Annals of Clinical Biochemistry: An international journal of biochemistry and laboratory medicine, 2015
Familial dysalbuminaemic hyperthyroxinaemia (FDH) is an important cause of discordant thyroid fun... more Familial dysalbuminaemic hyperthyroxinaemia (FDH) is an important cause of discordant thyroid function test (TFT) results (due to an inherited albumin variant) however, the diagnosis can be challenging. A 51 year-old man had persistently elevated free thyroxine (T4), with discordant normal TSH and normal free triiodothyronine. He was clinically euthyroid and had a daughter with similar TFTs. We aimed to apply a whole protein mass spectrometry (MS) method to investigate this case of suspected FDH. Intact serum albumin was assessed directly using electrospray time-of-flight (TOF) MS. Results were confirmed using tryptic peptide m/z mapping and targeted DNA sequencing (exons 3 and 7 of the albumin gene). We also used this sequencing to screen 14 archived DNA samples that were negative for thyroid hormone receptor mutations (in suspected thyroid hormone resistance). MS analysis demonstrated heterozygosity for an albumin variant with a 19 Da decrease in mass, indicative of an Arg→His substitution. The FDH variant was confirmed with peptide mapping (showing the precise location of the substitution, 218Arg→His) and DNA sequencing (showing guanine to adenine transition at codon 218 of exon 7). The same FDH variant was identified in one additional screened sample. TOF MS is a novel procedure for diagnosing FDH. The test is rapid (<10 min), can be performed on <2 µL of serum and requires minimal sample preparation. KEY: TERMS: : familial dysalbuminaemic hyperthyroxaemia, mass spectrometry, albumin variant, discordant thyroid function tests.
Objectives The aim of this study is to compare a new improved point of care cardiac troponin assa... more Objectives The aim of this study is to compare a new improved point of care cardiac troponin assay (new POC-cTnI) with 1. its predecessor (old POC-cTnI) and 2. a high sensitivity assay (hs-cTnI) for the diagnosis of acute myocardial infarction (AMI) and for major adverse cardiac events (MACE) by 30 days. Methods This is a single centre observational study, set in Christchurch Hospital, New Zealand. Patients presenting to the emergency department with non-traumatic chest pain underwent blood sampling at 0 h and 2 h post presentation for analysis with the 3 cTnI assays for the outcome of AMI and for analysis using an accelerated diagnostic protocol (ADP-normal 2 h troponins, normal electrocardiograms and Thrombolysis In Myocardial Infarction (TIMI) score of 0 or ≤ 1) for 30 day MACE. Results Of 962 patients, 220 (22.9%) had AMI. Old POC-cTnI was least sensitive at 70.0% (65.4–73.9%) by 2 h (p < 0.001). New POC-cTnI, sensitivity 93.6% (89.9–96.2%) had similar sensitivity to hs-cTnI,...
Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement t... more Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human alpha 1-antitrypsin (alpha 1AT) cDNA in yeast. Alteration of the alpha 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-III. Alteration of two further residues, to give a five-residue sequence identical to AT-III, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglycosylated and have shorter in vivo half-lives (t1/2) than human alpha 1AT. These antithrombins should be suitable for therapeutic replacement of AT-III in cases of congenital deficiency and in conditions associated with acquired AT-III deficiency, such as disseminated intravascular coagulation.
Betaine supplementation decreases post-methionine hyperhomic [3]. This is thought to contribute t... more Betaine supplementation decreases post-methionine hyperhomic [3]. This is thought to contribute to the common mocysteinemia in chronic renal failure. occurrence and rapid progression of atherosclerosis in Background: Fasting and post-methionine load hyperhomothe ESRD population. Although there is no evidence cysteinemia are independent risk factors for vascular disease yet to show that lowering plasma tHcy reduces the risk that are common in chronic renal failure. Folate decreases but seldom normalizes fasting total homocysteine (tHcy) concen-of atherosclerosis, it is widely believed that reducing trations in such patients. Glycine betaine (GB) is known to tHcy levels should be a therapeutic target [4]. decrease tHcy in other clinical settings, but whether it is bene-The optimal treatment for hyperhomocysteinemia in ficial in chronic renal failure has not been established. chronic renal failure has not been determined. Folic acid Methods: We conducted a crossover-controlled trial in 36 patients with chronic renal failure to determine if oral GB Patients
The performance of fructosamine assays in 35 laboratories was assessed over a six month period in... more The performance of fructosamine assays in 35 laboratories was assessed over a six month period in 1987. While agreement between laboratories was poor in the range expected in diabetic patients, most laboratories were internally consistent. The interlaboratory differences are largely attributed to the calibration methods used for this test. Fructosamine results from any one laboratory must not be interpreted by comparison with data from other laboratories or the literature. Other common biochemical tests were found to suffer from similar problems, but to a less serious extent.
Classical Rett syndrome is a severe neurodevelopmental X-linked dominant disorder affecting 1/15,... more Classical Rett syndrome is a severe neurodevelopmental X-linked dominant disorder affecting 1/15,000 girls worldwide. MECP2 has been identified as the predominant gene associated with Rett syndrome. Approximately 65-85% of patients with classical Rett syndrome have identifiable MECP2 mutations. In comparison, up to 57% of patients with atypical Rett have mutations in the MECP2 gene. To investigate the spectrum and frequency of MECP2 mutations in New Zealand Rett syndrome patients and evaluate whether available clinical criteria were sufficient to direct molecular testing for Rett syndrome. and Methods MECP2 coding regions were analysed by direct automated DNA sequencing and multiplex ligation dependent probe assay (MLPA) in samples from 74 patients referred for investigation of possible Rett syndrome. Necessary clinical criteria were examined in detail in 18 patients, with 7/18 having identifiable MECP2 mutations. Fifteen patients (20%) carried MECP2 mutations, four of which were no...
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, Jan 15, 2015
The increasing availability of liquid chromatography tandem mass spectrometry (LC-MS/MS) in clini... more The increasing availability of liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories provides the opportunity to replace or complement present underperforming immuno- and chemometric assays. Amylase and lipase show limited specificity and sensitivity for pancreatic inflammation and lack the capacity of monitoring the disease due to their short half-lives. Previous findings suggested that cleavage products of the pancreatic enzyme carboxypeptidase A could be a more suitable indicator for defining and classifying pancreatic inflammation. The plasma proteins albumin and β-fibrinogen were digested with trypsin and truncated forms (des-Leu-albumin, and des-Gln-β-fibrinogen) quantified against their non-truncated forms by LC-MS/MS. Four hundred fifty eight samples from 83 patients were used to evaluate the novel method and affirm its suitability for detecting acute pancreatitis. A robust, selective, precise and accurate LC-MS/MS method was set up to measure th...
Patients with porphyria present in a diverse and unusual variety of ways and most clinicians will... more Patients with porphyria present in a diverse and unusual variety of ways and most clinicians will see only a few cases, if any, during their professional lives. Porphyria may present (1) with acute symptoms, which may be abdominal pain, neurological or psychiatric; (2) with skin rash or photosensitivity; or (3) with a putative family history. Screening for latent porphyria has been greatly facilitated by fluorescence emission scanning of plasma and by mutational analysis. Our reference laboratory has recently diagnosed several cases of the less common types of porphyria, which we postulate is due to the availability of these methods and to the changing population of New Zealand. Accurate screening and diagnosis of porphyria is important, as an acute porphyric attack is life-threatening and preventable. Retrospective diagnosis may be difficult.
To determine the frequency of HLA-H gene mutations in New Zealand patients with haemochromatosis.... more To determine the frequency of HLA-H gene mutations in New Zealand patients with haemochromatosis. The Cys282Tyr and His63Asp mutations in the HLA-H gene were analyzed by polymerase chain reaction, restriction enzyme digestion and electrophoresis in two separate patient groups. The first was a group of 20 Christchurch patients with a definite clinical diagnosis of haemochromatosis. The second group consisted of 33 patients, with a provisional diagnosis of haemochromatosis, attending Dunedin Hospital for therapeutic venesection. All 20 Christchurch patients and 25 of the 33 (76%) Dunedin patients were homozygous for the Cys282Tyr mutation. After review of the clinical data, histology and response to venesection a diagnosis of haemochromatosis could be confidently excluded in six of the remaining eight patients. Despite atypical features, a diagnosis of haemochromatosis could not be excluded in the final two patients, one of whom was a compound heterozygote for the two mutations. Homoz...
Cowden disease is an autosomal dominant disorder associated with an elevated risk of breast, thyr... more Cowden disease is an autosomal dominant disorder associated with an elevated risk of breast, thyroid and skin cancers, in which germline mutations of a tumour suppressor gene (PTEN) have been identified. PTEN has a dual-specificity tyrosine phosphatase domain thought to be essential for tumour suppression. Previous genotype/phenotype correlations have identified several potential associations, for example that truncating mutations result in increased breast cancer risk. Such associations are useful in evaluating the phenotypic functions of PTEN sub-domains. However, genotype/phenotype correlations are likely to be complicated by nonsense mediated mRNA degradation. We report here that three out of four mutations do not significantly affect PTEN transcript stability. Furthermore, we show that manual sequencing methods are better than current dye-based sequencing technologies for detecting heterozygous mutations in PTEN transcripts.
Trace elements are commonly measured by inductively coupled mass spectrometry (ICP-MS). A 30-year... more Trace elements are commonly measured by inductively coupled mass spectrometry (ICP-MS). A 30-year-old man had a plasma selenium (Se) concentration on ICP-MS of 66 µmol/L (reference interval 0.45-1.40), a potentially lethal level, despite no history of Se exposure or toxicity symptoms. He had earlier undergone magnetic resonance imaging with a gadolinium (Gd) contrast agent, which is known to interfere with Se on ICP-MS. We aimed to adjust our method by monitoring a second Se isotope that is unaffected by Gd to detect this preanalytical interference. Plasma samples referred for trace metal testing had Se measured on ICP-MS (monitoring (78)Se), which we modified to also monitor a second isotope ((82)Se). The modified method was then applied to a specimen with known Gd contamination. Plasma Se results (n = 41) derived from monitoring the two different Se isotopes were similar with a good correlation (R (2 )= 0.991) over a range of 0.23-2.21 µmol/L. On repeat analysis, our patient had a...
High sensitivity troponin Point of care troponin Acute myocardial infarction Major adverse cardia... more High sensitivity troponin Point of care troponin Acute myocardial infarction Major adverse cardiac events Accelerated diagnostic protocol Objectives: The aim of this study is to compare a new improved point of care cardiac troponin assay (new POC-cTnI) with 1. its predecessor (old POC-cTnI) and 2. a high sensitivity assay (hs-cTnI) for the diagnosis of acute myocardial infarction (AMI) and for major adverse cardiac events (MACE) by 30 days. Methods: This is a single centre observational study, set in Christchurch Hospital, New Zealand. Patients presenting to the emergency department with non-traumatic chest pain underwent blood sampling at 0 h and 2 h post presentation for analysis with the 3 cTnI assays for the outcome of AMI and for analysis using an accelerated diagnostic protocol (ADP-normal 2 h troponins, normal electrocardiograms and Thrombolysis In Myocardial Infarction (TIMI) score of 0 or ≤1) for 30 day MACE. Results: Of 962 patients, 220 (22.9%) had AMI. Old POC-cTnI was least sensitive at 70.0% (65.4-73.9%) by 2 h (p b 0.001). New POC-cTnI, sensitivity 93.6% (89.9-96.2%) had similar sensitivity to hs-cTnI, sensitivity 95.0% (91.5-97.3%) (p = 0.508). There were 231 (24.0%) patients with 30 day MACE. When used as part of the ADP, all assays had 100% (98.0-100%) sensitivity using TIMI = 0. Sensitivities of new POC-cTnI ADP, 98.3% (95.4-99.4%), old POC-cTnI, 96.5% (93.2-98.4%) and hs-cTnI, 98.7% (96.0-99.7%) were similar (p = 0.063-0.375) using TIMI ≤ 1. Conclusions: A new POC-cTnI has improved sensitivity for AMI and MACE compared with its predecessor and comparable sensitivity to a high sensitivity assay. Now that sensitivities of the POC assay are improved, the new assay may be a useful alternative to central laboratory assays when rapid turn-around times are not possible.
Extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is the ... more Extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is the method of choice when it comes to the accurate quantification of 25-OH-vitamin D in blood samples. It is generally assumed that the addition of exogenous internal standard allows for the determination of the endogenous analyte concentration. In this study we investigated the extraction properties of endogenous and exogenous 25-OH-vitamin D. Eight samples were used for the evaluation of the extraction procedure and 59 patients' samples for a method comparison. The methanol-to-sample ratio (v/v) and the sample-to-hexane ratio (v/v) were varied and the LC-MS/MS signals of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and internal standard of the extracts recorded. The optimized 'in-house' LC-MS/MS assay was compared to two automated chemiluminescence immunoassays from DiaSorin and Abbott. Mathematical analysis of the data revealed a differential extraction of endogenous 2...
We recruited nondiabetic subjects (n = 158) attending a lipid disorders clinic, a subset of whom ... more We recruited nondiabetic subjects (n = 158) attending a lipid disorders clinic, a subset of whom (n = 46) had established cardiovascular disease. Glycine betaine, N,N-dimethylglycine, and carnitine were measured in fasting plasma and urine samples. The concentrations and excretions were related to known cardiovascular risk factors in multivariate regression models. The relationships between homocysteine and plasma and urinary glycine betaine were highly significant (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .002), comparable with the known relationships with folate and plasma creatinine. The regression coefficient for plasma glycine betaine was consistently approximately -0.1 in 5 different regression models (3 best-subsets and forward and backward stepwise regression models) for predicting homocysteine using 23 variables. Plasma glycine betaine was higher in males than in females, and the difference was associated with a difference in percentage of body fat. Its concentration included a constant factor of approximately 20 micromol/L that was independent of any of the variables investigated here. In the total group, body fat, homocysteine, and carnitine were significant predictors of plasma glycine betaine. Carnitine, an important betaine that is involved in lipid metabolism positively correlated with both homocysteine and glycine betaine. In our sample, the urinary excretion of glycine betaine was outside the reference range in 14 of the 158 subjects and the betaine fractional clearances were above the reference range in 23 subjects. Fractional clearance correlated strongly with plasma homocysteine (r = 0.50), and this relationship may be stronger in patients with known vascular disease. Urinary loss of glycine betaine may contribute to hyperhomocysteinemia and the development of cardiovascular disease.
Aim To establish an assay service for thiopurine methyl transferase (TPMT) activity in order to f... more Aim To establish an assay service for thiopurine methyl transferase (TPMT) activity in order to facilitate dose initiation of thiopurine drug therapy and to define appropriate reference intervals and optimal cut-offs for the New Zealand population. Methods 407 patients underwent radio-enzymatic assay testing of TPMT activity prior to initiation of thiopurine drug therapy. Those with low activity also underwent genotyping
Annals of Clinical Biochemistry: An international journal of biochemistry and laboratory medicine, 2015
Familial dysalbuminaemic hyperthyroxinaemia (FDH) is an important cause of discordant thyroid fun... more Familial dysalbuminaemic hyperthyroxinaemia (FDH) is an important cause of discordant thyroid function test (TFT) results (due to an inherited albumin variant) however, the diagnosis can be challenging. A 51 year-old man had persistently elevated free thyroxine (T4), with discordant normal TSH and normal free triiodothyronine. He was clinically euthyroid and had a daughter with similar TFTs. We aimed to apply a whole protein mass spectrometry (MS) method to investigate this case of suspected FDH. Intact serum albumin was assessed directly using electrospray time-of-flight (TOF) MS. Results were confirmed using tryptic peptide m/z mapping and targeted DNA sequencing (exons 3 and 7 of the albumin gene). We also used this sequencing to screen 14 archived DNA samples that were negative for thyroid hormone receptor mutations (in suspected thyroid hormone resistance). MS analysis demonstrated heterozygosity for an albumin variant with a 19 Da decrease in mass, indicative of an Arg→His substitution. The FDH variant was confirmed with peptide mapping (showing the precise location of the substitution, 218Arg→His) and DNA sequencing (showing guanine to adenine transition at codon 218 of exon 7). The same FDH variant was identified in one additional screened sample. TOF MS is a novel procedure for diagnosing FDH. The test is rapid (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;10 min), can be performed on &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;2 µL of serum and requires minimal sample preparation. KEY: TERMS: : familial dysalbuminaemic hyperthyroxaemia, mass spectrometry, albumin variant, discordant thyroid function tests.
Objectives The aim of this study is to compare a new improved point of care cardiac troponin assa... more Objectives The aim of this study is to compare a new improved point of care cardiac troponin assay (new POC-cTnI) with 1. its predecessor (old POC-cTnI) and 2. a high sensitivity assay (hs-cTnI) for the diagnosis of acute myocardial infarction (AMI) and for major adverse cardiac events (MACE) by 30 days. Methods This is a single centre observational study, set in Christchurch Hospital, New Zealand. Patients presenting to the emergency department with non-traumatic chest pain underwent blood sampling at 0 h and 2 h post presentation for analysis with the 3 cTnI assays for the outcome of AMI and for analysis using an accelerated diagnostic protocol (ADP-normal 2 h troponins, normal electrocardiograms and Thrombolysis In Myocardial Infarction (TIMI) score of 0 or ≤ 1) for 30 day MACE. Results Of 962 patients, 220 (22.9%) had AMI. Old POC-cTnI was least sensitive at 70.0% (65.4–73.9%) by 2 h (p < 0.001). New POC-cTnI, sensitivity 93.6% (89.9–96.2%) had similar sensitivity to hs-cTnI,...
Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement t... more Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human alpha 1-antitrypsin (alpha 1AT) cDNA in yeast. Alteration of the alpha 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-III. Alteration of two further residues, to give a five-residue sequence identical to AT-III, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglycosylated and have shorter in vivo half-lives (t1/2) than human alpha 1AT. These antithrombins should be suitable for therapeutic replacement of AT-III in cases of congenital deficiency and in conditions associated with acquired AT-III deficiency, such as disseminated intravascular coagulation.
Betaine supplementation decreases post-methionine hyperhomic [3]. This is thought to contribute t... more Betaine supplementation decreases post-methionine hyperhomic [3]. This is thought to contribute to the common mocysteinemia in chronic renal failure. occurrence and rapid progression of atherosclerosis in Background: Fasting and post-methionine load hyperhomothe ESRD population. Although there is no evidence cysteinemia are independent risk factors for vascular disease yet to show that lowering plasma tHcy reduces the risk that are common in chronic renal failure. Folate decreases but seldom normalizes fasting total homocysteine (tHcy) concen-of atherosclerosis, it is widely believed that reducing trations in such patients. Glycine betaine (GB) is known to tHcy levels should be a therapeutic target [4]. decrease tHcy in other clinical settings, but whether it is bene-The optimal treatment for hyperhomocysteinemia in ficial in chronic renal failure has not been established. chronic renal failure has not been determined. Folic acid Methods: We conducted a crossover-controlled trial in 36 patients with chronic renal failure to determine if oral GB Patients
The performance of fructosamine assays in 35 laboratories was assessed over a six month period in... more The performance of fructosamine assays in 35 laboratories was assessed over a six month period in 1987. While agreement between laboratories was poor in the range expected in diabetic patients, most laboratories were internally consistent. The interlaboratory differences are largely attributed to the calibration methods used for this test. Fructosamine results from any one laboratory must not be interpreted by comparison with data from other laboratories or the literature. Other common biochemical tests were found to suffer from similar problems, but to a less serious extent.
Classical Rett syndrome is a severe neurodevelopmental X-linked dominant disorder affecting 1/15,... more Classical Rett syndrome is a severe neurodevelopmental X-linked dominant disorder affecting 1/15,000 girls worldwide. MECP2 has been identified as the predominant gene associated with Rett syndrome. Approximately 65-85% of patients with classical Rett syndrome have identifiable MECP2 mutations. In comparison, up to 57% of patients with atypical Rett have mutations in the MECP2 gene. To investigate the spectrum and frequency of MECP2 mutations in New Zealand Rett syndrome patients and evaluate whether available clinical criteria were sufficient to direct molecular testing for Rett syndrome. and Methods MECP2 coding regions were analysed by direct automated DNA sequencing and multiplex ligation dependent probe assay (MLPA) in samples from 74 patients referred for investigation of possible Rett syndrome. Necessary clinical criteria were examined in detail in 18 patients, with 7/18 having identifiable MECP2 mutations. Fifteen patients (20%) carried MECP2 mutations, four of which were no...
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, Jan 15, 2015
The increasing availability of liquid chromatography tandem mass spectrometry (LC-MS/MS) in clini... more The increasing availability of liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories provides the opportunity to replace or complement present underperforming immuno- and chemometric assays. Amylase and lipase show limited specificity and sensitivity for pancreatic inflammation and lack the capacity of monitoring the disease due to their short half-lives. Previous findings suggested that cleavage products of the pancreatic enzyme carboxypeptidase A could be a more suitable indicator for defining and classifying pancreatic inflammation. The plasma proteins albumin and β-fibrinogen were digested with trypsin and truncated forms (des-Leu-albumin, and des-Gln-β-fibrinogen) quantified against their non-truncated forms by LC-MS/MS. Four hundred fifty eight samples from 83 patients were used to evaluate the novel method and affirm its suitability for detecting acute pancreatitis. A robust, selective, precise and accurate LC-MS/MS method was set up to measure th...
Patients with porphyria present in a diverse and unusual variety of ways and most clinicians will... more Patients with porphyria present in a diverse and unusual variety of ways and most clinicians will see only a few cases, if any, during their professional lives. Porphyria may present (1) with acute symptoms, which may be abdominal pain, neurological or psychiatric; (2) with skin rash or photosensitivity; or (3) with a putative family history. Screening for latent porphyria has been greatly facilitated by fluorescence emission scanning of plasma and by mutational analysis. Our reference laboratory has recently diagnosed several cases of the less common types of porphyria, which we postulate is due to the availability of these methods and to the changing population of New Zealand. Accurate screening and diagnosis of porphyria is important, as an acute porphyric attack is life-threatening and preventable. Retrospective diagnosis may be difficult.
To determine the frequency of HLA-H gene mutations in New Zealand patients with haemochromatosis.... more To determine the frequency of HLA-H gene mutations in New Zealand patients with haemochromatosis. The Cys282Tyr and His63Asp mutations in the HLA-H gene were analyzed by polymerase chain reaction, restriction enzyme digestion and electrophoresis in two separate patient groups. The first was a group of 20 Christchurch patients with a definite clinical diagnosis of haemochromatosis. The second group consisted of 33 patients, with a provisional diagnosis of haemochromatosis, attending Dunedin Hospital for therapeutic venesection. All 20 Christchurch patients and 25 of the 33 (76%) Dunedin patients were homozygous for the Cys282Tyr mutation. After review of the clinical data, histology and response to venesection a diagnosis of haemochromatosis could be confidently excluded in six of the remaining eight patients. Despite atypical features, a diagnosis of haemochromatosis could not be excluded in the final two patients, one of whom was a compound heterozygote for the two mutations. Homoz...
Cowden disease is an autosomal dominant disorder associated with an elevated risk of breast, thyr... more Cowden disease is an autosomal dominant disorder associated with an elevated risk of breast, thyroid and skin cancers, in which germline mutations of a tumour suppressor gene (PTEN) have been identified. PTEN has a dual-specificity tyrosine phosphatase domain thought to be essential for tumour suppression. Previous genotype/phenotype correlations have identified several potential associations, for example that truncating mutations result in increased breast cancer risk. Such associations are useful in evaluating the phenotypic functions of PTEN sub-domains. However, genotype/phenotype correlations are likely to be complicated by nonsense mediated mRNA degradation. We report here that three out of four mutations do not significantly affect PTEN transcript stability. Furthermore, we show that manual sequencing methods are better than current dye-based sequencing technologies for detecting heterozygous mutations in PTEN transcripts.
Trace elements are commonly measured by inductively coupled mass spectrometry (ICP-MS). A 30-year... more Trace elements are commonly measured by inductively coupled mass spectrometry (ICP-MS). A 30-year-old man had a plasma selenium (Se) concentration on ICP-MS of 66 µmol/L (reference interval 0.45-1.40), a potentially lethal level, despite no history of Se exposure or toxicity symptoms. He had earlier undergone magnetic resonance imaging with a gadolinium (Gd) contrast agent, which is known to interfere with Se on ICP-MS. We aimed to adjust our method by monitoring a second Se isotope that is unaffected by Gd to detect this preanalytical interference. Plasma samples referred for trace metal testing had Se measured on ICP-MS (monitoring (78)Se), which we modified to also monitor a second isotope ((82)Se). The modified method was then applied to a specimen with known Gd contamination. Plasma Se results (n = 41) derived from monitoring the two different Se isotopes were similar with a good correlation (R (2 )= 0.991) over a range of 0.23-2.21 µmol/L. On repeat analysis, our patient had a...
High sensitivity troponin Point of care troponin Acute myocardial infarction Major adverse cardia... more High sensitivity troponin Point of care troponin Acute myocardial infarction Major adverse cardiac events Accelerated diagnostic protocol Objectives: The aim of this study is to compare a new improved point of care cardiac troponin assay (new POC-cTnI) with 1. its predecessor (old POC-cTnI) and 2. a high sensitivity assay (hs-cTnI) for the diagnosis of acute myocardial infarction (AMI) and for major adverse cardiac events (MACE) by 30 days. Methods: This is a single centre observational study, set in Christchurch Hospital, New Zealand. Patients presenting to the emergency department with non-traumatic chest pain underwent blood sampling at 0 h and 2 h post presentation for analysis with the 3 cTnI assays for the outcome of AMI and for analysis using an accelerated diagnostic protocol (ADP-normal 2 h troponins, normal electrocardiograms and Thrombolysis In Myocardial Infarction (TIMI) score of 0 or ≤1) for 30 day MACE. Results: Of 962 patients, 220 (22.9%) had AMI. Old POC-cTnI was least sensitive at 70.0% (65.4-73.9%) by 2 h (p b 0.001). New POC-cTnI, sensitivity 93.6% (89.9-96.2%) had similar sensitivity to hs-cTnI, sensitivity 95.0% (91.5-97.3%) (p = 0.508). There were 231 (24.0%) patients with 30 day MACE. When used as part of the ADP, all assays had 100% (98.0-100%) sensitivity using TIMI = 0. Sensitivities of new POC-cTnI ADP, 98.3% (95.4-99.4%), old POC-cTnI, 96.5% (93.2-98.4%) and hs-cTnI, 98.7% (96.0-99.7%) were similar (p = 0.063-0.375) using TIMI ≤ 1. Conclusions: A new POC-cTnI has improved sensitivity for AMI and MACE compared with its predecessor and comparable sensitivity to a high sensitivity assay. Now that sensitivities of the POC assay are improved, the new assay may be a useful alternative to central laboratory assays when rapid turn-around times are not possible.
Extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is the ... more Extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is the method of choice when it comes to the accurate quantification of 25-OH-vitamin D in blood samples. It is generally assumed that the addition of exogenous internal standard allows for the determination of the endogenous analyte concentration. In this study we investigated the extraction properties of endogenous and exogenous 25-OH-vitamin D. Eight samples were used for the evaluation of the extraction procedure and 59 patients' samples for a method comparison. The methanol-to-sample ratio (v/v) and the sample-to-hexane ratio (v/v) were varied and the LC-MS/MS signals of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and internal standard of the extracts recorded. The optimized 'in-house' LC-MS/MS assay was compared to two automated chemiluminescence immunoassays from DiaSorin and Abbott. Mathematical analysis of the data revealed a differential extraction of endogenous 2...
We recruited nondiabetic subjects (n = 158) attending a lipid disorders clinic, a subset of whom ... more We recruited nondiabetic subjects (n = 158) attending a lipid disorders clinic, a subset of whom (n = 46) had established cardiovascular disease. Glycine betaine, N,N-dimethylglycine, and carnitine were measured in fasting plasma and urine samples. The concentrations and excretions were related to known cardiovascular risk factors in multivariate regression models. The relationships between homocysteine and plasma and urinary glycine betaine were highly significant (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .002), comparable with the known relationships with folate and plasma creatinine. The regression coefficient for plasma glycine betaine was consistently approximately -0.1 in 5 different regression models (3 best-subsets and forward and backward stepwise regression models) for predicting homocysteine using 23 variables. Plasma glycine betaine was higher in males than in females, and the difference was associated with a difference in percentage of body fat. Its concentration included a constant factor of approximately 20 micromol/L that was independent of any of the variables investigated here. In the total group, body fat, homocysteine, and carnitine were significant predictors of plasma glycine betaine. Carnitine, an important betaine that is involved in lipid metabolism positively correlated with both homocysteine and glycine betaine. In our sample, the urinary excretion of glycine betaine was outside the reference range in 14 of the 158 subjects and the betaine fractional clearances were above the reference range in 23 subjects. Fractional clearance correlated strongly with plasma homocysteine (r = 0.50), and this relationship may be stronger in patients with known vascular disease. Urinary loss of glycine betaine may contribute to hyperhomocysteinemia and the development of cardiovascular disease.
Aim To establish an assay service for thiopurine methyl transferase (TPMT) activity in order to f... more Aim To establish an assay service for thiopurine methyl transferase (TPMT) activity in order to facilitate dose initiation of thiopurine drug therapy and to define appropriate reference intervals and optimal cut-offs for the New Zealand population. Methods 407 patients underwent radio-enzymatic assay testing of TPMT activity prior to initiation of thiopurine drug therapy. Those with low activity also underwent genotyping
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Papers by Peter George