Optimization of a novel approach for the analysis of blood using Fourier transform infrared (FTIR) spectroscopy and chemometric analysis

Blood is one of the most common biological fluids encountered at crime scenes and is therefore constantly being tested for in the laboratory. Confirming the presence of blood can illuminate essential elements of a case as well as allow for identification via downstream DNA analysis. This significant investigative value is why it is crucial to use robust forensic testing techniques for blood detection. In the forensic laboratory, blood is identified using serological techniques. A presumptive test, such as a colorimetric test, is performed first. A confirmatory test, such as an immunochromatographic assay, is often performed following a presumptive positive result. While both types of tests have numerous advantages, they have several limitations as well. These limitations have served as the basis for exploring alternative techniques for forensic blood detection, such as FTIR. FTIR spectroscopy is a qualitative, non-destructive, confirmatory analytical technique. This technique uses infrared light to characterize organic compounds based on molecular structure. There are also several different FTIR techniques, such as ATR and DRIFTS. ATR-FTIR analysis has been widely researched for the detection of blood and other biological fluids, across several applications. ATR-FTIR may be preferable to serological blood detection because it can be quicker than combined serological blood testing, it requires minimal sample preparation, it does not damage DNA downstream, and it can detect multiple biological fluids at once. Despite all the advantages that ATR-FTIR analysis has over traditional forensic blood techniques, it has not yet been implemented in casework. This may be due to skepticism in using subjective and complex spectroscopic data that results from ATR-FTIR analysis of body fluids. The initial objective of this research was to develop an optimized protocol using ATR-FTIR and chemometric analysis to identify blood on cotton round substrates. Using these techniques together would allow for a rapid, nondestructive, confirmatory approach, that would be more objective than serological testing or FTIR analysis alone. However, due to complications throughout the research process, this objective was altered. The revised objective was to develop an optimized protocol using DRIFTS and chemometric analysis to identify blood on cotton round substrates. An optimized DRIFTS protocol for forensic blood identification was successfully developed. Blood samples from multiple donors were tested using this protocol, and all samples showed similar data. Human biological samples other than blood as well as non-human samples were also tested. These samples showed dissimilar data from the donors’ blood sample data. Chemometric analysis was then performed using AnalyzeIQ Lab software. After testing 93 pair-wise combinations of pre-processing methods and algorithms, a model was developed. Unfortunately, this model was not completely optimized. It had a 9.09% error rate, resulting from the misclassification of one sample. Future research is needed before implementation into casework. Alternative cotton substrates and data collection software should be considered. Additional time should be spent using AnalyzeIQ Lab software, to develop a model with a 0% error rate. If this cannot be achieved, an alternative chemometric analysis software should be considered