Internal Validation of the Anti-Human Hemoglobin Antibody for Enhanced Human Blood Detection at the PFSA DNA and Serology Laboratory, Pakistan.

Biological fluids collected from crime scenes play a crucial role in solving serious crimes such as murder, rape, burglary, and theft. Identifying human blood using various methods is crucial for linking disparate pieces of evidence and solving crimes. In this study, the Anti-Human Hemoglobin antibody (Ah-HB) from Sigma-Aldrich® USA was internally validated for human blood identification using the Ouchterlony Double Immunodiffusion (ODD) technique at the DNA and Serology Department of the Punjab Forensic Science Agency in Lahore, Pakistan. Additionally, a comparative analysis was conducted with Seratec® HemDirect (S_HD) strips to evaluate the economic feasibility of both methods. The internal validation included assessments of sensitivity, specificity, and an analysis of real-case work samples to determine the viability of the antibody as a confirmatory test for human blood. Although Ah-HB had lower sensitivity in detecting human blood at higher dilutions (1:10,000), it offered a more cost-effective solution per sample compared with S_HD, which demonstrated higher sensitivity (1:2,000,000) but at a significantly higher cost per sample. Specificity tests revealed no cross-reactivity with nonhuman blood for both Ah-HB and S_HD. This study emphasizes the importance of selecting suitable antibodies for forensic analysis by evaluating sensitivity, specificity, and cost-effectiveness. Ah-HB emerges as a valuable tool for forensic laboratories, providing reliable results at a lower cost compared with S_HD, thereby enhancing the efficiency and effectiveness of criminal investigations.