Analytical and Bioanalytical Chemistry (v.393, #5)

Quality assurance challenge 7 by Manfred Reichenbächer; Jürgen W. Einax (1377-1379).

Solution to spectroscopy challenge 13 by Reinhard Meusinger (1381-1382).

Diagnostic assays by Erwin Schleicher (1391-1392).
is Professor of Clinical Chemistry and Head of the Clinical Chemistry Unit at the University Clinic Tübingen, Germany. His major interests focus on the molecular mechanism of insulin signal transduction and its impairment, leading to insulin resistance and type 2 diabetes, and on the pathogenesis of diabetic nephropathy. In clinical chemistry his major interests are the search for laboratory results indicating the development of diabetes and its complications and on the miniaturization of diagnostic assays. His scientific work is published in more than 200 contributions.

Point-of-care testing of proteins by Axel Warsinke (1393-1405).
Point-of-care testing (POCT) is a fast developing area in clinical diagnostics that is considered to be one of the main driving forces for the future in vitro diagnostic market. POCT means decentralized testing at the site of patient care. The most important POCT devices are handheld blood glucose sensors. In some of these sensors, after the application of less than 1 µl whole blood, the results are displayed in less than 10 s. For protein determination, the most commonly used devices are based on lateral flow technology. Although these devices are convenient to use, the results are often only qualitative or semiquantitative. The review will illuminate some of the current methods employed in POCT for proteins and will discuss the outlook for techniques (e.g., electrochemical immunosensors) that could have a great impact on future POCT of proteins.
Keywords: POCT; In vitro diagnostics; Lateral flow; Immunosensors; Biosensors; Bioanalytical methods; Immunoassays/ELISA; Clinical/biomedical analysis; Electrochemical sensors/mass-sensitive sensors; Point-of-care

Protein microarrays for diagnostic assays by Michael Hartmann; Johan Roeraade; Dieter Stoll; Markus F. Templin; Thomas O. Joos (1407-1416).
Protein microarray technology has enormous potential for in vitro diagnostics (IVD). Miniaturized parallelized immunoassays are perfectly suited to generating a maximum of diagnostically relevant information from minute amounts of sample whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first products are already on the market. This article reviews the current state of protein microarrays and discusses developments and future demands relating to protein arrays in their role as multiplexed immunoassays in the field of diagnostics.
Keywords: Protein microarrays; Multiplexed diagnostics; In-vitro diagnostics; Focused protein-profiling; Analytical microarrays

Biosensor analyses of serum autoantibodies: application to antiphospholipid syndrome and systemic lupus erythematosus by Markus Thaler; Alexander Buhl; Harald Welter; Anita Schreiegg; Marcus Kehrel; Bettina Alber; Jochen Metzger; Peter B. Luppa (1417-1429).
Autoimmune disorders are rare human diseases characterized by the presence of circulating autoantibodies that bind the body’s own structural compounds as target antigens. The detection of autoantibodies is important for the diagnostic process. Immunofluorescence and immunoassay methods do not allow a reliable characterization of binding characteristics. Therefore, novel analytical techniques should be considered. This review describes the application of surface plasmon resonance biosensor systems for the diagnosis of autoimmune disorders. The covalent attachment of native antigens to the sensor chip is a suitable method for obtaining highly reproducible analyses of autoantibodies, allowing the evaluation of kinetic rate and affinity constants, and it may enable the identification of disease-relevant autoantibodies linked to disease progression. The autoantibody microarray is another future-oriented technique. Patterns of differential antigen recognition should allow early diagnosis. This is due to the fact that a broad range of autoreactive B cell responses in autoimmune disorders can only be mirrored by including a sufficient number of antigens in a microarray format.
Keywords: Antiphospholipid syndrome; β2-glycoprotein I; Cardiolipin; Double-stranded DNA; Systemic lupus erythematosus; Surface plasmon resonance

The development of noninvasive methods for diagnosis and prognosis of many diseases still remains a challenging task in clinical medicine. In recent years, protein separation and detection techniques have been refined such that they allow proteome screening of biological fluids with good reproducibility, accuracy, and real-time performance. The advancements in this field resulted in the initiation and conduction of many clinical studies aimed towards defining sensitive and selective biomarkers for disease or a pathological condition. In this review, we focus on the description of the state of the art proteomic platforms established or adapted to use urine as sample source for biomarker discovery. In the second part of this review, we will give an overview of recent clinical studies that used capillary electrophoresis–mass spectrometry (CE-MS) in urinary proteomics/peptidomics. Owing to its fast and accurate one-step screening method CE-MS allows resolution of the urinary small molecular weight proteome in high-throughput mode. This makes CE-MS well suited for use in large proteomic studies. The fact that urinary proteomic studies are not restricted to renal and urological disorders, but also enable assessment of systemic diseases like cardiovascular or infectious diseases, clearly demonstrates the considerably high potential that urine offers for biomarker discovery and for diagnosis, staging, prognosis, and progression monitoring of diseases.
Keywords: Biomarker discovery; Capillary electrophoresis–mass spectrometry; Urinary proteome analysis

Recent and future trends in blood group typing by Wim Malomgré; Birgid Neumeister (1443-1451).
Blood group typing is the process of testing red blood cells to determine which antigens are present and which are absent. It is standard practice to test for A, B, and D (Rh) antigens and to perform tests for other antigens in selected cases. ABO blood group typing is confirmed by reverse grouping that detects expected isoagglutinins. Unexpected antibodies can be demonstrated by antibody screening tests. For transfusion, donor units compatible with the patient are selected. Prior to transfusion, a crossmatch is performed as a final check for incompatibility. This article describes the recent and future methods of blood group typing and testing of serological compatibility. In addition, methods for blood bank automation are presented.
Keywords: Blood groups; Blood group typing; Automation

Cardiac markers: a clear cause for point-of-care testing by Ulrich Friess; Maik Stark (1453-1462).
Point-of-care testing (POCT) in patients with ischemic heart disease is driven by the time-critical need for fast, specific, and accurate results to initiate therapy instantly. According to current guidelines, the results of the cardiac marker testing should be available to the physician within 30 min (“vein-to-brain” time) to initiate therapy within 60–90 min (“door-to-needle” time) after the patient has arrived at the emergency room or intensive care unit. This article reviews the current efforts to meet this goal (1) by implementing POCT of established biochemical markers such as cardiac troponins, creatine kinase MB, and myoglobin, in accelerated diagnosis and management workflow schemes, (2) by improving current POCT methods to obtain more accurate, more specific, and even faster tests through the integration of optical and electrochemical sensor technology, and (3) by identifying new markers for the very early and sensitive detection of myocardial ischemia and necrosis. Furthermore, the specific requirements for cardiac POCT in regard to analytical performance, comparability, and diagnostic sensitivity/specificity are discussed. For the future, the integration of new immunooptical and electrochemical chip technology might speed up diagnosis even further. However, every new development will have to meet the stringent method validation criteria set for corresponding central laboratory testing.
Keywords: Point-of-care testing; Cardiac markers; Acute myocardial infarction; Biological samples; Biosensors

Patient self-testing and self-management of oral anticoagulation by Siegmund Braun; Michael Spannagl; Heinz Völler (1463-1471).
The development of portable instruments for measuring the prothrombin time (PT) in capillary whole blood has made it possible for patients on lifelong coumarin therapy to monitor the effect of the anticoagulant themselves (patient self-testing) and to adjust the dosage if necessary on their own (patient self-management). Dosage variations between individual patients and the narrow therapeutic range of the anticoagulant account for the need to monitor its effect, in order to minimize the risk of thromboembolism resulting from inadequate anticoagulation as well as the risk of bleeding due to overanticoagulation. This review focuses on the prerequisites for the successful implementation of patient self-testing and self-management, such as the selection of eligible patients, appropriate training and the reliability of available instruments. The outcomes of relevant clinical studies involving patient self-testing and self-management are also discussed.
Keywords: Bioanalytical methods; Clinical/biomedical analysis; Pharmaceuticals

Inflammation markers in point-of-care testing (POCT) by Albrecht Pfäfflin; Erwin Schleicher (1473-1480).
Inflammation is a central issue in medicine. Inflammatory processes may be local or systemic, acute or chronic, and they may be benign or fatal. In bacterial or viral infections fast and reliable diagnosis is essential for appropriate treatment, e.g. antimicrobial therapy. The time to diagnosis is critical because uncontrolled infections may lead to sepsis with a mortality rate close to 50%. Beside clinical signs, laboratory markers are important in detecting, differentiating, and monitoring inflammation, particularly acute infections. Currently several inflammation markers including leukocyte count and leukocyte differentiation, C-reactive protein (CRP), procalcitonin (PCT), and interleukins (IL) 6 and 8, is available, and potential future serum markers are under development. In this article the clinical use of these markers in routine laboratory and in point-of-care testing is described and the diagnostic value of the four groups of laboratory marker is compared. Current data show that leukocyte count or, better, neutrophil count, CRP, and PCT are well suited to support of rapid diagnosis of inflammation and infections in children and adults whereas measurement of IL-6 and 8 are preferable for detection of sepsis in neonates.
Keywords: Point-of-care testing; Inflammation; Infection; Sepsis; CRP; Procalcitonin; Leukocyte; Granulocyte; Interleukin cost-efficiency; Clinical outcome

Newborn screening for inborn errors of metabolism and endocrinopathies: an update by Ralph Fingerhut; Bernhard Olgemöller (1481-1497).
Newborn screening for inborn errors of metabolism and endocrinopathies has expanded during the last two decades, mainly owing to the introduction of new technologies such as tandem mass spectrometry and DNA analysis. However, every expansion of the screening panel requires critical review, discussion, and pilot studies. Different legal regulations and ethical concerns may lead to different decisions. Without claiming to be comprehensive, this review tries to give an overview of newborn screening, including its main problems and target diseases.
Keywords: Newborn screening; Genetic screening; Preventive medicine; Inborn errors of metabolism

In terms of testing, modern laboratory medicine can be divided into centralized testing in central laboratories and point-of-care testing (POCT). Centralized laboratory medicine offers high-quality results, as guaranteed by the use of quality management programs and the excellence of the staff. POCT is performed by clinical staff, and so such testing has moved back closer to the patient. POCT has the advantage of shortening the turnaround time, which potentially benefits the patient. However, the clinical laboratory testing expertise of clinical staff is limited. Consequently, when deciding which components of laboratory testing must be conducted in central laboratories and which components as POCT (in relation to quality and timeliness), it will be medical necessity, medical utility, technological capabilities and costs that will have to be ascertained. Provided adequate quality can be guaranteed, POCT is preferable, considering its timeliness, when testing vital parameters. It is also preferred when the central laboratory cannot guarantee the delivery of results of short turn-around-time (STAT) markers within 60 or (even better) 30 min. POCT should not replace centralized medical laboratory testing in general, but it should be used in cases where positive effects on patient care have been clearly demonstrated.
Keywords: Centralized laboratory testing; Laboratory medicine; Medical laboratory testing; Point-of-care testing; Quality; Timeliness; Turn-around-time; Clinical/Biomedical analysis; Sampling; Bioanalytical methods; Biological samples

The GeXP genetic analysis system allows for multiplexed detection and quantitation of up to 35 genes in 192 samples in a single analysis. The analytical procedure includes modified reverse transcription and PCR amplification, followed by capillary electrophoretic separation. RNA material from multiple sample types can be used, including blood, cell lines, and tissue material. This instrumentation has a lower limit of detection of <2 ng with a dynamic range greater than two orders of magnitude, as tested in this validation. Precision experiments demonstrate a within-run coefficient-of-variation (CV) = 11.1% at 25 ng and 12.9% at 12.5 ng total RNA for the complete workflow and CV = 4.8% at 25 ng and 6.7% at 12.5 ng levels for the GeXP analysis alone. The between-run precision for the entire workflow was determined to be 25% at 25 ng. We have devised an optimized protocol and use it to successfully identify a gene expression signature capable of discriminating prostate tumor and non-tumor tissue samples. We used a combination of multiplex gene panels to interrogate ~70 genes in our primary screen. Our results demonstrate that a subset of these genes can be used to separate normal and tumor prostate tissue samples. This protocol using the GeXP analyzer allows for a high-throughput, robust, and reproducible assessment of multiplexed gene expression analysis, and can be used for biomarker discovery to compare different sample groups. With a dynamic linear range and satisfactory precision, this technology holds promise for rapidly identifying gene expression signatures from multiplexed reactions of up to 35 genes in large numbers of samples with limited amounts of starting material.
Keywords: GeXP; Multiplexed gene expression; Cancer biomarkers; Analytical validation; Biomarker discovery

Characterization of lipid extracts from brain tissue and tumors using Raman spectroscopy and mass spectrometry by Milena Köhler; Susanne Machill; Reiner Salzer; Christoph Krafft (1513-1520).
Brain tissue is characterized by high lipid content. The amount of lipids decreases, and its composition changes in the most frequent primary brain tumor, the glioma. Scope of the current paper was to extract quantitatively lipids from porcine and human brain tissue as well as from five human gliomas using a modified protocol according to Folch. The lipid extracts were studied by Raman spectroscopy with 785 nm excitation and by mass spectrometry with electron impact ionization. Porcine and human brain tissues have similar water and lipid content and show similar Raman and mass spectra. In contrast, gliomas are characterized by increased water content and decreased lipid content. Elevated phosphatidylcholine to cholesterol ratios in lipid extracts of gliomas were indicated by Raman bands of the choline group and cholesterol. Due to its higher sensitivity, mass spectrometry detected increased levels of cholesterol ester relative to cholesterol in lipid extracts of gliomas. For comparison, thin tissue sections were prepared from the glioma specimens before lipid extraction; infrared spectroscopic images were recorded and analyzed by a supervised classification model. This study demonstrates how to improve the analysis of brain tumors and to complement the diagnosis of brain pathologies using a multimodal approach.
Keywords: Biopolymers/lipids; IR spectroscopy/Raman spectroscopy; Mass spectrometry/ICP-MS

Novel fluoroimmunoassay for ovarian cancer biomarker CA-125 by Davin Sok; Lisa-Jo A. Clarizia; Leslie R. Farris; Melisenda J. McDonald (1521-1523).
Cancer antigen 125 (CA-125) is a glycoprotein biomarker that denotes the presence of ovarian and reproductive cancers in women, with serum concentrations of CA-125 greater than 35 U/ml considered indicative of potential malignancies. A fluorescent immunoassay recently developed in our laboratory employing the ALYGNSA antibody-orientation system has been used to measure CA-125 levels. This system displayed significantly increased sensitivity with a detection limit of 1.5 U/ml compared to that of a commercial CA-125 enzyme-linked immunosorbent assay (15 U/ml) This tenfold lower level of detection of the ALYGNSA CA-125 assay should permit better identification and monitoring of ovarian cancer.
Keywords: Fluoroimmunoassay; ELISA; Cancer biomarker; CA-125

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts are formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN). NNK and NNN are considered carcinogenic to humans by the International Agency for Research on Cancer. Existing analytical methods for determination of HPB-releasing DNA adducts require 0.3–2.0 g of human target tissues such as lung and esophagus. For adduct determination in milligram amounts of biopsy samples, an ultrasensitive and specific method is presented using capillary gas chromatography coupled to a high-resolution mass spectrometer operated in the negative chemical ionization mode (GC-NCI-HRMS). The method has a limit of detection of 4.6 fmol HPB, a limit of quantification of 14.9 fmol HBP and a recovery of 45 ± 15%. Intra- and inter-day imprecision for N = 6 samples were calculated with coefficients of variation of <3.1%. Method applicability was evaluated with biopsies of esophageal mucosa (N = 14) yielding 5.6 ± 1.9 mg tissue and a mean adduct level of 6.13 ± 9.35 pmol HPB/mg DNA.
Keywords: Esophagus; High-resolution GC-NCI-HRMS; Tobacco-specific nitrosamines; DNA adducts

Antibody orientation enhanced by selective polymer–protein noncovalent interactions by Lisa-Jo A. Clarizia; Davin Sok; Ming Wei; Joey Mead; Carol Barry; Melisenda J. McDonald (1531-1538).
A unique interaction has been found between protein G’ (a truncated recombinant bacterial “alphabet” protein which aligns by noncovalent attachment to the antibody stem) and poly(methyl methacrylate), a thermoplastic polymer substrate, which can be easily fabricated using high-rate processes. Significantly improved orientation efficiency with traditional passive adsorption for this system (termed ALYGNSA) has been achieved as compared to the same assay performed on a polystyrene substrate with protein G’. Results were consistent with an average alignment of 80% of the human immunoglobulin G capture antibody which translated into a 30% to 50% improved alignment over an array of industry standards tested. Laser scanning confocal microscopy confirmed the immunological results. Studies of additional poly(methyl methacrylate) polymer derivatives and protein biolinker (A and AG) combinations have been conducted and have revealed different degrees of antibody alignment. These findings may lead to additional novel noncovalent methods of antibody orientation and greater sensitivity in immunological assays.
Keywords: IgG antibody orientation; Poly(methyl methacrylate); Protein G’; Laser scanning confocal microscopy

A capacitive biosensor for detection of staphylococcal enterotoxin B by Mahmoud Labib; Martin Hedström; Magdy Amin; Bo Mattiasson (1539-1544).
A sensitive method for the detection of staphylococcal enterotoxin B (SEB) using a flow-injection capacitive biosensor is presented. SEB was purified from a crude culture filtrate of Staphylococcus aureus through three chromatographic steps. The first two steps were based on ion-exchange chromatography, and the last step was carried out on a gel filtration column. The SEB recovery values after the purification stages were 88%, 74%, and 12%, respectively. A horseradish peroxidase labeled antistaphylococcal enterotoxin B was prepared by the periodate method and was further employed in a sandwich-enzyme-linked immunosorbent assay (ELISA) for the determination of SEB concentrations in different samples obtained during the processing of the crude filtrate. The capacitive biosensor could detect SEB concentrations as low as 0.3 pg ml−1 with a linearity ranging from 2.8 pg ml−1 to 2.8 ng ml−1 under optimized conditions. The response time was about 10 min. A good agreement was achieved between the developed capacitive biosensor system and ELISA as a reference method for detection of SEB levels in different purification samples. The newly developed sensor has the benefits of simplicity, high sensitivity, and multiple use capability.
Keywords: Staphylococcal enterotoxin B; Capacitive biosensor; Self-assembled monolayers; ELISA; Horseradish peroxidase

The potentialities of new ionic liquids (ILs) based on choline were evaluated as an electrophoretic medium in capillary electrophoresis for the analysis of alkaline and alkaline earth cations (Li+, K+, Na+, Cs+, Mg2+, Ba2+, Ca2+, and Sr2+) with indirect UV detection. Two types of capillaries were tested: an untreated fused silica and fused silica coated with a film of polyvinylalcohol. The coated capillary proved to be the best adapted for the metal ions studied. Moreover, it appeared that the nature of the ionic liquid anion influenced the baseline stability, and the bis(trifluoromethylsulfonyl) imide (NTf2 ) anion seemed to be the most efficient. These preliminary studies led us to synthesize a new ionic liquid, 2-hydroxy-N,N,N-trimethyl-1-phenylethanaminium NTf2 (phenylcholine NTf2). This liquid was able to act as the running electrolyte and probe, generating the background signal in indirect UV light and consequently simplifying the electrophoretic medium. Excellent baseline stability, good reproducibility, as well as good sensitivity of detection were obtained with this new ionic liquid. Thus, 510,000 plates/meter for Li+ with 40 mM IL were successfully obtained. The optimal concentration of IL was 20 mM with a detection limit ranging from 28 μg L−1 for Li+ to 1,000 μg L−1 for Cs+. This method (phenylcholine NTf2 with polyvinylalcohol capillary) was applied to analyze different commercial source and mineral waters. Finally, the potentiality of this ionic liquid in nonaqueous capillary electrophoresis was explored. The use of phenylcholine NTf2 with a fused silica capillary, in pure methanol medium and in the presence of acetic acid, made it possible to obtain separation selectivity different from that obtained in aqueous medium.
Keywords: Alkali cations; Alkaline earth cations; Ionic liquid; 2-Hydroxy-N,N,N-trimethyl-1-phenylethanaminium bis(trifluoromethylsulfonyl)imide; Capillary electrophoresis