bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoglobulin G, A and M Light Chain Ratio in Children
Authors: Haraldsson Á , Weemaes C M R , Kock-Jansen M J H , Eck-Arts P B J M v , de Boo T , Bakkeren J A J M , Stoelinga G B A
Published: February 2014

Values for the κ/λ light chain ratio in immunoglobulins G, A and M and the total κ/λ ratio, measured by enzyme linked immunosorbent assay, were evaluated in serum samples from different age groups (114 children, aged from 1 month to 15 years, and 20 adults).

The IgG κ/λ ratio decreased in the first 6 months and subsequently increased slowly during childhood towards the adult value of 2·0. The IgM κ/λ ratio increased at a greater rate than IgG κ/λ ratio in the first years of life and thereafter rose slightly throughout childhood to reach an adult value of 1·7. A decreasing IgA κ/λ ratio was found from 1 month of age onwards to an adult value of 1·1. The pattern of total κ/λ ratio was similar to the IgG κ/λ ratio with an adult value of 2·0.
Optimization and Characterization of Capture ELISA Methodology for Lp(a) Lipoprotein Quantification
Authors: Doetsch Karl M , Roheim Paul S , Thompson James J
Published: February 2014

In order to better characterize and optimize a typical capture ELISA system for Lp(a) lipoprotein, we have analysed kinetic details of the reaction. Plate coating with polyclonal antibody, recognition of captured analyte with monoclonal antibody, and detection of monoclonal antibody with alkaline phosphatase-labeled antiglobulin were essentially complete after one hour, probably being driven forward by a relative excess of reagent. However, complete capture of the Lp(a) analyte required about 6 h at low input concentrations. Shorter time periods for capture might therefore result in decreased sensitivity and reproducibility. Deviations from linearity in the assay dose response were associated with incomplete capture of Lp(a) and significant depletion of the monoclonal recognition antibody. With the final reaction conditions described, no significant differences in immunochemical reactivity between samples were found by analysis of dose response slopes. Finally, interferences from plasminogen, −20°C storage, anticoagulants, LDL, haemolysis, and bilirubin were minimal.
Direct Measurement of Low Density Lipoprotein in Whole Blood by Silver-Enhanced Gold-Labelled Immunoassay
Authors: Patel Nishith , Rocks Bernard F , Iversen S Andrew
Published: February 2014

A competitive silver-enhanced gold-labelled immunoassay has been developed for the direct measurement of low density lipoprotein (LDL) in whole blood. Immobilized LDL and sample LDL compete for added antibody. Quantitation of the bound antibody/antigen complex is achieved by the addition of gold-labelled antiimmunoglobulin G followed by enhancement of absorbance by addition of silver ions. Whole-blood samples from fasting patients were assayed directly for LDL by the procedure and the corresponding plasma samples were assayed for total cholesterol, high density lipoprotein and triglycerides followed by the indirect calculation of LDL cholesterol. The correlation between the two methods was good (r = 0·82) and the SEGLISA exhibited good precision.
Plasma Creatinine and the Effect of Delay in Separation of Samples
Authors: Simpson William G
Published: February 2014

There is a prevailing opinion that creatinine is an analyte which may be assayed with confidence in the plasma of ageing whole-blood samples. This is true of continuous flow methods employing dialysis, but in this study, where creatinine was measured in old samples by a kinetic Jaffé method on a discrete analyser, the concentration of creatinine was falsely raised. When plasma was left in contact with cells for 48 h, there was an apparent rise in the plasma creatinine by over 100 μmol/L compared with the 2 h concentration. This method-dependent error is therefore clinically significant.
Alleviation of Non-Islet Cell Tumour Hypoglycaemia by Growth Hormone Therapy is Associated with Changes in IGF Binding Protein-3
Authors: Teale J D , Blum W F , Marks V
Published: February 2014

The hypoglycaemia caused by non-islet cell tumours is often associated with an increase in plasma insulin-like activity. In many cases there is a relative if not always absolute increase in plasma insulin-like growth factor II (IGF-II). Growth hormone (GH) secretion is almost invariably depressed as is the plasma insulin response to oral glucose.

Despite the high concentration of IGFs (i.e. IGF-I and IGF-II) normally found in the plasma of healthy people their potential hypoglycaemic effect is not manifest due to the tightness with which they are bound to specific binding proteins (IGFBPs). Plasma levels of the major binding protein (IGFBP-3), which is GH-dependent, were depressed in three patients with tumour induced hypoglycaemia. Treatment with biosynthetic GH restored IGFBP-3 to levels which were approximately equimolar to total plasma IGF concentrations, alleviated the hypoglycaemia and restored the plasma insulin responses to oral glucose.

We suggest that after GH treatment IGF-II is sequestered by stimulated IGFBP-3 in association with a pre-existing acid-labile subunit to form high molecular weight complexes which prevent IGF-II gaining access to tissues receptors through which it exerts its hypoglycaemic effects.
Measure of Satisfaction of General Practitioners with the Chemical Pathology Services in Leeds Western Health District
Authors: Allen K R , Harris C M
Published: February 2014

The satisfaction of general practitioners with the District Chemical Pathology Services in Leeds Western Health District was assessed by means of a postal questionnaire. The areas of interest covered included communication, test turn-round times, format of reports, advice about tests undertaken in the practice and chemical pathology information services. Communication with the laboratory was the major area of dissatisfaction (21% GPs), followed by test turn-round times (14%) and format of reports (10%). More than half the respondents were interested in an offer of help in selecting equipment for their surgeries and subsequent quality control.

Customer satisfaction will become an important issue as a result of the NHS reforms and laboratories may find this exercise useful.
Dual Analyte Immunoassay—a New Approach to Neural Tube Defect and Down's Syndrome Screening
Authors: Macri J N , Spencer K , Anderson R
Published: February 2014

A microtiter plate based Dual Analyte enzymeimmunoassay method for the simultaneous measurement of α-fetoprotein (AFP) and Free-β human chorionic gonadotrophin (hCG) was evaluated. This rapid assay, which has application in both Neural Tube Defect screening and Down's screening, shows good precision with between assay coefficients of variation between 5 and 7·5% for AFP and 3·7 to 5·8% for Free-β(hCG). Correlation with single analyte procedures is good, with correlation coefficients being greater than 0·91 in both cases. Clinical discrimination in detecting both types of abnormalities is not compromised by this new simultaneous Dual Analyte assay. We conclude that the Dual Analyte approach, which combines analytes achieving the highest known detection efficiency, will bring about improvements in the efficiency of screening, reduce costs and improve report turnaround, all leading to better quality of patient care.
Within-Subject and Between-Subject Biological Variation of Prothrombin Time and Activated Partial Thromboplastin Time
Authors: Dot Dolors , Miró Jaume , Fuentes-Arderiu X
Published: February 2014

The within-subject and between-subject biological variation of prothrombin time (PT) and activated partial thromboplastin time (APTT) have been studied in a group of 19 women and 20 men over a period of 9 months. All measurements were performed with a mechanical coagulometer. The between-subject coefficients of variation were 6·8% for PT and 8·9% for APTT. The medians and the 90th percentiles of within-subject coefficients of variation were 1·7% and 5·9%, respectively, for PT, and 0·0% and 6·1% for APTT. These data were used to calculate the critical difference for significant change detection and the desirable between-day imprecision.
Serum Fructosamine Concentration in Patients with Nephrotic Syndrome and with Cirrhosis of the Liver: The Influence of Hypoalbuminaemia and Hypergammaglobulinaemia
Authors: Constanti C , Simo J M , Joven J , Camps J
Published: February 2014

We have investigated the influence of variation of the concentrations of serum albumin and immunoglobulins on serum fructosamine concentration in 33 patients with nephrotic syndrome, and 18 patients with cirrhosis of the liver. Protein alterations were evident in these patients and they were compared with 109 normal subjects, 43 patients with type II diabetes mellitus and nine diabetic patients with nephrotic syndrome.

The mean serum fructosamine concentration in diabetic patients (2·76±0·53 mmol/L) was significantly increased ( P < 0·001) by comparison with normal subjects (1·93 ±0·20 mmol/L) and the other patients studied. Patients with diabetic nephropathy had higher ( P < 0·01) serum fructosamine concentrations (2·23± 0·54 mmol/L) than non-diabetic patients with the nephrotic syndrome (1·57 ± 0·37 mmol/L) but remained with the normal range. Positive correlations were observed between fructosamine and immunoglobulins G and M in nephrotic and cirrhotic patients. Serum immunoglobulin A was also directly correlated with serum fructosamine in patients with cirrhosis of the liver. An inverse correlation between albumin and fructosamine in serum of patients with cirrhosis of the liver was also noted. We conclude that the fructosamine assay is not useful in the assessment of glycemic control in patients with cirrhosis of the liver, nephrotic syndrome or in any other clinical situation in which protein metabolism is altered.
Dependence of Measured Ionized Calcium on Protein Concentration as Measured by Three Ion-Selective Electrodes
Authors: McEnroe Eamonn , McKenna Malachi J , Diamond Dermot , Covington Arthur K , Freaney Rosemarie
Published: February 2014

A positive effect of protein on the measurement of ionized calcium in serum by ion-selective electrodes (ISEs) has been previously reported and the present study confirms this finding. Ionized calcium in serum was measured in the presence of increasing protein concentrations induced by venous stasis in 17 healthy volunteer subjects. Ionized calcium was measured using two commercial analysers, a Radiometer ICA2 analyser and a Baker Analyte + 2 analyser, and a calcium cell devised by Covington for the calcium reference method (CRM). Both commercial analysers used charged ionophores and the CRM used a neutral carrier ionophore in the selective membrane. A small but significant rise in ionized calcium with increasing protein was measured on all analysers. Substitution of isotonic KCl for saturated KCl in the reference electrode of the CRM resulted in significantly reduced values for ionized calcium in paired serum samples when measured using the isotonic salt bridge. This study supports the premise that the positive effect of protein is related to the salt bridge concentration of the reference electrode rather than the ISE membrane composition.
Urinary Albumin and Low Molecular Weight Protein Excretion in the Nephrotic Syndrome—Sequential Studies during Corticosteroid Treatment
Authors: Beetham Robert , Newman David
Published: February 2014

In a preliminary investigation into the behaviour of low molecular weight proteins in the nephrotic syndrome, we have measured urinary concentrations of albumin, α-1-microglobulin (α1-m) and retinol-binding protein (RBP) in six children for up to 11 days during the course of steroid therapy for nephrotic syndrome.

The results in part support the concept of independent proximal tubular absorption of albumin and low molecular weight proteins, and indicate that in the nephrotic syndrome the excretion of RBP and α1-m, two generally accepted markers of tubular proteinuria, is anomalous.
Auditing Quality Control Procedures in a Chemical Pathology Laboratory—a Multiple Regression Analysis
Authors: Tillyer C R , Gobin P T , Ray A K , Rimanova H
Published: February 2014

We undertook a retrospective analysis of the monthly test rejection rates and the monthly external quality assessment scheme performance indices for our laboratory's two automated analysers, and examined the association of these variables with measures of laboratory workload, manpower, staff training, instrument servicing, seasonal and temporal factors and changes of calibration, method and assigned internal quality control values. Using multiple linear regression and stepwise multiple linear regression, we found that test rejection rates differed significantly between instruments, and were highest on the instrument performing the widest variety and lowest volume of tests. On that instrument, rejection rates were significantly associated with the introduction of new staff and laboratory manpower levels, and also showed a highly significant trend upwards over the study period, independent of the effects of the other variables examined. External quality assessment scheme performance indices showed small trends over the study period. They were not related to the test rejection rates on either analyser but also showed a significant association with the introduction of new staff and a small but significant association with laboratory workload. We conclude that the training and introduction of new staff and decreased laboratory manpower levels may significantly increase the level of test rejection, and adherence to appropriate quality control protocols effectively maintains the quality of the laboratory's results, but may not be completely successful in filtering out the effects of some assignable causes of variation in test results. It is suggested that clinical laboratories use the statistical approach adopted here to identify factors which may be adversely affecting quality performance and running costs and to provide evidence that quality control procedures are both cost- and quality-effective.
Highly Sensitive Sandwich Enzyme Immunoassay for Alpha-Fetoprotein in Human Saliva
Authors: Yio Xian Yang , Jiang Jing , Yin Fen Zhi , Ruan Ke-He
Published: February 2014

To determine α-fetoprotein (AFP) in human saliva, a highly sensitive sandwich enzyme immunoassay for saliva AFP was developed. AFP standards and saliva samples were added into the wells of a polystyrene plate coated with goat IgG antibody against human AFP. After incubation, the wells were washed and horseradish peroxidase-labelled antibody was added. The enzyme activity specifically bound to the well was assayed using 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide as substrate. The reaction was stopped by addition of 2 M sulphuric acid and the AFP concentration was determined from the absorbance at 450 nm. The minimum detectable concentration was 8 ng/L. The recovery of AFP mixed with human saliva was 91·1–102·4%. The within-assay and between-assay coefficients of variation were 6·5–8·9% and 7·6–10·8%, respectively. The assay correlated well with a radioimmunoassay for human AFP ( r = 0·985, n = 13, P < 0·001). The mean concentration of AFP in normal human saliva was 14·3 ng/L (SEM = 4·9 ng/L, n = 10) and significantly higher levels of saliva AFP were observed in hepatocellular carcinoma patients with positive serum AFP (mean 1367·8 ng/L, SEM 595·4 ng/L, n = 6; P < 0·001). Strong correlation was observed between saliva AFP and serum AFP ( r = 0·978, P < 0·01, n = 13).
Reference Values of Serum and Urine Creatinine, and of Creatinine Clearance by a New Enzymatic Method
Authors: Sugita O , Uchiyama K , Yamada T , Sato T , Okada M , Takeuchi K
Published: February 2014

A new, totally enzymatic procedure for the determination of creatinine in serum and urine, using creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase and formaldehyde dehydrogenase is described. The assay was adapted to a discontinuous analyser with each analysis requiring only 20 μL of serum or 3 μL of urine. Analytical recovery of creatinine in serum and urine averaged 100·6%. Within-run and between-run precision studies gave coefficients of variation of 1·1% and 1·8%, respectively, for a serum with mean values of 83 μmol/L (9·4 mg/L) creatinine.

Creatinine concentrations in serum and urine were measured by this procedure, in Japanese children and adults. The reference intervals for serum creatinine concentrations in adults were 55–96 μmol/L (6·2–10·9 mg/L) in men and 40–66 μmol/L (4·5–7·5 mg/L) in women, and for urine, 9·46–19·01 mmol/day (1070–2150 mg/day) in men and 6·75–10·61 mmol/day (764–1200 mg/day) in women. The reference intervals of creatinine clearance were 88·0–176·4 mL/min in men and 75·7–173·0 mL/min in women.
Pyrophosphate in Synovial Fluid and Urine and its Relationship to Urinary Risk Factors for Stone Disease
Authors: Roberts N B , Dutton J , Helliwell T , Rothwell P J N , Kavanagh J P
Published: February 2014

Inorganic pyrophosphate (PPi) measurement in urine and synovial fluid has been established using the PPi-dependent phosphorylation of fructose-6-phosphate and subsequent reduction of dihydroxyacetone phosphate by NADH. The assay is linear up to 200 μmol/L, easy to perform and gives results comparable to more complex methods.

Daily urinary output of PPi was independently related to both age ( P = 0·0014) and sex ( P = 0·0002). Men had higher values than women and older individuals excreted greater amounts. Male stone formers, younger than 45 years, had lower values than age matched male controls ( P = 0·012). Younger female stone formers also tended to have lower values. In stone formers' urine significant and independent correlations were found of PPi excretion with urine volume ( P = 0·004) and with phosphate excretion ( P = 0·008). Oxalate excretion and that of other urine constituents and the degree of supersaturation with common stone-forming salts were not correlated with PPi. PPi excretion was markedly elevated in the urine of two patients with hypophosphatasia. The PPi concentration in synovial fluid from painful, swollen knee joints was elevated, but unrelated to the presence or absence of PPi or urate crystals.
The Steatocrit: An Improved Procedure
Authors: Lloyd D R , Rawashdeh M O , Booth I W , Brown G A
Published: February 2014

The steatocrit is a simple and easily repeated assay for measuring the fat content of infants' stools. However, we and others have experienced technical difficulties in its use. Three modifications were therefore made to the original procedure: incorporation of a lipid-soluble dye, improved homogenization and a heating step. The modified method was used to measure the stool fat content of young children with and without clinical steatorrhoea. Validation of the modified steatocrit is presented, together with examples of its application to both clinical research and clinical practice.
Investigation of the Use of Impermeable Fluid Barriers between Pelleted and Supernatant Enzyme Activity in a Pseudohomogeneous Enzyme Immunoassay
Authors: Maguire Gerald A , Horsfield Julie A
Published: February 2014

We have investigated the feasibility of performing an automated pseudohomogeneous enzyme immunoassay in which a separation step is not apparent to the user. This was achieved using a layer of silicone fluid, which is immiscible with aqueous solutions, as a physical barrier between the pelleted and supernatant enzyme. Initially we produced a manual assay based on the Serozyme T4 assay to demonstrate the feasibility of the approach. This assay showed good precision, parallelism and correlated with ‘in house’ results on patients' samples. Production of a fully automated assay was made difficult by the design of available equipment but none the less we demonstrated the feasibility of the automated approach by producing a standard curve for T4.
Enzyme Activities of the Mitochondrial Energy Generating System in Skeletal Muscle Tissue of Preterm and Fullterm Neonates
Authors: Sperl W , Sengers RCA , Trijbels J M F , Ruitenbeek W , Doesburg W H , Smeitink JAM , Kollée L A A , Boon J M
Published: February 2014

Quadriceps muscle specimens from autopsy of 28 neonates (gestational age 25–42 weeks) were investigated to determine pyruvate and malate oxidation rates and several enzymes of the mitochondrial oxidative process. In general, the levels of all mitochondrial parameters measured, including carnitine levels, were lower in the neonates who died within the first week of life than those in the control group (age > 5 years). Pyruvate and malate oxidation rates ( P < 0·05), activities of pyruvate dehydrogenase complex ( P < 0·10) and succinate: Cytochrome c oxidoreductase ( P < 0·05) increased significantly with gestational age. Pyruvate oxidation rates ( P < 0·05) as well as activities of citrate synthase ( P < 0·05) and NADH:Q1 oxidoreductase ( P < 0·05) were significantly lower in the group of very preterm infants at an age of 1–7 days compared with very preterm infants at an age between 3–8 weeks.

We conclude from our study that special reference values are necessary for a correct biochemical diagnosis of mitochondrial encephalomyopathies in the neonatal period. Differences between preterm and fullterm children of the same age (1 week) indicate a maturational process in human muscle tissue during gestation. Comparison of two different age groups within the very preterm neonates point to a postnatal maturation of the mitochondrial energy metabolism, at least in preterm neonates.
Glycine Absorption from Irrigation Fluid during Endoscopic Resection of Rectal Tumours
Authors: Ball M J , Clarke P J , Kettlewell M G W
Published: February 2014

A significant rise in plasma glycine concentration was observed in nine elderly patients undergoing endoscopic transanal resection of rectal tumours using glycine solution for irrigation. Despite the obvious absorption of glycine, plasma sodium concentration and osmolality were not significantly altered, except in a patient whose rectal wall was perforated. In this case plasma sodium and osmolality fell, and the patient developed hyperammonaemia. Potentially adverse metabolic consequences may occur when there are surgical complications, but in the absence of such problems the absorption of glycine appears to cause little effect.
Troponin T for the Differential Diagnosis of Ischaemic Myocardial Damage
Authors: Collinson P O , Moseley D , Stubbs P J , Carter G D
Published: February 2014

The diagnostic performance of a new enzyme linked immunosorbent assay for the cardiac structural protein troponin T in the differential diagnosis of ischaemic cardiac damage was assessed. A well documented set of patients admitted to the coronary care unit of a district general hospital were studied. At a cutoff value of 0·2μ/L, troponin T measurements 12–24 h after admission or 12–16 to 24–48 h from onset of chest pain showed an overall efficiency of 97 · 6% for diagnosis of proven myocardial infarction. Troponin T was not detectable in patients when ischaemic heart disease could be excluded but was present in four patients with angina. Detectable troponin T in these angina patients was associated with subsequent cardiac events.
Protein Excretion after Prolonged Exercise
Authors: Robertshaw M , Cheung C K , Fairly I , Swaminathan R
Published: February 2014

Random urine samples were obtained from 16 healthy subjects (nine men and seven women) before and after a 100 Km hill walk for the estimation of total protein, albumin, N-acetyl-β-glucosaminidase (NAG), retinol binding protein (RBP) and creatinine. The excretion of total protein, albumin and NAG (expressed in relation to creatinine excretion) increased significantly after the walk. The relative clearance of protein and albumin also increased. In four subjects serial measurements were made for 4 days and the excretion of albumin and NAG on the fourth day were similar to the pre-walk values. We conclude that proteinuria of prolonged exercise is at least partly due to reduced tubular reabsorption.
Plasma and 24 H-Urinary Catecholamine Concentrations in Normal and Patient Populations
Authors: Ross G A , Newbould E C , Thomas J , Bouloux P M G , Besser G M , Perrett D , Grossman A
Published: February 2014

Using a single HPLC-ECD methodology, plasma catecholamine data were compiled from 545 patients and 144 healthy subjects, and 24 h urinary free catecholamine data from 106 patients. From these data, normal reference ranges were derived for the clinical investigation of suspected phaeochromocytomas, specifically to address the question as to when a result is normal and when further investigation may be required. We have shown that noradrenaline and adrenaline concentrations in plasma and urine are not normally distributed, and basal plasma noradrenaline is significantly lower in normal volunteers than in hospital patients. We have also demonstrated that a reference range which does not take account of these factors will give a significant number of false-positive results. Age and sex did not appear to be significant variables for either plasma or urinary catecholamines.

In the investigation of phaeochromocytoma, 95% confidence limits may be used as a warning to repeat sampling and 99% confidence limits as rendering the diagnosis extremely probable. In patients with phaeochromocytomas, urinary catecholamines had a 99·5% specificity and 100% sensitivity in the diagnosis, and thus provide for a rapid and efficient diagnostic screen.
The Role of Expert Systems in Improving the Test Requesting Patterns of Clinicians
Authors: Peters Margaret , Broughton P M G
Published: February 2014

The many strategies proposed for influencing the test requesting patterns of clinicians have had only limited success, largely because they are labour intensive and depend on motivation and commitment. Clinical protocols which have been locally agreed between laboratory staff and clinicians are potentially one of the more successful strategies, but detailed study of their application in different clinical settings has been limited by practical problems. Expert systems offer a way of implementing locally agreed protocols and, consequently, of assisting the identification, audit and refinement of laboratory testing strategies. Where these systems have been applied in specialist units they have resulted in savings in time by both clinical and laboratory staff, and an overall reduction in the number of clinical chemistry tests done within and out of hours. These systems offer promise as a method of improving laboratory utilization.
Immunoaffinity Purification of Prostaglandin E2 and Leukotriene C4 Prior to Radioimmunoassay: Application to Human Synovial Fluid
Authors: Matsumoto Shuichi , Hayashi Yoko , Kinoshita Isamu , Ikata Takaaki , Yamamoto Shozo
Published: February 2014

When human synovial fluid as such was subjected to radioimmunoassays of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), there was no linear increase in PGE2 and LTC4 as the amount of synovial fluid was raised. For removal of substances thus disturbing the assay we developed a method of immunoaffinity purification of PGE2 and LTC4. A monoclonal antibody against PGE2 or LTC4 was coupled to BrCN-activated Sepharose 4B. When synovial fluid mixed with radiolabelled PGE2 or LTC4 was applied to the column of immobilized antibody, the ligand was adsorbed to the column and eluted with a mixture of methanol/water in a recovery of about 80%. The purified material showed a linearity between the amount of the sample and the value of radioimmunoassay. The one-step method was applied to synovial fluid from patients with rheumatoid arthritis, osteoarthritis and other joint diseases.
A Time-Resolved Fluoroimmunoassay for Recombinant Human Interleukin-3
Authors: Knopf Hans-Peter , Duffy Frances , Papoian Ruben
Published: February 2014

We describe a time-resolved fluoroimmunoassay (TRFIA) for recombinant human interleukin-3 (IL-3). The assay design uses two different monoclonal anti-IL-3 antibodies, giving a two-site immunometric assay. The TRFIA for IL-3 is a direct adaptation from our existing ELISA for IL-3. By using the same assay configuration and exchanging a europium-streptavidin conjugate for alkaline phosphatase-streptavidin, we were able to minimize the time required to develop a TRFIA, but still increase the working range of the TRFIA over the ELISA by 20-fold. The limit of detection is 5 ng/L, with the limits of quantification set at 5 and 30 000 ng/L human plasma.
The Presence of Antibodies against Extractable Nuclear Antigens in Serum: A Comparison of Immunoblotting versus Radial Immunodiffusion
Authors: Scharpe S , Uyttenbroeck W , Cooreman W
Published: February 2014

A commercial immunoblotting kit has recently been introduced to determine auto-antibodies against extractable nuclear antigens. We compared this new test with radial immunodiffusion for its usefulness in the routine laboratory procedures of a general hospital. Antigen preparation in immunoblotting includes a protein denaturation step prior to electrophoretic separation of the different proteins. In this way antigenic determinants that depend heavily on the protein superstructure are lost. In theory, auto-antibodies against these epitopes may be missed. In our series of 100 samples that had tested positively for antinuclear antibodies, radial immunodiffusion was able to detect one SSA positive sample that was negative by immunoblotting. However, 47 samples positive in immunoblotting, 37 positive for UBP, seven for anti-SSA, are for anti-Jo-1, one for anti-RNP and one for anti-Sm were missed by radial immunodiffusion. Most of these samples had low antinuclear antibody titres (1/80 or 1/160).
The Effect of the Concentration Ratio of Avidin and Biotin on a Single Step Sandwich Enzyme Immunoassay
Authors: Farina L , Iacobello C , Orlandini S , Ius A , Albertini A
Published: February 2014

The interaction between biotin and avidin, used in a single-step enzyme-immunoassay for ferritin determination, has been studied. The antigen is simultaneously bound by an antibody coated to a polystyrene bead and by an antibody coupled to biotin which reacts with avidin conjugated to peroxidase. We have assessed the optimal ratio between avidin, conjugated to peroxidase, and biotin, coupled to antibodies, to give rise to the best signal for a quantitative enzyme-immunoassay. We have found that a careful balance between biotinylated antibody and conjugated avidin is necessary for our purpose and a biotinylated antibody excess should be avoided since it causes a signal decrease. This ratio is uninfluenced by both the presence and the absence of the antigen. Thus, an avidin-biotin single-step methodology, which has proved to be reliable for routine use, was developed.
Identification of Sources of Lead Exposure among Children in Arar, Saudi Arabia
Authors: Al-Saleh Iman A , Fellows Craig , Delves Trevor , Taylor Andrew
Published: February 2014

The concentrations of lead and the isotopic ratios of lead, 206Pb:207Pb, were measured by inductively coupled plasma-mass spectrometry in environmental specimens, cosmetics and traditional remedies. The ratios were compared with those found in blood samples of Saudi children who had increased concentrations of total blood lead. The isotopic ratios in the blood specimens (1·144±0·027) were not significantly different from those determined in cosmetics and remedies (1·152±0·031) and indicated that these were the likely sources of exposure rather than the lead contained in petrol which had an isotopic ratio of 1·207.
Purification and Catalytic Properties of Liver Alcohol Dehydrogenase Isoenzymes in Patients with Hepatoma in Nigeria
Authors: Ogunleye I O , Olusi S O
Published: February 2014

Alcohol dehydrogenase isoenzymes were isolated from the liver of patients with hepatoma and healthy control individuals. Whereas only a single form of the enzyme was obtained in healthy control liver, two distinct forms of the enzyme were found in hepatoma liver. These two forms were named AD-I and AD-II according to their elution pattern in CM-cellulose chromatography and mobility towards the anode in polyacrylamide gel electrophoresis. Both isoenzymes resembled normal liver enzyme with respect to their molecular properties. However, the two forms had distinct kinetic properties, although AD-I had some properties similar to the normal liver enzyme. The Km values of AD-I for ethanol, n-butanol and m-nitrobenzyl alcohol were 61μM, 90μM and 292μM, respectively, as against the values for AD-II which were 473μM, 100μM and 60μM for the respective substrates. Pyrazole inhibited the activity of AD-II but not that of AD-I. These kinetic properties of alcohol dehydrogenase in patients with hepatoma could be of clinical importance particularly in the tropics where the disease is prevalent.
Analysis of Apolipoprotein Size Distribution by Electroimmunoblotting from Non-Denaturing Composite Gels
Authors: Lee Lucille T , Maldonado Blanca R , Roheim Paul S , Thompson James J
Published: February 2014

An exponential gradient gel with 0–35% acrylamide and 0·5% agarose was developed for electrophoresis of intact lipoproteins with subsequent electroimmunoblotting. The system resolved in a single gel lipoprotein-associated proteins of sizes from ‘free’ apoproteins to VLDL. Reproducibility between gels was good (coefficient of variation <8%). Examination of the effect of mild glutaraldehyde fixation on immunodetection showed variable results (lack of effect on apos (a), AII, and AIV; inhibition of apoB; enhancement of apos AI and E). The composite gel system described here will simplify analysis of apolipoprotein distributions in both health and disease and therefore will likely be useful in future clinical applications.
An Evaluation of the Measurement of Drugs of Abuse by Commercial and In-House Horizontal Thin Layer Chromatography
Authors: Wolff K , Sanderson M J , Hay A W M , Barnes I
Published: February 2014

We have compared an in-house horizontal thin layer chromatography (TLC) method with a commercial TLC screening kit (Toxi-Lab) to find the most suitable method for screening urine for opioid drugs. The in-house TLC procedure is cheaper and more efficient than the commercial TLC method and is ideal as a confirmatory method when used in conjunction with other established laboratory methods. The in-house TLC method is useful for one-off requests and is able to differentiate between small quantities (0·1–0·5 mg/L) of the different, commonly abused opioids and their metabolites, which means that it is well suited for use at a local Drug Addiction Unit as a routine screening service.
Glycohaemoglobin: Comparison of 12 Analytical Methods, Applied to Lyophilized Haemolysates by 101 Laboratories in an External Quality Assurance Programme
Authors: Weykamp Cas W , Penders Theo J , Muskiet Frits A J , van der Slik Willem
Published: February 2014

Stable lyophilized ethylenediaminetetra-acetic acid (EDTA)-blood haemolysates were applied in an external quality assurance programme (SKZL, The Netherlands) for glycohaemoglobin assays in 101 laboratories using 12 methods. The mean intralaboratory day-to-day coefficient of variation (CV), calculated from the assay of 12 unidentified pairs over a period of 1 year, was 5·2% (range: 0·2–28·7). Forty-seven per cent of laboratories did not meet the criterion of CV < 5%, whereas 68% did not meet the clinically more desirable 3·3–3·6%. Linearity, as derived from the analysis of five combinations of two haemolysates with low and high glycohaemoglobin percentages over 6 months, was excellent (mean correlation coefficient 0·9953; range: 0·9188–0·9999). Analysis of two samples with high and low glycohaemoglobin percentages gave mean interlaboratory coefficients of variation of 10% for one method performed by several laboratories and 22% for all methods performed by all laboratories. It is concluded that the majority of laboratories do not meet the clinically desirable intralaboratory precision and that an unacceptably high interlaboratory precision exists.
The Relationship between Serum Cholesterol and Serum Thyroid Hormones in Male Patients with Suspected Hypothyroidism
Authors: Johnston J , McLelland A , O'Reilly D StJ
Published: February 2014

The relationship between serum cholesterol, thyrotropin, thyroxine and triiodothyronine was investigated in 456 male patients with suspected hypothyroidism. The correlation between serum cholesterol and serum thyroxine ( r = 0·0572) and between serum cholesterol and serum triiodothyronine ( r = 0·1136) were not significant but the correlation between serum cholesterol and TSH ( r = 0·0376) was significant ( P< 0·001). The mean serum cholesterol was only significantly increased in the patient groups with a serum TSH greater than 20 mU/L. In 26 patients treated for hypothyroidism with thyroxine replacement there was a significant correlation between the decrease in serum cholesterol and the decrease in serum TSH ( r = 0·5334, P<0·01) but there was poor correlation between the decrease in cholesterol and either the increase in serum triiodothyronine or the increase in serum thyroxine.
Purified Haemoglobin Preparations in the Evaluation of HbA1c Determination by Ion Exchange Chromatography
Authors: Cohen Margo P , Witt Janice , Wu Van-Yu
Published: February 2014

The use of ion exchange resins for the estimation of HbA1c in clinical samples rests on the assumption that HbA1c is effectively and efficiently separated from other N-terminally modified haemoglobins and from HbAo. To test this assumption, we applied highly purified preparations of HbA1a+1b, HbA1c and HbAo to ion exchange minicolumns, using conditions of application simulating actual blood samples and the first and second elution buffers provided by the manufacturer. The authenticity and purity of the applied haemoglobin preparations were documented by high performance liquid chromatography, gel electrophoresis and carbohydrate content. About 40% of the applied HbA1a+1b eluted in the first fraction; 45% eluted in the second fraction, and 10% to 15% required 1 mol/L NaCl to elute from the column. Of the applied HbA1c, 65–80% eluted where expected in the second fraction, about 20% required 1 mol/L NaCl to elute from the column, and the remainder eluted with HbA1a+1b. Some 3–6% of pure HbAo applied to minicolumns emerged in the second fraction, with the remainder eluting as expected after making the buffer 1 mol/L in NaCl. The results indicate that the fraction eluting from ion exchange minicolumn chromatography that is designated ‘HbA1c’ contains HbA1a+1b, and that a substantial portion of the HbA1c in an applied sample does not elute in this fraction.
Clinical Relevance of Serum Amyloid a Protein Monitoring in Urinary Tract Infections
Authors: Časl Martin-Tino , Sabljar-Matovinović Mirjana , Kovačević Sandra , Počanić Darko , Preden-Kereković Vladimira , Jagarinec Nada
Published: February 2014

We have evaluated the clinical relevance of monitoring acute phase proteins in severe urinary tract infection. Body temperature, white blood cell count, erythrocyte sedimentation rate, serum amyloid A protein (SAA), C-reactive protein (CRP), α-1-antichymotrypsin (ACT) and α-1-acid glycoprotein (AGP) were determined daily in sera from 18 treated patients.

Two patterns of response could be identified: responders and non-responders whose therapy had to be changed. Mean values for each acute phase protein were calculated daily in both responders and non-responders. Statistical evaluation of the significance between the means for each protein was also performed on a daily basis and showed P < 0·01 for SAA and CRP on day 3, for ACT on day 5, and for AGP on day 6.

SAA and CRP appear to be the most reliable markers for antimicrobial therapy monitoring in patients with urinary tract infections.
A Simple Spectrophotometric Assay for Long-Chain Acyl-CoA Dehydrogenase Activity Measurements in Human Skin Fibroblasts
Authors: Ijlst Lodewijk , Wanders Ronald J A
Published: February 2014

Long-chain acyl-CoA dehydrogenase (LCAD) deficiency is an autosomal recessive disorder of fatty acid metabolism characterized by hypoglycemia, muscle weakness and hepato- and cardiomegaly to varying extents. Analysis of organic acids in urine usually reveals dicarboxylic aciduria with elevated levels of adipic, suberic and sebacic acids as well as longer chain dicarboxylic acids. Correct diagnosis of suspected patients requires measurement of LCAD in tissue or preferably, white blood cells and/or cultured skin fibroblasts. In this paper we present a simple spectrophotometric enzyme assay based on the use of ferricenium hexafluorophosphate as electron acceptor. Under optimized conditions the method presented allowed unequivocal identification of LCAD-deficiency in Fibroblast homogenates.
Between-Country Comparability of Clinical Chemistry Results: An International Quality Assessment Survey of 17 Analytes in Six European Countries through Existing National Schemes
Authors: Jansen R T P , Bullock D G , Vassault A , Baadenhuijsen H , De Leenheer A , Dumont G , de Verdier C-H , Zender R
Published: February 2014

Two lyophilized control sera were distributed through seven national external quality assessment schemes in six European countries—Belgium, Switzerland, France, The Netherlands, Sweden and the United Kingdom—participated in the study. The results for 17 routine analytes were obtained from almost 5000 laboratories for the two sera. The organizers of the schemes were asked to process the results according to a common outlier removal procedure, and submit method-related data if available. The two sera were also distributed through the external/internal scheme of The Netherlands, and the within-laboratory standard deviations calculated in this scheme have been used in a scaling procedure for the external mean values and between-laboratory standard deviations of the participating countries.

The results show remarkable agreement in the national mean values for practically all analytes, but considerable differences in the between-laboratory variation. Data from comparable method groups was obtained for 12 analytes from Belgium, France, The Netherlands and the UK. Though revealing some specific differences between methods and countries, the method-related data are generally in agreement with the all-method data. In this study reference method values were only available for cholesterol. The high degree of agreement found suggests, however, that mutual recognition of all-method mean values in national schemes could be acceptable, especially for analytes for which reliable reference methods are not available. The major element of variation is between-laboratory rather than between-country.
Dual Analyte Immunoassay in Neural Tube Defect and Down's Syndrome Screening: Results of a Multicentre Clinical Trial
Authors: Spencer K , Macri J N , Anderson R W , Aitken D A , Berry E , Crossley J A , Wood P J , Coombes E J , Stroud M , Worthington D J , Doran J , Barbour H , Wilmot R
Published: February 2014

We report a multicentre clinical field trial of a novel dual analyte enzyme immunoassay method for the simultaneous measurement of alpha-fetoprotein (AFP) and free β-human choriogonadotropin (hCG) in the same microtitre well. The assay was shown to have good technical performance in the hands of all trial centres, with between assay coefficients of variation better than 10% for both analytes across the whole of the assay ranges. The method compared well with single analyte measuring procedures and produced acceptable performance as judged by external quality assurance criteria. Recovery of added analyte and analyte dilution curves also showed acceptable performance.

In clinical evaluation of a large set of neural tube defect cases, good clinical discrimination from unaffected cases was observed using APP. With over 150 Down's syndrome cases, the combination of AFP and free (3 hCG confirmed the high detection rates achievable using this marker combination, with detection rates in excess of 70% in early gestation.

We conclude that the combination of clinically superior markers coupled with technologically innovative assay design will lead to more efficient Down's screening programmes.
Plasma Prekallikrein Levels in Patients with Hepatocellular Carcinoma and Liver Cirrhosis: A Pilot Study
Authors: Ciechanowicz Andrzej , Kawiak Jerzy , Miks Barbara , Dutkiewicz Tomasz , Dlugosz Aldona , Syczewska-Wawrzynowicz Marta , Marzȩcka Justyna
Published: February 2014

The concentration of plasma prekallikrein (PK) in five patients with hepatocellular carcinoma (HCC) has been measured and related to levels in 18 patients with liver cirrhosis (LC) and 30 healthy subjects. It was found that the mean PK level was significantly increased in patients with HCC, while patients with LC demonstrated lower concentrations, as compared with healthy subjects. The results indicate that PK might be useful in screening cirrhotic patients for HCC. Longitudinal studies of PK in a larger group of patients at risk of developing HCC are therefore recommended.
Renal Isoamylase Clearance as a Measure of Altered Renal Charge Selectivity in Patients with Diabetes Mellitus
Authors: Thum C N , Oelbaum R S , Foo A Y , Rosalki S B
Published: February 2014

Microalbuminuria is well established as a marker for early renal damage in diabetic patients. Differences in charge selectivity in glomerular protein filtration may also be an early marker of renal damage. We investigated the possible usefulness of the renal clearances of pancreatic and salivary amylases, and the ratio of the two, as markers of early renal damage in 55 diabetic subjects and 21 healthy controls. Diabetic patients with established albuminuria and microalbuminuria had increased clearance of salivary amylase and a trend toward lower pancreatic/salivary amylase clearance ratios compared to healthy controls and diabetic subjects without albuminuria, but the overlap with controls and diabetics without albuminuria was too large for the test to be useful.
Isoelectric Focusing in an Immobilized pH Gradient for the Detection of Intrathecal IgG in Cerebrospinal Fluid: Sensitivity and Specificity for the Diagnosis of Multiple Sclerosis
Authors: Cowdrey G N , Tasker P J , Gould B J , Rice-Oxley M , Firth G B
Published: February 2014

Cerebrospinal fluid and serum from 192 patients was analysed for the presence of intrathecally synthesized oligoclonal IgG bands using isoelectric focusing in an immobilized pH gradient pH7–10. The sensitivity of this method for the diagnosis of multiple sclerosis (MS) was 95% (21 of 22), or 75% if patients with suspected MS were included. The specificity for the diagnosis of MS was 98%, or 96% if the suspected MS patients were included. The very high specificity may be because the intrathecally synthesized oligoclonal IgG associated with MS is more alkaline than IgG from serum and is better detected in an immobilized alkaline pH gradient.
Successful Treatment of Short Stature and Delayed Puberty in Congenital Magnesium-Losing Kidney
Authors: Taylor A J , Dornan T L
Published: February 2014

Bartter's syndrome is a well described but uncommon disease characterized by hypokalaemia and hyperplasia of the juxtaglomerular apparatus of the kidney. It may present in infancy with failure to thrive and muscle weakness; it commonly causes short stature. Lesions at different sites within the renal tubule have been proposed as the cause of the syndrome. However, the biochemical abnormalities in many cases can be explained by defective reabsorption of chloride in the ascending loop of Henle, with loss of sodium and water and a secondary increase in renin and aldosterone concentrations. Less severe cases have been described which present in adolescence and have tetany as a prominent feature. Primary renal loss of magnesium associated with potassium wasting has been described in such cases and it has been suggested that these can be distinguished from classical Bartter's syndrome by hypocalciuria. This less well characterized disease has been named Welt, Gitelman-Welt or Gitelman syndrome and may include deficient tubular reabsorption of chloride, but the sites of magnesium and potassium loss in the kidney are uncertain. We describe a patient with this syndrome who presented with short stature, delayed puberty and tetany and responded well to magnesium replacement.