Storage Of Clinical Samples Research Articles

Monkeypox virus diagnosis and laboratory testing.

The multi-country outbreak of monkeypox virus (MPXV) infection, while the coronavirus disease 2019 pandemic is still an ongoing issue, has caused a new challenge. The re-emergence of MPXV and the rising incidence in non-endemic countries is turning into an upcoming threat to global health. Hence, rapid identification of the virus with appropriate methodology with the lowest false results plays a critical role in estimating the global extent of the crisis and providing preventive measures. This review summarised the main applicable strategies for primary detection and confirmation of MPXV and highlighted available data in biosafety, requirements, standard operating procedures, specimen collection, transportation and storage of clinical samples, and waste disposal of the viral agent. Also, various assays including molecular techniques, immunoassays, histopathological methods, electron microscopy, genomic sequencing, and cell culture have been illustrated. Moreover, we reflected on current knowledge of the advantages and disadvantages of each approach.

Variation in Thermal Stability among Respiratory Syncytial Virus Clinical Isolates under Non-Freezing Conditions.

Virus isolates are not only useful for diagnosing infections, e.g., respiratory syncytial virus (RSV), but can also facilitate many aspects of practical viral studies such as analyses of antigenicity and the action mechanisms of antivirals, among others. We have been isolating RSV from clinical specimens from patients with respiratory symptoms every year since our first isolation of RSV in 1964, and isolation rates have varied considerably over the years. As collected clinical specimens are conventionally stored in a refrigerator from collection to inoculation into cells, we hypothesized that certain storage conditions or associated factors might account for these differences. Hence, we evaluated the thermal stability of a total of 64 viruses isolated from 1998 to 2018 upon storage at 4 °C and 20 °C for a defined duration. Interestingly, and contrary to our current understanding, 22 strains (34%) showed a greater loss of viability upon short-term storage at 4 °C than at 20 °C. Thirty-seven strains (57%) showed an almost equal loss, and only five strains (8%) were more stable at 4 °C than at 20 °C. This finding warrants reconsideration of the temperature for the temporary storage of clinical samples for RSV isolation.

Virus Isolation and Propagation from H3N2 Influenza Infected Human Clinical Samples Under Distinct Sample Storage Conditions

Isolation and propagation of influenza virus from Influenza Like Illness (ILI) clinical sample is essential for the surveillance of circulating virus, such as antigenic and genetic analyses, antiviral sensitivity surveillance, as well as annual influenza vaccine selection. Madin-Darby canine kidney (MDCK) cell is conventionally used for virus isolation in public health laboratories. Throat swap samples of Influenza like Illness (ILI) were collected from two sentinel hospitals and screened seasonal influenza by real-time reverse transcription polymerase chain reaction (RT–PCR). H3N2 positive samples were performed virus isolation in MDCK cells. Samples were stored under different conditions before inoculation, 1-2 days at 2-8°C, 4-5 day or 8-9 days at 2-8°C, and no less than two months at -80°C. The results showed that long term (>2 month) -80°C storage of clinical samples (15.12%) had significantly lower virus isolation rate compare to short term (1-2 days and 4-9 days) under 2-8°C storage (88.37% for 1-2 days and 52.33% for 4-9 days). For those samples stored at 4°C, the shorter of the storage time, the better of sample quality and virus activity could be obtained, resulting in higher isolation rate. This study provides evidence for influenza surveillance and sample quality control.

Assessment of the Stability of Supraphysiological Ascorbate in Human Blood: Appropriate Handling of Samples from Clinical Trials for Measurements of Pharmacological Ascorbate.

Based on encouraging results from several early-phase clinical trials, there is renewed interest in the use of pharmacological ascorbate (i.e., intravenous administration resulting in >≈10 mM plasma ascorbate concentrations) in combination with standard-of-care cancer treatments including radiation and/or chemotherapy. Under normal, healthy physiological conditions, humans maintain plasma ascorbate concentrations in the range of 40-80 lM. However, in vivo antitumor activity requires supraphysiological plasma concentrations on the order of ≈20 mM. The stability of ascorbate in whole blood has been well studied. The goal of this work was to determine the appropriate handling methods of blood samples, after treatment with pharmacological ascorbate, which allow for the optimal measurement of ascorbate in plasma for dosing verification. Our findings indicate that ascorbate concentrations (mM) are relatively stable in whole blood collected in sodium heparin tubes and stored on ice (or at 4°C) for up to 24 h. After 24 h, ascorbate levels in plasma are relatively stable at 4°C for up to 72 h. At -20°C, plasma concentrations are relatively stable for 2-3 weeks, while at -80°C, ascorbate concentrations in plasma are stable for at least one month. In contrast, patient samples showed better stability when stored as whole blood compared to plasma at 4°C but increasing hemolysis over time may significantly skew ascorbate measurements. Additionally, patient samples can be reliably stored as plasma at -20°C for up to three weeks in either a frost-containing or frost-free environment. This information can guide the collection, processing and storage of clinical samples after pharmacological ascorbate infusions amenable to multi-center clinical trials.

Determining the effect of different environmental conditions on Ebola virus viability in clinically relevant specimens

In 2013–2016, West Africa experienced the largest and longest Ebola virus disease outbreak ever documented. The wide geographic spread and magnitude of the outbreak often limited the timely and rapid testing of diagnostic samples from patients with suspected Ebola virus disease, raising questions regarding the optimal storage and shipping conditions of clinically relevant specimens, including EDTA-whole blood, plasma, capillary blood, urine and seminal fluid (associated with sexual transmission of the Ebola virus after recovery from the disease). Therefore, the aim of our study was to identify the extent to which storage temperature and clinical specimen type influence Ebola virus viability. Virus infectivity was determined using a fluorescent focus-forming assay. In our study, we show that Ebola virus was the most stable in EDTA-whole blood and plasma samples, whereas rapid decay of infectivity was observed in simulated capillary blood, urine and semen samples, especially when these samples were stored at higher temperatures. The analysis of variance results demonstrated that both temperature and clinical specimen type have significant effects on virus viability, whereas donor differences were not observed. Repeated freeze and thaw cycles of the samples also had a notable impact on virus viability in EDTA-whole blood and urine. Due to the rapid temperature- and specimen-dependent degradation of the virus observed here, our study highlights the importance of proper clinical sample storage at low temperatures during transportation and laboratory analysis.

Molecular analysis of single circulating tumour cells following long‐term storage of clinical samples

The CellSearch® semiautomated CTC enrichment and staining system has been established as the ‘gold standard’ for CTC enumeration with CellSearch® CTC counts recognized by the FDA as prognostic for a number of cancers. We and others have gone on to show that molecular analysis of CellSearch® CTCs isolated shortly after CellSearch® enrichment provides another valuable layer of information that has potential clinical utility including predicting response to treatment. Although CellSearch® CTCs can be readily isolated after enrichment, the process of analysing a single CellSearch® patient sample, which may contain many CTCs, is both time‐consuming and costly. Here, we describe a simple process that will allow storage of all CellSearch®‐enriched cells in glycerol at −20 °C for up to 2 years without any measurable loss in the ability to retrieve single cells or in the genome integrity of the isolated cells. To establish the suitability of long‐term glycerol storage for single‐cell molecular analysis, we isolated individual CellSearch®‐enriched cells by DEPArray™ either shortly after CellSearch® enrichment or following storage of matched enriched cells in glycerol at −20 °C. All isolated cells were subjected to whole‐genome amplification (WGA), and the efficacy of single‐cell WGA was evaluated by multiplex PCR to generate a Genome Integrity Index (GII). The GII results from 409 single cells obtained from both ‘spike‐in’ controls and clinical samples showed no statistical difference between values obtained pre‐ and postglycerol storage and that there is no further loss in integrity when DEPArray™‐isolated cells are then stored at −80 °C for up to 2 years. In summary, we have established simple yet effective ‘stop‐off’ points along the CTC workflow enabling CTC banking and facilitating selection of suitable samples for intensive analysis once patient outcomes are known.

Abstract 3145: Clinical evaluation of streck cell-free DNA blood collection tubes for liquid profiling in oncology

Abstract Liquid profiling based on circulating tumor DNA (ctDNA) extracted from blood has become a powerful diagnostic approach in oncology. Making ctDNA analysis broadly available requires shipping whole blood to a clinical laboratory while ensuring cell-free DNA (cfDNA) and blood cell integrity to prevent dilution of ctDNA with cellular genomic DNA. Since standard EDTA blood collection tubes cannot retain this integrity for a prolonged time, several laboratories have proposed Streck Cell-Free DNA blood collection tubes (Streck cfDNA BCTs) as an alternative for sample collection. Qualification data for the use of Streck cfDNA BCTs in oncology is limited and mainly based on blood collected from healthy individuals as well as data extrapolated from the prenatal field. However, the data generated from these samples may not represent the unique dynamics of clinical oncology specimens and therefore a study of true clinical oncology samples is required to support the use of Streck cfDNA BCT tubes in routine practice. In this study, we performed a clinical evaluation of cfDNA sample integrity from matched Streck cfDNA BCTs vs standard EDTA tubes from colorectal, pancreatic and non-small cell lung cancer patients (N > 50). Blood drawn into Streck cfDNA BCTs was either processed immediately or 3 days after phlebotomy. DNA quantification was followed by BEAMing digital PCR (OncoBEAM™) on KRAS, NRAS and EGFR mutations and compared to matching specimens collected in EDTA tubes. Our results suggest that cfDNA yield as well as the genomic DNA background is not affected by prolonged storage of clinical samples in Streck cfDNA BCTs for up to 3 days. In all sample sets containing mutated ctDNA, the detected mutational load was comparable between Streck cfDNA BCTs and EDTA tubes. In conclusion, this study represents a comprehensive clinical evaluation of Streck cfDNA BCTs vs EDTA tubes for ctDNA profiling. In conjunction with the findings of our previously presented Streck cfDNA BCT shipping condition studies, our data supports the compatibility of clinical oncology specimens collected in Streck cfDNA BCTs with the BEAMing technology. Citation Format: Inga Medina Diaz, Stefan Holdenrieder, Annette Nocon, Makbule Kobilay, Dirk Skowasch, Stefanie Held, Johanna Weiland, Claudia Stamm, Frank Diehl, Frank Holtrup. Clinical evaluation of streck cell-free DNA blood collection tubes for liquid profiling in oncology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3145.

Urinary microRNA can be concentrated, dried on membranes and stored at room temperature in vacuum bags.

Urine accumulates traces of changes that occur in the body and can potentially serve as a better biomarker source. Urinary microRNA is a promising class of non-invasive disease biomarkers. However, long-term frozen human urine samples are not a good source for the extraction of urinary microRNA. In this paper, we demonstrate that urinary microRNA can be concentrated, dried on membranes and stored in vacuum bags at room temperature for several months. The amount of total RNA on the membranes after storage at room temperature for three months was unchanged. The levels of miR-16 and miR-21 exhibited no significant differences (P = 0.564, 0.386). This simple and economical method makes the large-scale storage of clinical samples of urinary microRNA or other nucleic acids possible.

Abstract P2-03-17: Assessing reproducibility of copy number arrays to assist breast cancer biomarker discovery

Abstract Introduction: Large-scale interrogation of the genome has emerged as an attractive method for identifying useful characteristics of cancer biology; in particular, the study of copy number aberrations (CNA) has recently received tremendous attention. A number of different technologies have been developed to assess the copy-number landscape, allowing us to better understand the role of CNA in cancer cells. The OncoScan CNA platform (Affymetrix Inc.) has been particularly appealing for oncology due of its ability to work well with formalin-fixed, paraffin-embedded (FFPE) materials, which is the primary form for storage of clinical samples. In addition, its high resolution, rapid analysis time and ability to interrogate different genomic characteristics (CNA, loss of heterozygosity or mutation) make the OncoScan platform highly popular: it has been widely cited in the literature for use in biomarker discovery, clonal evolution and sub-clonal detection, as well as population-based analyses. While CNAs identified by the OncoScan platform have shown good concordance with fluorescence in-situ hybridization (FISH) results, to date, no studies have been conducted to thoroughly assess the reproducibility of the assay. In this study, we have assessed the reproducibility of the OncoScan platform using identical samples performed in replicates across multiple chip batches. Moreover, we have assessed the effect on reproducibility of DNA treatment, including elution in water or TE buffer, as well as in the use of varying amounts of DNA. Methods: Affymetrix OncoScan FFPE Express 3.0 SNP Arrays were performed using the optimal input DNA as recommended by the manufacturer as well as fewer input amounts for comparison. CNAs were called using BioDiscovery Nexus Copy Number™ software (http://www.biodiscovery.com/software/nexus-copy-number/) using the SNP-FASST2 algorithm with modified parameters (significance threshold of 1 x 10-9 and minimum number of probes per segment of 10). Results: Initial reproducibility analysis involving 12 samples repeated either 2, 4 or 6 times both within a single batch and across different batches has revealed that CNA calls were concordant between replicates for the majority of the genome (ranges between 81% to 100%), suggesting high precision of the assay. In addition, we are in the process of assessing and comparing mutation calls across replicates to gain a more in-depth understanding of the platform. Conclusion: This is the first study examining the reproducibility of OncoScan FFPE assays; initial results have suggested that the assay is precise and has the potential for robust biomarker discovery. Additional characterizations would be interesting for evaluating its use as a clinical tool in the long term. Citation Format: Cindy Q Yao, Cheryl Crozier, Mary Anne Quintayo, Jane Bayani, Melanie Spears, Julie Livingstone, Esther Jung, Clement Fung, Victoria Sabine, Paul C Boutros, John MS Bartlett. Assessing reproducibility of copy number arrays to assist breast cancer biomarker discovery [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-17.

Significant Decline in Galactomannan Signal during Storage of Clinical Serum Samples

Galactomannan (GM) is widely used for detection of invasive aspergillosis in high-risk haemato-oncology patients. Recent publications have reported a lack of repeatability of GM detection. The objective of this retrospective study was to assess the repeatability of GM levels during storage of clinical samples. In a GM screening strategy, positive sera were repeat tested as per manufacturer’s recommendations. Short-term (ST) storage of samples was at +4 °C while long-term (LT) storage was at −80 °C. Bronchoalveolar (BAL) fluid was also repeating tested after ST storage and LT storage. Wilcoxon Signed Ranks Test was employed to assess the repeatability of GM levels. In a subset of 14 GM positive sera, repeat testing was performed on both the original serum and ethylenediaminetetraacetic acid (EDTA) pre-treated sample. There was a significant reduction in GM signals on repeat testing following ST storage (median GM index: 0.65 vs. 0.19; p < 0.001) and LT storage (median GM index: 0.56 vs. 0.10; p < 0.001) of serum samples. Of samples that were initially GM positive, an average GM index reduction of 50% was seen, with approximately two-thirds becoming GM negative on repeat testing of the same sample. In contrast, GM signal loss was not seen on repeat testing of BAL fluid following ST or LT storage. When GM positive serum samples were repeat tested using EDTA pre-treated serum from the first step of the testing protocol, all samples remained GM positive. In contrast, when the same samples were repeat tested from the original collected serum, 9 samples (64%) became GM negative. The significant reduction in GM signals during ST and LT storage of serum samples has implications for clinical management. Although the reasons for GM decline are unknown, they occur prior to the EDTA pre-treatment stage, indicating that the time from phlebotomy to testing should be minimized. BAL fluid GM index values remain stable.

Determination of a novel gamma-secretase inhibitor in human plasma and cerebrospinal fluid using automated 96 well solid phase extraction and liquid chromatography/tandem mass spectrometry

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid ( A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10–30 μL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite ( C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20 μL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard ( B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi ® Polar RP analytical column (50 mm × 3.0 mm, 4 μm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5 mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441 → 175 for A and 469 → 175 for B, respectively. The assays were validated over the concentration range of 0.5–200 ng/mL for human plasma and CSF. Replicate analyses ( n = 5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.

Determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine using high-performance liquid chromatography with fluorescence detection

A method for the determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine is described. Plasma and urine samples were extracted using 3M Empore extraction disk cartridges in the C 18 and MPC (mixed-phase cation-exchange) formats, respectively. The extract was analyzed using HPLC with fluorescence detection (ex 248 nm, em 300 nm), and included a column switching procedure to reduce run-time. The assay was linear in the concentration range 5 to 1000 ng/ml when 1-ml aliquots of plasma and urine were extracted. Recoveries of L-756 423 were greater than 84% over the calibration curve range using the described sample preparation procedures. Intra-day precision and accuracy for this assay was less than 9% RSD and within 7%, respectively. Inter-day variabilities for the plasma ( n=17) and urine ( n=10) were less than 5% and 3% for low (15 ng/ml) and high (750 ng/ml) quality control samples. Bovine serum albumin (0.5%) was used as an additive to urine to prevent precipitation of L-756 423 during the storage of clinical samples. The assay was used in support of human clinical trials.

Immunoelectrophoretic Analysis of C4 Split Products Expressing D but Not C Epitopes: Influence of Storage, Ca2+ and Ca2+-Chelating Agents

Based on immunoelectrophoretic methods a heterogeneity in the electrophoretic mobility of C4d was observed. C4d was defined immunochemically as C4 molecules expressing D but lacking C epitopes. A beta-mobile form was observed when EDTA or heparin was not added to the sample prior to electrophoretic analysis. This component was generated during electrophoresis. Another C4d component migrating to the post-albumin region probably represented an in vivo generated split product. However, this C4d form was also produced during storage of serum or plasma at room temperature and its formation was enhanced in the presence of EDTA. Based on these findings standard conditions for collection and storage of clinical samples for quantification of C4d by electroimmunoassay are suggested.