Topic: Cell Free Plasma DNA as a Predictor of Outcome in Severe Sepsis and Septic Shock

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Introduction: The research is a clinical and correlation experiment performed by a Finland study group to find out the effect of increased concentration of cell free DNA found in 255 patients 18yrs or older who were seriously ill with whole body inflammatory state in both Intensive care unit and hospital mortality, as well as to see if there was a relation between cell free plasma DNA and organ failure.

Procedure:Real time quantitative PCR assay for Beta globin gene was used to measure blood samples Age: 18yrs and older patients with severe sepsis or septic shock.

Time: 72hrs after their blood was collected over a period of 4months in 24hrsInstrument: Centrifugation was used to separate the plasma fraction and the blood samples.

Storage: stored at -20 degree C and later sent to Helsinki University Hospital, where they were stored at -80 degree C.

Data Collection: Simplified acute physiologic score (SAPS II), acute physiology and chronic health evaluation (APACHE II), Sequential organ failure assessment (SOFA), lactate concentration and creatine clearance score using Cockcroft Gault formula were used to study the amount of cell free plasma DNA in survivors and non survivors in both ICU (intensive care unit) and Hospital patients.

METHODS:1. The QIA amo DNA Blood Mini Kit (Qiagen) was used to extract DNA in order to ensure there weren’t any residual cells left behind.

2. The DNA extracted produced the following sequence: forward primer 5’-GCA CCT….GAA-3’. Reverse primer 5’CAC CAA……TCA-3’ as well as a single labeled fluorescent MGB probe 5’-FAM-TCT….MGB-NFQ. (Minor grove binding molecule and Nonfluorescent quencher molecule respectively).

Precision of Data:The data was ran 8times in multiple duplicates to ensure precision of the real time quantitative PCR method.

A standard curve of 10 fold serial dilution of human genomic DNA (Roche) was used in the analysis of the data as well as chi-square to test for variables.

The area under the curve with a 95% CIs was used to study the sensitivity and specificity to ensure a greater predictive value with P < 0.05 being significant.

Result:Cell free plasma DNA for survivors at admission: 8070 GE/mL 72hrs later 7457 Ge/mL wCell free plasma DNA for non survivors: 15904 GE/mL with P< 0.001and 72hrs later it was 15904 with P = 0.004Plasma DNA concentration for non survivors was 12386 GE/mL with P= 0.009 while those of the survivors = 7678Ge/mL.

SOFA score (r=0.30, P<0.001), APACHE II score(r=0.18, P=0.005), SAPII score(r=0.22, P=0.001), lactate concentration(r=0.40, P<0.001). Plasma creatine concentration (r=0.15, P=0.0018)In conclusion it was observed by the Finland study group that cell free plasma DNA was higher in non survivors compared to survivors in both ICU and hospital patients. The discrepancy between this new result and their old result that produced a plasma DNA median of 9366GE/mL was probably due to pre analytical error, the PCR method and differences in patient selection. There was no correlation on liver and renal failure with cell free plasma DNA. I am convinced about the finding and don’t think there was any place in the report that suggested bias. Their sample collection, method, data statistics and procedure seem appropriate, although they could have used control with healthy patients to compare their result. However the experiment seemed to be well done.

Authors: Katri Saukkonen, Paivi Lakkisto, Ville Pettila, Marjut Varpula, Sari Karisson, Esko Ruokonen, Kari Pulkki,Published: Department of Medicine and Emergency Care, Helsinki University Central Hospital, Helsinki FinlandPages 1000-1007