Research has demonstrated that most laboratory errors occur in the preanalytical phase of testing. In view of the paucity of studies examining preanalytical errors, we evaluated our laboratory request forms for the frequency and impact of incomplete data.
This study examined all request forms received at our laboratory during a five-day period. The forms were scrutinized for the presence of specific parameters. The impact of abbreviated diagnoses was analysed, as well as how lack of ward or telephone details affects the communication of critical results to clinicians.
A total of 2550 request forms were analysed. Medication(s) used by the patient (89.6%) and doctor’s contact number (61.2%) were the most incomplete parameters. No diagnosis was provided on 19.1% of forms, and when a diagnosis was present it was an abbreviated form in 37.3%. This resulted in 35.5% of diagnoses not being recorded by reception staff. Incomplete ward information was found on 4.9% of forms. In a separate search, the impact of 151 request forms (collected over a period of eight months), with incomplete ward location information and corresponding to critical results was assessed. Critical results were not communicated by telephone to clinicians in 19.9% of cases.
As laboratory data influences 70% of medical diagnoses, incorrect or incomplete data provided to the laboratory could significantly impact the success and cost of overall treatment.
Reference intervals are used to distinguish between healthy and diseased state. Ideally, they are defined using specimens only from ‘healthy’ individuals, but this is often difficult or impossible. In order to use routine clinical laboratory data, outliers must be removed before the underlying distribution and changes related to age and sex can be modelled. This paper illustrates the process for plasma alkaline phosphatase (ALP). ALP levels are high in infancy and childhood, peak in adolescence, are stable from the early 20s and rise after the fourth decade. Three types of normalizing transformations (Logarithmic, Box-Cox and Cole's LMS) are compared.
Single ALP results from 75,328 individuals aged 0–80 years were binned by sex and age. The normalizing transformations were applied to each bin, outliers were removed and the normalizing transformations were reapplied to the remaining data. The normality of the transformed data was assessed by normal score plots and the Kolmogorov-Smirnov test. Fractional polynomials were used to model the underlying parameters of the transformations and the derived parametric reference intervals (mean ± 1.96 standard deviations), separately for each sex as a whole and partitioned into two or three age ranges, with overlapping to give smooth transitions.
All transformations yielded acceptably normal data, but the LMS method gave the closest approximation to normal. Outlier rates were similar for each method. The derived reference ranges were similar for all the three methods. Splitting the data-set into several segments resulted in a better fit with the peak seen in adolescence.
Routine clinical laboratory specimens can be used to derive reference intervals.
We aimed to establish the relationship between glycated haemoglobin (HbA1c), hypertension and microalbuminuria onset in type 2 diabetes. We also intended to ascertain the metabolic action of homocysteine on LDL fatty acids and on renal function.
The study was carried out on 200 patients with type 2 diabetes and 200 healthy subjects. HbA1c, apolipoprotein B (apo B) and microalbuminuria were measured using immunoturbidimetric methods. Cholesterol, peroxide, urea and uric acid were assayed using colorimetric methods. Creatinine clearance was calculated using the Cockroft-Gault equation. Homocysteine was measured by immunological fluorescence polarization. LDL fatty acids were quantified by gas chromatography.
Creatinine and microalbuminuria significantly increased in type 2 diabetes when compared with controls. Microalbuminuria was significantly correlated with HbA1c and with the presence of high blood pressure. Homocysteinaemia significantly correlated with creatinine clearance in diabetes. Linoleic acid (C18:26) did not differ between groups. C18:26/C18:33 ratio was three times higher in diabetics than in controls. Total saturated fatty acids, homocysteine, H2O2 and LDL-thiobarbituric reactive substances significantly increased in microalbuminuric when compared with normoalbuminuric diabetes. Total polyunsaturated fatty acids, arachidonic acid (C20:46), LDL-cholesterol, apo B and creatinine clearance significantly decreased in microalbuminuric when compared with normoalbuminuric diabetes.
Microalbuminuria onset is associated with renal protein oxidation that is preceded by LDL fatty acid oxidation. The latter is initiated by H2O2 produced from an auto-oxidation of homocysteine and increased metabolism of arachidonic acid towards its pro-inflammatory eicosanoids. An oxidative stress state is the common ground of diffused vasculopathy.
For nearly 20 years, we and others have used a three-parameter power function as a direct estimation error model for immunoassays. The main application is imprecision profile plots (after translating from variance to coefficient of variation) but other uses include weighting functions for regression analysis and variance stabilizing transformations. Although generally successful, the intrinsic monotonicity of the function means that it fails to describe small but distinct increases in variance that occasionally occur near the assay detection limit.
A systematic comparison of five variance functions was undertaken, using randomly drawn samples from a large body of real immunoassay data.
Variance function accuracy (hence imprecision profile accuracy) can be markedly improved, particularly near the assay detection limit, by employing a pair of complementary three-parameter power functions, together with a constrained four-parameter function, which provides for a variance turning point.
A set of rules, based on an objective goodness-of-fit statistic, can be used to automate presentation of the most appropriate function for any particular data-set. Flexibility is easily incorporated into the selection rules and is actually highly desirable to encourage ongoing evaluation with a wider variety of data. A Win32 computer program that performs the variance function estimation and plotting is freely available.
A protocol exists for the collection of samples to investigate unexplained hypoglycaemia, termed the ‘hypopack’. These packs are kept in Accident and Emergency departments and neonatal special care baby units throughout Northern Ireland and most wards of the Regional Children's Hospital. A retrospective audit of 107 hypopacks received between July 2001 and December 2003 highlighted a number of problems: samples collected when the patient was receiving dextrose, incomplete clinical history provided, insufficient and haemolysed samples received and poor filing of reports in charts. These were addressed by re-designing the request form, updating the protocol and introducing a summative report. The new protocol was introduced in April 2006.
The aim of this study is to assess whether the revised protocol improved utility of the hypopack. A retrospective re-audit of 100 hypopacks received between April 2006 and May 2007 was performed.
Forty-nine percent of patients were hypoglycaemic (<2.6 mmol/L) compared with 35% in the original audit. In both audits, 33% of laboratory reports were missing from patients' charts. One case of medium-chain acyl-CoA dehydrogenase deficiency, three cases of hyperinsulinism and two endocrine-related cases were identified.
The new hypopack protocol has increased the number of appropriately performed investigations. Provision of information concerning dextrose infusion has assisted the interpretation of the hypopack results.
β2-Microglobulin (β2m) has been identified as the precursor protein of dialysis-related amyloidosis (DRA), which is a serious complication for haemodialysis (HD) patients. However, mechanisms underlying β2m amyloid fibril formation remains to be elucidated. We previously demonstrated, in amyloid deposits from HD patients, a conformational isoform of β2m with an unfolded C-terminus. However, no direct experiments have previously been performed to address whether unfolded β2m in the C-terminus may be prone to form amyloid fibrils.
To evaluate roles of C-terminal amino acids in β2m-induced amyloid formation, we generated six types of recombinant β2m with amino acid substitutions in the C-terminal region. To investigate their conformational change and amyloidogenicity, we measured circular dichroism spectra, the fluorescence intensity of tryptophan and thioflavin-T (ThT) of the recombinant β2m. To analyse morphological change of β2m, we performed electron microscopy (EM) on the samples with elevated ThT fluorescence intensity. We used ultrasonication to enhance β2m destabilization of the protein.
β2M Trp95Leu and Arg97Ala showed conformational changes and increased their amyloidgenicity compared with β2m wild-type (WT). With ultrasonication, β2m Trp95Leu and Arg97Ala generated more amyloid fibrils than did β2m WT even in physiological solution. EM showed that β2m formed amorphous debris containing typical amyloid fibrils at 24 hours, when ThT fluorescence intensity was three-fold lower than that at six hours.
Conformational changes in the C-terminus of β2m may play an important role in DRA and that ultrasonication is useful for analysis of β2m amyloidogenesis.
Adiponectin links obesity with insulin resistance, which causes various metabolic abnormalities including dyslipidaemia. Apolipoprotein E (apoE) phenotypes also affect lipoprotein profiles. We aimed to determine whether low adiponectin concentrations are associated with insulin resistance and downstream metabolic abnormalities in obese children.
We measured fasting concentrations of lipids, apoE, glucose, insulin and adiponectin, as well as anthropometric parameters, in 191 obese children aged 6–15 years. ApoE phenotypes were determined by isoelectric focusing. Boys (n = 79) and girls (n = 39) with apoE3/3 were classified into tertiles according to their adiponectin concentrations. Metabolic parameters, were compared among these three groups in boys and girls separately.
The low adiponectin groups had higher median homeostasis model assessment of insulin resistance (HOMA-IR) than the middle and high adiponectin groups in both boys [5.3 (low) versus 3.1 (middle; P < 0.05) and 3.5 (high; P < 0.05)] and girls [5.0 (low) versus 4.4 (middle) and 3.0 (high; P < 0.05)]. However, only boys who were in the low adiponectin group exhibited significantly higher concentrations of blood pressure, triglycerides, LDL-cholesterol, and remnant-like particle-cholesterol, and lower concentrations of HDL-cholesterol compared with the middle or high adiponectin groups.
Low adiponectin concentration is associated with insulin resistance in obese children. Furthermore, decreased adiponectin with E3/3 exhibited more prominent downstream metabolic abnormalities in obese boys than in obese girls.
Recent recommendation advocates the reporting of HbA1c in terms of mean plasma glucose. We examined the impact of improving patients' interpretation of a given HbA1c value on glycaemic control.
We conducted a questionnaire survey among 111 patients attending a hospital diabetes clinic. Patients were provided with information relating to the association between HbA1c and mean plasma glucose levels. Glycaemic control among 80 patients with poor glycaemic control was assessed before and approximately seven months after such intervention.
Of the respondents, 40.5% (45/111) were familiar (F) (31 type 1, 14 type 2) and 59.5% (66/111) were unfamiliar (U) (23 type 1, 43 type 2) with the term HbA1c. Following information about the interpretation of HbA1c, patients with poorly controlled diabetes (HbA1c >9%) showed a significant reduction in HbA1c levels if they were from group U (10.7% vs. 9.5%, P = 0.04) but not from group F (10.5 vs. 9.8, P = 0.28). Patients with moderately poor glycaemic control (HbA1c 7.5–9%) showed no significant change in HbA1c levels following intervention (8.3% vs. 8.2%, P = 0.57 group U; 8.3% vs. 8.2%, P = 0.79 group F).
Patients' knowledge of HbA1c is poor, especially among persons with type 2 diabetes. Improvement in patients' understanding of HbA1c, particularly among those with very poorly controlled diabetes with no prior knowledge of HbA1c is associated with improvement in their glycaemic control. Strategies to engage patients to know and interpret their HbA1c values should be encouraged within routine clinical practice.
Platelets are involved in the pathogenesis of aspirin-induced asthma (AIA). AIA patients suffer from an active disease despite avoidance of aspirin, and it has been suggested that administration of aspirin to these patients increases the generation of immediate oxygen products of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), in their platelets. 12-HPETE further activates the 5-lipoxygenase of leukotriene B4-producing inflammatory macrophages precipitating an acute asthmatic attack. Glutathione peroxidase (GPX) has the antioxidant capacity to reduce 12-HPETE, and thus modulate the arachidonic acid metabolic cascade. There is evidence that selenium (Se) nutrition can influence asthma but Se status in AIA patients has not received much attention.
We measured Se concentrations and GPX activities in platelets and plasma from 13 patients with AIA. Age- and sex-matched healthy individuals served as the control group.
Patients with AIA had significantly higher median platelet Se concentration (102 ng/mg platelet protein) when compared with controls (49 ng/mg platelet protein, P = 0.003). Plasma Se concentrations in patients with AIA and controls were not significantly different (P = 0.59). Median platelet GPX activity was significantly higher in patients with AIA (102.7 mU/mg platelet protein) than in controls (66 mU/mg protein) (P = 0.05). The patient and control groups when combined showed weak, but significant correlation between platelet Se concentration and platelet GPX activity (r = 0.44; P = 0.03).
It is proposed that the higher platelet Se concentration observed in AIA patients contributed to the higher platelet GPX activity seen in these patients. Such an enhanced antioxidant defence system might represent an adaptive response to protect against increasing free radical production by inflammatory cells in AIA and help decelerate ongoing respiratory hypersensitivity.
Alterations in the copy number of mitochondrial DNA (mtDNA) play a role in the pathogenesis of mitochondrial diseases and other many common diseases. Recently, the copy number of leukocyte mtDNA has been considered to serve as a biomarker to monitor or chase such diseases. Therefore, reproducible mtDNA measurement is required.
Peripheral blood mononuclear cells were prepared by a density-based method. The mtDNA/cell was measured by quantitative realtime polymerase chain reaction.
The degree of platelet contamination varied to a large extent among preparations. The mtDNA copy numbers per mononuclear cell were 269 ± 51 and 146 ± 14 in the samples before and after the platelet depletion, respectively.
A density-based mononuclear cell preparation causes heavy platelet contamination. The platelet depletion from a sample is particularly important for comparing the mtDNA contents between different dates or between different patients.
Estimated glomerular filtration rate (eGFR) is now routinely reported by most National Health Service laboratories. There are many limitations when interpreting eGFR: thyroid dysfunction is not widely recognized as being one of these.
We extracted from the pathology computer system results of all patients with hypothyroidism and hyperthyroidism who also had serum creatinine measured. We also extracted creatinine data on euthyroid patients. eGFR was calculated using the simplified Modification of Diet in Renal Disease Study equation.
The median eGFRs of the hypothyroid, euthyroid and hyperthyroid patients were 64, 77 and 107 mL/min/1.73 m2, respectively; all groups were significantly different from each other (P < 0.001).
Thyroid dysfunction is associated with significant alterations in eGFR and actual GFR.
The analytical performance characteristics of an enzyme-linked immunosorbent assay for the receptor for advanced glycation end-products (RAGE) were evaluated. The within- and between-subject components of biological variation were also estimated.
Blood was sampled from healthy volunteers into K2-ethylenediamine tetraacetic acid (EDTA) and serum separator tubes (SST) and the stability of RAGE in whole blood, plasma and serum examined. Performance characteristics of the assay were assessed using quality control materials. Three samples were obtained from each of 21 healthy volunteers one-week apart, RAGE measured and components of biological variation estimated.
RAGE concentrations in blood specimens collected into K2-EDTA and SST were stable for at least 6 hours and, after centrifugation, both plasma and serum were stable for at least 24 hours. The RAGE assay had the following characteristics: inter-assay imprecision: coefficient of variation ≤7.3%, working range: 26–5000 ng/L, linearity: r = 0.9977 and detection limit: 26 ng/L. Overall within- and between-subject biological variations were 14.6% and 56.5%, the index of individuality was 0.31 and the reference change value was 49.0% at P < 0.05.
Samples for RAGE analyses in serum or plasma can be collected without significant difficulties with the assay showing acceptable analytical performance characteristics. Conventional population-based reference values are of limited utility in diagnosis, indicating that RAGE is likely to be more useful in monitoring disease.