How can we use proteomics to learn more about platelets?

Proteomics tools provide a powerful means to identify, detect, and quantify protein-related details in studies of platelet phenotype and function. Here, we consider how historical and recent advances in proteomics approaches have informed our understanding of platelet biology, and, how proteomics tools can be used going forward to advance studies of platelets. It is now apparent that the platelet proteome is comprised of thousands of different proteins, where specific changes in platelet protein systems can accompany alterations in platelet function in health and disease. Going forward, many challenges remain in how to best carry out, validate and interpret platelet proteomics experiments. Future studies of platelet protein post-translational modifications such as glycosylation, or studies that take advantage of single cell proteomics and top-down proteomics methods all represent areas of interest to profiling and more richly understanding platelets in human wellness and disease.

What is the context?Platelets are well known as cellular mediators of hemostasis and drivers of thrombosis and inflammation. Thousands of different proteins come together to make up the molecular structure of platelets and support their functions in health and disease.What is new?"Proteomics" refers to laboratory approaches that study many proteins simultaneously to provide details on proteomes and the biology of a given system of interest. Many different high-throughput biochemical methods can be considered as proteomics, including mass spectrometry analysis.What is the impact?Proteomics has already identified more than 5,000 individual proteins in human platelets and provided important insights into how platelets may serve as biomarkers or therapeutic targets in human health and disease. In coming years, advances in proteomics technologies will likely help to characterize additional proteins and molecular systems in platelets to shed light on platelet-mediated mechanisms of physiology and pathology that have been largely unapproachable with other methods.

Preanalytical variables influencing the interpretation and reporting of biological tests on blood samples of living and deceased donors for human body materials.

With the present paper, the Working Group on Cells, Tissues and Organs and other experts of the Superior Health Council of Belgium aimed to provide stakeholders in material of human origin with advice on critical aspects of serological and nucleic acid test (NAT) testing, to improve virological safety of cell- and tissue and organ donation. The current paper focusses on a number of preanalytical variables which can be critical for any medical biology examination: (1) sampling related variables (type of samples, collection of the samples, volume of the sample, choice of specific tubes, identification of tubes), (2) variables related to transport, storage and processing of blood samples (transport, centrifugation and haemolysis, storage before and after centrifugation, use of serum versus plasma), (3) variables related to dilution (haemodilution, pooling of samples), and (4) test dependent variables (available tests and validation). Depending on the type of donor (deceased donor (heart-beating or non-heart beating) versus living donor (allogeneic, related, autologous), and the type of donated human material (cells, tissue or organs) additional factors can play a role: pre- and post-mortem sampling, conditions of sampling (e.g. morgue), haemodilution, possibility of retesting.

Actual approaches to the transportation of biological samples at low temperatures.

Taking into account the impact of shipment method of biosamples is necessary for obtaining high-quality biological samples in biobanking and laboratory research. The impact of liquid nitrogen, dry ice and cold accumulators on the quality of biological markers was considered, as well as recommendations to reduce the impact of these methods of shipment. The liquid nitrogen provides the best preservation of samples, however, dry ice is used much more often during their transportation. When transporting certain types of cells using dry ice, there is the way to use CryoStor CS1 and Cell Banker 1 cryoprotectors. The dry ice has a significant effect on both the pH of liquid biological samples and the coagulological parameters of plasma samples. The penetration of CO2 into the sample leads to changes in the parameters of PTT and APPT, as well as to decrease the protein C and fibrinogen level under certain conditions. Serum and plasma samples exposed to dry ice for more than 16 hours should be thawed open at room temperature, or instead of it should be kept at -80 °C for 24 hours to avoid changes in coagulation parameters, The use of cold accumulators is unacceptable for long-term shipment of serum and plasma containing unstable biomarkers because of insufficiently low temperature (increase over time to -25 °C and above). Besides, metal pellets can be used as cold storage batteries at low temperatures (up to -80 ° C), but they are not as effective as dry ice, since it is able to hold the required temperature for much longer.

Application and optimization of minimally invasive cell-free DNA techniques in oncogenomics.

The conventional method of measuring biomarkers in malignant tissue samples has already given subversive growth in cancer diagnosis, prognosis, and therapy selection. However, the regression and heterogeneity associated with tumor tissue biopsy have urged for the development of an alternative approach. Considering the limitations, cell-free DNA has emerged as a surrogate alternative, facilitating preoperative chemoradiotherapy (p < 0.0001) treatment response in rectal cancer and detection of biomarker in lung cancer. This potential of cell-free DNA in several other cancers has yet to be explored based on clinical relevance by optimizing the preanalytical factors. This review has highlighted the crucial parameters from blood collection to cell-free DNA analysis that has a significant impact on the accuracy and reliability of clinical data. The quantity of cell-free DNA is also a limiting factor. Therefore, a proper preanalytical factor for blood collection, its stability, centrifugation speed, and plasma storage condition are to be optimized for developing cancer-specific biomarkers useful for clinical purpose. Liquid biopsy-based origin of cell-free DNA has revolutionized the area of cancer research. Lack of preanalytical and analytical procedures may be considered for identification of novel biomarkers through next-generation sequencing of tumor-originated cell-free DNA in contradiction to tissue biopsy for cancer-specific biomarkers.

Circulating tumoral DNA: Preanalytical validation and quality control in a diagnostic laboratory.

We present the results of our technical validation process in establishing the analysis of circulating tumor DNA (ctDNA) as a diagnostic tool. Like most cells in our body, tumor cells shed DNA in the blood flow. Analysis of ctDNA mutational content can provide invaluable information on the genetic makeup of a tumor, and assist oncologists in deciding on therapy, or in following residual disease. However, low absolute amounts of circulating DNA and low tumor fraction constitute formidable analytical challenges. A key step is to avoid contamination with genomic DNA from cell lysis. Several brands of specialized blood collection tubes are available to prevent leukocyte lysis. We show that they are not equally efficient, depending on storage temperature and time before plasma preparation. We report our analysis of preanalytical factors pertaining to ctDNA analysis (tubes, transportation time, temperature) and our conclusions in terms of instructions to prescribing physicians. We also stress the importance of proper DNA quality control and compare several methods, including a differential amplicon length PCR technique which allows determination of multiple QC parameters from minimal amounts of DNA. Altogether, these data provide useful practical information to diagnostic laboratories wishing to implement the assay of ctDNA in clinical practice.

Effect of preanalytical and analytical variables on the clinical utility of mean platelet volume.

BACKGROUND: The study endpoint was to analyze the effect of preanalytical (time, temperature, anticoagulant) and analytical (imprecision, correlation between volume and platelet concentration) variables on mean platelet volume (MPV). A further aim was to calculate in an adult population the reference intervals using the Sysmex XE-5000 analyzer. A critical evaluation was also made of the clinical utility of these parameters. METHODS: Analyses of the above values were performed in duplicate in 170 healthy adults of both sexes: (1) within 30 min from collection, and (2) after 4 h. To evaluate stability over time, the value of the platelet parameters of 20 subjects were determined, a re-analysis being performed for a period of up to 24 h on samples maintained at room temperature and 4°C using either K2-EDTA or Na-citrate as anticoagulants. RESULTS: The stability over time of MPV closely depends on the anticoagulant used, storage temperature and time interval between venipuncture and analysis. An inverse, non-linear correlation between MPV and platelet count was also found. CONCLUSIONS: In view of their effect on MPV and other related indices, the preanalytical and analytical variables make them, little more than experimental.

The impact of preanalytical variables on measuring cerebrospinal fluid biomarkers for Alzheimer's disease diagnosis: A review.

INTRODUCTION: Cerebrospinal fluid (CSF) biomarkers have the potential to improve the diagnostic accuracy of Alzheimer's disease, yet there is a lack of harmonized preanalytical CSF handling protocols. METHODS: This systematic review summarizes the current literature on the influence of preanalytical variables on CSF biomarker concentration. We evaluated the evidence for three core CSF biomarkers: ß-amyloid 42, total tau, and phosphorylated tau. RESULTS: The clinically important variables with the largest amount of conflicting data included the temperature at which samples are stored, the time nonfrozen samples can be stored, and possible effects of additives such as detergents, blood contamination, and centrifugation. Conversely, we discovered that there is consensus that tube material has a significant effect. DISCUSSION: A unified CSF handling protocol is recommended to reduce preanalytical variability and facilitate comparison of CSF biomarkers across studies and laboratories. In future, experiments should use a gold standard with fresh CSF collected in low binding tubes.

Deciphering Sources of Variability in Clinical Pathology.

The objectives of this session were to explore causes of variability in clinical pathology data due to preanalytical and analytical variables as well as study design and other procedures that occur in toxicity testing studies. The presenters highlighted challenges associated with such variability in differentiating test article-related effects from the effects of experimental procedures and its impact on overall data interpretation. These presentations focused on preanalytical and analytical variables and study design-related factors and their influence on clinical pathology data, and the importance of various factors that influence data interpretation including statistical analysis and reference intervals. Overall, these presentations touched upon potential effect of many variables on clinical pathology parameters, including animal physiology, sample collection process, specimen handling and analysis, study design, and some discussion points on how to manage those variables to ensure accurate interpretation of clinical pathology data in toxicity studies. This article is a brief synopsis of presentations given in a session entitled "Deciphering Sources of Variability in Clinical Pathology-It's Not Just about the Numbers" that occurred at the 35th Annual Symposium of the Society of Toxicologic Pathology in San Diego, California.

Evaluating the effects of preanalytical variables on the stability of the human plasma proteome.

High quality clinical biospecimens are vital for biomarker discovery, verification, and validation. Variations in blood processing and handling can affect protein abundances and assay reliability. Using an untargeted LC-MS approach, we systematically measured the impact of preanalytical variables on the plasma proteome. Time prior to processing was the only variable that affected the plasma protein levels. LC-MS quantification showed that preprocessing times <6h had minimal effects on the immunodepleted plasma proteome, but by 4 days significant changes were apparent. Elevated levels of many proteins were observed, suggesting that in addition to proteolytic degradation during the preanalytical phase, changes in protein structure are also important considerations for protocols using antibody depletion. As to processing variables, a comparison of single- vs double-spun plasma showed minimal differences. After processing, the impact ⩽3 freeze-thaw cycles was negligible regardless of whether freshly collected samples were processed in short succession or the cycles occurred during 14-17 years of frozen storage (-80 °C). Thus, clinical workflows that necessitate modest delays in blood processing times or employ different centrifugation steps can yield valuable samples for biomarker discovery and verification studies.

The impact of preanalytical variables on the analysis of cell-free DNA from blood and urine samples.

Cell-free DNA (cfDNA), a burgeoning class of molecular biomarkers, has been extensively studied across a variety of biomedical fields. As a key component of liquid biopsy, cfDNA testing is gaining prominence in disease detection and management due to the convenience of sample collection and the abundant wealth of genetic information it provides. However, the broader clinical application of cfDNA is currently impeded by a lack of standardization in the preanalytical procedures for cfDNA analysis. A number of fundamental challenges, including the selection of appropriate preanalytical procedures, prevention of short cfDNA fragment loss, and the validation of various cfDNA measurement methods, remain unaddressed. These existing hurdles lead to difficulties in comparing results and ensuring repeatability, thereby undermining the reliability of cfDNA analysis in clinical settings. This review discusses the crucial preanalytical factors that influence cfDNA analysis outcomes, including sample collection, transportation, temporary storage, processing, extraction, quality control, and long-term storage. The review provides clarification on achievable consensus and offers an analysis of the current issues with the goal of standardizing preanalytical procedures for cfDNA analysis.

Extrinsic and intrinsic preanalytical variables affecting liquid biopsy in cancer.

Liquid biopsy, through isolation and analysis of disease-specific analytes, has evolved as a promising tool for safe and minimally invasive diagnosis and monitoring of tumors. It also has tremendous utility as a companion diagnostic allowing detection of biomarkers in a range of cancers (lung, breast, colon, ovarian, brain). However, clinical implementation and validation remains a challenge. Among other stages of development, preanalytical variables are critical in influencing the downstream cellular and molecular analysis of different analytes. Although considerable progress has been made to address these challenges, a comprehensive assessment of the impact on diagnostic parameters and consensus on standardized and optimized protocols is still lacking. Here, we summarize and critically evaluate key variables in the preanalytical stage, including study population selection, choice of biofluid, sample handling and collection, processing, and storage. There is an unmet need to develop and implement comprehensive preanalytical guidelines on the optimal practices and methodologies.

Preanalytical Variables in Hemostasis Testing.

Hemostasis testing performed in clinical laboratories are critical for assessing hemorrhagic and thrombotic disorders. The assays performed can be used to provide the information required for diagnosis, risk assessment, efficacy of therapy, and therapeutic monitoring. As such, hemostasis tests should be performed to the highest level of quality, including the standardization, implementation, and monitoring of all phases of the testing, which include the preanalytical, analytical, and post-analytical phases. It is well established that the preanalytical phase is the most critical component of the testing process, being the hands-on activities, including patient preparation for blood collection, as well as the actual blood collection, including sample identification and the post-collection handling to include sample transportation, processing, and storage of samples when testing is not performed immediately. The purpose of this article is to provide an update to the previous edition of coagulation testing-related preanalytical variables (PAV) and, when properly addressed and performed, can reduce the most common causes of errors in the hemostasis laboratory.

Effects of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens: A systematic quantitative assessment.

Introduction: Blood samples are the most common biospecimen in biobanks, and RNA from such blood samples is an important material for biomedical research. High-quality RNA is essential for consistent, reliable results. Preanalytical environmental conditions can affect the quality of blood RNA. Here, we carried out a quantitative assessment of the influence of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens in biobanks. Methods: Before RNA purification, blood samples from volunteers were exposed to 4 °C for 2, 6, 12, 24, or 48 h, 3 days, or 1 week, or exposed to room temperature (22-30 °C) for 1, 2, 6, 12, or 24 h. Hemolyzed samples were collected from laboratory department and some of them were prepared using the freeze-thaw method. After exposure to different preanalytical environmental conditions, the RNA simple Total RNA Kit was used to purify the RNA, following which a NanoDrop™ One and Qsep 100 Bio-Fragment Analyzer were used to assess RNA concentration, purity, and integrity. In addition, a part of the RNA was immediately reverse transcribed into cDNA when it was purified, then the relative expression levels of 18S, ACTB, HIF1α, HMOX1, and MKI67 were determined by real-time quantitative PCR. Finally, 30 blood samples were collected from the surplus samples in our laboratory department to assess their RNA quality without knowledge of their storage conditions (duration/temperature). Results: For blood samples stored at 4 °C, there was a significant difference between the RNA integrity after 1 week compared to after 2 h. For blood samples stored at room temperature (22-30 °C), the RNA integrity was also significantly different at 6 h and 0 h. Hemolysis caused by freeze-thawing severely affected RNA quality, whereas clinical hemolysis generally produced no significant effects. Moreover, expression of 18S, ACTB, HIF1α, HMOX1, and MKI67 in whole blood stored under different conditions showed irregular changes, suggesting that preservation conditions are also important for gene expression. Conclusion: RNA integrity was qualified for blood samples stored at 4 °C for up to 72 h or at room temperature (22-30 °C) for up to 2 h. Hemolysis usually does not affect the RNA quality of blood samples unless the hemolysis method damages leukocytes.

Banking of Tumor Tissues: Effect of Preanalytical Variables in the Phase of Pre- and Postacquisition on RNA Integrity.

Background: RNA integrity of tumor tissues from 12 common organs was measured, and tumor tissues from liver were found to have the best RNA integrity in our previous study. The effects of preanalytical variables in the phase of pre- and postacquisition on RNA integrity were further assessed in hepatocellular carcinoma (HCC) tissues in this study. Methods: RNA integrity number (RIN) was measured in tissues from 146 HCC patients. First, 42 fresh HCC tumor tissues were newly collected to assess the effect of various preanalytical variables in the phase of preacquisition on RNA integrity. Second, eight paired HCC tumor and normal tissues were newly collected and used in the gradient course study of ex vivo ischemia time and freeze-thaw cycles on RNA integrity. Finally, 96 stock-frozen tumor tissues with various years of frozen storage were used to assess the effect of cryopreservation time. Results: RNA integrity was found to be independent of patient age, sex, clinical stage, tumor location, HBV infection status, tumor diameter, and surgical approach, but affected by tumor grade. Tumor tissues with a greater tumor grade had lower RIN. With the prolongation of ex vivo ischemia time, freeze-thaw cycles, and cryopreservation time, the RIN of HCC tissues showed decreasing trends. Significant decreases in RIN of the tumor and normal tissues were observed at 6 and 2 hours of ex vivo ischemia time, respectively, and significantly decreased RIN of tumor tissues was observed after six freeze-thaw cycles and 6 years of cryopreservation. Conclusions: Preanalytical variables in the phase of preacquisition such as tumor grade, and in the postacquisition phase such as ex vivo ischemia time, freeze-thaw times, and freeze-storage time both have effects on RNA integrity of HCC tissues. Tissue-based translational research should pay attention to preanalytical variables when collecting and utilizing tumor tissues.

High Counts in Hematologic Malignancies Predict Low Metaphase Yield for Cytogenetic Analysis.

OBJECTIVES: This study examined the impact of various preanalytical variables on metaphase yield in hematologic malignancies. METHODS: Marrow samples from patients with hematologic malignancies that were subjected to cytogenetic analysis were categorized into two groups: one with samples that yielded an adequate number of metaphases, defined as at least 20, and a second with a low number of metaphases (LNM), having fewer than 20 metaphases. Age, sex, bone marrow nucleated cell (MNC) count, and peripheral blood counts (hemoglobin, total WBC count, and platelet count) were analyzed for an association with LNM. RESULTS: Of 455 samples, 17% (79/455) belonged to the LNM group, including 6% (27/455) that yielded no metaphases. MNCs and WBCs were higher in the LNM group (P < .001 for both). MNCs were higher in LNM groups in both acute myeloid leukemia (P = .008) and acute lymphoblastic leukemia (P = .001). Receiver operating characteristic curves showed moderate prediction of MNC and WBC counts for LNM with areas under the curves of 0.7. Other analyzed parameters showed no significant associations with LNM. CONCLUSIONS: Low metaphase yields occur frequently in hematologic malignancies with high counts. This could reflect biological characteristics of these malignancies that merit further investigation.

Detection of preanalytical errors in arterial blood gas analysis.

Introduction: Blood gas analysis (BGA) is an essential test used for years to provide vital information in critically ill patients. However, the instability of the blood gases is a problem. We aimed to evaluate time and temperature effects on blood gas stability. Materials and methods: Arterial blood was collected from 20 patients into syringes. Following BGA for baseline, syringes were divided into groups to stand at 4°C and 22°C for 30, 60, 90, 120 minutes. All were tested for pH, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), oxygen saturation (sO2), oxyhemoglobin (O2Hb), sodium, potassium, glucose, lactate, oxygen tension at 50% hemoglobin saturation (p50), and bicarbonate. A subgroup analysis was performed to detect the effect of air on results during storage. Percentage deviations were calculated and compared against the preset quality specifications for allowable total error. Results: At 4°C, pO2 was the least stable parameter. At 22°C, pO2 remained stable for 120 min, pH and glucose for 90 min, lactate and pCO2 for 60 min. Glucose and lactate were stable when chilled. Air bubbles interfered pO2 regardless of temperatures, whereas pCO2 increased significantly at 22°C after 30 min, and pH decreased after 90 min. Bicarbonate, sO2, O2Hb, sodium, and potassium were the unaffected parameters. Conclusions: Correct BGA results are essential, and arterial sample is precious. Therefore, if immediate analysis cannot be performed, up to one hour, syringes stored at room temperature will give reliable results when care is taken to minimize air within the blood gas specimen.

When Tissue Is the Issue: Expanding Cell-Free DNA "Liquid Biopsies" to Supernatants and Nonplasma Biofluids.

While tissue biopsy remains the gold standard for tumor biomarker testing, assays using plasma-derived cfDNA, aka circulating-tumor DNA (ctDNA), have recently demonstrated validity in the setting of limited tissue or recurrent disease. Tumor-derived cfDNA is also present in nonplasma biofluids and supernatants procured through interventional procedures. Evaluation of cfDNA extracted from these fluids may have benefits at nearly every stage of cancer patient management, from diagnosis and prognosis to monitoring disease progression and predicting therapeutic response. This review will focus on preanalytical, analytical, and postanalytical variables that must be considered when analyzing "liquid biopsies" outside the plasma compartment.

Review of coagulation preanalytical variables with update on the effect of direct oral anticoagulants.

There are many preanalytical variables (PAV) that are known to affect coagulation testing. The more commonly acknowledged PAV addressed by the clinical laboratory tend to start with their influence on blood collection, but realistically coagulation PAV starts with the patient, where the laboratory has less influence or control. Patient selection and appropriate timing for blood collection may be integral for assuring proper diagnosis and management. Laboratory control and assurance for ideal phlebotomy practice would mitigate most PAVs related to blood collection to minimize suboptimal sample collection. Laboratory oversight of sample transportation, processing and storage will assure sample integrity until testing can be facilitated. The purpose of this document is to review common PAV that should be taken into consideration when ordering, performing and interpreting a coagulation test result, with additional attention to the effect of direct oral anticoagulants (DOACs).

The influence of delay in mononuclear cell isolation on acute myeloid leukemia phosphorylation profiles.

Mass-spectrometry (MS) based phosphoproteomics is increasingly used to explore aberrant cellular signaling and kinase driver activity, aiming to improve kinase inhibitor (KI) treatment selection in malignancies. Phosphorylation is a dynamic, highly regulated post-translational modification that may be affected by variation in pre-analytical sample handling, hampering the translational value of phosphoproteomics-based analyses. Here, we investigate the effect of delay in mononuclear cell isolation on acute myeloid leukemia (AML) phosphorylation profiles. We performed MS on immuno-precipitated phosphotyrosine (pY)-containing peptides isolated from AML samples after seven pre-defined delays before sample processing (direct processing, thirty minutes, one hour, two hours, three hours, four hours and 24 h delay). Up to four hours, pY phosphoproteomics profiles show limited variation. However, in samples processed with a delay of 24 h, we observed significant change in these phosphorylation profiles, with differential phosphorylation of 22 pY phosphopeptides (p < 0.01). This includes increased phosphorylation of pY phosphopeptides of JNK and p38 kinases indicative of stress response activation. Based on these results, we conclude that processing of AML samples should be standardized at all times and should occur within four hours after sample collection. SIGNIFICANCE: Our study provides a practical time-frame in which fresh peripheral blood samples from acute myeloid patients should be processed for phosphoproteomics, in order to warrant correct interpretation of in vivo biology. We show that up to four hours of delayed processing after sample collection, pY phosphoproteomic profiles remain stable. Extended delays are associated with perturbation of phosphorylation profiles. After a delay of 24 h, JNK activation loop phosphorylation is markedly increased and may serve as a biomarker for delayed processing. These findings are relevant for biomedical acute myeloid leukemia research, as phosphoproteomic techniques are of particular interest to investigate aberrant kinase signaling in relation to disease emergence and kinase inhibitor response. With these data, we aim to contribute to reproducible research with meaningful outcomes.