Abscopal Effects, Clastogenic Effects and Bystander Effects: 70 Years of Non-Targeted Effects of Radiation.

In vitro and in vivo observations accumulated over several decades have firmly shown that the biological effects of ionizing radiation can spread from irradiated cells/tissues to non-targeted cells/tissues. Redox-modulated intercellular communication mechanisms that include a role for secreted factors and gap junctions, can mediate these non-targeted effects. Clearly, the expression of such effects and their transmission to progeny cells has implications for issues related to radiation protection. Their elucidation is also relevant towards enhancing the efficacy of cancer radiotherapy and reducing its impact on the development of normal tissue toxicities. In addition, the study of non-targeted effects is pertinent to our basic understanding of intercellular communications under conditions of oxidative stress. This review will trace the history of non-targeted effects of radiation starting with early reports of abscopal effects which described radiation induced effects in tissues distant from the site of radiation exposure. A related effect involved the production of clastogenic factors in plasma following irradiation which can induce chromosome damage in unirradiated cells. Despite these early reports suggesting non-targeted effects of radiation, the classical paradigm that a direct deposition of energy in the nucleus was required still dominated. This paradigm was challenged by papers describing radiation induced bystander effects. This review will cover mechanisms of radiation-induced bystander effects and the potential impacts on radiation protection and radiation therapy.

Effect of pre-analytical variables on Raman and FTIR spectral content of lymphocytes.

The use of Fourier transform infrared (FTIR) and Raman spectroscopy (RS) for the analysis of lymphocytes in clinical applications is increasing in the field of biomedicine. The pre-analytical phase, which is the most vulnerable stage of the testing process, is where most errors and sample variance occur; however, it is unclear how pre-analytical variables affect the FTIR and Raman spectra of lymphocytes. In this study, we evaluated how pre-analytical procedures undertaken before spectroscopic analysis influence the spectral integrity of lymphocytes purified from the peripheral blood of male volunteers (n = 3). Pre-analytical variables investigated were associated with (i) sample preparation, (blood collection systems, anticoagulant, needle gauges), (ii) sample storage (fresh or frozen), and (iii) sample processing (inter-operator variability, time to lymphocyte isolation). Although many of these procedural pre-analytical variables did not alter the spectral signature of the lymphocytes, evidence of spectral effects due to the freeze-thaw cycle, in vitro culture inter-operator variability and the time to lymphocyte isolation was observed. Although FTIR and RS possess clinical potential, their translation into a clinical environment is impeded by a lack of standardisation and harmonisation of protocols related to the preparation, storage, and processing of samples, which hinders uniform, accurate, and reproducible analysis. Therefore, further development of protocols is required to successfully integrate these techniques into current clinical workflows.

Raman Spectroscopy for Early Detection of Cervical Cancer, a Global Women's Health Issue-A Review.

This review focuses on recent advances and future perspectives in the use of Raman spectroscopy for cervical cancer, a global women's health issue. Cervical cancer is the fourth most common women's cancer in the world, and unfortunately mainly affects younger women. However, when detected at the early precancer stage, it is highly treatable. High-quality cervical screening programmes and the introduction of the human papillomavirus (HPV) vaccine are reducing the incidence of cervical cancer in many countries, but screening is still essential for all women. Current gold standard methods include HPV testing and cytology for screening, followed by colposcopy and histopathology for diagnosis. However, these methods are limited in terms of sensitivity/specificity, cost, and time. New methods are required to aid clinicians in the early detection of cervical precancer. Over the past 20 years, the potential of Raman spectroscopy together with multivariate statistical analysis has been shown for the detection of cervical cancer. This review discusses the research to date on Raman spectroscopic approaches for cervical cancer using exfoliated cells, biofluid samples, and tissue ex vivo and in vivo.

Application of Advanced Non-Linear Spectral Decomposition and Regression Methods for Spectroscopic Analysis of Targeted and Non-Targeted Irradiation Effects in an In-Vitro Model.

Irradiation of the tumour site during treatment for cancer with external-beam ionising radiation results in a complex and dynamic series of effects in both the tumour itself and the normal tissue which surrounds it. The development of a spectral model of the effect of each exposure and interaction mode between these tissues would enable label free assessment of the effect of radiotherapeutic treatment in practice. In this study Fourier transform Infrared microspectroscopic imaging was employed to analyse an in-vitro model of radiotherapeutic treatment for prostate cancer, in which a normal cell line (PNT1A) was exposed to low-dose X-ray radiation from the scattered treatment beam, and also to irradiated cell culture medium (ICCM) from a cancer cell line exposed to a treatment relevant dose (2 Gy). Various exposure modes were studied and reference was made to previously acquired data on cellular survival and DNA double strand break damage. Spectral analysis with manifold methods, linear spectral fitting, non-linear classification and non-linear regression approaches were found to accurately segregate spectra on irradiation type and provide a comprehensive set of spectral markers which differentiate on irradiation mode and cell fate. The study demonstrates that high dose irradiation, low-dose scatter irradiation and radiation-induced bystander exposure (RIBE) signalling each produce differential effects on the cell which are observable through spectroscopic analysis.

MiRNA-Mediated Fibrosis in the Out-of-Target Heart following Partial-Body Irradiation.

Recent reports have shown a link between radiation exposure and non-cancer diseases such as radiation-induced heart disease (RIHD). Radiation exposures are often inhomogeneous, and out-of-target effects have been studied in terms of cancer risk, but very few studies have been carried out for non-cancer diseases. Here, the role of miRNAs in the pathogenesis of RIHD was investigated. C57Bl/6J female mice were whole- (WBI) or partial-body-irradiated (PBI) with 2 Gy of X-rays or sham-irradiated (SI). In PBI exposure, the lower third of the mouse body was irradiated, while the upper two-thirds were shielded. From all groups, hearts were collected 15 days or 6 months post-irradiation. The MiRNome analysis at 15 days post-irradiation showed that miRNAs, belonging to the myomiR family, were highly differentially expressed in WBI and PBI mouse hearts compared with SI hearts. Raman spectral data collected 15 days and 6 months post-irradiation showed biochemical differences among SI, WBI and PBI mouse hearts. Fibrosis in WBI and PBI mouse hearts, indicated by the increased deposition of collagen and the overexpression of genes involved in myofibroblast activation, was found 6 months post-irradiation. Using an in vitro co-culture system, involving directly irradiated skeletal muscle and unirradiated ventricular cardiac human cells, we propose the role of miR-1/133a as mediators of the abscopal response, suggesting that miRNA-based strategies could be relevant for limiting tissue-dependent reactions in non-directly irradiated tissues.

Development and Validation of a Raman Spectroscopic Classification Model for Cervical Intraepithelial Neoplasia (CIN).

The mortality associated with cervical cancer can be reduced if detected at the precancer stage, but current methods are limited in terms of subjectivity, cost and time. Optical spectroscopic methods such as Raman spectroscopy can provide a rapid, label-free and nondestructive measurement of the biochemical fingerprint of a cell, tissue or biofluid. Previous studies have shown the potential of Raman spectroscopy for cervical cancer diagnosis, but most were pilot studies with small sample sizes. The aim of this study is to show the clinical utility of Raman spectroscopy for identifying cervical precancer in a large sample set with validation in an independent test set. Liquid-based cervical cytology samples (n = 662) (326 negative, 200 cervical intraepithelial neoplasia (CIN)1 and 136 CIN2+) were obtained as a training set. Raman spectra were recorded from single-cell nuclei and subjected to a partial least squares discriminant analysis (PLSDA). In addition, the PLSDA classification model was validated using a blinded independent test set (n = 69). A classification accuracy of 91.3% was achieved with only six of the blinded samples misclassified. This study showed the potential clinical utility of Raman spectroscopy with a good classification of negative, CIN1 and CIN2+ achieved in an independent test set.

Micro-RNA and Proteomic Profiles of Plasma-Derived Exosomes from Irradiated Mice Reveal Molecular Changes Preventing Apoptosis in Neonatal Cerebellum.

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.

Women's contributions to radiobiology in Ireland; from small beginnings .

OBJECTIVES: To describe the contribution of women radiobiologists in Ireland to the development of the discipline internationally and at home and to discuss the history of radiobiology in Ireland to date. This parallels the history of the evolution of a small radiobiology group in Kevin Street, Dublin Institute of Technology (DIT) which was formerly part of the City of Dublin Vocational Education Committee. There followed years of development first as a radiobiological research center which evolved in the FOCAS Research Institute now embedded within Technological University Dublin (TU Dublin). CONCLUSIONS: Over the last 45 years, the women of the Radiation and Environmental Science Centre (RESC) contributed to the major paradigm shift in low dose radiobiology contributing exciting new research concerning non-targeted effects, including discovery of lethal mutations, medium transfer bystander mechanisms, and signaling pathways. They also developed translational research using human explant culture systems with unique immunocytochemical methods and more recently evolved to molecular and spectroscopic analysis of clinical samples. The RESC also developed unique in vitro research methods into effects of radiation on non-human species of concern in ecosystems.

Classification of cytological samples from oral potentially malignant lesions through Raman spectroscopy: A pilot study.

The potential of Raman microspectroscopy of exfoliated cells has been demonstrated for oral cancer diagnosis. In this study, brush biopsies were collected from the buccal mucosa/tongue of healthy donors (n = 31) and from oral mucosal dysplastic lesions (n = 31 patients). Raman spectra were acquired and subjected to partial least squares-discriminant analysis (PLS-DA). The patient samples could be differentiated from healthy donor samples with 96% sensitivity and 95% specificity. Furthermore, PLS-DA models were developed based on cytopathological and histopathological assessment. Low and high grade dysplasia could be discriminated with 64% sensitivity and 65% specificity based on cytopathological assessment, while 81% sensitivity and 86% specificity could be achieved when histopathological assessment was within six months of the brush biopsy sampling. Therefore, this explorative study has successfully demonstrated that Raman spectroscopy may have a role in monitoring patients with dysplasia and may reduce the need for multiple biopsies.

A 4-Gene Signature of CDKN1, FDXR, SESN1 and PCNA Radiation Biomarkers for Prediction of Patient Radiosensitivity.

The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.

Raman spectral cytopathology for cancer diagnostic applications.

Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer.

Raman Spectroscopy of Liquid-Based Cervical Smear Samples as a Triage to Stratify Women Who Are HPV-Positive on Screening.

The role of persistent high-risk human papillomavirus (HPV) infection in the development of cervical precancer and cancer is now well accepted, and HPV testing has recently been introduced for primary cervical screening. However, the low specificity of HPV DNA testing can result in large numbers of women with an HPV-positive result, and additional triage approaches are needed to avoid over-referral to colposcopy and overtreatment. The aim of this study was to assess Raman spectroscopy as a potential triage test to discriminate between transient and persistent HPV infection. HPV DNA status and mRNA status were confirmed in ThinPrep® cervical samples (n = 60) using the Cobas 4800 and APTIMA HPV test, respectively. Raman spectra were recorded from single-cell nuclei and subjected to partial least squares discriminant analysis (PLSDA). In addition, the PLSDA classification model was validated using a blinded independent test set (n = 14). Sensitivity of 85% and specificity of 92% were achieved for the classification of transient and persistent HPV infection, and this increased to 90% sensitivity and 100% specificity when mean sample spectra were used instead of individual cellular spectra. This study showed that Raman spectroscopy has potential as a triage test for HPV-positive women to identify persistent HPV infection.

Out-of-Field Hippocampus from Partial-Body Irradiated Mice Displays Changes in Multi-Omics Profile and Defects in Neurogenesis.

The brain undergoes ionizing radiation exposure in many clinical situations, particularly during radiotherapy for brain tumors. The critical role of the hippocampus in the pathogenesis of radiation-induced neurocognitive dysfunction is well recognized. The goal of this study is to test the potential contribution of non-targeted effects in the detrimental response of the hippocampus to irradiation and to elucidate the mechanisms involved. C57Bl/6 mice were whole body (WBI) or partial body (PBI) irradiated with 0.1 or 2.0 Gy of X-rays or sham irradiated. PBI consisted of the exposure of the lower third of the mouse body, whilst the upper two thirds were shielded. Hippocampi were collected 15 days or 6 months post-irradiation and a multi-omics approach was adopted to assess the molecular changes in non-coding RNAs, proteins and metabolic levels, as well as histological changes in the rate of hippocampal neurogenesis. Notably, at 2.0 Gy the pattern of early molecular and histopathological changes induced in the hippocampus at 15 days following PBI were similar in quality and quantity to the effects induced by WBI, thus providing a proof of principle of the existence of out-of-target radiation response in the hippocampus of conventional mice. We detected major alterations in DAG/IP3 and TGF-ß signaling pathways as well as in the expression of proteins involved in the regulation of long-term neuronal synaptic plasticity and synapse organization, coupled with defects in neural stem cells self-renewal in the hippocampal dentate gyrus. However, compared to the persistence of the WBI effects, most of the PBI effects were only transient and tended to decrease at 6 months post-irradiation, indicating important mechanistic difference. On the contrary, at low dose we identified a progressive accumulation of molecular defects that tended to manifest at later post-irradiation times. These data, indicating that both targeted and non-targeted radiation effects might contribute to the pathogenesis of hippocampal radiation-damage, have general implications for human health.

The Potential of Raman Spectroscopy in the Diagnosis of Dysplastic and Malignant Oral Lesions.

Early diagnosis, treatment and/or surveillance of oral premalignant lesions are important in preventing progression to oral squamous cell carcinoma (OSCC). The current gold standard is through histopathological diagnosis, which is limited by inter- and intra-observer errors and sampling errors. The objective of this work was to use Raman spectroscopy to discriminate between benign, mild, moderate and severe dysplasia and OSCC in formalin fixed paraffin preserved (FFPP) tissues. The study included 72 different pathologies from which 17 were benign lesions, 20 mildly dysplastic, 20 moderately dysplastic, 10 severely dysplastic and 5 invasive OSCC. The glass substrate and paraffin wax background were digitally removed and PLSDA with LOPO cross-validation was used to differentiate the pathologies. OSCC could be differentiated from the other pathologies with an accuracy of 70%, while the accuracy of the classifier for benign, moderate and severe dysplasia was ~60%. The accuracy of the classifier was lowest for mild dysplasia (~46%). The main discriminating features were increased nucleic acid contributions and decreased protein and lipid contributions in the epithelium and decreased collagen contributions in the connective tissue. Smoking and the presence of inflammation were found to significantly influence the Raman classification with respective accuracies of 76% and 94%.

Biomedical applications of vibrational spectroscopy: Oral cancer diagnostics.

Vibrational spectroscopy, based on either infrared absorption or Raman scattering, has attracted increasing attention for biomedical applications. Proof of concept explorations for diagnosis of oral potentially malignant disorders and cancer are reviewed, and recent advances critically appraised. Specific examples of applications of Raman microspectroscopy for analysis of histological, cytological and saliva samples are presented for illustrative purposes, and the future prospects, ultimately for routine, chairside in vivo screening are discussed.

Vibrational spectroscopy of liquid biopsies for prostate cancer diagnosis.

BACKGROUND: Screening for prostate cancer with prostate specific antigen and digital rectal examination allows early diagnosis of prostate malignancy but has been associated with poor sensitivity and specificity. There is also a considerable risk of over-diagnosis and over-treatment, which highlights the need for better tools for diagnosis of prostate cancer. This study investigates the potential of high throughput Raman and Fourier Transform Infrared (FTIR) spectroscopy of liquid biopsies for rapid and accurate diagnosis of prostate cancer. METHODS: Blood samples (plasma and lymphocytes) were obtained from healthy control subjects and prostate cancer patients. FTIR and Raman spectra were recorded from plasma samples, while Raman spectra were recorded from the lymphocytes. The acquired spectral data was analysed with various multivariate statistical methods, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and classical least squares (CLS) fitting analysis. RESULTS: Discrimination was observed between the infrared and Raman spectra of plasma and lymphocytes from healthy donors and prostate cancer patients using PCA. In addition, plasma and lymphocytes displayed differentiating signatures in patients exhibiting different Gleason scores. A PLS-DA model was able to discriminate these groups with sensitivity and specificity rates ranging from 90% to 99%. CLS fitting analysis identified key analytes that are involved in the development and progression of prostate cancer. CONCLUSIONS: This technology may have potential as an alternative first stage diagnostic triage for prostate cancer. This technology can be easily adaptable to many other bodily fluids and could be useful for translation of liquid biopsy-based diagnostics into the clinic.

A pilot study for early detection of oral premalignant diseases using oral cytology and Raman micro-spectroscopy: Assessment of confounding factors.

This study demonstrates the efficacy of Raman micro-spectroscopy of oral cytological samples for differentiating dysplastic, potentially malignant lesions from those of normal, healthy donors. Cells were collected using brush biopsy from healthy donors (n = 20) and patients attending a Dysplasia Clinic (n = 20). Donors were sampled at four different sites (buccal mucosa, tongue, alveolus, gingiva), to ensure matched normal sites for all lesions, while patient samples were taken from clinically evident, histologically verified dysplastic lesions. Spectra were acquired from the nucleus and cytoplasm of individual cells of all samples and subjected to partial least squares-discriminant analysis. Discriminative sensitivities of 94% and 86% and specificity of 85% were achieved for the cytoplasm and nucleus, respectively, largely based on lipidic contributions of dysplastic cells. Alveolar/gingival samples were differentiated from tongue/buccal samples, indicating that anatomical site is potentially a confounding factor, while age, gender, smoking and alcohol consumption were confirmed not to be.

Raman microspectroscopic study for the detection of oral field cancerisation using brush biopsy samples.

Field cancerisation (FC) is potentially an underlying cause of poor treatment outcomes of oral squamous cell carcinoma (OSCC). To explore the phenomenon using Raman microspectroscopy, brush biopsies from the buccal mucosa, tongue, gingiva and alveolus of healthy donors (n = 40) and from potentially malignant lesions (PML) of Dysplasia Clinic patients (n = 40) were examined. Contralateral normal samples (n = 38) were also collected from the patients. Raman spectra were acquired from the nucleus and cytoplasm of each cell, and subjected to partial least squares-discriminant analysis (PLS-DA). High discriminatory accuracy for donor and PML samples was achieved for both cytopalmic and nuclear data sets. Notably, contralateral normal (patient) samples were also accurately discriminated from donor samples and contralateral normal samples from patients with multiple lesions showed a similar spectral profile to PML samples, strongly indicating a FC effect. These findings support the potential of Raman microspectroscopy as a screening tool for PML using oral exfoliated cells.

MicroRNA Analysis of ATM-Deficient Cells Indicate PTEN and CCDN1 as Potential Biomarkers of Radiation Response.

Genetic and epigenetic profile changes associated with individual radiation sensitivity are well documented and have led to enhanced understanding of the mechanisms of the radiation-induced DNA damage response. However, the search continues to identify reliable biomarkers of individual radiation sensitivity. Herein, we report on a multi-biomarker approach using traditional cytogenetic biomarkers, DNA damage biomarkers and transcriptional microRNA (miR) biomarkers coupled with their potential gene targets to identify radiosensitivity in ataxia-telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines (LCL); ATM-proficient cell lines were used as controls. Cells were 0.05 and 0.5 Gy irradiated, using a linear accelerator, with sham-irradiated cells as controls. At 1 h postirradiation, cells were fixed for γ-H2AX analysis as a measurement of DNA damage, and cytogenetic analysis using the G2 chromosomal sensitivity assay, G-banding and FISH techniques. RNA was also isolated for genetic profiling by microRNA (miR) and RT-PCR analysis. A panel of 752 miR were analyzed, and potential target genes, phosphatase and tensin homolog (PTEN) and cyclin D1 (CCND1), were measured. The cytogenetic assays revealed that although the control cell line had functional cell cycle checkpoints, the radiosensitivity of the control and AT cell lines were similar. Analysis of DNA damage in all cell lines, including an additional control cell line, showed elevated γ-H2AX levels for only one AT cell line. Of the 752 miR analyzed, eight miR were upregulated, and six miR were downregulated in the AT cells compared to the control. Upregulated miR-152-3p, miR-24-5p and miR-92-15p and all downregulated miR were indicated as modulators of PTEN and CCDN1. Further measurement of both genes validated their potential role as radiation-response biomarkers. The multi-biomarker approach not only revealed potential candidates for radiation response, but provided additional mechanistic insights into the response in AT-deficient cells.

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma.

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)-FTIR and HT-Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.