Jonathan Berg

Vitamin D concentrations in an UK inner-city multicultural outpatient population

Background: Vitamin D deficiency is still thought to be widespread in the UK and in recent years the number of cases of rickets reported in children has increased. In this study, the distribution of vitamin D and the prevalence of vitamin D deficiency have been determined for a multi-ethnic population from the inner-city area of Birmingham, UK, where a vitamin D testing service has been readily available for over 10 years. Methods: Serum 25-hydroxyvitamin D concentration was determined using an automated platform (Nichols Advantage Speciality System) for 830 outpatient samples collected randomly at the end of summer (September). Results: In our total study population, prevalence of vitamin D deficiency, defined as a 25-hydroxyvitamin D concentration < 10 μg/L, was high (24%): one in eight Caucasians, one in four Black Afro-Caribbeans and one in three Asians were found to be deficient. Levels of deficiency were much higher in Asian women, with almost one in two individuals (43%) found to have a vitamin D level below 10 μg/L. Conclusion: Our study has shown that widespread vitamin D deficiency in a UK inner-city population remains an issue. In concordance with other studies, we found a high prevalence of vitamin D deficiency in Afro-Caribbean and Asians, and, in particular, women. It is clear that more routine screening of vitamin D is needed.

Ethnic variation of thiopurine S-methyltransferase activity: a large, prospective population study

UNLABELLED The use of azathioprine (and its metabolite mercaptopurine) is limited by toxicity, especially myelosuppression, which is related to activity of the enzyme thiopurine S-methyltransferase (TPMT). TPMT activity varies between individuals and is considered deficient in one in 300 cases. AIMS & METHODS We identified TPMT activity within an ethnically diverse population of patients attending an inner-city hospital phlebotomy service. A total of 1000 subjects were recruited and analyzed with respect to age, sex and ethnicity. RESULTS Samples were analyzed from 456 Caucasians, 342 South Asians and 180 Afro-Caribbeans. Six subjects had deficient TPMT activity (0.6%: four women, two men; four Caucasians, one Afro-Caribbean, one South Asian). TPMT activity (nmol 6-methylthioguanine (6-MTG)/gHb/h) ranged 0-76 (median [interquartile range]: 33 [28-39]). Enzyme activity was lower in Afro-Caribbeans (30 [25-37.5]) than Caucasians (34 [29-40]) and South Asians (33 [29-38]), which was significant after adjustment for age and sex (p < 0.0001). Activity was lower in women (p = 0.022), especially South Asian females (n = 194; 32 [28-36]), compared with (35 [30-40]) in men (n = 148; p = 0.002). CONCLUSIONS A higher prevalence of TPMT deficiency was recorded than in previous studies. Afro-Caribbeans have lower activity than Caucasians and South Asians. TPMT enzyme activity was lower among females, especially in South Asians.

Pathology Harmony; a pragmatic and scientific approach to unfounded variation in the clinical laboratory

It was five years ago that the idea of Pathology Harmony was conceived in Birmingham, UK. At that time it was realized that three major organizations undertaking clinical biochemical analyses in the city all used the same analytical platforms and reagents but offered different reference intervals to help interpret the results they produced. On initial inspection, it was not possible to discern any scientific explanation for these differences. While this variation was not unexpected and was longstanding, it was seen as an issue that needed to be addressed. Indeed, the specific area of reference range non-uniformity has recently been highlighted in the Journal. While Pathology Harmony is a UK initiative, its basis, drivers and implementation will resonate in other countries.

The value of measuring serum cholesterol-adjusted vitamin E in routine practice

Background: If the lipid concentration is not taken into account, then in certain situations the vitamin E status of a patient may be wrongly assigned based on serum measurement alone. In this study, the utility of using the calculated vitamin E:cholesterol ratio to determine vitamin E status was compared to the measurement of vitamin E alone. Methods: The vitamin E:cholesterol ratio was measured in 457 patient samples received for routine vitamin E analysis. Serum vitamin E concentration was determined by high-performance liquid chromatography (HPLC) with UV detection and cholesterol concentration, using a Roche analyser and reagents. Results: The mean vitamin E concentration and vitamin E:cholesterol ratio for the total study population was 22.0 μmol/L and 5.9 μmol/mmol, respectively. Of the 457 patient samples analysed, 57 (12.5%) were found to have a low vitamin E concentration, but only 25 (47%) of these patients had a low vitamin E:cholesterol ratio as well. Two patients both with cholestasis had a normal vitamin E concentration but low vitamin E:cholesterol ratio. Conclusion: The determination of vitamin E:cholesterol ratios can be used to better define the vitamin E status of patients with disease states or disorders likely to raise LDL cholesterol, for example cholestasis. For the majority of samples, measurement of serum vitamin E concentration alone is sufficient to establish patient vitamin E status.

Whose TPMT activity is it anyway

Determination of thiopurine S-methyltransferase (TPMT) activity prior to starting azathioprine therapy is used to identify individuals with low or deficient TPMT activities who are at risk of severe complications and even death This case describes a patient treated with azathioprine without prior knowledge of TPMT status. Pancytopaenia developed over several months and at this point TPMT activity was determined and found to be low at 16 nmol 6-methyl thioguanine (6-MTG)/g Hb/h, which is within the reference interval associated with heterozygosity for TPMT mutant alleles. On repeating the TPMT measurement 3 months later, the TPMT activity was 2 nmol 6-MTG/g Hb/h, consistent with deficient TPMT activity (homozygosity for TPMT mutant alleles), suggesting the patient is at high risk of myelosuppression if treated with thiopurine drugs. Retrospectively, it was found that the patient had received transfusions of red blood cell and platelets 6 days before TPMT activity was first measured. This case underlines the importance of determining TPMT activity status prior to azathioprine treatment, rather than taking a dose incrementation approach. It also highlights the caution that must be taken in interpreting TPMT activity in patients who have recently been transfused.

Thiopurine S-methyltransferase genotype-phenotype concordance: used as a quality assurance tool to help control the phenotype assay

Background As part of the quality control system for our TPMT phenotyping service we monitor the genotype-phenotype concordance for patient samples with deficient and low TPMT activity. We have studied the genotype-phenotype concordance over the last year to demonstrate its effectiveness as a quality assurance tool. Methods From July 2007 to July 2008 TPMT genotyping was performed on all routine samples analysed using our phenotypic assay with an activity of ≤40 nmol 6-MTG/gHb/h. The monthly genotype-phenotype concordance was calculated between: all deficient TPMT activity results and a homozygous mutant or compound heterozygote genotype, low TPMT activity and a heterozygote genotype, normal TPMT activity and a wild-type genotype. Results A total of 14,832 samples were analysed by TPMT phenotyping and 1769 of these by genotyping. The monthly mean concordance between low TPMT activity and a mutant heterozygote genotype was 83%, ranging from 67–90%. The number of individuals with deficient TPMT activity identified by phenotyping was 44. For two of these individuals only one mutant allele was detected, and for one no common mutations were identified. Conclusions Monitoring the genotype-phenotype concordance is an effective quality assurance tool for the TPMT phenotyping assay. As demonstrated in this study current genotyping assays risk missing some deficient patients.

Rapid measurement of anticonvulsant drug concentrations in the out-patient clinic, using HPLC with direct injection of plasma.

A manual column-switching technique is described for the measurement of phenytoin, phenobarbitone, carbamazepine, and carbamazepine 10,11-epoxide. The analytical system is designed to be portable for use at the out-patient clinic and comprises an isocratic pump, UV detector and injection valve, together with a preparation column. Diluted plasma or serum is injected, without pre-extraction, onto a preparation column which replaces the sample loop on the injection valve. After washing unwanted material to waste, the preparation column is switched in-line with the analytical column, where separation of analytes occurs. The precision, accuracy and carryover of this extra-laboratory system are comparable with those obtained with laboratory-based immunoassay systems. Operation of the system allows the reporting of results within 5 min of sample injection and requires no specialist skills. The technique should be of particular interest to district general hospital laboratories where workload does not justify the cost of an automated HPLC system as the total capital cost is comparable to that of a portable glucose analyser. In contrast to immunoassay systems consumable costs are minimal. The equipment is easy to transport and may be used in the out-patient department to provide an analytical service similar to that provided for the determination of prothrombin time at the anticoagulant clinic.

Reference intervals for thiopurine S-methyltransferase activity in red blood cells using 6-thioguanine as substrate and rapid non-extraction liquid chromatography

Background: Although widely used, thiopurine drugs have a narrow therapeutic index and treatment can result in life-threatening toxicity, the basis being pharmacogenetic variation in thiopurine metabolism by thiopurine S-methyltransferase (TPMT). We recently developed a modified phenotyping assay to determine TPMT activity in red blood cells. Here we describe improvements to the method and establish reference intervals in a large prospective study. Methods: A modified enzyme assay for TPMT activity is reported. It uses 6-thioguanine as substrate with heat treatment of the incubate to stop the reaction and precipitate protein prior to high-performance liquid chromatographic (HPLC) analysis. Measurement of the reaction product, 6-methylthioguanine (6-MTG), uses HPLC with fluorimetric detection. Results: The assay shows excellent characteristics, with clear discrimination of patients who are deficient in TPMT activity (< 5 nmol 6-MTG per g Hb per h) from heterozygotes (5-24 nmol 6-MTG per g Hb per h) and patients with normal activity (>25 nmol 6-MTG per g Hb per h). Conclusion: A modified TPMT assay is described which is suited for routine analysis in a regional centre. The method overcomes the need for extraction and has speeded up the chromatographic determination of 6-MTG, enabling large numbers of samples to be analysed. A prospective study of 1000 individuals has established the distribution of TPMT activity using the assay.

Analysis of legal high materials by ultra-performance liquid chromatography with time of flight mass spectrometry as part of a toxicology vigilance system: what are the most popular novel psychoactive substances in the UK?

Introduction Legal highs also known as novel psychoactive substances mimic the effects of classic drugs of abuse. Challenges to developing screening services for novel psychoactive substances include identifying which novel psychoactive substances are available to target. Using new techniques such as exact mass time of flight can help identify common novel psychoactive substances to target for screening patient samples by routine methods such as tandem mass spectrometry. We demonstrate this strategy working in our own clinical toxicology laboratory after qualitative analysis of 98 suspect materials for novel psychoactive substances by ultra-performance liquid chromatography with time of flight mass spectrometry. Results From July 2014 to July 2015 we received 98 requests to test a range of different suspect materials for novel psychoactive substances including herbs, tobacco, liquids, pills and powders. Overall, 87% of the suspect materials tested positive for novel psychoactive substances, and 15% for controlled drugs. Three common novel psychoactive substances were present in 74% of the suspect materials: methiopropamine, a methamphetamine analogue; ethylphenidate, a cocaine mimic; and the third generation synthetic cannabinoid 5F-AKB-48. For the 55 branded products we tested only 24% of the stated contents matched exactly the compounds we detected. Conclusion Testing suspect materials using ultra-performance liquid chromatography with time of flight mass spectrometry has identified three common novel psychoactive substances in use in the UK, simplifying the development of a relevant novel psychoactive substances screening service to our population. By incorporating this into our routine liquid chromatography tandem mass spectrometry drugs of abuse screen, then offers a clinically relevant novel psychoactive substances service to our users. This strategy ensures our clinical toxicology service continues to remain effective to meet the challenges of the changing drug use in the UK.

Serum Total Bile Acid Levels in Patients Receiving Rifampicin and Isoniazid

Serum bile acid levels in 61 patients receiving daily doses of rifampicin and isoniazid for the treatment of tuberculosis have been investigated. Bile acids were measured using 3α-hydroxysteroid dehydrogenase in a continuous-flow system. Abnormally elevated levels were found in 44 patients (72%) during the period of study up to 80 days after onset of treatment. The results showed a mean of 24·9 μmol/l and a positively skewed distribution. Whilst marginally raised levels of bilirubin were seen in some samples (mean 8·2 μmol/l), these did not reflect the marked changes observed in bile acids. Patients receiving rifampicin and isoniazid may therefore have markedly elevated levels of total serum bile acids, while other tests used to assess liver function can remain normal.