Multiple Body Fluids Research Articles

Mass spectrometry-based proteomics for source-level attribution after DNA extraction

Biological traces recovered from crime scenes serve as vital evidence in forensic investigations. While DNA evidence is frequently used to address the sub-source level of the hierarchy of propositions, the biological source of the DNA can be highly probative at the source level. Current body fluid detection methods pose certain limitations, such as reports of false positive results from some of the presumptive and/or confirmatory tests in current use. These tests are also individual tests for the detection of one body fluid, meaning that if the sample is suspected to be a mixture of multiple body fluids, then different tests would need to be conducted to confirm the body fluid(s) present, which may exhaust small amounts of available biological trace. Proteomics applications for the identification of body fluids have been previously explored, and potential biomarkers indicative of body fluids discovered from liquid-chromatography tandem mass spectrometry (LC-MS/MS) methods have been reported. This work focuses on developing a mass spectrometry-based proteomics approach for the identification of body fluids by targeting discriminating peptide biomarkers from the non-DNA component left over after DNA extraction of samples. The non-DNA component is typically a waste product but with unappreciated evidential value. Our methodology for the purification of proteins from the post-DNA extraction waste includes an acetone precipitation and single-pot solid-phase-enhanced sample preparation (SP3) technique, microwave-assisted trypsin digestion, and LC-MS/MS analysis of the resultant peptides. Preliminary results from this proof-of-concept study include a list of potentially discriminating proteins and peptides for blood, saliva, and semen developed from the analysis of post-DNA extraction waste. Our method allows for multiple analytes to be targeted simultaneously from a DNA profiling waste stream and we anticipate that it could eventually be incorporated into standard forensic laboratory workflows.

Exploring Circular RNA Profile and Expression in Extracellular Vesicles.

Extracellular vesicles (EVs) are small vesicles secreted by various cell types and are enriched in multiple body fluids. EVs containing RNA have the potential to modulate biological processes and are being investigated for their diagnostic and therapeutic applications. Circular RNAs (circRNAs), generated through back-splicing of exons, are enriched in EVs. Given their unique characteristics and diverse functions, EV-circRNAs are important players in disease pathology. This chapter describes a workflow for investigating the expression profile of EV-circRNAs, which includes EVs separation, library preparation, and bioinformatics analysis. This workflow can aid the investigation of EV-circRNAs and their potential role in disease pathology.

Metabolic Communication by SGLT2 Inhibition.

SGLT2 (sodium-glucose cotransporter 2) inhibitors (SGLT2i) can protect the kidneys and heart, but the underlying mechanism remains poorly understood. To gain insights on primary effects of SGLT2i that are not confounded by pathophysiologic processes or are secondary to improvement by SGLT2i, we performed an in-depth proteomics, phosphoproteomics, and metabolomics analysis by integrating signatures from multiple metabolic organs and body fluids after 1 week of SGLT2i treatment of nondiabetic as well as diabetic mice with early and uncomplicated hyperglycemia. Kidneys of nondiabetic mice reacted most strongly to SGLT2i in terms of proteomic reconfiguration, including evidence for less early proximal tubule glucotoxicity and a broad downregulation of the apical uptake transport machinery (including sodium, glucose, urate, purine bases, and amino acids), supported by mouse and human SGLT2 interactome studies. SGLT2i affected heart and liver signaling, but more reactive organs included the white adipose tissue, showing more lipolysis, and, particularly, the gut microbiome, with a lower relative abundance of bacteria taxa capable of fermenting phenylalanine and tryptophan to cardiovascular uremic toxins, resulting in lower plasma levels of these compounds (including p-cresol sulfate). SGLT2i was detectable in murine stool samples and its addition to human stool microbiota fermentation recapitulated some murine microbiome findings, suggesting direct inhibition of fermentation of aromatic amino acids and tryptophan. In mice lacking SGLT2 and in patients with decompensated heart failure or diabetes, the SGLT2i likewise reduced circulating p-cresol sulfate, and p-cresol impaired contractility and rhythm in human induced pluripotent stem cell-derived engineered heart tissue. SGLT2i reduced microbiome formation of uremic toxins such as p-cresol sulfate and thereby their body exposure and need for renal detoxification, which, combined with direct kidney effects of SGLT2i, including less proximal tubule glucotoxicity and a broad downregulation of apical transporters (including sodium, amino acid, and urate uptake), provides a metabolic foundation for kidney and cardiovascular protection.

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids.

Body fluid identification is an essential step in the forensic biology workflow that can assist DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the specificity that molecular techniques can achieve; therefore, molecular methods, including microRNA (miRNA) and microbial signature characterization, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase the discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance (blood and semen). This project synergized the benefits of microRNAs and microbial DNA to identify multiple body fluids using DNA extracts. A reverse transcription (RT)-qPCR duplex targeting miR-891a and let-7g was validated, and miR-891a differential expression was significantly different between blood and semen. The miRNA duplex was incorporated into a previously reported qPCR multiplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius to presumptively identify vaginal/menstrual secretions, feces, and saliva, respectively. The combined classification regression tree model resulted in the presumptive classification of five body fluids with 94.6% overall accuracy, now including blood and semen identification. These results provide proof of concept that microRNAs and microbial DNA can classify multiple body fluids simultaneously at the quantification step of the current forensic DNA workflow.

Lassa Fever Infection among Healthcare Workers during the 2018 Outbreak in Nigeria

Introduction: Healthcare workers (HCWs) are potentially exposed to infection during viral hemorrhagic fever outbreaks. In the wake of 2018, Nigeria experienced an unprecedented surge in cases of Lassa fever (LF), which affected HCWs. To guide infection prevention and control (IPC) strategies in similar settings, we characterize HCWs' infection and describe the gaps in IPC standards and practices during the outbreak. Methods: Data was collected using a structured questionnaire, interview, and review of case notes of 21 HCW with laboratory-confirmed Lassa fever who were treated at the Irrua Specialist Teaching Hospital (ISTH) Irrua and the Alex-Ekwemen Federal Teaching Hospital, Abakaliki (AEFETHA), between 1st January and 27th May 2018. Information collected was the patients' socio-demographic characteristics, date of potential exposure and onset of illness, nature, and type of exposure, clinical features, outcome, use of personal protective equipment (PPE), and personnel IPC training and were analyzed using descriptive statistics with Microsoft Excel. Results: The study included 21 HCWs, and 12 (57.14%) were doctors. The case fatality rate was 23%. Nearly two-thirds (62%) of the HCWs could describe a likely procedure leading to their exposure and infection. Among 13 HCWs, 85% had multiple blood and body fluids exposure, while 15% had needle stick injury or scalpel cut. About one-fifth of the participants had received some IPC training. Conclusion: Limited IPC adherence and inappropriate risk assessment were identified as factors leading to Lassa fever exposure and infection among HCWs. There is an urgent need to provide IPC training for all HCWs and to ensure an adequate supply of IPC materials to all healthcare facilities as part of emergency preparedness, especially in LF endemic areas.

Assessment of Changes in Physiological Markers in Different Body Fluids at Rest and after Exercise.

Physiological and biological markers in different body fluids are used to measure the body's physiological or pathological status. In the field of sports and exercise medicine, the use of these markers has recently become more popular for monitoring an athlete's training response and assessing the immediate or long-term effects of exercise. Although the effect of exercise on different physiological markers using various body fluids is well substantiated, no article has undertaken a review across multiple body fluids such as blood, saliva, urine and sweat. This narrative review aims to assess various physiological markers in blood, urine and saliva, at rest and after exercise and examines physiological marker levels obtained across similar studies, with a focus on the population and study methodology used. Literature searches were conducted using PRISMA guidelines for keywords such as exercise, physical activity, serum, sweat, urine, and biomarkers, resulting in an analysis of 15 studies for this review paper. When comparing the effects of exercise on physiological markers across different body fluids (blood, urine, and saliva), the changes detected were generally in the same direction. However, the extent of the change varied, potentially as a result of the type and duration of exercise, the sample population and subject numbers, fitness levels, and/or dietary intake. In addition, none of the studies used solely female participants; instead, including males only or both male and female subjects together. The results of some physiological markers are sex-dependent. Therefore, to better understand how the levels of these biomarkers change in relation to exercise and performance, the sex of the participants should also be taken into consideration.

Precise and comprehensive determination of multiple body fluids by applying statistical cutoff values to a multiplex reverse transcription-PCR and capillary electrophoresis procedure for forensic purposes

Body fluid identification from crime scene evidence is an essential procedure in forensic investigations. Among various procedures, multiplex reverse transcription PCR assays have a clear advantage over conventional methods because different types of body fluids can be analyzed simultaneously. For more precise, comprehensive, and objective identification of forensically relevant body fluids, 15 target genes for blood, saliva, semen, vaginal fluid, and nasal secretion were selected; their primers were re-designed and multiplex PCR conditions were optimized to prioritize specificity for those body fluids. Multiple amplicons were separated and determined by the SeqStudio Genetic Analyzer with an all-in-one and easy-to-use cartridge. Then, the cutoff value was set for each marker to eliminate the detection of slight amplification in non-targeted body fluids. As a result, the targeted body fluid specificities of the developed procedure were drastically improved. Although successful determination of the target gene depends on sample condition and marker sensitivity, our procedure was applicable for the precise determination of body fluids in mixed body fluid stains, aged samples, and various mock casework samples. Therefore, it could be a powerful and convenient tool for the precise identification of multiple body fluids in forensic laboratories.

Extracellular phosphoprotein regulation is affected by culture system scale-down

BackgroundPhosphorylated proteins are known to be present in multiple body fluids in normal conditions, and abnormally accumulated under some pathological conditions. The biological significance of their role in the extracellular space has started being elucidated only recently, for example in bone mineralization, neural development, and coagulation. Here, we address some criticalities of conventional culture systems for the study of the extracellular regulation of phosphorylation. MethodsWe make use of microfluidics to scale-down the culture volume to a size comparable to the interstitial spaces occurring in vivo. The phosphoprotein content of conditioned media was analyzed by a colorimetric assay that detects global phosphorylation. ResultsWe found that miniaturization of the culture system increases phosphoprotein accumulation. Moreover, we demonstrated that in conventional culture systems dilution affects the extent of the phosphorylation reactions occurring within the extracellular space. On the other hand, in microfluidics the phosphorylation status was not affected by addition of adenosine triphosphate (ATP) and FAM20C Golgi Associated Secretory Pathway Kinase (FAM20C) ectokinase, as if their concentration was already not limiting for the phosphorylation reaction to occur. ConclusionsThe volume of the extracellular environment plays a role in the process of extracellular phosphorylation due to its effect on the concentration of substrates, enzymes and co-factors. General significanceThus, the biological role of extracellular phosphoregulation may be better appreciated within a microfluidic culture system.

Usefulness of Liquid Biopsy for Intraocular Malignancies

Retinoblastoma (RB) is the most common primary intraocular malignancy in children, whereas Uveal melanoma (UM) is the most common intraocular malignancy in adults [1,2]. Tissue biopsy is the standard gold technique for diagnosing the malignant neoplasm but an incisional tissue biopsy or fine needle aspiration biopsy (FNAB) is contraindicated for the intraocular malignancy [3]. Clinical diagnosis and imaging study are the only way to diagnose the intraocular malignancy due to the risk and fear of extraocular spread [4]. Recently, liquid biopsy has gained in popularity in the ophthalmic field. Liquid biopsy allows retinoblastoma diagnosis and a better understanding of the metastatic spread of uveal melanoma. Recently, the USA Food and Drug Administration (FDA) approved the make use of liquid biopsy (LB) as an appropriate diagnosis, prognosis, and also for monitoring tool in non-small cell lung carcinoma to keep away from invasive tissue biopsy in designated cases [5-7]. Liquid biopsy (LB) utilizes biofluid to evaluate for tumor-derived cells or cell-free DNA. LB is a relatively non-invasive technique rather than a tissue biopsy. In LB, material collected from multiple body fluids such as aqueous humor (AH), blood, cerebrospinal fluid, urine, and saliva for molecular diagnosis [8] and detecting of cancer biomarkers such as circulating tumor cells (CTC), tumor derived cell free DNA (ct-DNA), circulating tumor RNA (ct-RNA), microRNA (miRNA), tumorrelated exosomes (TREs), and extracellular vescicles (EVs) [7]. Aqueous humor samples for RB (Ocular LB) and Venous blood samples for UM (systemic LB) are utilizing for analyzing the molecular characteristics [8]. In others ophthalmic malignancies like conjunctival melanoma or squamous cell carcinoma, the role of LB is still not studied because tissue biopsy is routinely done for confirming the diagnosis and also for mutational status [9-11].

Peptide-Affinity Isolation of Extracellular Vesicles and Cell-Free DNA From Human Plasma.

Extracellular vesicles (EVs) are membrane-bound nanoparticles that carry DNA, RNA, and protein cargoes and are found in a variety of biofluids. EVs, along with cell-free DNA (cfDNA), have attracted interest as a source of biomarker material for liquid biopsy, a process in which a sample of body fluid is used for the detection or monitoring of disease. The Vn96 synthetic peptide facilitates the isolation of both EVs and cfDNA from multiple body fluids, including human plasma, placing it as a versatile tool for the capture of multiple biomarker materials for disease detection and/or treatment monitoring. In this chapter, we describe an optimized protocol for Vn96-mediated isolation of EVs and cfDNA from human plasma samples, as well as downstream methods for EV enumeration and DNA, RNA, and protein extraction from the material captured by Vn96 for use in biomarker discovery or detection.

Detection of bloodstains using attenuated total reflectance-Fourier transform infrared spectroscopy supported with PCA and PCA-LDA.

The most important task in a criminal investigation is to detect and identify the recovered biological stains beyond reasonable scientific doubt and preserve the sample for further DNA analysis. In the light of this fact, many presumptive and confirmatory tests are routinely employed in the forensic laboratories to determine the type of body fluid. However, the currently used techniques are specific to one type of body fluid and hence it cannot be utilized to differentiate multiple body fluids. Moreover, these tests consume the samples in due process, and thus it becomes a great limitation especially considering the fact that samples are recovered in minute quantity in forensic cases. Therefore, such limitations necessitate the use of non-destructive techniques that can be applied simultaneously to all types of bodily fluids and allow sample preservation for further analysis. In the current work, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy has been used to circumvent the aforementioned limitations. The important factors which could influence the detection of blood such as the effect of substrates, washing/chemical treatment, ageing, and dilution limits on the analysis of blood have been analysed. In addition, blood discrimination from non-blood substance (biological and non-biological in nature) has also been studied. Chemometric technique that is PCA-LDA has been used to discriminate blood from other body fluids and it resulted in 100% accurate classification. Furthermore, blood and non-blood substances including fake blood have also been classified into separate clusters with a 100% accuracy, sensitivity, and specificity. All-inclusive, this preliminary study substantiates the potential application of ATR-FTIR spectroscopy for the non-destructive identification of blood traces in simulated forensic casework conditions with 0% rate of false classification.

A data-driven, high-throughput methodology to determine tissue-specific differentially methylated regions able to discriminate body fluids.

Tissue-specific differentially methylated regions (tDMRs) are regions of the genome with methylation patterns that modulate gene expression in those tissue types. The detection of tDMRs in forensic evidence can permit the identification of body fluids at trace levels. In this report, we have performed a bioinformatic analysis of an existing array dataset to determine if new tDMRs could be identified for use in body fluid identification from forensic evidence. Once these sites were identified, primers were designed and bisulfite modification was performed. The relative methylation level for each body fluid at a given locus was then determined using qPCR with high-resolution melt analysis (HRM). After screening 127 tDMR's in multiple body fluids, we were able to identify four new markers able to discriminate blood (2 markers), vaginal epithelia (1 marker) and buccal cells (1 marker). One marker for each target body fluid was also tested with pyrosequencing showing results consistent with those obtained by HRM. This work successfully demonstrates the ability of in silico analysis to develop a novel set of tDMRs capable of being differentiated by real time PCR/HRM. The method can rapidly determine the body fluids left at crime scenes, assisting the triers of fact in forensic casework.

Non-Coding RNAs in the Brain-Heart Axis: The Case of Parkinson's Disease.

Parkinson’s disease (PD) is a complex and heterogeneous disorder involving multiple genetic and environmental influences. Although a wide range of PD risk factors and clinical markers for the symptomatic motor stage of the disease have been identified, there are still no reliable biomarkers available for the early pre-motor phase of PD and for predicting disease progression. High-throughput RNA-based biomarker profiling and modeling may provide a means to exploit the joint information content from a multitude of markers to derive diagnostic and prognostic signatures. In the field of PD biomarker research, currently, no clinically validated RNA-based biomarker models are available, but previous studies reported several significantly disease-associated changes in RNA abundances and activities in multiple human tissues and body fluids. Here, we review the current knowledge of the regulation and function of non-coding RNAs in PD, focusing on microRNAs, long non-coding RNAs, and circular RNAs. Since there is growing evidence for functional interactions between the heart and the brain, we discuss the benefits of studying the role of non-coding RNAs in organ interactions when deciphering the complex regulatory networks involved in PD progression. We finally review important concepts of harmonization and curation of high throughput datasets, and we discuss the potential of systems biomedicine to derive and evaluate RNA biomarker signatures from high-throughput expression data.

Noncoding RNAs in oral premalignant disorders and oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) has the highest mortality rate among all head and neck cancers and a relatively low five-year survival rate. Generally, the development of an oral mucosal malignancy represents a multistep process beginning with normal oral mucosa epithelium and culminating in OSCC after transitioning through intermediary oral premalignant disorders (OPMDs), during which dysplasia is often observed. Noncoding RNAs (ncRNAs) are RNAs that are not translated into proteins, but still can participate in regulating neoplastic cell behavior. Recently, data have emerged on the role of ncRNAs in the progression of oral mucosal malignant diseases, but the exact mechanisms through which ncRNAs are involved remain to be elucidated. Knowledge on ncRNAs has added an extra layer of complexity to our understanding of the malignant progression of oral mucosal diseases. The identification of ncRNAs in multiple body fluids as biomarkers may provide new diagnostic options that can be used for the diagnosis and prognosis of OPMDs and OSCC, respectively. Despite overall advances that have been made in cancer treatment, the treatment options for OPMDs and OSCC are still limited. Several studies have shown that ncRNA-based treatment regimens may hold promise as alternative methods for treating OPMDs and OSCC. The use of ncRNAs as therapeutic agents, including miR-155, miR-34 and lncRNA HOTAIR, appear promising.

Evaluating the effect of body fluid mixture on the relative expression ratio of blood-specific RNA markers

The estimation of the time elapsed since a biological stain was deposited at a crime scene can provide crucial information to a forensic investigation, indicating either when a crime was committed, or whether the biological evidence was deposited at the time of a known crime event. This would enable the investigators to limit the number of suspects and to assess alibis. The relative expression ratios (RERs) of body fluid-specific RNA markers are promising molecular tools for indicating the age of biological stains. However, the nature of some forensic samples found at crime scenes could be challenging, as they frequently occur in a mixture of different body fluid types. The research presented here has utilised reverse transcription quantitative PCR (RT-qPCR) to explore the impact of bloodstains being present in mixtures with other body fluids (saliva or semen) on the resulting RERs of blood-specific markers. The expression level of three blood-specific markers (HBA, HBB and miR16) along with two reference genes (18S and U6) were analysed across multiple ageing time points in pure and mixed bloodstains. For some markers, no significant differences were found when comparing RERs in pure and mixed bloodstains, however some RERs were altered in mixed stains. This indicates that the presence of body fluid mixtures may have a significant effect on the RERs of some blood-specific markers. This should therefore be considered when selecting markers for estimating the age of stains, particularly when multiple body fluids are thought to be present.

Research progress on the functions of exosomes in colorectal cancer

Exosomes are a class of extracellular vesicles that secreted by a variety of cells, with the length of 40-100 nm and a lipid bilayer membrane structure. They are often observed in multiple body fluids and are important mediators in intercellular communication. Several researches demonstrated that exosomes are closely linked to the development, invasion, metastasis, and drug resistance of colorectal cancer (CRC). In this review, the role of CRC-derived exosomes in the pathogenesis, diagnosis, prognosis and treatment of CRC were briefly summarized to provide a strategy for new therapeutic targets in CRC. Key words: Colorectal neoplasms; Exosomes; Biomarkers, tumor

The role of liquid biopsy in oral squamous cell carcinoma and oral potentially malignant disorders

Background: Oral squamous cell carcinoma (OSCC) is a common malignancy of the head and neck region and a major cause of morbidity and mortality worldwide. It is associated with a poor survival rate for late-stage disease, and a high rate of reoccurrence. OSCC commonly develops from oral potentially malignant disorders (OPMD), such as oral leukoplakia, and is commonly attributed to smoking, alcohol, betel nut chewing, HPV infection and other environmental factors. Currently, there are limited screening strategies in place to detect OPMD lesions, especially those with an increased likelihood of malignant transformation to OSCC. Liquid biopsy is an emerging option for detection of cancers as it is minimally invasive and can provide accurate information about cancers. Circulating tumor DNA (ctDNA), circulating tumor cells (CTCs) and microRNAs are present in multiple body fluids such as saliva and plasma, and are very promising biomarkers for cancer detection, treatment planning and disease monitoring for.

Multiplex body fluid identification using surface plasmon resonance imaging with principal component analysis

Body fluid identification is a key component in forensic casework, providing important information for the reconstruction of criminal events. Body fluid identification in combination with DNA analysis allows the linking of individuals to criminal acts and can therefore be of great importance in determining the outcome of criminal court cases. However, none of the current body fluid identification methods meet all forensic requirements, such as a high sensitivity, a high specificity and the ability to analyse multiple body fluids in a single run. In this pilot-study, we explore, for the first time, surface plasmon resonance imaging (SPRi) with antibody-based detection to serve as a novel multiplex body fluid identification assay for blood, semen, saliva, urine and sweat using minimal sample preparation. A training set consisting of ten donors per body fluid was analysed to determine whether body fluid specific response signals could be obtained. Principal component analysis (PCA) was performed as a statistical tool to cluster the body fluid samples by response signal pattern reduction and to uncover the sources of variation between the body fluids. Four principal components allowed complete clustering of all body fluid types. Blind testing of body fluid samples revealed that five out of eight unknown samples could correctly be clustered to their corresponding group, three out of eight samples were identified as inconclusive. Although optimization of the current SPRi method is required for use in the forensic field, this pilot-study demonstrates the feasibility of SPRi to differentiate five forensically relevant body fluids.

Developmental validation of the ParaDNA® Body Fluid ID System—A rapid multiplex mRNA-profiling system for the forensic identification of body fluids

Identifying the biological origin of forensic traces can provide crucial evidence to aid criminal investigations. Current forensic practice for the identification of body fluids mostly uses protein-based presumptive tests. Such tests cannot identify all of the forensically relevant fluids and have issues of cross-reactivity. More recently, messenger RNA methods have been developed that have expanded the range of body fluids that can be positively identified. However, these methods are slow and require expert scientists to run the processes and interpret the results. The ParaDNA® Body Fluid ID System has been designed to provide a simple, fast and robust way to identify forensically relevant body fluids in a lab or field-deployable manner. The system can analyse and detect mRNA targets for six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual blood and peripheral blood. Utilising the ParaDNA Sample Collector and instruments, minimal training is required to enable both forensic scientists and non-specialist personnel to analyse biological samples directly, without prior sample processing, in approximately 90 min. The developmental validation studies described here were designed to address requirements of end users, based on the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Body Fluid ID System on a range of mock evidence items. The data collected demonstrate that the Body Fluid ID System can automatically determine the presence of specific body fluid mRNA markers in single-source and mixed samples on multiple substrate types and body fluids could be identified with as little as 0.05ng total RNA and 1μl of the relevant fluid. Results can either be used to support confirmation of source from previously obtained STR DNA profiling results or to improve sample success rates by making better informed evidential submissions.

Identification and detection of protein markers to differentiate between forensically relevant body fluids

The identification of body fluids at a crime scene is an important aspect of forensic casework analysis, being a source for investigative leads and contributing to case evidence. Yet, current methods for the forensic identification of body fluids suffer from several limitations, ranging from poor sensitivity and specificity, to sample destruction and interference with subsequent DNA analysis. Moreover, current identification assays target only one body fluid at the time. Besides being inefficient in terms of time, money and sample consumption, poor identification methods can also negatively influence the outcome of a (court) case. In this study, eleven potential protein biomarkers and antibodies were selected and assessed on their suitability for serving as identification markers, as a first step towards the development of a new multiplex protein-based body fluid identification assay relying on antigen–antibody interactions. Performing antibody-based dot blot assays, the specificity of the biomarkers for their target body fluids was evaluated, and biomarker detection was studied in diluted, mixed, aged and simulated casework samples. Hereby, nine out of eleven markers were identified as promising biomarkers to identify blood, semen, saliva, urine and sweat. With the identification of these targets and detection antibodies, a major step forward has been taken towards the development of a highly sensitive and specific, fast and non-labour-intensive protein-based body fluid identification assay, suitable for on-site analysis and able to test for multiple body fluids in a single reaction.