Purpose
To facilitate the transition of MALDI–MS Imaging (MALDI–MSI) from basic science to clinical application, it is necessary to analyze formalin‐fixed paraffin‐embedded (FFPE) tissues. The aim is to improve in situ tryptic digestion for MALDI–MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites.
Experimental Design
FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three different sites) are prepared for MALDI–MSI. Samples are coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples are sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50 µm spatial resolution. Data are analyzed using flexImaging, SCiLS, and R.
Results
Trypsin application and digestion are identified as sources of variation and loss of spatial resolution in the MALDI–MSI of FFPE samples. Using the described workflow, it is possible to discriminate discrete histological features in different tissues and enabled different sites to generate images of similar quality when assessed by spatial segmentation and PCA.
Conclusions and Clinical Relevance
Spatial resolution and site‐to‐site reproducibility can be maintained by adhering to a standardized MALDI–MSI workflow.
Background: Amplification of viral ribonucleic acid (RNA) by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is the gold standard to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the initial outbreak, strategies to detect and isolate patients have been important to avoid uncontrolled viral spread. Although testing capacities have been upscaled, there is still a need for reliable high throughput test systems, specifically those that require alternative consumables. Therefore, we tested and compared two different methods for the detection of viral PCR products: rRT-PCR and mass spectrometry (MS). Methods: Viral RNA was isolated and amplified from oro- or nasopharyngeal swabs. A total of 22 samples that tested positive and 22 samples that tested negative for SARS-CoV-2 by rRT-PCR were analyzed by MS. Results of the rRT-PCR and the MS protocol were compared. Results: Results of rRT-PCR and the MS test system were in concordance in all samples. Time-to-results was faster for rRT-PCR. Hands-on-time was comparable in both assays. Conclusions: MS is a fast, reliable and cost-effective alternative for the detection of SARS-CoV-2 from oral and nasopharyngeal swabs.
Many
studies have demonstrated that tissue phenotyping (tissue
typing) based on mass spectrometric imaging data is possible; however,
comprehensive studies assessing variation and classifier transferability
are largely lacking. This study evaluated the generalization of tissue
classification based on Matrix Assisted Laser Desorption/Ionization
(MALDI) mass spectrometric imaging (MSI) across measurements performed
at different sites. Sections of a tissue microarray (TMA) consisting
of different formalin-fixed and paraffin-embedded (FFPE) human tissue
samples from different tumor entities (leiomyoma, seminoma, mantle
cell lymphoma, melanoma, breast cancer, and squamous cell carcinoma
of the lung) were prepared and measured by MALDI-MSI at different
sites using a standard protocol (SOP). Technical variation was deliberately
introduced on two separate measurements via a different sample preparation
protocol and a MALDI Time of Flight mass spectrometer that was not
tuned to optimal performance. Using standard data preprocessing, a
classification accuracy of 91.4% per pixel was achieved for intrasite
classifications. When applying a leave-one-site-out cross-validation
strategy, accuracy per pixel over sites was 78.6% for the SOP-compliant
data sets and as low as 36.1% for the mistuned instrument data set.
Data preprocessing designed to remove technical variation while retaining
biological information substantially increased classification accuracy
for all data sets with SOP-compliant data sets improved to 94.3%.
In particular, classification accuracy of the mistuned instrument
data set improved to 81.3% and from 67.0% to 87.8% per pixel for the
non-SOP-compliant data set. We demonstrate that MALDI-MSI-based tissue
classification is possible across sites when applying histological
annotation and an optimized data preprocessing pipeline to improve
generalization of classifications over technical variation and increasing
overall robustness.
Aims Human papilloma virus (HPV) infection is a causative agent for approximately 5% of all new cancer cases in humans. The virus is detected in cervical, anal, vaginal, penile, vulvar and head and neck cancers and has prognostic implications. Thus, test systems are required to detect high-risk but also low-risk HPV subtypes with high specificity and sensitivity in a timeeffective and cost-effective manner. In the present study we developed a new mass spectrometry (MS)-based test system for the detection of HPV infections in formalinfixed paraffin-embedded (FFPE) tissue samples. Methods A high-throughput matrix-assisted laser desorption ionisation time of flight MS-based assay was applied to genotype 19 HPV types in FFPE tissue specimens (n=46). The results from the MS assay were compared with the results obtained from two hybridisation-based test systems: the HPV 3.5 LCD-array kit and the EuroArrayHPV system. Results In 36 out of 46 (78%) tissue samples, a HPV infection could be detected by the MS-based HPV assay. In 16 samples (44%) only one and in 20 samples (56%) two to six HPV subtypes were identified. The overall agreement of all three assays was almost perfect (Cohen's k value: 0.83). Conclusions The MS-based assay is highly sensitive, reliable as well as cost-effective and represents a suitable technology for the detection of HPV infections in FFPE tissue material.
Among neonates, tested positive for SARS-CoV-2, the majority of infections occur through postpartum transmission. Only few reports describe intrauterine or intrapartum SARS-CoV-2 infections in newborns. To understand the route of transmission, detection of the virus or virus nucleic acid in the placenta and amniotic tissue are of special interest. Current methods to detect SARS-CoV-2 in placental tissue are immunohistochemistry, electron microscopy, in-situ hybridization, polymerase chain reaction (PCR) and next-generation sequencing. Recently, we described an alternative method for the detection of viral ribonucleic acid (RNA), by combination of reverse transcriptase-PCR and mass spectrometry (MS) in oropharyngeal and oral swabs. In this report, we could detect SARS-CoV-2 in formal-fixed and paraffin-embedded (FFPE) placental and amniotic tissue by multiplex RT-PCR MS. Additionally, we could identify the British variant (B.1.1.7) of the virus in this tissue by the same methodology. Combination of RT-PCR with MS is a fast and easy method to detect SARS-CoV-2 viral RNA, including specific variants in FFPE tissue.
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