Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

Whenever a child presents to hospital with a reduced level of consciousness, admitting clinicians have to decide the underlying cause rapidly so that the correct emergency treatment can be initiated. Unfortunately, the clinical presentations of many of the possible diagnoses are very similar. The diagnosis often results from investigations within the clinical biochemistry laboratory. In the past, clinicians have had limited guidance on which tests to request when presented with a child with a reduced level of consciousness. Guidelines have recently been developed relating to all aspects of management of the child in a coma. Due to a lack of evidence in the literature regarding the most appropriate first line tests for children with a reduced level of consciousness, a formal consensus process ('Delphi consensus') was performed using a large multidisciplinary panel of experts. The recommendations reached by this process include the list of initial ('core') tests to request for all children with a reduced level of consciousness (excluding those immediately after suffering a convulsion and those involved in obvious trauma). Depending upon the results of these 'core' tests and the clinical condition of the child, further tests may be requested later. The key point is that all the samples have been taken at the time of presentation to provide the best chance of reaching a diagnosis and correctly treating the child. The article reviews the recommended core investigations and further tests and discusses how individual laboratories can help to implement the guidelines jointly with their Emergency and Paediatric Departments.

Background: The purpose of this study was to compare The Binding Site serum free light chain assay on two analytical platforms; the Dade Behring BNII and the Olympus AU400. Reference intervals were subsequently established on each analyser.

Methods: In total, 112 serum samples routinely submitted to the laboratory for protein electrophoresis were used for the comparison study. Reference interval data was generated from 126 ostensibly healthy anonymous individuals.

Results: Serum free light chain results on the BNII and AU400 analysers are not directly comparable. The BNII produces results which are significantly higher than the AU400.

Conclusions: Laboratories using The Binding Site serum free light chain assay are strongly advised to establish local reference intervals.

Background: We investigated how sensitive serum free light chain (FLC) analysis was for the detection of Bence-Jones protein (BJP) and whether a serum κ/λ ratio could replace urine electrophoresis as part of the investigation algorithm for monoclonal gammopathy.

Methods: Serum κ and λ FLC analysis was performed on 932 consecutive patients investigated for monoclonal gammopathy, along with serum electrophoresis, serum immunofixation where appropriate, and in 483 individuals urine immunofixation. A reference interval for the κ/λ ratio was derived from all patients who had normal serum and urine electrophoresis and no B-cell dyscrasia (n = 312).

Results: The 100% reference interval was 0.21-1.44 (median 0.50), lower than that previously published. From the 483 patients who provided urines, BJP was detected in 34, with an abnormal κ/λ ratio in 26 of these. Using the reference interval, sensitivity, specificity, negative and positive predictive values of an abnormal κ/λ ratio for the presence of BJP were 0.76, 0.96, 0.98 and 0.59 respectively. Seven patients had an abnormal κ/λ ratio with no paraprotein.

Conclusion: All κ/λ results should be interpreted against a reference interval appropriate for the analyser in use and the population under study. Some urines will be positive for BJP when the serum κ/λ ratio is normal, but there is little difference in outcome whether urine for BJP or serum FLC analysis is included in the screening strategy for monoclonal gammopathies. On balance, we believe that it is not yet time to dispense with urine BJP in the investigation of patients with suspected monoclonal gammopathy.

Aims: To investigate whether hTERT mRNA expression could be used as a diagnostic and prognostic marker of bladder transitional cell carcinoma.

Methods: SYBR green I real-time fluorescence quantitative PCR method was used to quantify hTERT mRNA expression in exfoliated cells from patients with bladder transitional cell carcinomas.

Results: A significant correlation was observed between the level of hTERT mRNA expression in exfoliated cells collected in urine with pathological grade and clinical stage.

Conclusions: Our results show that SYBR green real-time fluorescence quantitative evaluation of hTERT mRNA could be a useful marker of tumor progression for the early diagnosis and prognosis of bladder cancers.

Background: Specific proteins have traditionally been analyzed using methodologies such as immunonephelometry on specialized analyzers. It is now possible to perform specific protein testing on clinical chemistry analyzers using immunoturbidimetry. Performance characteristics of turbidimetric assays for eight common specific proteins were evaluated against a nephelometric method.

Methods: The Abbott Architect ci8200 and the Beckman Immage were used to perform IgA, IgG, IgM, C3, C4, haptoglobin, and transferrin testing. Abbott, Sentinel and Roche reagents were used to perform CRP testing on the ci8200. The specific protein assays were evaluated for precision, linearity, limit of detection, functional sensitivity, method comparison, prozone, and workflow.

Results: Total precision for the immunoturbidimetric assays was consistently better than 2% CV and linear throughout the dynamic range of the assays (recovery ± 10% of target values). The limits of detection, functional sensitivities, and accuracy (as determined by performance against target values for proficiency testing samples and reference materials) are suitable for clinical purposes. Method comparison, as determined by correlation coefficients, and performance against proficiency testing samples, demonstrated good agreement between the turbidimetric and nephelometric tests. Both the turbidimetric and nephelometric assays provided reliable results under conditions of antigen excess. Specific protein test requests could be consolidated within our routine chemistry workload without impacting analytical test throughput.

Conclusions: As demonstrated by their performance characteristics, the Architect ci8200 immunoturbidimetric specific protein assays are suitable for routine use and correlate well with representative immunonephelometric assays on the Beckman Immage analyzer. The ability to perform specific protein analyses on an integrated clinical chemistry/immunoassay system can allow for consolidation of testing on a single platform, resulting in improved laboratory operations efficiency.

Background: Oxidative stress has been implicated in the pathogenesis of coeliac disease. The aim of this study was to examine the modulation of the biochemical response to oxidative stress in untreated and treated coeliac disease.

Methods: The study involved peripheral blood samples from 39 paediatric patients (18 with active, 11 with silent form of the disease, 10 on gluten-free diet [GFD]) and 30 control subjects. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the concentrations of total glutathione (GSH) and lipid hydroperoxides (LOOH) were determined in patients and controls.

Results: In comparison to the controls, a significant increase in SOD activity was found in the active group (P<0.05), while CAT activity was elevated in GFD group (P<0.05). GPx activity was lower in patients than in controls (active and silent, P<0.001; GFD, P<0.01). GSH contents were significantly reduced in all patient groups (P<0.001) as well, while the concentration of LOOH was elevated in active and silent group (P<0.001). The concentration of LOOH correlated negatively with the activity of GPx (r = -0.32, P<0.01) and the concentration of GSH (r = -0.70, P<0.001). A significant positive correlation was found between the concentration of GSH and the activity of GPx (r = 0.57, P<0.001).

Conclusions: The results show evidence of increased oxidative stress in untreated coeliac disease. Although LOOH were not significantly elevated in the GFD group, changes in antioxidant enzyme activities and GSH content demonstrate that oxidative stress persists even in treated patients.

Background: Intestinal alkaline phosphatase (IAP) isozymes in the healthy human serum samples appears in two isoforms: normal-molecular-weight IAP (NIAP) and high-molecular-weight IAP (HIAP). We have demonstrated that the reference range for serum alkaline phosphatase (ALP) activity is higher in blood group B and O antigen secretors than in the tested other blood groups, for the appearance of these isoforms is depended on blood group B or O antigen secretors.

Methods: We assessed a diethanolamine-L-phenylalanine (DEA-Phe) method for measuring tissue non-specific alkaline phosphatase (TNAP). This assays the sum of liver alkaline phosphatase and bone alkaline phosphatase activities as determined by an inhibiting IAP activity method with Phe. We classified 420 healthy subjects into two groups, a group of subjects who had blood group B or O antigen secretors (n = 184) and a group of subjects who had other blood groups (n = 236).

Results: ALP activity was higher in the B or O secretor group than in the other group: 20.9% higher (P<0.001) by the N-methyl-D-glucamine method, 13.7% higher (P<0.002) by 2-amino-2-methyl-1-propanol method, and 9.6% higher (P<0.05) by the diethanolamine method, but there was no significant difference in TNAP activity between the two blood group when measured by the DEA-Phe method.

Conclusions: These results of this study support the expectation that the DEA-Phe method would be specific for TNAP activity. In addition, the reference range for TNAP activity did not vary with the differences in the tested all blood groups.

Background: It is now established that the bioactive lipid lysophosphatidic acid (LPA) contributes to cancer initiation, progression and metastasis, including those of prostate cancer. LPA is produced in the serum and plasma mainly by conversion from lysophospholipids through the action of lysophospholipase D (lysoPLD), which is identical to the soluble form of autotaxin (ATX) originally isolated as a tumour cell motility-stimulating factor. In this study, we evaluated the usefulness of lysoPLD/ATX activity as a diagnostic marker.

Methods: The serum lysoPLD activity, assessed by measuring choline liberation from the substrate lysophosphatidylcholine, was measured in patients with prostate cancer and compared with the concentrations of prostate-specific antigen (PSA) and conventional nutritional assessment markers.

Results: The serum lysoPLD activity in prostate cancer patients was not statistically different from that in the controls. Consistent with this, there was no correlation between the serum lysoPLD activity and the serum PSA concentrations. However, the lysoPLD/ATX activity did decrease after operation in the prostate cancer patients and seemed to reflect the postoperative damage or the nutritional status.

Conclusions: We postulate that while the serum lysoPLD/ATX may not be a marker of prostate cancer, it promises to instead be a new marker of nutritional status.

Background: Surveyor^TM nuclease is a new single-strand-specific endonuclease that cleaves heteroduplex DNA at a base-mismatch site in both DNA strands. We applied this enzyme to detection of p53 gene mutations in haematological malignancy.

Methods: DNA fragments including exons 5 and 6, and those including exons 7 and 8, of the p53 gene, were amplified by polymerase chain reaction using DNA samples extracted from bone marrow aspirates. Denaturation followed by gradual annealing of the amplified fragments formed a heteroduplex with a base-mismatch if a mutation existed because the DNA samples contained wild-type DNA derived from coexisting non-malignant cells. After cleavage by Surveyor nuclease, mutations were simply detected by gel electrophoresis as extra bands of shorter size.

Results: Somatic mutations were clearly detected by this method in three of 39 different samples and confirmed by sequencing. The limit of detection estimated by changing the proportion of heteroduplexes in hetero/homoduplexes was between 1/8 and 1/16.

Conclusion: We suggest that our method is not only simple, but also sensitive, compared with other complicated methods, and would therefore be useful in current clinical laboratory settings.

Background: Protein 1 (P1)/Clara cell 16 kDa protein (CC16, previously named CC10), a potentially immunosuppressive protein secreted by non-ciliated cells of the tracheobronchial epithelium, has been found to be a new useful lung-specific biomarker in several pathological lung conditions. Particularly, urinary P1 (uP1) may reflect the altered lung functions in pneumoconiosis.

Methods: We investigated the relationship between uP1 values and lung functions in 31 non-smoking pneumoconiotic males (mean age 73 years) with a history of dust exposure work in shipbuilding. The protein was measured using an originally prepared enzyme-linked immunosorbent assay system. The forced expiratory volume in 1 s % (FEV_1.0%) and % vital capacity (%VC) were tested with a spirometer.

Results: The mean values of uP1 were 4.62 ± 4.82 (mean ± standard deviation) ng/mol creatinine. A univariable correlation test showed a significant positive correlation between uP1 and %VC (r = 0.356, P = 0.049). Also, a multiple regression analysis, when adjusted for age, disease duration, FEV_1.0% and %VC, showed a significant correlation of uP1 with %VC (β = 0.467, P = 0.030).

Conclusion: The results suggest that a decreased uP1, corroborated by a decreased %VC, may be the result of damage to secretory cells. Measurement of uP1 may become a possible index of fibrotic changes in pneumoconiosis.

Background: Rapid and sensitive tests for detecting buprenorphine and its metabolites for drug-screening situations have been long awaited. From the tests available, we have evaluated two on-site drugs-of-abuse testing devices using competitive binding immunoassays and one homogeneous enzyme immunoassay measured on an automated analyser.

Methods: A total of 49 urine specimens were tested using three different kits. Two were point-of-care devices, a cassette test, QuikPac II OneStep Buprenorphine Test, and a strip test, QuikStrip OneStep Buprenorphine Test. The other was the CEDIA^® Buprenorphine Assay performed on a Roche Modular P analyser. The confirmation analyses were performed using liquid chromatography-mass spectrometry.

Results: The sensitivities of the three methods ranged from 88% to 100% and specificities from 91% to 100%. All three kits, especially the cassette and strip devices differed markedly from each other with respect to interpretation of the test result and to clarity of the test performance. Increasing the read time of the QuikStrip device from 5 to 30 min resulted in an increase in false-negative test results.

Conclusions: Our results indicate that special care should be taken when selecting immunology-based point-of-care methods for measurement of buprenorphine.

The clinical diagnosis of mixed cryoglobulinaemia is difficult due to heterogeneity in presentation. Symptoms include the classical triad of purpura, arthralgia and weakness, with one or more other organs involved. We discuss a case of cryoglobulinaemia that presented with sensory motor neuropathy and with features of mononeuritis multiplex syndrome, but which lacked other classical features. Laboratory testing revealed a profile typical of mixed cryoglobulins: immunoglobulin M (IgM) paraprotein, low fourth carbon (C4) and positive rheumatoid factor. Subsequent investigations failed to reveal an underlying infectious or neoplastic cause. This case demonstrates the need to include cryoglobulinaemia in the differential diagnosis for peripheral neuropathy, and the critical importance of using the correct collection procedure to isolate cryoglobulins.

We investigated a patient with type 1 diabetes mellitus undergoing pancreatic islets transplantation. In this patient, we evaluated the clinical usefulness of serial measurement of serum S100A8/A9 complex levels for detecting acute inflammatory responses associated with rejection of transplanted pancreatic islets. The serum S100A8/A9 complex was a more sensitive marker for acute inflammation associated with islet transplant rejection than the serum C-reactive protein. Thus, the serial measurement of the serum S100A8/A9 complex concentration is useful for monitoring the patients with pancreatic islet transplantation.

We previously reported a case of heterozygous β-thalassaemia with IVS1-1G > C substitution in the β-globin gene and a non-detectable level of mutant mRNA in the patient's reticulocytes. The purpose of this study was to determine whether the transcription and RNA splicing and processing of the mutant gene occurred. We analysed the expression of the mRNA encoded by the cloned mutant gene in COS-1 cells by reverse transcription-polymerase chain reaction followed by agarose gel electrophoresis and nucleotide sequencing. The G > C mutation completely inactivated the normal 5- splice site and resulted in the activation of two cryptic 5- splice sites, located 16 and 38 nt upstream of the normal site. The usage of these two cryptic sites accords with the findings of reports on IVS1-1G > A or IVS1-1G > C substitution of exon 1 of the β-globin gene. Additional experiments that involved transfection of equal amounts of both normal and mutant vectors into COS-1 cells indicated the presence of mutant mRNAs. In conclusion, the β-thalassaemia gene (IVS1-1G > C) was expressed in transfected cells, but showed aberrant RNA splicing. Further studies will be required to clarify the molecular mechanism that results in severe reduction in the mutant mRNA level in vivo.