To Develop an Algorithm for Predicting the Unfavorable Course of Sepsis in Children Based on a Comprehensive Assessment of Immunological, Biochemical and Molecular Genetic Markers
Conditions: Sepsis
Sponsor: The Republican Research and Practical Center for Epidemiology and Microbiology

Version 1, 06 June 2024 
 
 
 
 
 
 
 
 
 
 
 
PROTOCOL 
 
Protocol ID:             
NCT05908162 
Brief Title:                   
Algorithm 
for 
Predicting 
the 
Unfavorable Course of Sepsis in 
Children 
Acronym:                 
RRPCEM_SEPSIS 
Official Title:           
To Develop an Algorithm for 
Predicting the Unfavorable Course 
of Sepsis in Children Based on a 
Comprehensive 
Assessment 
of 
Immunological, Biochemical and 
Molecular Genetic Markers         
Principal Investigator: E.G.Fomina 
Sponsor:                                      
The Republican Research and 
Practical Center for Epidemiology 
and Microbiology                              
 
Document Date:  
 
June 06, 2024 
 
 

Version 1, 06 June 2024 
 
Collection of peripheral blood for the study 
We collected 5-10 ml of venous blood under aseptic conditions into two tubes. One 
tube with anticoagulant (heparin solution or ethylenediaminetetetraacetate (EDTA)), 
the second tube with blood coagulation activator. The closed vacutainer with blood 
is inverted several times to mix the blood with the anticoagulant. The patient's blood 
was delivered to the Immunology and Cell Biotechnology Laboratory within no 
more than 4 hours of collection. 
Investigation of cellular immunity indicators 
Sample preparation includes the following steps 
- Spreading of antibodies according to the developed panel in test tubes; 
- Adding 100 µl of blood to the antibody mixture and mixing thoroughly; 
- Incubation for 15 min at +4 - +12°C in the dark; 
- Add 2 ml of lysis solution to lyse the erythrocytes, mix the tube contents; 
- Incubate for 10 minutes at +16 to +30°C in the dark; 
- Centrifuge the tubes (200-300 g - 5 minutes) to precipitate the leucocytes; 
- Add 150 µl DPBS and mix. 
  - Count on a flow cytometer. 
Counting of cell samples on a FACSCalibur cytofluorimeter included the following 
mandatory steps  
- Performance control/calibration of the instrument; 
- Adjustment of light scattering parameters on the cytogram plotted in FSC\\SSC 
coordinates; 
- adjustment of the 'threshold' parameters on the FSC channel to exclude cell debris 
from the analysis; 
- Adjusting the voltage on the fluorescence channels using an unstained control 
sample; 
- Adjusting the spectral compensation using single stain controls; 
- Counting of the test sample at 50,000 leukocytes.  
Data were analysed using Flowing Software version 2.5.1 or BD FACSDiva 7.0. 
 
 

Version 1, 06 June 2024 
 
Sample Preparation for the Study of Spontaneous and Stimulated Production 
of Oxygen Radicals (ROS) by Blood Neutrophils 
Sample preparation involves the following steps: 
- Labelling (positive '+' - stimulated sample and negative '-' - unstimulated sample) 
and serial numbering of sample tubes; 
- Place the cytometer tubes in a rack and number them; 
- Add 1 µl of DHR to the tubes; 
- Add 50 µl of RPMI medium; 
- Add 50 µl of blood and mix thoroughly; 
- Incubate for 10 minutes at +37°C; 
- Add 20 µl RPMI to '+' samples only and mix the contents of the tube; 
- Incubate for 30 min at +37°C in the thermostat; 
- Add 2 ml lysis solution, mix tube contents; 
- Incubate for 10 min at +16 - +30°C in the dark; 
- Centrifuge the tubes (200-300 g - 5 minutes) to precipitate the leucocytes; 
- Add 100 µl DPBS and mix. 
 Sample preparation for the spontaneous necrosis and apoptosis of peripheral 
blood cells (PI, Annexin V FITC) assay. 
Sample preparation involves the following steps: 
 - Appropriately label two tubes (stained and unstained sample) for the cytometer; 
 - Prepare a 1X lysis solution by diluting a 10X stock solution; 
 - Prepare a single annexin binding buffer by diluting the 10x stock solution; 
 - Add 1 ml of blood to a 15 ml tube; 
 - adding 14 ml of lysing solution to the tube containing blood, mixing the contents 
of the tube;  
 - Incubate for 10 minutes at room temperature in the dark; 
 - Centrifuge the tubes (200-300g - 5 minutes) to precipitate the leucocytes; 
 - Wash the precipitate with 2.5 ml DPBS: add 2.5 ml DPBS, tip to resuspend the 
cells; 
- Centrifuge the tubes (200-300g - 5 minutes) to precipitate the cells; 

Version 1, 06 June 2024 
 
- Collect the supernatant; 
 - Dissolve the precipitate in 200 µl of cold single annexin binding buffer; 
 - Transfer 100 µl of cell suspension to two cytometer tubes; 
 - diluting the volume of the cell suspension to 500 µl with cwith cold single annexin-
binding buffer; 
- adding 2.5 µl Annexin V FITC and 2.5 µl PI only to the stained sample tube; 
- gentle stirring on a vortex; 
- incubation at room temperature for 20 min in a dark place. 
- counting on a flow cytometer. 
Statistical processing of results 
Statistical processing of the data obtained was performed using Statistica version 12 
(StatSoft, USA). Values are presented as Me (25 - 75), where Me is the median and 
25 and 75 are the interquartile ranges as 25th and 75th percentiles. The normality of 
the distribution of values was assessed using the Shapiro-Wilk W criterion. Given 
the lack of normal distribution in the samples studied, non-parametric methods were 
used to compare groups of data and study correlations. The Mann-Whitney U-
criterion was used to compare two independent samples. A significance level of p 
<0.05 was used as the criterion for reliability of differences between indicators.