Automation and validation of a MALDI-TOF MS (Mass-Fix) replacement of immunofixation electrophoresis in the clinical lab

2021 ◽  
Vol 59 (1) ◽  
pp. 155-163
Author(s):  
Mindy Kohlhagen ◽  
Surendra Dasari ◽  
Maria Willrich ◽  
MeLea Hetrick ◽  
Brian Netzel ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Turnaround Time ◽  
Automated System ◽  
Serum Samples ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Automated Method ◽  
Positive Specimen ◽  
Specimen Identification ◽  
Tof Ms

AbstractObjectivesA matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method (Mass-Fix) as a replacement for gel-based immunofixation (IFE) has been recently described. To utilize Mass-Fix clinically, a validated automated method was required. Our aim was to automate the pre-analytical processing, improve positive specimen identification and ergonomics, reduce paper data storage and increase resource utilization without increasing turnaround time.MethodsSerum samples were batched and loaded onto a liquid handler along with reagents and a barcoded sample plate. The pre-analytical steps included: (1) Plating immunopurification beads. (2) Adding 10 μl of serum. (3) Bead washing. (4) Eluting the immunoglobulins (Igs), and reducing to separate the heavy and light Ig chains. The resulting plate was transferred to a second low-volume liquid handler for MALDI plate spotting. MALDI-TOF mass spectra were collected. Integrated in-house developed software was utilized for sample tracking, driving data acquisition, data analysis, history tracking, and result reporting. A total of 1,029 residual serum samples were run using the automated system and results were compared to prior electrophoretic results.ResultsThe automated Mass-Fix method was capable of meeting the validation requirements of concordance with IFE, limit of detection (LOD), sample stability and reproducibility with a low repeat rate. Automation and integrated software allowed a single user to process 320 samples in an 8 h shift. Software display facilitated identification of monoclonal proteins. Additionally, the process maintains positive specimen identification, reduces manual pipetting, allows for paper free tracking, and does not significantly impact turnaround time (TAT).ConclusionsMass-Fix is ready for implementation in a high-throughput clinical laboratory.

scholarly journals Prospective Evaluation of Light Scatter Technology Paired with Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Rapid Diagnosis of Urinary Tract Infections

2017 ◽  
Vol 55 (6) ◽  
pp. 1802-1811 ◽  
Author(s):  
Sandra Montgomery ◽  
Kiana Roman ◽  
Lan Ngyuen ◽  
Ana Maria Cardenas ◽  
James Knox ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Automated System ◽  
Light Scatter ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tract Infections ◽  
Organism Identification ◽  
Tof Ms

ABSTRACTUrinary tract infections are one of the most common reasons for health care visits. Diagnosis and optimal treatment often require a urine culture, which takes an average of 1.5 to 2 days from urine collection to results, delaying optimal therapy. Faster, but accurate, alternatives are needed. Light scatter technology has been proposed for several years as a rapid screening tool, whereby negative specimens are excluded from culture. A commercially available light scatter device, BacterioScan 216Dx (BacterioScan, Inc.), has recently been advertised for this application. Paired use of mass spectrometry (MS) for bacterial identification and automated-system-based susceptibility testing straight from the light scatter suspension might provide dramatic improvement in times to a result. The present study prospectively evaluated the BacterioScan device, with culture as the reference standard. Positive light scatter specimens were used for downstream rapid matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS organism identification and automated-system-based antimicrobial susceptibility testing. Prospective evaluation of 439 urine samples showed a sensitivity of 96.5%, a specificity of 71.4%, and positive and negative predictive values of 45.1% and 98.8%, respectively. MALDI-TOF MS analysis of the suspension after density-based selection yielded a sensitivity of 72.1% and a specificity of 96.9%. Antimicrobial susceptibility testing of the samples identified by MALDI-TOF MS produced an overall categorical agreement of 99.2%. Given the high sensitivity and negative predictive value of results obtained, BacterioScan 216Dx is a reasonable approach for urine screening and might produce negative results in as few as 3 h, with no downstream workup. Paired rapid identification and susceptibility testing might be useful when MALDI-TOF MS results in an organism identification, and it might decrease the time to a result by more than 24 h.

scholarly journals Rapid identification of respiratory bacterial pathogens from bronchoalveolar lavage fluid in cattle by MALDI-TOF MS

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Laura Van Driessche ◽  
Jade Bokma ◽  
Piet Deprez ◽  
Freddy Haesebrouck ◽  
Filip Boyen ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Turnaround Time ◽  
Rapid Identification ◽  
Antimicrobial Use ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tract Infections ◽  
Tof Ms

AbstractRespiratory tract infections are a major health problem and indication for antimicrobial use in cattle and in humans. Currently, most antimicrobial treatments are initiated without microbiological results, holding the risk of inappropriate first intention treatment. The main reason for this empirical treatment is the long turnaround time between sampling and availability of identification and susceptibility results. Therefore the objective of the present study was to develop a rapid identification procedure for pathogenic respiratory bacteria in bronchoalveolar lavage fluid (BALf) samples from cattle by MALDI-TOF MS, omitting the cultivation step on agar plates to reduce the turnaround time between sampling and identification of pathogens. The effects of two different liquid growth media and various concentrations of bacitracin were determined to allow optimal growth of Pasteurellaceae and minimise contamination. The best procedure was validated on 100 clinical BALf samples from cattle with conventional bacterial culture as reference test. A correct identification was obtained in 73% of the samples, with 59.1% sensitivity (Se) (47.2–71.0%) and 100% specificity (Sp) (100–100%) after only 6 hours of incubation. For pure and dominant culture samples, the procedure was able to correctly identify 79.2% of the pathogens, with a sensitivity (Se) of 60.5% (45.0–76.1%) and specificity (Sp) of 100% (100–100%). In mixed culture samples, containing ≥2 clinically relevant pathogens, one pathogen could be correctly identified in 57% of the samples with 57.1% Se (38.8–75.5%) and 100% Sp (100–100%). In conclusion, MALDI-TOF MS is a promising tool for rapid pathogen identification in BALf. This new technique drastically reduces turnaround time and may be a valuable decision support tool to rationalize antimicrobial use.

scholarly journals Detection of M-Protein in Acetonitrile Precipitates of Serum Using MALDI-TOF Mass Spectrometry: A Novel Methodology

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-37
Author(s):  
Nikita Mehra ◽  
Gopal Gopisetty ◽  
Jayavelu S ◽  
Arivazhagan Rajamanickam ◽  
Shirley Sundersingh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mass Spectra ◽  
The Other ◽  
M Protein ◽  
Light Chains ◽  
Serum Samples ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms ◽  
Apparently Healthy

Background: Multiple myeloma (MM) and plasmacytoma(s) belong to a group of clonal plasma cell dyscrasias. In 97-98% of all cases, they are characterised by the detection of a monoclonal protein (M-protein) in the blood, and sometimes in the urine. MALDI-TOF-mass spectrometry (MS) has demonstrated excellent analytical sensitivity for the screening and detection of M-protein. We present the results of a novel methodology for M-protein analysis by MALDI-TOF MS. Patients and Methods: Blood samples from patients and controls were collected after obtaining Institutional Ethics Committee approval. The work was carried out in accordance with the Declaration of Helsinki after obtaining written informed consent. Patients with confirmed multiple myeloma or plasmacytoma and M-protein detected by serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE), and serum free light chains (FLC) were included for MALDI-TOF MS analysis. IFE and FLC analysis were sent to independent laboratories for external validation of the MALDI-TOF MS results. Reagent-based extraction The serum fraction was separated from whole blood by centrifugation at 5000 rpm for 15 minutes and stored at -80oC until further analysis. Twenty-five μL of the serum sample was mixed with 50% acetonitrile (ACN) to form a precipitate. After precipitation and incubation, the mixture was centrifuged. The protein precipitate was washed with 20% ACN. After centrifugation, the supernatant was discarded, and the precipitate was reconstituted in a buffer comprising 10% formic acid (FA) and 50 mmol/L tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The MALDI-TOF MS results were validated using immunoenrichment by anti-kappa (κ) and anti-lambda (λ) biotin-labelled antibodies immobilised on streptavidin magnetic beads. MALDI-TOF MS measurements were obtained for intact proteins using alpha-cyano-4-hydroxycinnamic acid as a matrix. The images obtained were overlaid on apparently healthy serum samples to confirm the presence of M-protein. The samples were then analysed using UltraflexTM LT, Bruker MALDI/TOF-TOF mass spectrometer. The mass spectra for each sample was exported to FlexAnalysis 3.3 (Bruker Daltonics) and background subtracted. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range- κ m/z- [M+2H]2+: 11550-12300 Da; [M+H]+: 23100-24600 Da), and λ m/z- [M+2H]2+: 11100-11500 Da; [M+H]+: 22200-23100 Da. All the images were acquired at a m/z range of 10000-29000 Da. Mass measurement was analysed with a summation of 500-5000 shots depending on the intensity of the M-peak. Results: Twenty-seven patient samples: 24- multiple myeloma, and 3- plasmacytoma with an M-protein identified by other biochemical tests, were chosen for ACN precipitation and analysed by MALDI-TOF MS. The median age was 62 years (range:44-72); males-12 (44%). A mass spectrometrist, S.J was blinded to the IFE and FLC results- blinded analyst. N.M was the unblinded analyst. Neat sample (without dilution) was spotted on the MALDI plate for all the control and patient samples. The Gaussian distribution of κ and λ light chains were obtained by analysing 20 serum samples of apparently healthy blood donors. All the 27 samples (100%) with M-protein confirmed by the other biochemical techniques, demonstrated a peak suggestive of M-protein with mass/charge (m/z) falling within the κ or λ range on MALDI-TOF MS: 24 patients with κ peak, and 3 with λ peak. (Fig. 1a and 1b) Immunoenrichment was performed on two samples- 1 with κ peak, and the other with λ peak, analysed by MALDI-TOF-MS by ACN precipitation. The mass spectra by immunoenrichment and ACN precipitation were found to be identical with the light chain m/z falling within their respective range. (Fig. 2 and 3) Three samples were labelled as confounders due to low peak intensity. However, their peaks matched their corresponding IFE and FLC reports. Concordance between MALDI-TOF MS and IFE was observed in 21/23 patients (91%); concordance between MALDI-TOF MS and FLC was observed in 23/24 patients (96%). Conclusions: We report the results of a low-cost, reagent-based extraction process using ACN precipitation to enrich for κ and λ light chains, which can be used for screening and for qualitative analysis of M-protein. Further studies are required to identify the immunoglobulin isotype, and to quantify the M-protein by this methodology. Disclosures No relevant conflicts of interest to declare.

scholarly journals MALDI-TOF MS: the proteomic approach, the future of fungal identification in India

2020 ◽  
Vol 8 (7) ◽  
pp. 2575
Author(s):  
Mallika Fonseca ◽  
Sarala Menon ◽  
Praveen Rahi ◽  
Prachi Patekar ◽  
Abhay S. Chowdhary
Keyword(s):  
Yeast Species ◽  
Fungal Infections ◽  
Tertiary Care ◽  
Turnaround Time ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Proteomic Approach ◽  
Conventional Methods ◽  
Tof Ms

Background: India, being a country where fungal infections are rampant, is urgently in need of effective tools for early and accurate diagnosis of fungal infections. Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) is a recent method which has shown potential in identifying clinically important bacterial pathogens as well as clinically important fungi. The main objective of this study was to compare the utility of MALDI-TOF MS for the identification of fungi against that of conventional methods.Methods: The project was carried out in a tertiary care government hospital in India. Fifty clinical isolates comprising mainly various yeast species were subjected to conventional identification (Phenotypic) as well as MALDI-TOF-MS. Their results were further compared.Results: MALDI-TOF MS showed a high concordance with conventional methods while identifying species like C. albicans, C. tropicalis, C. parapsilosis and C. neoformans, although the concordance for species such as Rhodotorula and Trichosporon could only be matched up to genus level.Conclusions: MALDI-TOF MS-based identification is both a rapid and a viable tool for identification of clinically relevant yeast species with good correlation to conventional methods and a quick turnaround time.

scholarly journals Detection of M-Protein in Acetonitrile Precipitates of Serum using MALDI-TOF Mass Spectrometry

2020 ◽  
Author(s):  
Nikita Mehra ◽  
Gopal Gopisetty ◽  
S Jayavelu ◽  
Rajamanickam Arivazhagan ◽  
Shirley Sundersingh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Extraction Process ◽  
M Protein ◽  
Newly Diagnosed ◽  
Serum Samples ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Chain Analysis ◽  
Immunofixation Electrophoresis ◽  
Tof Ms

AbstractPurpose of the researchMultiple myeloma and plasmacytomas belong to a group of disorders, namely plasma cell dyscrasias and are identified by the presence of a monoclonal protein (M-protein). MALDI-TOF-mass spectrometry (MS) has demonstrated superior analytical sensitivity for the detection of M-protein and is now used for screening of M-protein at some centres. We present the results of an alternative methodology for M-protein analysis by MALDI-TOF MS.MethodsSerum samples from patients with newly diagnosed multiple myeloma or plasmacytoma with positive M-protein detected by serum protein electrophoresis, immunofixation electrophoresis and serum free light chain analysis, underwent direct reagent-based extraction process using Acetonitrile (ACN) precipitation. Serum κ and λ light chains were validated using immunoenrichment by anti- κ and anti- λ biotin-labelled antibodies immobilised on streptavidin magnetic beads. MALDI-TOF MS measurements were obtained for intact proteins using alpha-cyano-4-hydroxycinnamic acid as matrix. The images obtained were overlaid on apparently healthy donor serum samples to confirm the presence of M-protein.Principle resultsCharacteristic M-protein peaks were observed in the ACN precipitates of serum in the predicted mass ranges for κ and λ. The κ and λ peaks were confirmed by immunoenrichment analysis. Twenty-seven patient samples with either newly diagnosed multiple myeloma or plasmacytoma with monoclonal gammopathy detected by the standard methods were chosen for Acetonitrile precipitation and analysed by MALDI-TOF MS. All 27 patient samples demonstrated a peak suggestive of M-protein with mass/charge (m/z) falling within the κ and λ range. The concordance rate with serum immunofixation electrophoresis and free light chain analysis was above 90%.Major conclusionsWe report the results of a low-cost reagent-based extraction process using Acetonitrile precipitation to enrich for κ and λ light chains, which can be used for the screening and qualitative analysis of M-protein.

895: Proteomic Analysis of Sera from Bladder Cancer Patients with MALDI-TOF-MS

2007 ◽  
Vol 177 (4S) ◽  
pp. 297-297
Author(s):  
Kristina Schwamborn ◽  
Rene Krieg ◽  
Ruth Knüchel-Clarke ◽  
Joachim Grosse ◽  
Gerhard Jakse
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Proteomic Analysis ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

HPTLC-MALDI/TOF/MS vs. HPLC-ESI/MS2 for identification to flavonoids in plant extracts

Planta Medica ◽  
2016 ◽  
Vol 81 (S 01) ◽  
pp. S1-S381
Author(s):  
L Fougère ◽  
D Da Silva ◽  
E Destandau ◽  
C Elfakir
Keyword(s):  
Plant Extracts ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

Infektionserreger beim Schwein – Analyse des kultivierbaren Mikrobioms mittels MALDI-TOF MS

2017 ◽  
Author(s):  
M Erhard ◽  
M Metzner ◽  
D Köhler-Repp ◽  
B Köhler ◽  
R Storandt
Keyword(s):  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

Quantitative metabolomics of Iranian saffron based on their HPLC-DAD and MALDI-TOF-MS

2019 ◽  
Author(s):  
M Hooshyari ◽  
H Rezadoost ◽  
P Ghezellou ◽  
A Ghassempour
Keyword(s):  
Maldi Tof Ms ◽  
Quantitative Metabolomics ◽  
Maldi Tof ◽  
Tof Ms
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