ failure (MOF) in critically ill patients and to dynamically assess the effectiveness of treatment. Prediction involves promptly identifying the tendency toward MOF development through consistent monitoring of biomarkers reflecting the extent of endothelial damage, enabling timely adjustments to treatment and reducing the risk of mortality.

Currently, multiple organ failure (MOD) is considered as a failure of two or more functional systems, a universal lesion of all organs and tissues by aggressive mediators with a temporary predominance of one or another organ failure [Zilber A.P. Etudes in Critical Medicine. Moscow: MEDpress-inform; 2006]. MOD after critical conditions is one of the main causes of mortality in intensive care patients.

According to experts, at the time of providing care to a patient in critical condition, there are no laboratory criteria indicating a high probability of developing multiple organ failure after recovery from critical condition. It is proposed to use a study of microcirculation disorders, which are often secondary to multiple organ failure and therefore cannot be considered specific signs used to prognosticate multiple organ failure. The methodology for assessing microcirculatory disorders in critical conditions is a complex task and should be based on the analysis of a set of clinical, biochemical, and, probably, instrumental methods. Considering that microcirculatory parameters have greater validity in terms of predicting an unfavorable outcome of critical condition and the development of multiple organ failure, timely diagnosis and development of algorithms for correcting microcirculation disorders can contribute to the prevention of multiple organ failure [T.O. Tokmakova, S.Yu. Permyakova, A.V. Kiseleva, D.L. Shukevich, E.V. Grigoriev, General Reanimatology 2012, Vol. 8, No. 2, pp. 74-78].

Currently, there are no precise markers predicting the progression of multiple organ failure in critical illness. Researchers primarily focus on sepsis markers and laboratory parameters of existing organ damage. These markers are not universal indicators of the likelihood of developing multiple organ failure. They lack sufficient sensitivity and specificity for predicting the development of multiple organ failure, as they reflect only the activity of the pathological process, whether infectious, toxic, inflammatory, or ischemic. Predicting the development of multiple organ failure using various assessment scales is often universal for all critical illnesses, but, unfortunately, is a retrospective factor in assessing existing multiple organ failure.

Specific examples of criteria for predicting MOF include: hemostatic system disorders (platelet count and activated partial thromboplastin time, fibrin/fibrinogen degradation products, procoagulant phospholipid activity, thrombomodulin activity, etc.) [Mihajlovic D, Lendak D, Mitic G, Cebovic T, Draskovic B, Novakov A, Br-kic S. Prognostic value of hemostasis-related parameters for prediction of organ dysfunction and mortality in sepsis. Turkish Journal of Medical Sciences. 2015;45(1):93-98. Itani R, Minami Y, Haruki S, Watanabe E, Hagiwara N. Prognostic impact of disseminated intravascular coagulation score in acute heart failure patients referred to a cardiac intensive care unit: a retrospective cohort study. Heart and Vessels. 2017;32(7):872-879. Van Dreden P, Woodhams B, Rousseau A, Dreyfus JF, Vasse M. Contribution of procoagulant phospholipids, thrombomodulin activity and thrombin generation assays as prognostic factors in intensive care patients with septic and non-septic organ failure. Clinical Chemistry and Laboratory Medicine. 2013;51(2):387-396. https://doi.org/10.1515/cclm-2012-0262], blood glucose and lactate concentrations [Sung J, Bochicchio GV, Joshi M, Bochicchio K, Tracy K, Scalea TM. Admission Hyperglycemia Is Predictive of Outcome in Critically Ill Trauma Patients. The Journal of Trauma. 2005;59(1):80-83], high-density lipoprotein levels [Cirstea M, Walley KR, Russell JA, Brunham LR, Genga KR, Boyd JH. Decreased high-density lipoprotein cholesterol level is an early prognostic marker for organ dysfunction and death in patients with suspected sepsis. Journal of Critical Care. 2017;38:289-294], acid-base balance and blood gas responsiveness (decrease in hemoglobin oxygen saturation in the blood in the superior vena cava ScvO2 to less than 70% during the first 6 hours of early goal-directed sepsis therapy) [Samraj RS, Zingarelli B, Wong HR. Role of biomarkers in sepsis care. Shock. 2013;40(5):358-365] and phospholipase A2 type 2 levels [Lausevic Z, Lausevic M, Trbojevic-Stankovic J, Krstic S, Stojimirovic B. Predicting multiple organ failure in patients with severe trauma. Canadian Journal of Surgery. 2008;51(2):97-102], as well as hematological parameters, thrombocytopenia, and monocytopenia [Takala A, Jousela I, Jansson SE, Olkkola KT, Takkunen O, Orpana A, Karonen SL, Repo H. Markers of systemic inflammation predicting organ failure in community-acquired septic shock. Clinical Science. 1999;97(5):529-538]

S. Margraf et al. [Margraf S, Lögters T, Reipen J, Altrichter J, Scholz M, Windolf J. Neutro-phil-derived circulating free DNA (cf-DNA/NETs): a potential prognostic marker for posttraumatic development of inflammatory second hit and sepsis. Shock. 2008;30(4):352-358], having studied the possibilities of predicting posttraumatic MOF, propose using for this purpose the level of lymphocytes, as well as the content of the lipid peroxidation marker myeloperoxidase. Studies of the activity of apoptosis markers and cytokines in the prognosis of MOF also did not find a positive solution. All these indicators are secondary to the development of organ damage, have low sensitivity and specificity, and are often associated with a specific critical condition, which deprives them of universality given the simultaneous influence of various etiological factors on the development of a critical condition and subsequent multiple organ failure.

Therefore, despite the huge variety of indicator substances and their combinations, the problem of identifying highly sensitive markers that are universal for predicting multiple organ failure regardless of the etiology of the critical condition remains unresolved.

Furthermore, elevated levels of any given marker do not necessarily indicate MOF and may be observed against the background of compensated interorgan interactions. The relationships between known inflammatory mediators, endotoxemia markers, or specific biomarkers can vary significantly at the serum, tissue, and cellular levels. The multifaceted nature of mediator involvement in generalized forms of inflammation and the complexity of the process explain the failure of attempts to link MOF pathogenesis to specific cytokines and the failure of "anti-mediator" therapy.

It should be noted that it is impossible to assess the relationship between the dynamics of certain pathogens, markers of organ damage, toxins of exogenous and endogenous origin with the dynamics of multiple organ failure in the early stages after a critical condition, as well as their use for a dynamic assessment of the effectiveness of specific treatment methods.

None of the biomarkers can adequately reflect the rapidly changing nature of the patient's condition with the risk of developing MOF, so our attention was drawn to the possibility of using circulating endothelial cells as a possible universal marker reflecting the dynamics of multiple organ failure in the immediate post-critical period and used for the dynamic assessment of treatment effectiveness.

The closest analogue (prototype) of the claimed invention is “A new approach to assessing endothelial dysfunction: determination of the number of circulating endothelial cells by flow cytometry” [Feoktistova VS, Vavilova TB, Sirotkina O.V., Boldueva S.A., Gaikovaya LB, Leonova I.A., Laskovets A.B., Ermakov A.I. Hematology, group of authors, 2015]. The prototype is a method for assessing endothelial dysfunction based on the quantitative determination of circulating endothelial cells (CEC) in peripheral blood using flow cytometry. The method involves collecting venous blood, processing the sample with fluorescently labeled monoclonal antibodies to CD146 (to identify CECs) and CD45 (a pan-leukocyte marker), and normalizing the CEC count to ^3⋅10 leukocytes. The presence of more than three CECs per 3⋅10 ^leukocytes has been associated with an increased risk of coronary heart disease and acute myocardial infarction in young and middle-aged women.

The disadvantages of the prototype are:

1. Lack of dynamic monitoring: the prototype provides for a single measurement of the CEC level, whereas predicting multiple organ failure requires consistent (dynamic) monitoring of changes in the indicator over time (upon admission, after 24 and 48 hours).

2. Different clinical focus: the prototype method is aimed at assessing the risk of coronary heart disease in women, rather than predicting the development of multiple organ failure in critically ill patients, which significantly changes the conditions of application.

3. Use of peripheral blood: the prototype is based on the analysis of peripheral blood, while for the assessment of critically ill patients it is preferable to use blood taken from a central venous catheter, which provides a more up-to-date reflection of microcirculation processes.

4. Static threshold values: established threshold values (more than 3 CEC per 3⋅10 ⁵ leukocytes) do not take into account the dynamics of changes, which is a key parameter for timely prediction and evaluation of the effectiveness of therapy in multiple organ failure.

Thus, although the prototype contains a basic element - the quantitative determination of CEC by flow cytometry - its methodology and parameters do not fully satisfy the requirements of the new invention, aimed at the dynamic prognosis of multiple organ failure and the assessment of the effectiveness of therapy in critical conditions.

The following essential features have been identified for the claimed invention in common with the prototype: quantitative determination of circulating endothelial cells in the blood using flow cytometry, in which the results are normalized by the number of leukocytes (for example, by 3⋅10 ⁵ leukocytes) and the threshold values of these cells are used as a prognostic marker for assessing the risk of developing a pathology associated with impaired endothelial function.

The essence of the invention.

The technical challenge lies in expanding the range of methods for predicting multiple organ failure and assessing the effectiveness of intensive care through dynamic assessment of the endothelial state in critically ill patients.

The technical result consists in the timely detection of the risk of developing PON and prompt control of the effectiveness of therapy through sequential monitoring of the level of CEC, reflecting the state of microcirculation and endothelial damage.

The technical result is achieved through sequential quantitative analysis of circulating endothelial cells in venous blood in critically ill patients using flow cytometry. The results are normalized to ^3⋅10 leukocytes. Measurements are performed in three stages: upon admission to the intensive care unit (initial measurement), 24 hours later, and 48 hours later after the start of intensive care. It has been established that a baseline value of at least 4 circulating endothelial cells per ^3⋅10 leukocytes, an increase in circulating endothelial cells by more than 25% after 24 hours and by more than 40% after 48 hours indicates a high risk of multiple organ failure, and a decrease in circulating endothelial cells by 50% or more after 24 hours indicates the effectiveness of the treatment and a reduced risk of multiple organ failure. A decrease in the level of circulating endothelial cells in venous blood by 50% or more 24 hours after the relief of a critical condition indicates a reduced risk of developing multiple organ failure.

The invention is implemented as follows.

Prediction of the development of multiple organ failure is achieved by determining the dynamics of changes in the level of circulating endothelial cells (CEC) in venous blood. Endothelial damage and impaired microcirculation are key factors in the pathogenesis of multiple organ failure, so quantitative changes in CEC serve as an objective indicator of the degree of endothelial dysfunction, which, in turn, correlates with the risk of developing multiple organ failure.

To achieve the desired technical result, the invention provides for the following set of actions (operations):

Methodology for taking measurements starting from the moment of development of a critical condition in a patient:

1. Primary measurement: upon admission of the patient to the intensive care unit or intensive care unit before the start of therapeutic measures.

1.1 Venous blood is collected from a central venous catheter. This provides the most accurate picture of the microcirculation.

1.2. Sample preparation: addition of anticoagulant, incubation with fluorescently labeled antibodies (CD146 and CD45) at established concentrations at room temperature in the dark for 15 minutes.

1.3. Lysis of erythrocytes using a buffer solution at an automated sample preparation station.

1.4. Determination of the baseline level of endothelial cells: analysis of the sample on a flow cytometer with specific markers for the identification of endothelial cells, with the adjustment of data collection modes (e.g., collecting 300,000 events) and using gating algorithms to identify CECs, normalization of the result to 3⋅10 ⁵ leukocytes (the result is expressed as the number of cells per 3⋅10 ⁵ leukocytes).

2. Secondary measurement: 24 hours after the start of therapy, a repeat blood sample is taken using a similar method (to determine the dynamics of changes after the start of intensive therapy).

3. Tertiary measurement: another measurement is taken 48 hours after the start of therapy (to confirm the dynamics and predict the further course of the disease).

These actions are carried out sequentially, which allows dynamic monitoring of changes in the level of the CEC.

This procedure allows us to obtain objective information about the dynamics of endothelial dysfunction, which is a key factor for timely prediction of the risk of developing multiple organ failure and taking corrective treatment measures.

Conditions for the implementation of actions and modes:

- Blood sampling is performed under sterile conditions using vacuum tubes containing an anticoagulant (e.g. 0.5 M EDTA).

- Sample analysis is performed no later than 3 hours after collection to maintain the stability of cellular parameters.

- Flow cytometry is performed on a specialized instrument (e.g., Cytomics FC 500 or similar equipment) with established analysis modes that include collecting at least 300,000 events, subsequent data selection using forward and side scattergrams, and the use of gating algorithms to exclude cellular fragments (debris).

Substances, reagents and devices used:

- Reagents: fluorescently labeled monoclonal antibodies for identification of CECs, such as antibodies to CD146 (labeled with a fluorochrome, such as phycoerythrin) and to CD45 (a pan-leukocyte marker labeled with a combination of fluorochromes, such as pc5).

- Devices: flow cytometer, vacuum tubes with anticoagulant, automated sample preparation system (erythrocyte lysis using a buffer solution).

Prediction of the development of multiple organ failure:

In this invention, prediction is understood to mean an analysis of the dynamics of changes in the CEC level to identify a tendency towards the development of multiple organ failure. If the initial value of the CEC level (determined upon patient admission) is at least 4 cells per 3⋅10 ⁵ leukocytes, and then an increase of more than 25% is observed after 24 hours and more than 40% after 48 hours, this indicates a high risk of developing MOF (sensitivity 82.2%, specificity 79.5%, ROC area - 0.81). Conversely, a decrease in the CEC level by 50% or more 24 hours after the start of therapy indicates the effectiveness of treatment measures and a reduced risk of developing multiple organ failure (sensitivity 76.2%, specificity 80.7%, ROC area - 0.73).

This method can be implemented in clinical practice in intensive care units for dynamic monitoring of critically ill patients. It does not require specialized equipment, as it utilizes standard laboratory flow cytometry equipment, and enables prompt corrective measures in therapy based on objective laboratory data.

Clinical example 1: myocardial infarction with complications.

The patient, a 58-year-old man, was urgently admitted to the intensive care unit with severe chest pain radiating to the left arm and lower jaw, accompanied by weakness, shortness of breath, nausea, and profuse cold sweats. His medical history included stage 2 hypertension, hyperlipidemia, overweight (BMI 31), and long-term smoking (1 pack per day for 30 years), as well as a family history of myocardial infarction.

During the initial examination:

- Heart rate - 110 beats/min, blood pressure - 90/60 mmHg, respiratory rate - 24/min, _SpO2 - 92%.

- ECG revealed ST segment elevation in leads II, III, aVF with mirror changes in leads V1-V3 and rare ventricular extrasystoles.

- Laboratory parameters upon admission:

Troponin I - 6.5 ng/ml, CPK-MB - 280 U/l, leukocytes - 12×10 ⁹ /l, glucose - 8.2 mmol/l, creatinine - 115 μmol/l, pH - 7.30, lactate - 6.5 mmol/l.

The level of circulating endothelial cells (CEC) was 7 cells per 300 thousand leukocytes, which indicated severe endothelial damage.

After emergency coronary angiography and percutaneous coronary intervention with stenting of the right coronary artery, complex drug therapy was started (antiplatelet agents, anticoagulants, beta-blockers, ACE inhibitors, statins).

Dynamic monitoring after 24 and 48 hours revealed persistently high CEC levels (7 cells/300,000 leukocytes), which correlated with worsening hemodynamic parameters, the development of cardiogenic shock, progression of multiple organ failure (SOFA score 14), and an increase in lactatemia to 8 mmol/L. The patient died 48 hours later from refractory cardiogenic shock. High CEC levels confirmed severe endothelial damage, predicting an unfavorable outcome and demonstrating the importance of dynamic monitoring of this indicator for the rapid prediction of the risk of multiple organ failure.

Case study 2: complex postoperative sepsis in an elderly patient.

The patient, an 86-year-old woman, underwent surgery for THA, which resulted in multiple complications: bilateral lower lobe pneumonia, sepsis, septic shock, hydrothorax, and posthemorrhagic anemia. In the early postoperative period, upon admission to the intensive care unit, the measured CEC level was approximately 8 cells per 300,000 white blood cells (twice the normal range), indicating significant endothelial dysfunction.

Over the following days, dynamic monitoring was performed, measuring CEC, inflammatory markers (CRP, IL-6), and assessing organ dysfunction using the SOFA scale. By day 5, a gradual decrease in CEC levels was observed (approximately twofold compared to baseline), which coincided with a decrease in SOFA scores and a decrease in CRP levels, despite a minor change in procalcitonin levels. This positive trend allowed for adjustment of therapy and contributed to the stabilization of the patient's condition. Dynamic monitoring of CEC proved to be a key indicator of treatment effectiveness and a predictor of a favorable outcome, despite high values of traditional inflammatory markers.

Clinical example 3: severe pulmonary embolism (PE).

The patient, a 52-year-old woman, was admitted to the intensive care unit with acute dyspnea, tachycardia (heart rate 120 bpm), hypotension (blood pressure 80/50 mmHg), and cyanosis. She had a history of long-standing venous insufficiency, smoking, and a family history of thromboembolic complications. Computed tomography with pulmonary angiography confirmed massive pulmonary embolism, affecting more than 50% of the pulmonary vascular bed, and echocardiography revealed right ventricular dilation and a decrease in its ejection fraction to 35%.

Upon admission, the CEC level was 4 cells per 300,000 leukocytes, but by the beginning of the second day, a sharp increase to 7 cells per 300,000 leukocytes was observed. Concurrently, a significant increase in IL-6 levels, deterioration in hemodynamic parameters, and an increase in the SOFA score (from 8 to 17 points) were observed. Despite the initial treatment, which included thrombolysis, oxygen, and vasopressor therapy, the patient's condition worsened, and she died 48 hours later. A sharp increase in CEC served as a strong predictor of the rapid development of multiple organ failure and a poor prognosis.

Clinical example 4: postoperative complications in urological diseases.

The patient, a 75-year-old woman, underwent elective endoscopic surgery (THA) for ureteral stones with a high risk of thromboembolic complications. Postoperatively, upon admission to the intensive care unit, CEC measurements were performed sequentially over time (on days 1, 2, 3, 4, and 7).

Initially, the CEC level was within the normal range (up to 4 cells per 300,000 leukocytes) or slightly elevated, corresponding to low SOFA scores (1-2). Meanwhile, other inflammatory markers (CRP, IL-6, procalcitonin) were significantly elevated. However, follow-up demonstrated persistently low CEC levels, which correlated with the absence of organ dysfunction progression, stable hemodynamics, and a favorable clinical outcome. This approach allowed for timely treatment adjustments and prevented the development of serious complications, despite the high risk associated with associated factors.

Clinical example 5: acute gangrene of the lower limb after trauma

The patient, a 68-year-old man, was admitted to the intensive care unit complaining of severe pain, swelling, and discoloration of the right lower leg, which had developed three days earlier following an injury sustained while gardening. Initial examination revealed gangrene of the right lower extremity, characterized by a mottled skin discoloration, blisters, and an absence of pulse in the peripheral arteries of the lower leg and foot. His medical history included long-term smoking (one pack of cigarettes per day for 40 years, quitting two years ago) and medication-controlled hypertension.

Additional tests upon admission:

- Chest X-ray - no pathological changes.

- ECG - sinus rhythm, heart rate 72 beats/min.

- Laboratory parameters: D-dimer - 750 ng/ml; leukocytes - 11×10 ⁹ /l; creatinine - 130 μmol/l; urea - 10 mmol/l; glucose - 6 mmol/l; arterial blood gases - within normal limits.

Measurement of the level of circulating endothelial cells (CEC) showed 1 cell per 300 thousand leukocytes.

A day later, the patient suddenly lost consciousness while smoking another cigarette. Examination revealed the absence of respiratory movements and carotid pulsations, skeletal muscle spasms with a characteristic arched back, and dilated pupils that did not respond to light. Resuscitation measures were immediately initiated: defibrillation (150 J), tracheal intubation, intravenous adrenaline, and initiation of cardiopulmonary resuscitation. Fifteen minutes after repeat defibrillation, sinus rhythm was restored, and the patient was transferred to the intensive care unit for further intensive care.

Laboratory data in dynamics:

- Upon admission to the intensive care unit: CEC - 1 cell/300 thousand leukocytes; IL-6 - 150 pg/ml.

- After 48 hours: CEC - 1 cell/300 thousand leukocytes; IL-6 - 190 pg/ml.

- After 72 hours: CEC - 0 cells/300 thousand leukocytes; IL-6 - 200 pg/ml.

- Six hours after cardiopulmonary resuscitation, consciousness was restored, according to the Glasgow scale - 15 points, after 12 hours the patient was transferred to independent breathing.

Further examination with lower extremity angiography revealed critical ischemia of the right leg with occlusion of the popliteal artery. Due to the lack of evidence of significant organ failure, it was decided to perform emergency surgery—fasciotomy and necrosectomy to remove nonviable tissue.

Postoperative progress: Following surgery, the patient received antibiotic therapy, antithrombotic therapy (low-molecular-weight heparin), and daily dressings with antiseptic solutions. Postoperatively, a gradual improvement in general condition was observed, swelling and pain decreased, and the wound began to clear. Follow-up measurements showed continued low CEC levels, indicating the absence of significant endothelial damage. The patient was discharged two months after surgery, and a six-month follow-up examination revealed complete wound healing.

Low levels of CEC and moderately elevated IL-6 values over time confirmed the absence of significant endothelial damage, which contributed to a favorable outcome and confirmed the effectiveness of timely resuscitation and surgical intervention in critical conditions.

These clinical examples demonstrate that dynamic monitoring of circulating endothelial cell levels, conducted according to the proposed method, enables timely prediction of the risk of developing multiple organ failure. High baseline or rising circulating endothelial cell levels are associated with deterioration of the patient's condition and the development of complications, while a decrease in circulating endothelial cell levels correlates with the effectiveness of therapy and improvement in clinical parameters. This approach facilitates timely adjustment of treatment measures, which can reduce mortality in critically ill patients.

Claims (1)

A method for predicting multiple organ failure in critically ill patients, which includes taking blood from patients, in which, from the moment the patient in critical condition is transferred to the intensive care unit, the number of circulating endothelial cells (CEC) in the blood is determined using flow cytometry, in which the results are normalized to 3⋅10 ⁵ leukocytes, characterized by the fact that an initial measurement of CEC is carried out at the time of the patient's admission to the intensive care unit, then a secondary measurement of CEC after 24 hours and a tertiary measurement of CEC after 48 hours from the start of treatment for the critical condition, while if the initial number of CEC is at least 4 cells per 3⋅10 ⁵ leukocytes, and its level in subsequent measurements after 24 and 48 hours is maintained or increases, the development of multiple organ failure is predicted, and a decrease in the level of CEC by more than 50% after 24 hours or more after the start of treatment indicates a reduced risk of developing multiple organ failure and the effectiveness of the therapy.