Blood Lysates Research Articles

A simple and cost-effective real-time PCR method using diluted and heat-treated whole blood lysate

Although DNA extraction is a crucial step before polymerase chain reaction (PCR), attempts to perform PCR without DNA isolation have been ongoing since the 1990s. While partial success has been achieved with direct conventional PCR, the DNA isolation step has remained indispensable for real-time PCR. Here, we developed a method that does not require complete DNA isolation by lysing EDTA-treated whole blood samples through the application of osmotic pressure and heat, followed by centrifugation to get a clear lysate. Blood samples were mixed with distilled water, incubated at 95 °C for 20 min before centrifugation at 14,000 rpm for 5 min. The resulting lysates were used as templates for real-time PCR. Real-time PCRs were performed under identical conditions with lysates and DNA samples using 9 different primer sets. Target genes were successfully amplified using 1:10 and 1:5 diluted blood lysates both at 60 °C and 61 °C. The PCR efficiency for ACTB and PIK3CA differed by 20% and 14%, respectively, between the DNA samples and blood lysates. We successfully amplified all selected genes using “GG-RT PCR”, greater temperature, greater speed method, without the need for additional enzymes or buffers. GG-RT PCR presents a cost effective option for applications such as SNP analysis and deletion detection if appropriate primer designs are made.

Molecular identification of haemoparasites in animals using blood lysate PCR: a quick and inexpensive alternative to purified whole genomic DNA

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay’s sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria, Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

Influence of Blood Components on Neuroinflammation, Blood-Brain Barrier Breakdown, and Functional Damage After Acute Subdural Hematoma in Rats.

A central component of injury development after acute subdural hematoma (ASDH) is the increased intracranial pressure and consecutive mechanical reduction of cerebral blood flow (CBF). However, the role of different blood constituents in ASDH as additional lesioning factors remains unclear. This study examines the influence of blood components on neuroinflammation, blood-brain barrier (BBB) breakdown, and functional deficits in a rat model of ASDH. We infused corpuscular (whole blood, whole blood lysate, and red cell blood) and plasmatic (blood plasma, anticoagulated blood plasma, and aqueous isotonic solution) blood components into the subdural space while CBF was monitored. Rats then underwent behavioral testing. Lesion analysis and immunohistochemistry were performed 2 days after ASDH. Inflammatory reaction was assessed using staining for ionized calcium-binding adaptor molecule 1 and glial fibrillary acidic protein, interleukin-1ß, tumor necrosis factor-alpha, and membrane attack complex. Integrity of the BBB was evaluated with albumin and matrix metalloproteinase 9 (MMP9) staining. We observed a significant drop in CBF in the corpuscular group (75% ± 7.5% of baseline) with distinct post-operative deficits and larger lesion volume compared to the plasmatic group (13.6 ± 5.4 vs. 1.3 ± 0.4 mm3). Further, inflammation was significantly increased in the corpuscular group with stronger immunoreaction. After whole blood infusion, albumin and MMP9 immunoreaction were significantly increased, pointing toward a disrupted BBB. The interaction between corpuscular and plasmatic blood components seems to be a key factor in the detrimental impact of ASDH. This interaction results in neuroinflammation and BBB leakage. These findings underscore the importance of performing surgery as early as possible and also provide indications for potential pharmacological targets.

Rapid, scalable assay of amylin‐β amyloid co‐aggregation in brain tissue and blood

AbstractBackgroundIslet amyloid polypeptide (amylin) secreted from the pancreas crosses the blood brain barrier into the brain parenchyma and interacts with Alzheimer’s disease (AD) associated cerebral β‐amyloid (Aβ) plaques, which are found in both sporadic and early‐onset familial AD. Immunoprecipitated amylin from human AD brain tissue analyzed by Western blot using an anti‐Aβ antibody detects Aβ immunoreactivity, which indicates that the amylin and Aβ peptides aggregate in the brains of humans with AD, forming amylin‐Aβ hetero‐oligomers that are sodium dodecyl sulfate (SDS)‐soluble. We report the development of an ELISA to detect amylin‐Aβ hetero‐oligomers in brain tissue and blood.MethodThe amylin‐Aβ ELISA relies on a monoclonal anti‐Aβ middomain antibody (detection) and a polyclonal anti‐amylin antibody (capture) designed to recognize an epitope that is distinct from the high affinity amylin‐Aβ binding sites. We screened temporal cortex homogenates obtained from 13 cognitively unaffected (CU) humans and 47 persons with sporadic AD (sAD) for amylin‐Aβ hetero‐oligomers. Individuals were of similar age (sAD, 85.65 ± 7.04 years vs. CU, 87.22 ± 7.30 years; p = 0.482). Brain tissue amylin and Aβ concentrations were measured using conventional ELISAs. Amylin‐Aβ oligomer levels were also measured in whole blood lysates from APP/PS1 rats and APP/PS1 rats expressing amyloid forming amylin in the pancreas (APP/PS1/HIP rats; all rats age‐matched, 16‐months).ResultThe brains of most AD patients had detectable amylin‐Aβ hetero‐oligomer concentrations, whereas about half of those CU had brain tissue amylin‐Aβ hetero‐oligomers below the detection limit (0.02 ng/mg total protein; one‐way ANOVA, P < 0.0001). Amylin‐Aβ hetero‐oligomers were more common in brains with frequent neuritic plaques (CERAD C). Higher brain tissue amylin‐Aβ hetero‐oligomer levels were associated with greater brain tissue amylin concentrations (r = 0.38; P < 0.05). There was no correlation between cerebral amylin‐Aβ hetero‐oligomer accumulation and age in the analysis of samples from sAD and CU groups (r = ‐0.22; P > 0.05). Levels of amylin‐Aβ hetero‐oligomers in whole blood lysates obtained from APP/PS1/HIP rats were higher than those from APP/PS1 littermates (P < 0.05).ConclusionStudies are needed to determine the extent to which amylin‐Aβ hetero‐oligomerization can be detected in human blood.

Rapid, scalable assay of amylin‐β amyloid co‐aggregation in brain tissue and blood

AbstractBackgroundIslet amyloid polypeptide (amylin) secreted from the pancreas crosses the blood brain barrier into the brain parenchyma and interacts with Alzheimer’s disease (AD) associated cerebral ß‐amyloid (Aß) plaques, which are found in both sporadic and early‐onset familial AD. Immunoprecipitated amylin from human AD brain tissue analyzed by Western blot using an anti‐Aß antibody detects Aß immunoreactivity, which indicates that the amylin and Aß peptides aggregate in the brains of humans with AD, forming amylin‐Aß hetero‐oligomers that are sodium dodecyl sulfate (SDS)‐soluble. We report the development of an ELISA to detect amylin‐Aß hetero‐oligomers in brain tissue and blood.MethodThe amylin‐Aß ELISA relies on a monoclonal anti‐Aß middomain antibody (detection) and a polyclonal anti‐amylin antibody (capture) designed to recognize an epitope that is distinct from the high affinity amylin‐Aß binding sites. We screened temporal cortex homogenates obtained from 13 cognitively unaffected (CU) humans and 47 persons with sporadic AD (sAD) for amylin‐Aß hetero‐oligomers. Individuals were of similar age (sAD, 85.65 ± 7.04 years vs. CU, 87.22 ± 7.30 years; p = 0.482). Brain tissue amylin and Aß concentrations were measured using conventional ELISAs. Amylin‐Aß oligomer levels were also measured in whole blood lysates from APP/PS1 rats and APP/PS1 rats expressing amyloid forming amylin in the pancreas (APP/PS1/HIP rats; all rats age‐matched, 16‐months).ResultThe brains of most AD patients had detectable amylin‐Aß hetero‐oligomer concentrations, whereas about half of those CU had brain tissue amylin‐Aß hetero‐oligomers below the detection limit (0.02 ng/mg total protein; one‐way ANOVA, P < 0.0001). Amylin‐Aß hetero‐oligomers were more common in brains with frequent neuritic plaques (CERAD C). Higher brain tissue amylin‐Aß hetero‐oligomer levels were associated with greater brain tissue amylin concentrations (r = 0.38; P < 0.05). There was no correlation between cerebral amylin‐Aß hetero‐oligomer accumulation and age in the analysis of samples from sAD and CU groups (r = ‐0.22; P > 0.05). Levels of amylin‐Aß hetero‐oligomers in whole blood lysates obtained from APP/PS1/HIP rats were higher than those from APP/PS1 littermates (P < 0.05).ConclusionStudies are needed to determine the extent to which amylin‐Aß hetero‐oligomerization can be detected in human blood.

Development of a Closed System qPCR Assay to Detect Mutations in IDH1/IDH2 in Patients with Acute Myeloid Leukemia (AML)

Background: Acute myeloid leukemia (AML) is a heterogenous disease characterized by clonal proliferation of poorly differentiated myeloid cells and is associated with mutations in several genes including the isocitrate dehydrogenase (IDH) genes IDH1 and IDH2. It has been reported that 20% of AML patients carry IDH1/IDH2 mutations (IDH1 - 8%, IDH2 - 12%) which are strongly associated with disease progression and relapse. Additionally, several IDH1/IDH2 inhibitors such as ivosidenib and enasidenib have been approved by the FDA for treatment of AML patients with IDH1 and IDH2 mutations respectively. Therefore, identification of these single nucleotide polymorphism (SNP) mutations in IDH1/IDH2 can help in disease classification and selection of effective targeted inhibitors. Objectives: Our goal is to develop a rapid and user-friendly cartridge-based qPCR assay that can detect 7 IDH1 and 10 IDH2 mutations in human genomic DNA from EDTA whole blood lysate. Using a single cartridge, any of the four codons (IDH1 R132/R100 or IDH2 R140/R172) will be called out when a mutation is detected. This reporting style is more than sufficient in deciding a course of treatment as current therapies only differ between a mutation in either IDH1 or IDH2. Methods: Six levels of custom generated plasmids (64%, 16%, 4%, 1%, 0.25%, and 0.0625%) for the four most prevalent IDH1/IDH2 mutations were spiked-into EDTA whole blood lysate and tested with prototype IDH1/IDH2 mutation assay. Standard curves of Ct vs. log % mutation were generated for R132C, R132H, R140Q, and R172K. Custom generated plasmids were also made for the remaining 13 mutations and have been tested around the LoD (1%). In addition to plasmids, human cell line DNA with 14 of the 17 mutations were also spiked into EDTA whole blood lysate and tested around the LoD (1%). A standard curve of Ct vs Log % mutation was generated with R132H human cell line DNA. The remaining 3 mutations were not commercially available as cell line DNA. Specificity of the IDH1/IDH2 mutation assay was assessed by testing EDTA whole blood lysate from 20 individual donors in quadruplicate. Results: The prototype IDH1/IDH2 assay demonstrated a dynamic range from 64% to 0.0625% with a limit of detection at 1% for all the mutations that were tested. Figure 1 shows the dynamic range curves for the most prevalent mutations: IDH1 R132C & R132H, and IDH2 R140Q & R172K. These dynamic range curves include data points for both plasmid DNA (pDNA in red) and human cell line DNA (gDNA in blue) to show equivalency between the two test materials. The performance of the remaining 13 mutations in plasmid DNA perform similarly in their respective codons with no more than 2Ct difference at 1% levels. The remaining 10 human cell line DNA mutations perform similarly to plasmid DNA. Conclusions: We have prototyped 18-plex assay designed to detect 7 IDH1 and 10 IDH2 mutations and call out its corresponding codon in EDTA whole blood samples using a single cartridge at an estimated LoD of 1%.

Identification of 17 Distinct IDH1/IDH2 Mutations from Acute Myeloid Leukemia Peripheral Blood Using a Multiplex Real-Time PCR Closed Cartridge System

Background: Acute myeloid leukemia (AML) is characterized by clonal expansion of poorly differentiated “blast cells” in peripheral blood and bone marrow causing ineffective erythropoiesis and bone marrow failure. It has been demonstrated that mutations in isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) are associated with disease relapse and reduced survival. There are 17 AML-related IDH1/IDH2 mutations located at R100, R132, R140, and R172 that are targeted by different IDH1/IDH2 selective inhibitors. The sensitivity of detecting IDH1/IDH2 needs to be improved for early cancer detection. Current PCR-based assays cannot detect all 17 IDH1/IDH2 mutations, and next generation sequencing approaches take longer, use complicated workflows, and require costly reagents as well as substantial data analysis. Therefore, the rapid identification of patients with AML who harbor IDH1/IDH2 mutations could improve the efficient selection of targeted inhibitors for these patients. Aim: To create a multiplex PCR assay that detects 17 IDH1/IDH2 mutations from peripheral blood with high sensitivity and specificity. Method: We will highlight five technical aspects of this qPCR assay that contribute to its high sensitivity and specificity: 1. the “anchor - bridge - foot” design of SuperSelective (SS) primers that can detect different mutations within the same codon. 2. the amplification-refractory mutation system (ARMS) primer design strategy was also tested which has comparable effects to SS primer. 3. a two-step nested PCR to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. 4. a short (20-25nt) blocking oligonucleotide (wild-type blocker, WTB) to reduce SS and ARMS primer binding at the wild-type codon site. 5. four probes carrying different fluorescence reporters were used to identify the four IDH1/IDH2 mutation codons. Results: We synthesized 17 plasmid DNA (pDNA) templates and 14 CRISPR-engineered cell lines, each carrying one IDH1 or IDH2 mutation and the housekeeping gene ABL. SS and ARMS primer were screened with 5·10 4 cps/test of mutation template in a whole blood background. 6 mutations of R132 Codon (R132C, G, H, L, S, V), R100Q, 4 mutations of R140 (R140Q, L, G, W), and 6 mutations of R172 (R172K, M, G, S1 [516G>T], S2 [516G>C], W) can successfully be identified from whole blood lysate at the LoD level (1%) as shown in Figure 1 in red. Moreover, adding WTB to the PCR reaction improved the specificity with a reduced background signal. Importantly, in addition to the plasmid, cell line genomic DNA (gDNA) that was spiked into blood lysate at the LoD level (1%) also performed similarly to plasmid DNA as shown in Figure 1 in blue. Summary/Conclusion: We present a prototype assay with design intention to detect 17 mutations of IDH1/IDH2 from the peripheral blood of AML patients with excellent sensitivity and specificity and a strong potential to outperform any products on the market. Utilizing our Xpert Instrument System a fully automated, single-use cartridge product with pre-loaded sample-processing reagents, the assay aims to report results in less than 3 hours, including hands-on time.

Streamlining the Detection of FLT3 Internal Tandem Duplication (FLT3-ITD) Using the Cepheid Genexpert® Automated System

Objectives: The development of targeted therapies for Acute Myeloid leukemia (AML) demands fast and reliable diagnostic tests. Specifically, FMS-like tyrosine kinase 3 (FLT3-ITD), comprising nearly 25% of all AML cases, is the most common targetable mutation in AML. Current diagnosis of FLT3-ITD by PCR involves numerous laborious steps, with bench-top steps involving time-consuming whole blood processing, DNA isolation, plate-based PCR and subsequent fragment analysis by capillary electrophoresis. Consequently this adds significant overhead, time-consuming personnel training, and numerous steps which could lead to errors, all of which increase the resource burden and time to result. The objective of this study was to create an automated FLT3-ITD diagnostic prototype that is faster, requires less hands-on time that can detect all clinically relevant ITD lengths. Methods: We have developed a simplified FLT3-ITD prototype assay using the Cepheid GeneXpert® automated platform for FLT3-ITD detection. The prototype substantially streamlines the process, using a less than 30 minutes off-board time to prepare a universal blood lysate that is compatible with all Cepheid Hematology Oncology products. Once the sample is loaded into the cartridge and inserted into the instrument, the cartridge performs automated DNA isolation, purification, and qPCR. A Taqman-based probe for ABL is utilized in the qPCR step as a PCR control to ensure that the blood processing and qPCR was successful and avoid wasted downstream steps. Quantitative fragment analysis can easily be performed on the PCR product using capillary electrophoresis (CE) for precise allelic ratio (AR) callout. Additionally, the PCR product is compatible be run on a bioanalyzer or standard Agarose Gel-Electrophoresis if available. Results: The developed automated FLT3-ITD diagnostic prototype demonstrated significant improvements over the existing methodologies with substantially reduced time to result and simplicity in use. To assess the sensitivity of this prototype, we used MV4-11 cancer cells harboring a 30 bp ITD and CRISPR-CAS engineered human cell lines with 3 bp, 21 bp, and 75 bp FLT3 ITD insertions, comprising the most commonly detected ITD lengths spiked into EDTA blood as a test material. For these ITD's, we observed sensitivity and linearity down to 0.01-0.02 by CE for the ITDs tested. Importantly, the prototype detected all lengths of ITD mutations with inserts from 3bp to 300 bp using plasmid test materials. Conclusion: We have successfully developed a novel FLT3-ITD diagnostic prototype that has several major advantages over existing methodologies. The prototype significantly reduces the time and hands-on effort required for analysis, streamlining the diagnostic process for same day results. The larger sample volume used allows improved sensitivity to ensure accurate detection, even at low allelic frequencies. Additionally, the prototype requires fewer reagents in that sample prep and qPCR are performed in a single cartridge and run hands-free. Downstream analysis of amplified products can be easily performed and validated using capillary electrophoresis, bioanalyzer, or gel electrophoresis. Finally, because this prototype uses the same universal blood lysate as other AML tests, it has the potential to improve future diagnostic therapeutic interventions in the clinic.

Indicators of oxidative stress in blood samples of indigenous residents and newcomers in the Arctic zone of Yakutia

Introduction. Oxidative stress is non-specific reaction of human organism in response to various damaging factors, including climatic. 
 The purpose of the study. To compare markers of oxidative stress and corresponding age dependences in blood samples of indigenous (evolutionarily adapted) and newcomer inhabitants of the Arctic zone of Yakutia. 
 Materials and methods. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and malondialdehyde content (MDA) were determined in blood lysates of indigenous (n=100) and newcomers (n=37) residents of Chokurdakh and Tiksi settlements. The compared subsamples did not differ in age (medians 34 and 37 years, p=0.407). 
 Results. No significant differences were found between newcomers and natives in terms of SOD, CAT and MDA content in the blood. The GPx activity of newcomers was 1.2 times higher than that of the natives (27.8 [22.4; 32.0] and 23.4 [19.2; 29.4] U/g Hb, p=0.042), but rapidly decreased with age (R= –0.549; p=0.001) 
 in parallel with the increase in MDA content (R=0.420; p=0.01), whereas the indigenous people had no age-related changes in GPx and MDA. 
 Limitations. Associated with a comparatively modest sample size (137 persons). 
 Conclusion. According to modern gerontology, age-related trends in enzyme activity arise due to changes in regulation of corresponding genes and reflect the rate of aging of the population. So it can be assumed that our data, which show accelerated aging of Arctic alien inhabitants compared to the indigenous ones, can be explained by genetic polymorphism of GPx1 transcription factors.

Separation and Washing of Candida Cells from White Blood Cells Using Viscoelastic Microfluidics

An early and accurate diagnosis of Candida albicans is critical for the rapid antifungal treatment of candidemia, a mortal bloodstream infection. This study demonstrates viscoelastic microfluidic techniques for continuous separation, concentration, and subsequent washing of Candida cells in the blood. The total sample preparation system contains two-step microfluidic devices: a closed-loop separation and concentration device and a co-flow cell-washing device. To determine the flow conditions of the closed-loop device, such as the flow rate factor, a mixture of 4 and 13 μm particles was used. Candida cells were successfully separated from the white blood cells (WBCs) and concentrated by 74.6-fold in the sample reservoir of the closed-loop system at 800 μL/min with a flow rate factor of 3.3. In addition, the collected Candida cells were washed with washing buffer (deionized water) in the microchannels with an aspect ratio of 2 at a total flow rate of 100 μL/min. Finally, Candida cells at extremely low concentrations (Ct > 35) became detectable after the removal of WBCs, the additional buffer solution in the closed-loop system (Ct = 30.3 ± 1.3), and further removal of blood lysate and washing (Ct = 23.3 ± 1.6).

Addition of exogenous γ-glutamyl hydrolase eliminates the need for lengthy incubation of whole blood lysate for quantitation of folate vitamers by high performance liquid chromatography-tandem mass spectrometry

BackgroundMeasurement of folate monoglutamates by HPLC-MS/MS in whole blood lysate (WBL) requires lengthy incubation prior to analysis, risking degradation of labile folate vitamers. ObjectiveWe explored whether addition of a commercially available recombinant exogenous γ-glutamyl hydrolase (exoGGH) enzyme reduced the required incubation time of WBL for measurement of folate as monoglutamates. MethodsFor conventional deglutamylation of polyglutamates, WBL was incubated 4 h at 37°C. Alternatively, we added exoGGH to WBL at varying concentrations (1–10 µg/mL) and incubation times (0–90 min). We also investigated modifications to the sample diluent (pH, ascorbic acid vs. sodium ascorbate, and ascorbate concentration). Finally, we tested the effect of the enzyme in different sample types: WBL from frozen whole blood vs. frozen WBL or vs. frozen washed red blood cells (RBC). Samples (n ≤ 15 per experiment) were analyzed by HPLC-MS/MS for 6 folate monoglutamates and 5-methyltetrahydrofolate diglutamate. ResultsOptimal deconjugation of folate polyglutamates was achieved using 1% ascorbic acid and 5 µg enzyme/mL WBL, requiring ≤ 30 min incubation time to achieve complete folate recovery as monoglutamates. This treatment resulted in similar folate concentrations as conventional deglutamylation (4 h at 37°C). The exoGGH enzyme was effective in samples stored frozen as whole blood and as WBL. However, extended thaw time of whole blood resulted in 5-methyltetrahydrofolate loss and unacceptable changes to non-methyl folate concentration. Total folate (with exoGGH) measured in washed RBC was ∼15% lower than RBC folate calculated from WBL concentrations (conventional deglutamylation). ConclusionsUse of exoGGH minimized incubation time and thus may avoid degradative losses of labile folate forms during sample preparation. The lower folate results in washed RBC may be due to inadequate packing of RBC among other unidentified factors. A larger study is required to confirm lack of differences in folate concentrations determined with and without the use of exoGGH.

A microfluidic finger-actuated blood lysate preparation device enabled by rapid acoustofluidic mixing.

For many blood-based diagnostic tests, including prophylactic drug analysis and malaria assays, red blood cells must be lysed effectively prior to their use in an analytical workflow. We report on a finger-actuated blood lysate preparation device, which utilises a previously reported acoustofluidic micromixer module. The integrated device includes a range of innovations from a sample interface, to the integration of blisters on a laser engraved surface and a large volume (130 μL) one-stroke manual pump which could be useful in other low-cost microfluidic-based point-of-care devices. The adaptability of the acoustic mixer is demonstrated on highly viscous fluids, including whole blood, with up to 65% percent volume fraction of red blood cells. Used in conjunction with a lysis buffer, the micromixer unit is also shown to lyse a finger-prick (approximately 20 μL) blood sample in 30 seconds and benchmarked across ten donor samples. Finally, we demonstrate the ease of use of the fully integrated device. Cheap, modular, but reliable, finger-actuated microfluidic functions could open up opportunities for the development of diagnostics with minimal resources.

Cerebrovascular accumulation of amyloid‐forming amylin secreted from the pancreas induces brain hypoxia

AbstractBackgroundIslet amyloid polypeptide (amylin) is a β‐cell hormone co‐secreted with insulin. Histological analyses of human brains identified amylin deposits co‐localized with parenchymal and vascular β‐amyloid in humans with vascular cognitive impairment and Alzheimer’s dementia. Because circulating amyloid‐forming amylin triggers systemic hypoxia signaling, we sought to determine the relationship between circulating amylin levels and brain hypoxia markers in a transgenic rat model of amylin‐mediated neurological deficits.MethodTo assess amylin‐related brain hypoxia signaling, we performed a 16‐month longitudinal study in which rats that express amyloid‐forming human amylin in pancreatic β‐cells (HIP rats) were compared to wild type (WT) littermates that express non‐amyloidogenic rodent amylin (n=10 rats/group).ResultBlood amylin levels measured by ELISA were ∼2‐fold higher in 16‐month old HIP rats compared to WT littermates (P<0.01). The Thiovlavin T (Th T) fluorescence signal intensity in blood lysates from HIP rats increased compared to that in WT littermates indicating the presence of amyloid‐forming in the circulation in HIP rats (P<0.01). This was associated with higher amylin concentration in brain microvessel lysates (P<0.05) and amylin immunoreactivity signal intensity in brain sections. Plasma erythropoietin, a marker of systemic hypoxia was higher in HIP rats than in WT littermates (P<0.05), which correlated with brain accumulation of HIF‐2α (P<0.05) and HIF‐1α (P=0.07), and with altered mitochondrial DNA in brain tissue (P<0.05).ConclusionIncreased circulating levels of amyloid‐forming amylin promotes cerebrovascular amylin deposition leading to brain hypoxic‐ischemic injury. Future studies need to address functional effects of amylin‐induced brain hypoxia signaling in the brain.

NPM1 Mutation Quantitation Evaluated By a Commercially Available Assay and the Cepheid Prototype‡ Xpert® NPM1 Mutation Assay

Aims The objective of this study was to compare the performance of the novel Cepheid Prototype Xpert® NPM1 Mutation Assay to the Qiagen ipsogen® NPM1 mut A, B&D MutaQuant Kits using samples contrived using cell line-spiked peripheral blood. Our goal was to evaluate the comparative performance of a faster and easier to use assay than commercially available products for use in the monitoring of minimal residual disease (MRD) in NPM1-positive AML patients. Background A common mutation in AML (20-30% of cases) is a four base pair insertion in the NPM1 gene.¹ NPM1 plays a role in ribosome biogenesis, maintaining genomic stability, and modulating growth suppression by binding and stabilizing p53.² NPM1 mutant A consists of about 80% of NPM1 mutations, while mutants B or D are present in a sizeable minority of patients.¹² Monitoring of NPM1 mutations can provide prognostic value and can assist with choice of treatment.³ Methods Three NPM1-positive cell lines, OCI-AML3 MutA, OCI-AML3 MutB, and OCI-AML3 MutD, which express NPM1 MutA, NPM1 Mut B, and NPM1 Mut D, respectively, were grown and isolated. After processing the cells into cell lysate, the cell-lysates were spiked into NPM1-negative EDTA whole blood lysate and serially diluted to target 13 or 14 NPM1/ABL levels from approximately 2500% (log reduction (LR) 1.4) to approximately 0.01% (LR -4.0). A subset of each level underwent hands-off sample preparation, total RNA extraction, and RT-qPCR all within the Cepheid GeneXpert automated cartridge system, and another subset had total RNA extracted using the Zymo Direct-zol™ RNA Miniprep Plus Kit. RNA was qualified and quantified using the Thermo Scientific NanoDrop™ 2000 Spectrophotometer before being tested per the comparator assay vendor's recommendations. The Cepheid Prototype assay utilized 4.5mL of lysate, whereas the Qiagen kits used 100 ng of RNA per test. Specimens spiked with cell lines expressing different NPM1 mutations were tested using the respective ipsogen kit designed for that mutation. The Cepheid Prototype assay was evaluated against the comparator assay for each subtype using Deming regressions. Bland-Altman plots were utilized to evaluate bias between assay quantitation outputs. Results Deming regressions and associated Bland-Altman plots showed a high degree of correlation and agreement between comparator assays and the Cepheid Prototype assay (Figure 1). Conclusions The novel Cepheid Prototype assay showed a high level of agreement with Qiagen kits in this study using whole blood EDTA lysate samples spiked with three NPM1-positive cell-lysates. Agreement tended to decline at low input levels, and a small but acceptable degree of nonlinearity was present in MutA and MutD. Total time to result (TTR) was minimized in the Cepheid Prototype assay to under 170 minutes, while the comparator assay had a TTR of approximately six hours plus RNA extraction. While RNA extraction is performed within Cepheid Prototype assay cartridge and is included in this 170-minute TTR, the Qiagen kit requires additional expensive and labor-intensive extraction steps that can last one-hour to multiple days, which are not included in the six-hour TTR estimate. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Метаболическая активность иммунокомпетентных клеток в оценке индивидуальной холодовой чувствительности

The rapid switch on of the transient short-term responses involved in adjustment of homeostasis plays a key role in human adaptation to low temperatures that is essential for adjustment to low-temperature environment. The network of signaling pathways together with metabolic regulators provide sufficient plasticity of the cells of immune system, the normal function of which is extremely important for successful human adaptation. Sufficient energy supply to immunocompetent cells makes it possible to form an adequate immune response to any negative factor and to ensure adaptive functional rearrangements. The study was aimed to assess the variants of the immunocompetent cell metabolic pathways involved in acquiring individual cold sensitivity. A total of 180 people aged 25–55 (130 females, 50 males) were assessed before and after the short-term whole body cooling. Enzyme immunoassay was used to define the levels of IL10, IL6, TNFα, irisin, transferrin, sTfR, HIF-1α, Sirt3 in peripheral blood and cell lysate. The levels of glycogen (cytochemical methods) and ATP (luciferin-luciferase assay) in lymphocytes were defined. The decrease in peripheral blood lymphocyte levels after cooling was indicative of the formation of immediate adaptive response and activation of glycolysis amid less intense inflammatory response. The increase in the levels of circulating lymphocytes after the cold esposure was associated with activation of inflammatory responses. The lower ratio of HIF-1α/SIRT3 metabolic regulators was found in the surveyed volunteers who showed no changes in the levels of lymphocytes. This indicated predominance of mitochondrial activity in adaptation to low temperatures.

Parallelization with Dual-Trap Single-Column Configuration Maximizes Throughput of Proteomic Analysis.

Proteomic analysis on the scale that captures population and biological heterogeneity over hundreds to thousands of samples requires rapid mass spectrometry methods, which maximize instrument utilization (IU) and proteome coverage while maintaining precise and reproducible quantification. To achieve this, a short liquid chromatography gradient paired to rapid mass spectrometry data acquisition can be used to reproducibly quantify a moderate set of analytes. High-throughput profiling at a limited depth is becoming an increasingly utilized strategy for tackling large sample sets but the time spent on loading the sample, flushing the column(s), and re-equilibrating the system reduces the ratio of meaningful data acquired to total operation time and IU. The dual-trap single-column configuration (DTSC) presented here maximizes IU in rapid analysis (15 min per sample) of blood and cell lysates by parallelizing trap column cleaning and sample loading and desalting with the analysis of the previous sample. We achieved 90% IU in low microflow (9.5 μL/min) analysis of blood while reproducibly quantifying 300-400 proteins and over 6000 precursor ions. The same IU was achieved for cell lysates and over 4000 proteins (3000 at CV below 20%) and 40,000 precursor ions were quantified at a rate of 15 min/sample. Thus, DTSC enables high-throughput epidemiological blood-based biomarker cohort studies and cell-based perturbation screening.

Ultrasensitive electrochemical genosensors for species-specific diagnosis of malaria

The absence of reliable species-specific diagnostic tools for malaria at point-of-care (POC) remains a major setback towards effective disease management. This is partly due to the limited sensitivity and specificity of the current malaria POC diagnostic kits especially in cases of low-density parasitaemia and mixed species infections. In this study, we describe the first label-free DNA-based genosensors based on electrochemical impedance spectroscopy (EIS) for species-specific detection of P. falciparum, P. malariae and P. ovale. The limits of detection (LOD) for the three species-specific genosensors were down in attomolar concentrations ranging from 18.7 aM to 43.6 aM, which is below the detection limits of previously reported malaria genosensors. More importantly, the diagnostic performance of the three genosensors were compared to quantitative real-time polymerase chain reaction (qPCR) assays using purified genomic DNA and the paired whole blood lysates from clinical samples. Remarkably, all the qPCR-positive purified genomic DNA samples were correctly identified by the genosensors indicating 100% sensitivity for each of the three malaria species. The specificities of the three genosensors ranged from 66.7% to 100.0% with a Therapeutic Turnaround Time (TTAT) within 30 min, which is comparable to the TTAT of current POC diagnostic tools for malaria. This work represents a significant step towards the development of accurate and rapid species-specific nucleic acid-based toolkits for the diagnosis of malaria at the POC.

Robustness of the CDC Folate Microbiologic Assay Kit During Simulated Delayed Shipping

ObjectivesThe WHO has recommended the accurate and practical microbiologic assay (MBA) to assess population folate status. To help laboratories in low-resource countries conduct folate measurements, the CDC developed a folate MBA kit containing critical reagents: calibrator, microorganism, quality control (QC), and reagent stock solutions to prepare culture medium. Shipping frozen kits internationally is complex and sometimes delayed, potentially causing materials to thaw. We investigated whether refrigerated or ambient kit temperatures affect folate results.MethodsWe conditioned 5-methyltetrahydrofolate calibrator stock solution, L. rhamnosus microorganism inoculation, serum and whole blood lysate (WBL) QC samples, and reagent stock solutions (ascorbic acid, chloramphenicol, and manganese sulfate) at 6°C ≤ 3 d and 20°C ≤ 2 d protected from light; we then compared conditioned vs. optimally stored materials (-70°C) by analyzing serum and/or WBL samples (≥3 runs).ResultsThe conditioned calibrator showed a mean folate difference (range from 3 runs for 43 WBL samples) of −0.3% (−4.0 to 4.1%) at 6°C/3 d and 0.9% (−1.6% to 2.7%) at 20°C/2 d. Similarly, the conditioned microorganism showed a mean difference of 4.3% (0.7% to 8.3%) at 6°C/3 d and 0.6% (−3.5% to 4.9%) at 20°C/2 d. The conditioned reagents showed a mean difference (range from 3 runs for 5 serum and 5 WBL samples) of 0.3% (−7.4% to 9.0%) at 6°C/3 d and 3.6% (0.1% to 9.0%) at 20°C/2 d. The 2 conditioned serum QC samples (∼13 and ∼40 nmol/L) showed a mean difference (range from 6 runs) of −0.6% (−6.0% to 2.3%) at 6°C/1 d, but higher differences of −5.8%, −5.9%, and −20.7% at 6°C/3 d, 20°C/1 d, and 20°C/2 d, respectively. The conditioned low WBL QC sample (∼300 nmol/L) showed small mean differences (−1.5%, −2.4%, −1.7%, and −0.3% at 6°C/1 d, 6°C/3 d, 20°C/1 d, and 20°C/2 d, respectively), while the conditioned high WBL QC sample (∼600 nmol/L) showed higher mean differences (−3.1%, −12.4%, −6.9%, and −23.7%, respectively).ConclusionsAll kit components except for the QC materials demonstrated adequate stability (<5% change) at 6°C ≤ 3 d or at 20°C ≤ 2 d. The QC materials should not be exposed to 6°C for > 1 d to ensure stability. These findings provide important and practical information for kit shipment management.Funding SourcesNone.

Robustness of the CDC Folate Microbiologic Assay Kit During Simulated Delayed Shipping

ObjectivesThe WHO has recommended the accurate and practical microbiologic assay (MBA) to assess population folate status. To help laboratories in low-resource countries conduct folate measurements, the CDC developed a folate MBA kit containing critical reagents: calibrator, microorganism, quality control (QC), and reagent stock solutions to prepare culture medium. Shipping frozen kits internationally is complex and sometimes delayed, potentially causing materials to thaw. We investigated whether refrigerated or ambient kit temperatures affect folate results.MethodsWe conditioned 5-methyltetrahydrofolate calibrator stock solution, L. rhamnosus microorganism inoculation, serum and whole blood lysate (WBL) QC samples, and reagent stock solutions (ascorbic acid, chloramphenicol, and manganese sulfate) at 6°C ≤ 3 d and 20°C ≤ 2 d protected from light; we then compared conditioned vs. optimally stored materials (-70°C) by analyzing serum and/or WBL samples (≥3 runs).ResultsThe conditioned calibrator showed a mean folate difference (range from 3 runs for 43 WBL samples) of −0.3% (−4.0 to 4.1%) at 6°C/3 d and 0.9% (−1.6% to 2.7%) at 20°C/2 d. Similarly, the conditioned microorganism showed a mean difference of 4.3% (0.7% to 8.3%) at 6°C/3 d and 0.6% (−3.5% to 4.9%) at 20°C/2 d. The conditioned reagents showed a mean difference (range from 3 runs for 5 serum and 5 WBL samples) of 0.3% (−7.4% to 9.0%) at 6°C/3 d and 3.6% (0.1% to 9.0%) at 20°C/2 d. The 2 conditioned serum QC samples (∼13 and ∼40 nmol/L) showed a mean difference (range from 6 runs) of −0.6% (−6.0% to 2.3%) at 6°C/1 d, but higher differences of −5.8%, −5.9%, and −20.7% at 6°C/3 d, 20°C/1 d, and 20°C/2 d, respectively. The conditioned low WBL QC sample (∼300 nmol/L) showed small mean differences (−1.5%, −2.4%, −1.7%, and −0.3% at 6°C/1 d, 6°C/3 d, 20°C/1 d, and 20°C/2 d, respectively), while the conditioned high WBL QC sample (∼600 nmol/L) showed higher mean differences (−3.1%, −12.4%, −6.9%, and −23.7%, respectively).ConclusionsAll kit components except for the QC materials demonstrated adequate stability (<5% change) at 6°C ≤ 3 d or at 20°C ≤ 2 d. The QC materials should not be exposed to 6°C for > 1 d to ensure stability. These findings provide important and practical information for kit shipment management.Funding SourcesNone.

VITAMIN B1 BIOLOGICAL FUNCTION IMPLEMENTATION IN THE BLOOD OF PATIENTS WITH STOMACH CANCER UNDER SURGICAL INTERVENTION

Background. The probability of undergoing surgery always predetermines the state of stress in a person; therefore, it is advisable to search for ways to optimize and (or) reduce this unfavourable effect. Objective. To find out the mechanism of vitamin B1 antistress activity during surgery. Material and methods. Metabolism intensity was investigated on donors’ blood lysates (n = 19) and those of patients with stage III stomach cancer (n = 64), referred to an elective surgery, aged 51-70. The blood was taken from the cubital vein three days before the operation, after premedication, during the most traumatic moment of the operation, after extubation, as well as on the first and third days of the postoperative period. The surgery was performed under combined multicomponent anesthesia using nitric oxide, sodium hydroxybutyrate, and epidural block. Thiamine and thiamine diphosphate kinase activities were assessed by the concentration of the formed thiamine di- and triphosphates of the vitamin. The activities of thiamine mono-, di- and triphosphatases were determined by the release of inorganic phosphate. The concentration of inorganic phosphate was recorded colorimetrically. The content of B1 and its derivatives in the blood was determined by the method of ion-pair reversed-phase HPLC. Results. There has been observed an increased content of thiamine monophosphate and that of free thiamine in the blood of donors and patients with stomach cancer. The registered rate of the hydrolytic thiamine monophosphatase reaction is not high. At the stages of premedication and maximum trauma of surgical exposure, the concentration of monophosphoric ester rapidly decreases alongside with monophosphatase activation. Therefore, the thiamine monophosphate hydrolysis is the rate-limiting link of vitamin B1 metabolism. The level of free thiamine remains persistently increased at all stages of surgical treatment. Thiamine monophosphatase activity is manifested at two pH optima – of 6.0 and 9.0. Thiamine monophosphate hydrolysis at pH of 9.0 is catalyzed by alkaline phosphatase. At pH of 6.0, in addition to thiamine monophosphoric ester, the enzyme hydrolyzes only p-nitrophenyl phosphate, flavin mononucleotide and phosphotyrosine, that allows it to be classified as hepatic acid phosphatase. The noted changes in B1 metabolism under stress concern mainly non-coenzyme forms - thiamine mono-, triphosphate, and free thiamine, which are used at the stages of thiol reduction as important components of insulin synthesis. Conclusions. The use of vitamin B1 allows to optimize the development of the stress response at all stages of surgical treatment. Its protective effect is achieved through the activation of the insulin-synthetic function of the pancreas, which increases the level of immunoreactive insulin in the blood. The formation of the most favorable physiological conditions for insulin synthesis provides an increased background of free thiamine, which is created due to the hydrolysis of noncoenzyme forms of the vitamin. The relationship between thiamine metabolism and B2 exchange and regulation of intracellular signaling pathways has been traced.