Quantitative Chemiluminescence Immunoassay

‡) The physician diagnoses sepsis including symptoms of infection, systemic toxicity consistent with sepsis (fever

> 38 0 C or hypothermia < 36 0 C , chills, hypotension [systolic blood pressure < 90 mmHg], tachycardia, leukocytosis, thrombocytopenia, acidosis, ...). For children ≤ 1 year: fever > 38 0 C or hypothermia < 37 0 C, suffocation or slow pulse [3], [76], [ 85].

‡) And/or positive blood culture at least once, excluding catheter culture [39], [76], [133].

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Catheter-associated sepsis was diagnosed when there was at least 1 positive blood culture from peripheral venous blood, clinical manifestations of sepsis (fever > 38 0 C, chills and/or hypotension) and no other source of sepsis other than a positive catheter culture [76].

Diagnosis of sepsis due to possible skin contamination (coagulase-negative staphylococci or viridan group streptococci) requires isolation of the same organism from at least 2 positive blood cultures or 1 positive blood culture in patients in whom only 1 blood culture has been performed [133].

Polymicrobial sepsis is diagnosed when at least two microorganisms are isolated from the same blood culture within 48 hours or one known pathogen and one or more likely skin-contaminating agents are isolated from at least two positive blood cultures [133].

†) Catheter infection: cases where sepsis is not diagnosed, blood cultures are negative multiple times but catheter cultures are positive, we classify them as catheter infection.

†) Central venous infection: diagnosed if the patient has signs of infection and/or positive blood cultures before or within 48 hours of central venous catheter removal and a positive catheter tip culture (≥ 1000 colony-forming units) [152].

†) Endocarditis: Postoperative intracardiac infection is diagnosed when at least one of the following criteria is met: 1) confirmed by echocardiography (vesicles) or positive blood culture. In suspected cases, other manifestations such as pulmonary or splenic infarction, thromboembolism are sufficient for diagnosis.

2) Patients with at least two of the following criteria: fever > 38 0 C , new or changed murmur, infarction, skin manifestations (petechiae, hemorrhagic plaques, painful subcutaneous nodules), congestive heart failure or abnormal electrocardiogram, physician treating endocarditis. For children ≤ 1 year old, additional manifestations of fever > 38 0 C or hypothermia < 37 0 C, asphyxia or bradycardia [39], [ 85].

†) Mediastinal infection: signs of infection, chest pain or sternal instability and at least 1 of the following criteria: 1) Mediastinal drainage or purulent discharge from the mediastinum, 2) Positive bacterial culture, 3) Mediastinal widening on radiograph or 4) Antibiotic or antifungal treatment [39], [85].

†) Sternal infection: signs of infection, sternal pain or sternal instability and at least 1 of the following criteria:

1) The incision is opened to drain fluid or the chest wound is debrided, 2) Bacterial culture is positive [39], [65].

†) Surgical site infection (superficial or deep): diagnosed if occurring within 30 days after surgery and having at least 1 of the following criteria: surgical site pus, bacterial isolation, and at least one of the signs and symptoms of inflammation (swelling, heat, redness, pain) [65], [152].

2.2.4.2. Paraclinical characteristics

- Echocardiography

Diagnosis of TOF and monitoring of left ventricular ejection fraction (%) before and after surgery.

- Test kits used in research

+ Routine tests: recorded in medical records for 8 days from 1 day before

surgery (N0), day of surgery (N1), day 2 (N2) to day 7 after surgery (N7) including:

* Blood cell count: red blood cells (normal: 4 - 5.8 x 10 9 /L),

hemoglobin (12 - 16.5 g/dL), hematocrit: 34 - 51%); white blood cells (4

- 10 x 10 6 /L), white blood cells (2.5 - 7.5 x 10 6 /L), lymphocytes (1.5 - 4 x 10 6 /L) and platelets (150 - 450 x 10 6 /L)

* Hemostasis: 2 times before and after surgery including 3 routine tests: prothrombin ratio (%) (normal: ≥ 70%); activated partial thromboplastin time (seconds) (normal: 28 - 42 seconds) and fibrinogen (g/L) (normal: 1.5 - 2 g/L) [15]

* Biochemical tests:

†) Liver function: total bilirubin (normal 0 - 19 μmoL/L), SGOT (0 - 38 U/L), SGPT (0 - 41 U/L)

†) Kidney function: urea (normal: 1.7 - 8.3 mmol/L), creatinine (normal: Male: 63 - 115; Female: 53 - 100 μmol/L)

†) Blood glucose (normal: children: 2.6 - 4.5; adults: 3.5 - 6.4 mmol/L) [134]

†) Blood gas: only post-operative tests include pH (normal 7.35 - 7.45), PaCO2 (35 - 45 mmHg), PaO2 (70 - 100 mmHg), alkalinity

residual (± 2 mmol/L), HCO3 - (22 - 30 mmol/L), SaO2 (75 - 99%) [127]. FiO2 (%)

only monitored by ventilator.

+ MD tests: the two main time points are 1 day before surgery (N0) and the second day after surgery (N2) when the inflammatory response is highest [50], [126], [167].

* CRP (mg/dL): normal range is 0.0 - 0.8 mg/dL [134]. CRP is divided into 3 groups: < 5 mg/dL, 5 - 10 mg/dL and > 10 mg/dL [36], [52].

* IL-6 (pg/mL): normal range from 0.0 - 5.9 pg/mL [158].

IL-6 is divided into 4 groups: < 250 pg/mL, 250 - 500 pg/mL,

> 500 - 1000 pg/mL and > 1000 pg/mL [112], [166].

* IL-10 (pg/mL): normal range from 0.0 - 9.1 pg/ mL [159].

IL-10 is divided into 4 groups: < 85 pg/mL, 85 - 125 pg/mL,

> 125 - 250 pg/mL and > 250 pg/mL [69], [112].

When two interleukin results are below the instrument's measurement threshold (IL-6 < 2 pg/mL and IL-10 < 5 pg/mL) [160], the result is calculated as zero.

* Use the IL-6/IL-10 ratio at each testing time point to assess cytokine imbalance [183].

+ Bacterial culture: culture of pus, blood, urine, ...

2.2.5. Research means

2.2.5.1. Research medical records

The research medical record is drafted according to a unified form (Appendix 1) .

2.2.5.2. Testing equipment

All tests were conducted at the Department of Hematology - Center for Hematology and Blood Transfusion, Department of Biochemistry and Department of Microbiology, Hue Central Hospital.

+ Hematology test

* Blood cell counter: Cell - Dyn 3200 machine of Abbott - USA. The machine has two channels: measuring cells in the optical channel according to the principle of flow cytometry and measuring hemoglobin according to the principle of colorimetry. Collect enough 2 mL of blood in a tube with EDTA anticoagulant. Platelet results

< 150 x 10 6 /L were all stained with Giemsa to rule out pseudothrombocytopenia.

by stopping the exercise with EDTA.

* Coagulation testing machine: automatic STA compact machine of Diagnostica Stago - France. The machine operates on the principle of electromagnetic probe to detect the clotting time. The machine has the advantage of avoiding errors due to test samples such as lipid-rich plasma, increased bilirubin. Test chemicals used are of

Genuine supply. Collect blood in a test tube with 3.2% sodium citrate anticoagulant at a ratio of 1/9 (1 volume of anticoagulant / 9 volumes of blood), adjust the anticoagulant according to the hematocrit. The test is performed within 4 hours, ensuring compliance with the blood collection standards of the coagulation laboratory.

+ Biochemical and immunological tests:

* Medica Easy Blood Gas blood gas analyzer from Medica USA: the machine is designed to directly measure pH (hydrogen ion activity), PaCO2 (partial pressure of CO2) and PaO2 (partial pressure of O2) on whole blood samples taken with syringes or heparin-coated capillaries. The results are usually adjusted for temperature, thus accurately reflecting in vivo conditions . Based on pH, PaCO2, PaO2 and additional parameters such as temperature, hemoglobin, ... the machine will calculate additional results such as HCO3 - , base excess, SaO2, ...

* Japanese OLYMPUS AU640 machine: tests bilirubin, creatinine, CRP and glucose.

†) Bilirubin quantification: colorimetric test for the quantitative determination of total bilirubin in human serum or plasma. Test principle: stable diazonium salt reacts directly with conjugated bilirubin and reacts with unconjugated bilirubin in the presence of a catalytic factor to form azobilirubin. The absorption at 540 nm is proportional to the total bilirubin concentration.

†) Creatinine quantification: using Raffe kinetic colorimetric method. Principle: Creatinine reacts with picric acid in alkaline environment to form an orange complex. The rate of change in optical density measured at 520/800 nm wavelength is proportional to the concentration of creatinine in the sample.

†) CRP quantification: immunophelometry method of measuring the antigen-antibody reaction between goat anti-human CRP antibody and CRP in the test sample.

†) Glucose quantification: colorimetric method, using enzymes

hexokinase (HK) works on the following principle:

Glucose + ATP HK G-6-P + ADP

G-6-P + NAD G6PDH 6-Phosphogluconate +NADH + H +

The change in optical density at 340/380 nm is proportional to the amount of glucose present in the sample.

* IMMULITE 1000 analyzer from Siemens - USA: used to quantify IL-6 and IL-10. Use the IL-6 and IL-10 quantification kit provided by the manufacturer.

Principle: solid-phase enzyme-linked chemiluminescent immunometric assay.

Blood samples can use serum or plasma (anticoagulated with heparin). Blood samples are centrifuged, serum is separated, serum is frozen and stored in a deep freezer (-30 0 C) of SANYO - Japan at the Regional Blood Bank - Hue Central Hospital before testing. The storage time of frozen samples can last up to 6 months [158], [159].

This is a sandwich immunoassay method, used to quantify low concentration analytes easily and quickly. The substance to be quantified (IL-6 or IL-10) is an antigen sandwiched between 2 specific antibodies in the reagent:

Anti-IL-6 + IL-6 + anti-IL-6 enzyme conjugate

The tracer is an enzyme conjugated to a chemiluminescent substrate that emits light. The intensity of the emitted light is proportional to the amount of enzyme present and is directly related to the amount of IL-6 in the test sample (Figure 2. 2).

Detection threshold of the machine: IL-6: 2 - 1000 pg/mL ;

IL-10: 5 - 1000 pg/mL [160].

For semi-quantitative results > 1000 pg/mL, the serum sample will be diluted 1/2 or 1/4, ... to repeat the test a second or third time.

3 until a quantitative result is obtained. The result is then inversely calculated with the dilution to give the test sample result.

Antibody captures immobilized antigen

Antigen supplementation

The second antibody is labeled

Substrate addition Signal detection and quantification

Signal strength

Wash Wash

Analyte concentration The signal intensity (light) is proportional to the analyte concentration

Figure 2.2. Quantitative chemiluminescence immunoassay

* Source: according to Rockland immunochemicals inc. (2012) [ 146]

+ Microbiological testing:

* Sputum, pus, urine: direct Gram staining method, white blood cell count, ... to find bacteria.

* Blood culture: on Mac Conkey medium on BacT/ALERT blood culture machine of BIORAD - France.

* Culture other specimens according to the routine procedures of the Department of Microbiology on nutrient agar and blood agar media.

* Fungal culture on Sabauraud medium.

* Antibiogram on Mueller - Hinton medium.

2.2.5.3. Functional exploration

Preoperative electrocardiogram, echocardiogram, and chest X-ray were performed at the Department of Cardiovascular Function Testing and the Department of Radiology, Hue Central Hospital. Postoperative measurements were performed at the bedside at the Department of Cardiac Resuscitation - Cardiovascular Center - Hue Central Hospital.

2.2.6. Research process

2.2.6.1. Surgical procedure

All TOF patients selected for the study were operated on according to standard procedures with the same anesthesia, resuscitation, surgery and CT team. Upon arrival in the ICU, patients were monitored and treated by the same team of doctors and nurses from the Department of Anesthesia and Cardiac Resuscitation - Hue Central Hospital.

- Preoperative preparation

+ Internal medicine: propanolol, hydroxyzine sedative (Atarax) is given the night before surgery and the morning before entering the operating room.

+ Temporary surgery: left or right aortopulmonary bypass surgery [25].

- Surgery:

+ Anesthesia

* Anesthesia with fantanyl, norcuron, diprivan, ketamine and morphine ...

* Use of GC: DEXA average dose of 1 mg/kg at induction of anesthesia or MP average dose of 30 mg/kg mixed into the THNCT priming fluid or no GC used.

* Prophylactic antibiotics: all patients are given prophylactic antibiotics before surgery and maintained until they leave the cardiac resuscitation room, except in cases of infection, which will be used for a longer period of time. Commonly used antibiotics are third-generation cephalosporins.

* Prophylactic thrombolysis with tranexamic acid, average dose 50 mg/kg divided into 2 times before and after THNCT. If there is excessive bleeding after surgery, use 1 - 2 additional doses.

+ Extracorporeal circulation and surgery

* Opening the sternum and removing part or all of the thymus gland helps expose the pulmonary artery bifurcation more easily.

* Place aortic and vena cava catheters to establish the THNCT loop.