Forensic Discrimination Research Articles

Forensic discrimination of blue fountain pen inks based on dielectric constant property

The present work aims to show the analysis and comparison of fountain pen inks that are crucial in forensic questioned document examination, particularly in developing nations where the likelihood of fraud is higher in situations involving checks, marriage papers, birth and death records, and similar documents via using dielectric properties of material. Dielectric constant measurement is a new and deep method for discriminating fountain pen inks. To achieve this goal the dielectric constant of the identification of fountain pen ink has been studied to differentiate commercially used blue colour fountain pen inks in Turkey. The data was obtained by designing and setting up an alternating current (AC), Function Generator, and Oscilloscope to measure the resistance of each fountain pen ink sample. The measurements were performed in the frequency range between 1 Hz and 3 MHz at room temperature. Then, resistance measurements were used to calculate the dielectric constant. To support the result, as a conventional method, TLC-IA (image analysis) was applied to the same samples to discriminate these samples based on the intensity profile of red, green, and blue (RGB) by using the software PyCharm Community 2024.1.1. The results from each method supported each other. The distinction between samples can be made based on their dielectric constants in the frequency range of 2–2.5 MHz, and their loss factors in the range of 0–1 MHz have also been found to be distinguishing variables. Also, for the result of TLC-IA similarity ratios, the mean was calculated as 51.72% while the minimum value was 9.66%. For example, Sample 6 was distinguished from other samples with these two methods. So, dielectric properties, the new method, allowed us to identify the different fountain pen inks with the obtained results.

RGB Analysis for Forensic Discrimination of Blue Fountain Pen Inks

RGB Analysis for Forensic Discrimination of Blue Fountain Pen Inks

Forensic Discrimination of Various Subtypes of Regenerated Cellulose Fibers in Clothing Available on the Consumer Market

The discrimination of five subtypes of regenerated cellulose fibers, i.e., viscose, bamboo, lyocell, modal, and cupro, from both men’s and women’s clothing available on the prevalent apparel market was described. The examinations were conducted using optical microscopy (in transmitted white light and polarized light), scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM–EDX), and Fourier Transform Infrared Spectroscopy (FTIR). The microscopic methods revealed characteristic features of the morphological structure of the examined fibers, enabling the identification of differences between the subtypes. As a result, the microscopic methods were found to be the most effective for identifying and distinguishing between the types of examined fibers. Although the FTIR technique did not allow for distinguishing between the fiber subcategories, it contributed to the enlargement of the IR spectra databases for regenerated cellulose fibers. Based on the findings, a general scheme of the procedure for identifying the tested fibers was proposed.

Forensic discrimination of fiber microspectrophotometry data by resampling and repeating two-sample hypothesis testing

Textile fibers are important forensic trace evidence in criminal investigations. Colored textile fibers are often analyzed using non-destructive microspectrophotometry. However, when the spectra of questioned and known samples are very similar, it is difficult to compare the spectra visually. Chemometric methods, such as principal component analysis and agglomerative hierarchical clustering, have been applied to microspectrophotometry data. In this study, we used two-sample hypothesis testing as a chemometric approach for discrimination of fiber samples without reducing or eliminating the dimensionality of the data. Cotton fibers dyed with vat dyes were analyzed by microspectrophotometry. We examined suitable data types and statistical methods for test fiber samples by resampling and repeating hypothesis testing. A combination of spectral data type and statistical method was identified that provided a good balance of type I (false exclusion) and type II (false inclusion) error rates in two-sample hypothesis testing of the fibers.The main advantage of our chemometric approach is that an appropriate method for samples can be selected by using the type I and II error rates. Furthermore, our method can use all the measured data for the calculation without reducing or eliminating the dimensionality of the data, so it is not necessary to reduce variables or smooth the spectrum. This statistical method also requires no training set. Therefore, two-sample hypothesis testing could be applied to fibers of different types, colors, or materials from those used in this study. Resampling and repeating two-sample hypothesis testing technique is promising as a new chemometric approach for comparing microspectrophotometry spectra.

Forensic Discrimination of White Architectural Paints via ATR FT-IR Spectroscopy and Chemometrics

The present work entailed the comparative examination and discrimination of white architectural paint samples of different brands/manufacturers. The analysis of this evidence can facilitate exhibiting a linkage between the victim, and the crime scene and assist in the reconstruction of the crime scene. Architectural paint traces have evidential value, especially in cases of burglary, acts of vandalism, property crimes, etc. In the present study, a total of 102 samples of 34 distinct brands (3 samples of each brand) of white commercially available architectural paints were purchased and analysed using ATR FT-IR spectroscopy and multivariate analytical tools. The use of visual analysis of FTIR spectral data resulted in 12 undifferentiated/ indistinguishable sample pairs with a discrimination power of 97.86%. Adding PCA and PLS-DA in concert with visual analysis of FTIR spectra resulted in 99.4% (3 sample pairs undifferentiated) and 100% discrimination power, respectively. In conclusion, samples of each brand were completely differentiated using ATR FT-IR spectra subjected to the PLS-DA model. To assess the impact of different substrates on the examination of paint chips, simulated samples were produced on different substrates and the obtained results from the substrates study have been summarized. The ATR-FTIR spectroscopy therefore suggests an objective and rapid approach for the discrimination of white architectural paints for forensic purposes.

Chemical and Imaging Analysis of Questioned Document Paper by FTIR, ICP and Video Spectral Comparator (VSC) for Forensic Discrimination

AbstractForensic document examination has been considered one of the most important fields in the forensic science. For this, a quick, effective, and cheap techniques for evaluation of forensic evidence is highly required. This study explores the comparison of A4 document paper by chemical and physical analysis. 16 of white A4 paper from different brands were analyzed by FTIR for investigation of structural characteristics. Video Spectral Comparator 8000 has been used to compare the A4 papers under different light source. The inorganic filler composition in the paper is also explored by ICP‐OES. The calcium carbonate content in document papers play a discriminant role which found to be between 16.02 % and 35.54 %. Also, water contact angle has been utilized in order to examine the surface of paper which is generally related to the inorganic filler and other additives. The WCA results ranged between 83° and 111° that enable quick and easy way to compare A4 document papers. The experimental results have demonstrated that besides FTIR methods, other methods such as ICP‐OES for elemental composition, WCA for wetting test, VSC for visualization under different light of source could have been applied for comparison of document papers in case of criminal cases.

Forensic Discrimination of condom lubricants using ATR-FTIR spectroscopy in combination with chemometrics- effect of matrices, pre-coital and post-coital conditions.

The escalating rates of rape and sexual assault cases underscore the essential need to analyze associative evidence of sexual assault to ensure justice. The condom, when used during the act of sexual assault/rape, leaves behind traces of lubricant that could be instrumental in establishing the link between the victim and perpetrator. This leading evidence is crucial especially when the traditional DNA analysis is challenging owing to the lack of biological evidence. The present study employs the use of ATR FTIR spectroscopy coupled with the chemometric, to discern the chemical discrimination of condom lubricants. The study also aimed to investigate simulated real-case scenarios by analysing the effect of different matrices such as vaginal fluid, menstrual blood, and undergarment substrates (cotton, polyester and nylon) on condom traces. The difference in the spectral profile of pre-coitus and post-coitus samples was also investigated. The results of the present study are noteworthy, unveiling the efficacy of discriminating the condom lubricants of different brands and other frequently used household lubricants with 100% sensitivity, specificity, and accuracy. Condom lubricants are detected despite the presence of contaminants such as menstrual blood, and vaginal fluid, across different substrates and, in post-coital conditions. In conclusion, this study presents a comprehensive approach to analysing condom lubricants, offering a potential breakthrough in the investigation of sexual assault cases, coupled with the ability to counter false accusations.

Application of ATR-FTIR spectroscopy and chemometrics for the forensic discrimination of aged peripheral and menstrual bloodstains

Accurately distinguishing between peripheral and menstrual bloodstains at crime scenes is a major challenge in forensic practice. The time since deposition (TSD) of bloodstains at the crime scene also plays an essential role in determining the postmortem interval or injury time. In this study, we used attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy combined with chemometric methods to differentiate peripheral and menstrual bloodstains considering the TSD and menstrual phase.The results of the principal component analysis (PCA) model showed that the fresh peripheral and menstrual bloodstains were relatively easy to distinguish, while the aged samples (2 W, 4 W, and 6 W TSD) caused a certain level of spectral signature convergence in the spectral features. Subsequently, the partial least squares discriminant analysis (PLS-DA) model and the genetic algorithm combined with PLS-DA (GA-PLSDA) model were constructed considering the TSD.The accuracy of internal cross-validation and external validation of the PLS-DA model demonstrated internal and external classification abilities of 88.3 % and 90.0 % accuracy, respectively. Additionally, the results of GA-PLSDA model showed that the accuracy of external and internal validation was 89.2 % and 90.0 %, respectively.The PCA, PLS-DA, and GA-PLSDA models showed that the critical differences between peripheral and menstrual bloodstains were in protein, phosphoric acid and glucose content. This preliminary study suggests that ATR-FTIR spectroscopy combined with chemometrics may be a potential method to distinguish between peripheral and menstrual bloodstains using complex conditions and provides support for further studies.

A complete pipeline enables haplotyping and phasing macrohaplotype in long sequencing reads for polyploidy samples and a multi-source DNA mixture.

Macrohaplotype combines multiple types of phased DNA variants, increasing forensic discrimination power. High-quality long-sequencing reads, for example, PacBio HiFi reads, provide data to detect macrohaplotypes in multiploidy and DNA mixtures. However, the bioinformatics tools for detecting macrohaplotypes are lacking. In this study, we developed a bioinformatics software, MacroHapCaller, in which targeted loci (i.e., short TRs [STRs], single nucleotide polymorphisms, and insertion and deletions) are genotyped and combined with novel algorithms to call macrohaplotypes from long reads. MacroHapCaller uses physical phasing (i.e., read-backed phasing) to identify macrohaplotypes, and thus it can detect multi-allelic macrohaplotypes for a given sample. MacroHapCaller was validated with data generated from our designed targeted PacBio HiFi sequencing pipeline, which sequenced ∼8-kb amplicon regions harboring 20 core forensic STR loci in human benchmark samples HG002 and HG003. MacroHapCaller also was validated in whole-genome long-read sequencing data. Robust and accurate genotyping and phased macrohaplotypes were obtained with MacroHapCaller compared with the known ground truth. MacroHapCaller achieved a higher or consistent genotyping accuracy and faster speed than existing tools HipSTR and DeepVar. MacroHapCaller enables efficient macrohaplotype analysis from high-throughput sequencing data and supports applications using discriminating macrohaplotypes.

Forensic discrimination between traditional and compressive imaging by blurring kernel investigation

AbstractImage forensics encompasses a set of scientific tests to investigation of a suspected event via intrinsic clues of imaging pipeline. Traditional image sensing at the Nyquist-Shannon rate as well as the new modality of compressive imaging below the rate are two main types of sensing in photography and imaging applications. Hence, for forensic investigators, it would importantly necessitate the ability to discriminate among images captured by them. However, due to the complex nonlinear nature of imaging processes, investigating imagers’ traces is a difficult task. To this intent, we first systematically model the imaging pipelines as an encoder-decoder pair. For exploring distinguishable traces, we mathematically simplify and linearize the pair for compressive imaging and two main forms of traditional image sensing with or without compression. Our theoretical analyses on the approximate linear models reveal blurring kernels of different imagers have discriminability. To validate it in real-world scenarios, we considered the whole imaging process as an inverse problem and estimated the blurring kernel based on a deconvolution approach, where the discriminability is also justified by information visualization. Then, we designed a pipeline classification system, where a deep convolutional neural network is trained by the estimated blurring kernels to be able to classify the three imaging systems. Our results in compressive imaging identification show an accuracy improvement about 3.7 % in comparison to the best result among compared methods. Implementation codes are available for research and development.

Comparison of Gap-Segment and Savitzky-Golay Algorithms in Forensic Discrimination of Soils Based on Ultra-Performance Liquid Chromatography Data

Comparison of Gap-Segment and Savitzky-Golay Algorithms in Forensic Discrimination of Soils Based on Ultra-Performance Liquid Chromatography Data

Characterization of metal components in white-based polyester single fibers by X-ray fluorescence spectrometry and X-ray absorption fine structure analysis utilizing synchrotron radiation for forensic discrimination

Discrimination of single fibers is important in forensic investigation. Conventional microscopic observation and analytical methods such as micro-Fourier transform-infrared spectroscopy, a suitable technique for polymer component identification, can discriminate single white fibers made from different polymers. Polyesters contain metal compounds derived from polymerization catalysts, transesterification catalysts, and matting agents. Here we propose a forensic method for discriminating white-based polyester single fibers. The X-ray intensity ratios, distribution of metal elements, and chemical states are identified by semi-microbeam X-ray fluorescence (XRF) spot analysis, nanobeam XRF imaging, and X-ray absorption fine structure analysis (XAFS) utilizing synchrotron radiation, respectively. The analytical specimens were white-based polyester single fibers from the standard forensic fiber collection (Microtrace LLC, USA) and commercially available polyester products. Semi-microbeam XRF spot analysis detected metallic elements such as Ti, Mn, Co, Ge, and Sb derived from polymerization catalysts, transesterification catalysts, and matting agents. The X-ray intensity ratios of Ti/Mn, Ti/Ge, Ti/Sb, and Mn/Sb were obtained with good reproducibility but those of Ti/Co, Mn/Co, and Co/Sb, which are related to cobalt, were lowly reproducible in some samples. The poor reproducibility of the cobalt-related X-ray intensity ratios was attributed to uneven distributions of the metallic elements in the polyester single fibers, as visualized by nanobeam XRF imaging. The images showing elemental heterogeneity have proven to be good markers for identification. The XAFS results showed that three types of Ti compounds and two types of cobalt compounds are utilized in different applications of polyester single fibers; moreover, the chemical states are useful indices of single-fiber discrimination.

High-Performance Thin-Layer Chromatography hyphenated with image processing and chemometrics as a tool for forensic discrimination of Cannabis sativa L. chemotypes

Cannabis sativa L. is the most widely cultivated, trafficked, and abused illicit drug. The reliable distinction between drug type and fiber type C. sativa remains a topic of interest for forensic chemists. For the first time, we applied a combination of simple, cost-effective, and reliable High-Performance Thin-Layer Chromatography (HPTLC) with image analysis as well as sophisticated multivariate tools for differentiating C. sativa drug, fiber and intermediate chemotypes. After extraction, major cannabinoids from 43 seized C. sativa were separated using the HPTLC method. Peak areas of Δ9-THC, CBN, and CBD were calculated by simple image processing, and Xfactor = [THC + CBN]/CBD was determined as criteria used to discriminate three C. sativa chemotypes. The obtained results were compared with Gas Chromatography with Flame Ionization Detection (GC-FID) as the reference method for the determination of Xfactor.Principal Component Analysis and Hierarchic Cluster Analysis were applied to classify three C. sativa chemotypes according to their chemical pattern. The proposed approach clearly distinguishes 26 drugs, 13 fiber, and 4 intermediate chemotypes in seized C. sativa, aligning with GC-FID analysis. The results showed that the HPTLC technique in combination with multivariate methods is an accurate and reliable tool for high-throughput and forensic screening of three C. sativa chemotypes. Compounds such as Δ9-THC and CBD were marked as the most important cannabinoids responsible for the classification of the seized C. sativa chemotypes.

Alleviating Luminescence and Quenching toward Discrimination of Ballpoint Pen Inks Using Spectroscopy and Chromatography Techniques

The phenomenon of luminescence and quenching is widely applied in the forensic examination of various ink materials. Here, we introduce a hybrid spectroscopic and chromatography approach to gain insights into the underlying cause of infrared luminescence (IRL) in blue ballpoint (BP) pen inks. A total of thirty BP pen ink samples from the Egyptian market were employed in this study. Insights into the origin of luminescence and quenching in the studied samples were gained based on video spectral comparator (VSC), thin layer chromatography (TLC), ultraviolet–visible (UV–Vis) spectrophotometry, and photoluminescence (PL) spectroscopy. Results showed that some of the studied inks possessed IRL due to the presence of crystal violet and some other triaryl methane dyes. Nevertheless, some ink samples did not possess IRL, despite the presence of the dyes responsible for IRL in their matrix. Interestingly, the inclusion of phthalocyanine dye in those non-luminescent ink matrices resulted in luminescence quenching, mainly due to the overlap between the absorption of phthalocyanine dye and the luminescence of the triarylmethane dyes. The IRL behavior of the ink sample under the first illumination wave band (400–485 nm) exerted control over the IRL activity across subsequent illumination wavebands, and the most effective differentiation was achieved by utilizing the first and second preset filters in VSC. The results revealed the luminescent components present in studied inks and unraveled their distinct luminescence behavior present within the ink matrix. The combination of optical spectroscopy and chromatography techniques could provide a distinctive tool to reveal the luminescence and quenching behaviors of ink dyes for the successful forensic discrimination of several BP writing pens.

Forensic Discrimination of Smokeless Powders Using Carbon and Nitrogen Isotope Ratios.

Improvised explosive devices pose a threat to the public by way of terrorism and criminal activities. In the United States a commonly used low explosive in improvised explosive devices is smokeless powder (SP), due to its ease of access. Traditionally, forensic examinations are often sufficient in determining the physical and chemical characteristics of SPs. However, these exams are limited in differentiating or associating SPs when comparing two materials which are physically and/or chemically consistent. Stable isotope analysis of carbon and nitrogen has been used for explosives to further forensic chemical comparisons and aid in sample differentiation. In this manuscript we explore the utility of stable isotope analysis of SPs to differentiate manufacturer and geographic origin. Both bulk isotope analysis and component isotope analysis of carbon and nitrogen via an extraction method using dichloromethane were evaluated to compare the overall isotope signature of individual SPs. Through the combination of bulk and component isotope measurements of SPs, we were able to identify geographic relationships; however, the manufacturer origins were not as clearly discriminated. This technique demonstrates a potential improvement to traditional forensic examinations of smokeless powder by adding additional information when explosives are chemically and/or physically consistent.

DNA Extraction from Heartwood and Quick Species Authentication Using Real-Time PCR: A Case Study of the Rosewood (<i>Pterocarpus Indicus</i>)

Illegal logging, felling and timber trade have continued to increase over the past two decades, leading to a decline in forest biodiversity and the extinction of some wood species. <i>Pterocarpus indicus</i> Willd., listed in the National Standards of the People’s Republic of China for Hongmu (GB/T 18107-2017), is widely used in production of high-end furniture, decorative flooring and musical instruments due to its high-quality timber. For molecular species identification, the quality and quantity of DNA extracted from wood samples should first be ensured. However, extracting DNA from dried, aged timber heartwood is difficult, as heartwood contains little fragmented DNA, along with lots of phenolic compounds known to impede sequence amplification. In order to protect <i>P. indicus</i> from over-exploitation and to achieve accurate species-level identification, we established a particular extraction method for obtaining amplifiable DNA from heartwood samples and the real-time PCR assay for species discrimination of <i>P. indicus</i> in this study. The quantity and quality of DNA extracted from dry heartwood samples using the modified CTAB method were 2.40-37.70 ng/μL and 1.55-2.12 demonstrated by OD_260/280, respectively. Primer set P9, targeting <i>P. indicus</i> specific microsatellite Pin2-20 sequence, was amplifiable in newly established real-time PCR. Through analysis, this real time PCR was shown to be specific and sensitive with a detection limit around 0.17 ng/μL. Hopefully, this study will contribute to heartwood DNA extraction and species identification of timber logs for forensic discrimination, law enforcement and natural resource conservation.

Forensic Discrimination of Drug Powder Based on Drug Mixing Condition Determined Using Micro Fourier Transform Infrared Spectroscopy.

The quantitative evaluation of the drug mixing condition was conducted for application in the forensic discrimination of drug powders using micro Fourier transform infrared (FT-IR) spectroscopy. Bromhexine hydrochloride (BHCl) and p-hydroxybenzoic acid (PHBA) were used as the simulated drug and additive, respectively. Equal masses of two chemicals were (1) simply mixed, (2) homogenized using agate mortar, or (3) dissolved in methanol and dried, and then (4) homogenized using agate mortar. The mixed powders dispersed on BaF2 plates were subjected to mapping analysis of micro FT-IR spectroscopy using synchrotron radiation (SR) or globar light in transmission mode with aperture sizes of 2.5 x 2.5 and 10 x 10μm2, and x-y scanning steps of 2.5 and 10 μm, respectively. The areas of the vibration bands specific to BHCl (C-N bending) and PHBA (C=O stretching) were converted to the molar contents (CBHCl, CPHBA), and the relative content ratio (RCR: CPHBA/[CBHCl + CPHBA]) was used as one mixing parameter. The resulting two-dimensional distribution map provided the relative spatial localizations of the two species, and frequency histograms with a horizontal axis of RCR were plotted to evaluate the RCR distribution. The percentage frequency of the extreme value in which RCR was 0 or 1 (%EV) was used as one mixing index. After excluding the extreme values, the coefficient of variation (CV) of the RCR distribution was used as another mixing index. The differentiation among four mixing modes could be evaluated from the standpoint of %EV and CV, and the discrimination capacity by SR instrument was superior to that by globe light instrument.

Selected Aspects of Forensic Discrimination of Blue and Black/Grey Cotton Fibres Derived from Denim Fabrics

Fragments of single textile fibres are one of the most commonly found microtraces at crime scenes. Among them, the widespread blue and black/grey cotton fibres should be recognized. The analytical methods routinely used in fibre examination mainly focus on color assessment and determination of the fibres’ morphological features as well as chemical composition. This publication presents the physicochemical characteristics of blue and black/grey denim fabrics and fibres as well as an overview of the non-destructive and destructive methods used in the discrimination of these fibres. Usually, such fibre microtraces are very difficult to distinguish in forensic examinations due to their widespread abundance, and, thus, their evidential value is not significant. As previous research shows, most denim material samples were colored with indigo dye. However, due to the changing trends in denim production and the fashion market, indigo derivatives may play a more critical role. The literature review shows significant shortcomings in the development of techniques focusing on the analysis of the dyes contained in denim fibres, and this is a research direction worth pursuing.

Quantitative Analysis of Kruppel-Like Factor 5-Related Messenger RNA Transcripts in Ischemic Myocardium for Discrimination of Death Causes

Background: Accumulated studies have demonstrated that Kruppel-like factor 5 (KLF5), a transcription factor, plays an important role in regulating cell proliferation and tissue remodeling through the expression of its downstream genes. KLF5-related factors are expected to be involved in the healing process after myocardial injury or myocardial ischemic changes, especially for the forensic diagnosis of myocardial ischemic physiopathology. Aim and Objectives: This study aimed to explore the discrimination ability and applicability of KLF5-related factors in SCD caused by MI compared with other causes of death to provide further insights into the forensic diagnosis of myocardial ischemic pathology. Materials and Methods: The relative quantification of F-Box and WD Repeat Domain Containing 7 (FBW7), KLF5, factor-binding protein (FGFBP) 1, and FGFBP2 messenger RNAs (mRNAs) in myocardial tissue samples was performed using real-time fluorescence quantitative reverse transcription polymerase chain reaction. KLF5 and FGFBP1/2 protein levels were examined using immunohistochemistry (IHC). The forensic autopsy cases (27 in total, autopsy within 72 h postmortem) included seven cases of acute myocardial infarction and 10 cases of acute myocardial ischemia. There were 10 cases in the control group, including four cases of traffic injury one case of injury by fall from height, one case of electric death, and four cases of blunt force injury. Results: Characteristic results were found in myocardial samples from three groups of deaths: KLF5 and FGFBP1 mRNA levels were significantly elevated in the infarction and ischemia groups, while FBW7 mRNA levels were significantly decreased. FBW7 is an important ubiquitin ligase that can mediate the degradation of KLF5 protein. In addition, FBW7 and FGFBP2 mRNA levels were decreased in the infarction group compared with the ischemia group. The IHC results were consistent with the observed mRNA expression patterns. Conclusions: Quantitative detection of FBW7, KLF5, FGFBP1, and FGFBP2 mRNA transcripts in myocardial tissues supports the pathophysiological study of myocardial ischemic diseases and provides molecular pathological evidence for forensic discrimination of death causes.

Developmental validation of the Microreader Group Y Direct ID System: A novel six-dye typing system with 54 Y-chromosomal loci for forensic application.

Y-chromosome, as a gender-determined biological marker, is inherited only between fathers and sons. The Y-chromosome short tandem repeats (Y-STRs) play an essential role in paternity lineage tracing as well as sexual assault cases. The Microreader Group Y Direct ID System as a six-dye multiplex amplification kit, including 53 Y-STR and one Y-Indel locus, would improve performance and aid in obtaining more information through a greater number of loci with high polymorphism. In the present study, to verify the accuracy and efficiency of the kit, developmental validation was conducted by investigating sensitivity, species specificity, PCR inhibition, male-male and male-female mixtures, and reproducibility. The kit was tested using 311 male samples from Han and Qiang populations in Sichuan Province. The results showed that this kit had fairly high power for forensic discrimination (Han: haplotype diversity [HD]=1, Qiang: HD=0.999944). Additionally, 44 confirmed father-son pairs were also genotyped, among which 69 distinct haplotypes could be obtained. These father-son pairs cannot be distinguished by commonly used Y-STR panels, indicating that adding these extra Y-STRs to a single panel can achieve better discrimination performance. Collectively, the Microreader Group Y Direct ID System is robust and informative for forensic applications.