A critical review of current laboratory methods used to evaluate mosquito repellents.

Pathogens transmitted by mosquitoes threaten human health around the globe. The use of effective mosquito repellents can protect individuals from contracting mosquito-borne diseases. Collecting evidence to confirm and quantify the effectiveness of a mosquito repellent is crucial and requires thorough standardized testing. There are multitudes of methods to test repellents that each have their own strengths and weaknesses. Determining which type of test to conduct can be challenging and the collection of currently used and standardized methods has changed over time. Some of these methods can be powerful to rapidly screen numerous putative repellent treatments. Other methods can test mosquito responses to specific treatments and measure either spatial or contact repellency. A subset of these methods uses live animals or human volunteers to test the repellency of treatments. Assays can greatly vary in their affordability and accessibility for researchers and/or may require additional methods to confirm results. Here I present a critical review that covers some of the most frequently used laboratory assays from the last two decades. I discuss the experimental designs and highlight some of the strengths and weaknesses of each type of method covered.

Automation of a Factor XIII Activity Assay Utilizing a Plasma Blank Measurement.

Factor XIII (FXIII) is an essential coagulation factor that stabilizes fibrin clots and allows the clot to resist fibrinolysis. Inherited or acquired FXIII deficiency is a severe bleeding disorder with manifestations that can include fatal intracranial hemorrhage. Accurate FXIII laboratory testing is necessary for diagnosis, subtyping, and treatment monitoring. The recommended first-line test is FXIII activity, most commonly performed by commercial ammonia release assays. In these assays, it is important to perform a plasma blank measurement to correct for FXIII-independent ammonia production, which can lead to clinically significant overestimation of FXIII activity. Automated performance of a commercial FXIII activity assay (Technoclone, Vienna, Austria), including blank correction, on the BCS XP instrument is described.

Current strategies to determine antifungal and antimicrobial activity of natural compounds.

Fungal and microbial infections are increasingly common diseases affecting not only humans, but also animals. Despite the fact that there are wide ranges of antifungal drugs that can be used as therapy against different types of mycosis, the large-scale needed for new antifungal and antimicrobial agents is undeniable. The reasons for a great demand for new agents are low effectiveness due to the development of resistance, host toxicity and various side effects of currently used therapeutics. In order to develop novel drugs against fungal infections, scientists need to search for new molecules that show antimicrobial activity. However, there are various methods to determine antifungal and antimicrobial activity such as diffusion methods, bioautography methods, dilution methods and other frequently used methods. This review aims to explain the methodologies mentioned, to highlight the functioning, usage, advantages and disadvantages and to compare the techniques using different sources of the last years. Additionally, some of the currently investigated natural compounds such as essential oils, which show promising results in the medication of fungal diseases, are mentioned.

Obstacles to Early Diagnosis and Treatment of Inherited von Willebrand Disease: Current Perspectives.

Von Willebrand disease (VWD), the most common inherited bleeding disorder, is highly heterogeneous, and its early diagnosis may be difficult, especially for mild cases and in qualitative von Willebrand factor (VWF) defects. Appropriate VWD diagnosis requires the combination of personal and/or family history of bleeding and abnormal VWF laboratory testing. The use of bleeding assessment tools has been helpful in standardizing bleeding history collection and quantification of bleeding symptoms to select patients who may benefit of further hemostatic testing. Type 1 and 3 VWD which represent quantitative VWD variants are relatively easy to diagnose. The diagnosis of type 2 VWD requires multiple assessments to evaluate the effects induced by the responsible abnormality on the heterogeneous functions of VWF. Sensitive and reproducible tests are needed to evaluate different VWF activities, starting from measuring VWF-platelet interaction. In the recent years, several increasingly sensitive, rapid and automated assays have been developed, but they are not widely available so far. Genetic testing for VWD diagnosis is not a common practice because VWF gene is very large and highly polymorphic and therefore it is used only in specific cases. It is evident that the early and correct VWD diagnosis allows optimal management of bleeding and situations at risk. Tranexamic acid, desmopressin, replacement therapy with plasma-derived concentrates with a variable content of VWF and FVIII, or the new recombinant VWF are the different therapeutic options available. Careful VWD classification guides treatment because desmopressin is widely used in type 1 while replacement therapy is the cornerstone of treatment for type 2 and 3 variants.

Variation in common laboratory test results caused by ambient temperature.

BACKGROUND: Laboratory tests measure important aspects of physiology, but their results also vary for idiosyncratic reasons. We explore an underappreciated source of variation: ambient temperature on the day blood is drawn. METHODS: In a sample of 4,877,039 individuals between 2009-2015, we model 215,234,179 test results as a function of temperature, controlling for individual and city-week fixed effects. This measures how day-to-day temperature fluctuations affect results over and above the individual's mean values, and seasonal variation. FINDINGS: 51 of 75 assays are significantly affected by temperature, including measures of kidney function (increased creatinine, urea nitrogen, and urine specific gravity), cellular blood components (decreased neutrophils, erythrocytes, and platelets), and lipids (increased high-density lipoprotein [HDL] and decreased total cholesterol, triglycerides, and low-density lipoprotein [LDL]). These small, day-to-day fluctuations are unlikely to correlate with long-term physiological trends; for example, lipid panels checked on cooler days look lower risk, but these short-term changes probably do not reflect stable changes in cardiovascular risk. Nonetheless, doctors appear to treat these individuals differently. We observe 9.7% fewer statin prescriptions for individuals checked on the coolest versus the warmest days (-0.42% versus baseline of 4.34%, p < 0.001). CONCLUSIONS: Ambient temperature affects the results of many laboratory tests. These distortions, in turn, affect medical decision-making. Statistical adjustment in reporting is feasible and could limit undesired temperature-driven variability. FUNDING: None.

Revisiting the activated protein C-protein S-thrombomodulin ternary pathway: Impact of new understanding on its laboratory investigation.

Although suspected conceptually in the 60 s, Protein C and Protein S activities in hemostasis were investigated and reported from the mid-80 s, followed by the discovery of Thrombomodulin, an endothelial cell membrane associated protein, playing the most important heamostatic role. These 3 proteins act in regulating thrombogenesis and protecting against thrombo-embolic events. When blood is activated, any trace of circulating thrombin is captured by Thrombomodulin in the microcirculation, making thrombin become an anticoagulant through its capacity to activate Protein C to Activated Protein C, which operates as a sentinel in blood coagulation, in the form of a complex with free Protein S, to block any new blood activation site, and more especially circulating activated Factors V and VIII. Protein S not only acts as the Activated Protein C cofactor, but also as the cofactor of Tissue Factor Pathway Inhibitor. In addition, it has some functions in the complement pathway through its binding to C4b-BP. Another capability of activated protein C is to lower fibrinolytic activity, as the Activated Protein C Inhibitor is also known as Plasminogen Activator Inhibitor 3. The Protein C-Protein S system becomes less efficient in the presence of mutated Factor V (Factor V-Leiden or other variants), which is resistant to its inactivating effect. Other pathologies linked to this system concern the development of allo- or auto-antibodies to Protein S or to thrombin, which can generate severe thrombotic complications in affected patients. Some antithrombotic drugs have originated from this regulatory system. Protein C or Protein S concentrates are used for treating deficient patients. Activated Protein C (especially in patients with sepsis) or Thrombomodulin are proposed as antithrombotic medications. Most importantly, congenital or acquired Protein C or Protein S deficiencies are associated with severe recurrent thrombotic events. From the clinical standpoint most of the patients are heterozygous, as homozygosity is almost incompatible with life in the absence of a continuous and efficient treatment. Laboratory investigation of this highly complex system involves many different specialized assays for measuring these 3 proteins' activities, their antigenic content or their genetic sequence. The Protein S in-vitro anticoagulant activity is weak and contrasts with its high antithrombotic role in-vivo, showing that diagnostic assays have not yet succeeded in reproducing all the natural mechanisms for evidencing the anticoagulant role of Protein S. This paradoxal notion is discussed and illustrated in this manuscript as well is a revisit of the major characteristics and pathophysiological functions of the Protein C-Protein S-Thrombomodulin system; the associated pathologies; and the main laboratory tools available for clinical diagnosis. In respect to future perspectives, we also focused on developing more significant and relevant assays, especially for Protein S, thanks to the understanding of its biological roles.

Extended Half-Life Coagulation Factors: A New Era in the Management of Hemophilia Patients

Despite effective factor replacement and various treatment schedules, there remain several challenges and unmet needs in the prophylactic treatment of hemophilia limiting its adoption and thereby posing an increased risk of spontaneous bleeding. In this regard, extended half-life (EHL) recombinant factor VIII (rFVIII) and factor IX (rFIX) products promise optimal prophylaxis by decreasing the dose frequency, increasing the compliance, and improving the quality of life without compromising safety and efficacy. EHL products might lead to higher trough levels without increasing infusion frequency, or could facilitate the ability to maintain trough levels while reducing infusion frequency. This paper aims to provide a comprehensive review of the rationale for developing EHL coagulation factors and their utility in the management of hemophilia, with special emphasis on optimal techniques for half-life extension and criteria for defining EHL coagulation factors, as well as indications, efficacy, and safety issues of the currently available EHL-rFVIII and EHL-rFIX products. Potential impacts of these factors on quality of life, health economics, and immune tolerance treatment will also be discussed alongside the challenges in pharmacokinetic-driven prophylaxis and difficulties in monitoring the EHL products with laboratory assays.

Microarray Immunodiagnostics for Aeroallergens.

PURPOSE OF REVIEW: The impact of new technologies, especially multiplexed molecular allergy diagnostics based on allergen arrays, on the management of complex patients with respiratory allergies has been important. RECENT FINDINGS: Currently, the detailed characteristics of the IgE profile of the patient, such as sensitization to genuine or cross-reacting components or the sensitization to potentially harmful allergens, allow an allergist to tailor treatment in the context of precision medicine rules. A number of relevant articles have been published in recent years on this topic, and, in this review, the new added values of allergen array-based diagnostics are reported.

Laboratory assay measurement of modified clotting factor concentrates: a review of the literature and recommendations for practice.

Over the past several years, novel modified clotting factor concentrates (CFCs) have been introduced into practice and are now widely prescribed in the countries where they are licensed. These products allow for less frequent infusions of CFC, thereby providing improved convenience and/or higher trough levels. They have been extensively studied for prophylaxis, episodic treatment of bleeding and for surgical prophylaxis. One issue that has emerged regarding the clinical application of these products revolves around the measurement of infused CFC in the clinical coagulation laboratory. Recent studies have demonstrated significant problems with the measurement of correct FVIII/IX levels following infusion of novel CF VIII/IX concentrates. The source of this problem appears to be related to the tremendous variability of the APTT reagents that are used in the one-stage clotting assay, the most commonly used assay for determining factor levels. More specifically, the issue is related to the type of activator used in the reagents. Depending on the combination of the CFC and the APTT activator, the observed results may be either under- or overestimated to degrees that would be clinically relevant. Recommendations based on a review of published information regarding the potential for incorrect measurements of factor VIII/IX levels following infusion of recently developed, novel factor VIII/IX CFCs are presented for the clinician to use in clinical practice.

Revisiting antithrombin in health and disease, congenital deficiencies and genetic variants, and laboratory studies on α and ß forms.

Antithrombin [AT] is the main inhibitor for activated plasma coagulation serine esterases, inhibiting thrombin, Factors Xa and IXa, but also Factors XIIa, XIa, VIIa, kallicrein, and plasmin. Its activity is highly enhanced by heparin, through binding to the pentasaccharide sequences, for inhibition of all coagulation proteases, except thrombin, which inhibition requires its additional binding to the heparin polysaccharide chain. However, AT is the major inhibitor of thrombin in the blood circulation. Congenital or acquired deficiencies of AT expose affected patients to an increased risk of developing unprovoked and recurrent thrombo-embolic diseases. Antithrombin can be measured with various laboratory techniques, by either immunological or functional methods. Earlier, a radial immunodiffusion immunoassay allowed measurement of the protein antigenic content. Functional assays are mainly designed with Anti-Thrombin or Anti-Factor Xa chromogenic methods and are useful for detecting genetic molecular mutations with decreased inhibitory activity and contributed to study the conformational changes of antithrombin and its variants, which potentially regulate the activity of this serine protease inhibitor. These assays are not equivalent in terms of diagnosing protein abnormalities, associated with increased thrombotic incidence, and they have variable performance for reflecting impaired antithrombin binding capacity for heparin, reduced progressive inhibition of serine proteases, or accelerated switch rates to the latent and less active forms. A small proportion of AT (<10%) is present in blood in the ß-form, with a lower oligosaccharide content, a lower Molecular Weight, a higher binding rate to endothelial glycosaminoglycans, and a higher anticoagulant activity, hence requiring specific laboratory methods for its measurement. The ß-AT form is then of critical importance for controlling blood activation by tissue injury and preventing development of thrombo-embolic diseases. This article reviews the performance characteristics of the currently available assays, and their usefulness for monitoring the use of AT concentrates in intensive care units, disseminated intravascular coagulation or severe infections, to restore the anticoagulant protective effect of heparin by supplementing the requested AT concentration. The issues of automation, harmonization and standardization are also revisited and discussed.

Factor XIII deficiency diagnosis: Challenges and tools.

Factor XIII deficiency (FXIIID) is a rare hereditary bleeding disorder arising from heterogeneous mutations, which can lead to life-threatening hemorrhage. The diagnosis of FXIIID is challenging due to normal standard coagulation assays requiring specific FXIII assays for diagnosis, which is especially difficult in developing countries. This report presents an overview of FXIIID diagnosis and laboratory methods and suggests an algorithm to improve diagnostic efficiency and prevent missed or delayed FXIIID diagnosis. Assays measuring FXIII activity: The currently available assays utilized to diagnose FXIIID, including an overview of their complexity, reliability, sensitivity, and specificity, as well as mutational analysis are reviewed. The use of a FXIII inhibitor assay is described. Diagnostic tools in FXIIID: Many laboratories are not equipped with quantitative FXIII activity assays, and if available, limitations in lower activity ranges are important to consider. Clot solubility tests are not standardized, have a low sensitivity, and are therefore not recommended as routine screening test; however, they are the first screening test in almost all coagulation laboratories in developing countries. To minimize the number of patients with undiagnosed FXIIID, test quality should be improved in less well-equipped laboratories. Common country-specific mutations may facilitate diagnosis through targeted genetic analysis in reference laboratories in suspected cases. However, genetic analysis may not be feasible in every country and may miss spontaneous mutations. Centralized FXIII activity measurements should also be considered. An algorithm for diagnosis of FXIIID including different approaches dependent upon laboratory capability is proposed.

Assays for estimating HIV incidence: updated global market assessment and estimated economic value.

INTRODUCTION: Accurate incidence estimates are needed to characterize the HIV epidemic and guide prevention efforts. HIV Incidence assays are cost-effective laboratory assays that provide incidence estimates from cross-sectional surveys. We conducted a global market assessment of HIV incidence assays under three market scenarios and estimated the economic value of improved incidence assays. METHODS: We interviewed 27 stakeholders, and reviewed journal articles, working group proceedings, and manufacturers' sales figures. We determined HIV incidence assay use in 2014, and estimated use in 2015 to 2017 and in 5 to 10-years under three market scenarios, as well as the cost of conducting national and key population surveys using an HIV incidence assay with improved performance. RESULTS: Global 2014 HIV incidence assay use was 308,900 tests, highest in Asia and mostly for case- and population-based surveillance. Estimated 2015 to 2017 use was 94,475 annually, with declines due to China and the United States discontinuing incidence assay use for domestic surveillance. Annual projected 5 to 10 year use under scenario 1 - no change in technology - was 94,475. For scenario 2 - a moderately improved incidence assay - projected annual use was 286,031. Projected annual use for scenario 3 - game-changing technologies with an HIV incidence assay part of (a) standard confirmatory testing, and (b) standard rapid testing, were 500,000 and 180 million, respectively. As HIV incidence assay precision increases, decreased sample sizes required for incidence estimation resulted in $5 to 23 million annual reductions in survey costs and easily offset the approximately $3 million required to develop a new assay. CONCLUSIONS: Improved HIV incidence assays could substantially reduce HIV incidence estimation costs. Continued development of HIV incidence assays with improved performance is required to realize these cost benefits.

Effects of direct oral anticoagulants on lupus anticoagulant assays in a real-life setting.

Laboratory diagnosis of lupus anticoagulant (LA) is based on prolongation in at least one coagulation assay (diluted Russell's viper venom time - dRVVT or activated partial thromboplastin time - aPTT), which normalises after addition of phospholipids. Both assays may be influenced by anticoagulants and therefore LA should not be tested during warfarin or heparin treatment. It has been shown (primarily in vitro) that direct oral anticoagulants (DOACs - dabigatran [DAB], rivaroxaban [RIV] and apixaban [API]) may also influence LA testing. We tested the effects of DOACs on assays routinely used for the diagnosis of LA in patients treated with these drugs in a real-life setting. Plasma from patients with atrial fibrillation treated with DAB (n=30), RIV (n=20) and API (n=17) and not known to have LA were tested using dRVVT (LA-screen and LA-confirm, Life Diagnostics) and aPTT (PTT-LA, Diagnostica Stago and aPTT Actin FS, Siemens Healthcare Diagnostics) assays. According to the diagnostics algorithm, dRVVT and aPTT ratios of <1.2 were considered negative, ratios of >1.4 positive, while if the ratios were 1.2-1.4 LA could not be ruled out. Plasma concentrations varied between 8-172 µg/l for DAB, 8-437 µg/l for RIV and 36-178 µg/l for API. LA diagnosis was negative in only eight (27 %) plasma samples from patients treated with DAB, and in five (25 %) and four samples (24 %) from patients treated with RIV and API, respectively. LA Positivity (dRVVT and aPTT ratios >1.4) was found in 5 cases (17 %) among patients treated with DAB, in 10 cases (50 %) treated with RIV and in 7 cases (41 %) treated with API. A concentration-dependent effect of DOACs on dRVVT-based parameters was observed, particularly as regards DAB. At lower concentrations, RIV and API had only minor effects on the confirmatory tests (below 100 µg/l and 70 µg/l, respectively). Our results suggest that a risk of overestimation of LA detection is present in samples from patients treated with DOACs. Therefore, LA testing should not be performed during treatment with DOACs. Prolongation in confirmatory assays may be helpful for the recognition of false positivity, especially as regards DAB.

The second Team Haemophilia Education Meeting, 2016, Frankfurt, Germany.

The first Team Haemophilia Education (THE) Meeting was held on 7-8 May 2015 in Amsterdam, The Netherlands. It aimed to promote the optimal care of patients with haemophilia through education of the multidisciplinary treatment team. This was achieved by reviewing the latest developments in haemophilia management, considering how these can be implemented in the clinic to improve patient care and providing a platform for networking and debate for all haemophilia treatment team members. The second THE Meeting was held on 19-20 May in Frankfurt, Germany, and participants included doctors, nurses, physiotherapists, patient representatives and data management staff from 20 different countries. Topics covered the role of the multidisciplinary team in delivering the best haemophilia care, challenges in the management of haemophilia across Europe, available clotting factor treatments, future treatments and the use of genetics in advising carriers of haemophilia. This report is a summary of the key developments in haemophilia care presented by various investigators and healthcare professionals at THE Meeting 2016.

Testing for Cytomegalovirus in Pregnancy.

Congenital cytomegalovirus (CMV) infection represents a relevant cause of deafness and neurological damage in newborns. Intrauterine CMV transmission might result after primary or nonprimary infections, though at different rates (30% versus 0.2%, respectively). At present, a prenatal diagnosis of CMV infection is based mainly on maternal serology, the detection of CMV-DNA in amniotic fluid and fetal blood, and ultrasound (US) and magnetic resonance imaging (MRI). Recent evidences suggest that congenital CMV infection may be an immune-mediated disease and that evaluation of humoral and especially T-cell immunities may improve the overall prenatal diagnosis. This review summarizes the most recent advancements in the diagnosis of maternal and prenatal CMV infections.

Anti-Xa bioassays for the laboratory measurement of direct Factor Xa inhibitors in plasma, in selected patients.

In the past decade Direct Oral Anti-Coagulants (DOACs), targeting Thrombin or Factor Xa, have enormously facilitated the daily treatment of all relevant patients, including those requiring lifelong therapy. These DOACs have considerable advantages over the use of oral Vitamin K Antagonist (VKA) treatments, in view of having little interferences with food and other medications and also not requiring adjustment for age, gender or weight, with some well-defined exceptions. In this current What's Happening Section we focus on measurements of DiXaIs in plasma using anti-Xa assays, with the objective of providing a tribute to Professor Michel Meyer Samama, who was not only a real leader in this field but, in the past, both authors benefited from his wisdom, as a teacher who dedicated his scientific and professional life (among many other interests in hemostasis, thrombosis and fibrinolysis) to develop and promote methods and strategies for laboratory monitoring of anticoagulants. This review presents the performance characteristics of the Anti-Factor Xa assays (measuring Factor Xa inhibition by drugs), which are available for measuring Direct Factor Xa Inhibitors in plasma, and show good compliance of the results with the reference LC:MS method (which measures the mass of Direct Factor Xa Inhibitors). We also present the preparation and validation of drug specific plasma calibrators and controls which are requested for drug measurements. These assays are convenient and practical laboratory tools which can be used in any laboratory setting, and meet the requirements of regulatory bodies for making smart, quantitative, sensitive, accurate and ease of use assays for measuring DOACs when needed. The manuscript focuses mainly on the following areas of current interest: interference in coagulation assays; anti-Xa laboratory methods; development of calibrators and controls for DiXaIs; method validation and comparison with reference techniques (LC:MS); regulatory requirements and method registrations; newer clinical applications and experience on DiXaIs with Anti-Xa assays, and future perspectives.

The first Team Haemophilia Education meeting, 2015, Amsterdam, The Netherlands.

Haemophilia remains a complex disorder to diagnose and manage, requiring close cooperation between multidisciplinary healthcare professionals. There are still many unmet challenges in haemophilia care. The first Team Haemophilia Education (THE) meeting, held on 7-8 May 2015 in Amsterdam, The Netherlands, aimed to promote the optimal care of haemophilia patients through education of the multidisciplinary treatment team. This was achieved by reviewing the latest developments in haemophilia management, considering how these can be implemented in the clinic to improve patient care and providing a platform for networking and debate for all haemophilia treatment team members. Haemophilia treatment centres from several countries were asked to complete a premeeting online questionnaire to establish the biggest challenges that they face when managing patients. The concerns expressed were used to develop the agenda, which comprised a combination of formal presentations, case studies and informal workshops covering such topics as pharmacokinetics, laboratory assays and tailoring of treatment to individual patients. This report is a summary of the key developments in haemophilia care presented by various investigators and healthcare professionals at THE meeting 2015.

Direct Oral Anticoagulants (DOACs) in the Laboratory: 2015 Review.

Direct oral anticoagulant therapies, including direct anti-Xa and thrombin inhibitors have recently been introduced and may have advantages over vitamin K antagonists such as warfarin. This review describes briefly the clinical utility and mechanism of action of these agents. Detailed information is provided on effect of these agents on routine assays including the APTT and PT as well as their impact on specialty laboratory assays. Also included are the use of drug specific assays and a discussion of alternative methods to determine relative drug concentration, such as evaluating drug calibrators in APTT and PT assays and using heparin calibrated anti-Xa assays to measure direct Xa inhibitors.

Statistical estimation of antibody concentration using multiple dilutions.

In medicine and chemistry, measurement of concentrations usually involves calibration that maps the observed responses to the underlying concentration using inversion of a standard curve. The Enzyme-linked ImmunoSorbent Assay (ELISA) is one example of such methods that is commonly used to measure antibody concentration. A problem in this and similar type of technology is that an accurate measurement is obtainable only if the observations fall within the optimal, near-linear range of the standard curve. It is common to conduct a series of doubling or tripling dilutions of the samples, so that at least some of the diluted samples are within the optimal range. A single dilution may then be selected for statistical analysis. This common practice does not fully utilize the data from multiple dilutions and reduces accuracy. We consider two weighted average estimators for fully utilizing the information from multiple dilutions. The first uses weights inversely proportional to the variances of the dilution-specific calibrated values; the second is a simplified form of the first. Simulation results demonstrated the superiority of this weighted estimation approach over the conventional approach of analyzing a single selected dilution. We apply the methods to an experimental study of malaria vaccine candidates.

Laboratory testing issues for protein C, protein S, and antithrombin.

Thrombophilia is a complex disease process, which clinically expresses as venous thrombosis. The presence of a genetic defect in one of the major contributing components (protein C [PC], protein S [PS], and antithrombin [AT]) to thrombophilia can be determined by clinical laboratory assays. However, understanding the limitations and problems associated with assays is paramount to an accurate analysis of the genetic status. This review will discuss the major analytical issues and provide recommendations for assaying PC, PS, and AT in plasma. Recommendations are also made about pre-analytical and postanalytical issues clinically affecting these assays.