Aysha Habib Khan

Comparison of high performance liquid chromatography, radio immunoassay and electrochemiluminescence immunoassay for quantification of serum 25 hydroxy vitamin D

OBJECTIVEnTo compare the performance of radioimmunoassay (RIA) with high-performance liquid chromatography (HPLC) and electrochemiluminescence (ECLIA) for the quantification of vitamin D (25OHD).nnnMETHODSnHPLC method for the determination of 25OHD in human biological samples was developed and compared in terms of accuracy and precision with a commercially available RIA assay. Performance of RIA assay with ECLIA technology for 25OHD analysis was further compared.nnnRESULTSnMedian 25OHD levels with HPLC vs. RIA were 50.1nmol/L (IQ=17.7-199.4nmol/L) and 51.1nmol/L (IQ=12.5-187.2nmol/L) respectively, whereas median 25OHD concentration with RIA vs. ECLIA was 32.4nmol/L (9.98-199.7nmol/L) and 29.9nmol/L (4.9-214.6nmol/L), respectively. Comparison data for HPLC vs. RIA showed RIA=-1.13+1.01 (HPLC) (RMSE=11.2nmol/L) and for RIA vs. ECLIA revealed, ECLIA=3.21+0.9 (RIA) (RMSE9.6nmol/L).nnnCONCLUSIONnAcceptable correlation was observed among HPLC and RIA and also with RIA and ECLIA in quantification of 25OHD.

Prevalence of vitamin D deficiency and its correlates: results of a community-based study conducted in Karachi, Pakistan

SummaryOf the 305 premenopausal females in a cross-sectional study in randomly selected communities of Karachi, Pakistan, 90.1xa0% showed to be vitamin D deficient. Age, town of residence, and housing structure were significant predictors of vitamin D levels. Measures to address D deficiency and its associated long latency effects are urgently needed.AimsThis study aims to find out the prevalence and risk factors of vitamin D deficiency in community-dwelling premenopausal females in Karachi.MethodsA cross-sectional study was conducted in randomly selected communities downtown (Saddar) and suburbs (Gulshan and Malir Town) in Karachi, Pakistan. Information related to sociodemographics (age, education, employment, and household income), housing structure, sunlight exposure, and skin pigmentation as well as dietary intake (using a food frequency questionnaire) was collected. Serum vitamins D3 levels were also measured. Mean and SD was computed for continuous variables and frequency and proportions were computed for categorical variables. Data were further analyzed by Chi-square test and ANOVA. Multiple linear regression analysis was done to find out determinants of vitamin D (VD) levels.ResultsTotal of 305 premenopausal females were recruited. Mean age, BMI, and waist circumference of the study participants was 31.97u2009±u20098xa0years, 25.06u2009±u20095.6xa0kg/m2, and 88.42u2009±u200913.3xa0cm, respectively. Majority of the females were vitamin D deficient (91.50xa0%) with mean vitamin D levels of 21.77u2009±u200921.66xa0nm/L. Mean vitamin D levels were significantly different among females residing in downtown and suburbs. High frequency of vitamin D deficiency was observed in females dwelling in downtown (Saddar). According to the results of multiple linear regression analysis, determinants of VD levels were age, town of residence, and housing structure.ConclusionHigh prevalence of vitamin D deficiency is seen in females in the community of Karachi, Pakistan. Age, town of residence, and housing structure were the significant predictors of vitamin D levels. Measures to combat the issue of D deficiency and its associated long latency effects are urgently needed.

Development and validation of sunlight exposure measurement questionnaire (SEM-Q) for use in adult population residing in Pakistan

BackgroundVitamin D deficiency has been identified as a major public health problem worldwide. Sunlight is the main source of vitamin D and its measurement using dosimeters is expensive and difficult for use in population-based studies. Hence, the aim of this study was to develop and validate questionnaires to assess sunlight exposure in healthy individuals residing in Karachi, Pakistan.MethodsTwo questionnaires with seven important items for sunlight exposure assessment were developed. Fifty four healthy adults were enrolled based on their reported sunlight exposure (highu2009=u200917, moderateu2009=u200918, lowu2009=u200919) from Aga Khan University, Karachi. Over four days, study participants were asked to wear a dosimeter between sunrise and sunset and report time spent and activities undertaken in the sun for questionnaire validation. Algorithm for item weightage was created as an average score based on ultraviolet B percentage received. Blood samples were obtained for serum vitamin D.ResultsThe mean time (minutes) spent in sun over 4u2009days (±SD) was 69.5 (±32) for low, 83.5 (±29.7) for moderate and 329 (±115) for high exposure group. The correlation between average time (minutes) spent in sun over 4u2009days and mean change in absorbance of UV dosimeters for 4u2009days was 0.60 (pu2009<u20090.01). Correlation between average score and vitamin D levels was found to be 0.36 (pu2009=u20090.01) for short term questionnaire score, 0.43 (pu2009=u20090.01) for long term questionnaire score in summers and 0.48 (pu2009=u20090.01) in winters.ConclusionsThe sunlight exposure measurement questionnaires were valid tools for use in large epidemiological studies to quantify sunlight exposure.

Ethnic disparity in 21-hydroxylase gene mutations identified in Pakistani congenital adrenal hyperplasia patients

BackgroundCongenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders caused by defects in the steroid 21 hydroxylase gene (CYP21A2). We studied the spectrum of mutations in CYP21A2 gene in a multi-ethnic population in Pakistan to explore the genetics of CAH.MethodsA cross sectional study was conducted for the identification of mutations CYP21A2 and their phenotypic associations in CAH using ARMS-PCR assay.ResultsOverall, 29 patients were analyzed for nine different mutations. The group consisted of two major forms of CAH including 17 salt wasters and 12 simple virilizers. There were 14 phenotypic males and 15 females representing all the major ethnic groups of Pakistan. Parental consanguinity was reported in 65% cases and was equally distributed in the major ethnic groups. Among 58 chromosomes analyzed, mutations were identified in 45 (78.6%) chromosomes. The most frequent mutation was I2 splice (27%) followed by Ile173Asn (26%), Arg 357 Trp (19%), Gln319stop, 16% and Leu308InsT (12%), whereas Val282Leu was not observed in this study. Homozygosity was seen in 44% and heterozygosity in 34% cases. I2 splice mutation was found to be associated with SW in the homozygous. The Ile173Asn mutation was identified in both SW and SV forms. Moreover, Arg357Trp manifested SW in compound heterozygous state.ConclusionOur study showed that CAH exists in our population with ethnic difference in the prevalence of mutations examined.

Ionized calcium measurement in serum and plasma by ion selective electrodes: comparison of measured and calculated parameters.

To determine the concentration of ionized calcium (iCa) collected in lithium heparin and gel tubes and to correlate the measured iCa with calculated iCa. Anaerobic fasting blood samples were simultaneously collected from healthy laboratory workers in lithium-heparin and gel tubes. iCa, pH, total calcium (CaT), total protein and albumin were measured. Ionized Ca was calculated with albumin and globulin values using an appropriate formula. Mean iCa in gel tubes showed a positive constant bias of 0.08xa0mmol/L (pxa0<xa00.001) when compared to lithium heparin results. The mean pH of blood taken in lithium heparin and gel tubes was non-significant (pxa0=xa00.3). Measured iCa poorly correlated with CaT (rxa0=xa00.2, pxa0=xa00.1) and calculated iCa (rxa0=xa00.2, pxa0=xa00.2). To evaluate the calcium status direct measurement of iCa must be done rather than using the formulae for iCa determination. In addition serum is recommended as the choice of sample for iCa determination in comparison to plasma samples.

Assessing the effect of dietary calcium intake and 25 OHD status on bone turnover in women in Pakistan

SummaryBone health assessed in three towns of Karachi, Pakistan in females showed poor calcium intake, vitamin D deficiency, secondary hyperparathyroidism, and high bone turnover. Correlates of high bone turnover included females residing in Saddar Town, underweight females less than 30xa0years of age from low socio-economic status, and secondary hyperparathyroidism.AimsTo assess bone health and association of dietary calcium and 25 hydroxy vitamin D with bone turnover in the community-dwelling females of Karachi.MethodsBone health was assessed in three randomly selected towns of Karachi, Pakistan. One premenopausal female fulfilling the inclusion criteria from each household was included in the study. Dietary calcium was assessed through a food frequency questionnaire and biochemical markers including calcium, phosphates, albumin, magnesium, creatinine, and SGPT, intact parathyroid hormone, 25 hydroxy vitamin D, and N-telopeptide of type I collagen were measured to assess the bone health.ResultsThree hundred and five females were included from three towns. Overall, 90.5xa0% of females had vitamin D deficiency with 42.6 and 23.3xa0% having secondary hyperparathyroidism and high bone turn over respectively. Prevalence of vitamin D deficiency, secondary hyperparathyroidism, and high bone turnover was significantly different among towns. Mean vitamin D levels were significantly low and iPTH levels significantly high in females with high bone turnover. Calcium intake was not significantly different among females with normal, high, and low bone turnover. Correlates of high bone turnover included females residing in Saddar Town, underweight females less than 30xa0years of age belonging to low socio-economic status, and secondary hyperparathyroidism.ConclusionCompromised bone health is seen in community-dwelling females of Karachi. There is a need to perform large-scale community-based studies in all age groups to understand the interplay of markers in our population to understand the impact of these variables translating into the risk of osteoporosis.

Error identification in a high-volume clinical chemistry laboratory: Five-year experience

Abstract Introduction. Quality indicators for assessing the performance of a laboratory require a systematic and continuous approach in collecting and analyzing data. The aim of this study was to determine the frequency of errors utilizing the quality indicators in a clinical chemistry laboratory and to convert errors to the Sigma scale. Materials and methods. Five-year quality indicator data of a clinical chemistry laboratory was evaluated to describe the frequency of errors. An ‘error’ was defined as a defect during the entire testing process from the time requisition was raised and phlebotomy was done until the result dispatch. An indicator with a Sigma value of 4 was considered good but a process for which the Sigma value was 5 (i.e. 99.977% error-free) was considered well controlled. Results. In the five-year period, a total of 6,792,020 specimens were received in the laboratory. Among a total of 17,631,834 analyses, 15.5% were from within hospital. Total error rate was 0.45% and of all the quality indicators used in this study the average Sigma level was 5.2. Three indicators – visible hemolysis, failure of proficiency testing and delay in stat tests – were below 5 on the Sigma scale and highlight the need to rigorously monitor these processes. Conclusions. Using Six Sigma metrics quality in a clinical laboratory can be monitored more effectively and it can set benchmarks for improving efficiency.

Bone health status of premenopausal healthy adult females in Pakistani females

SummaryBone health status in healthy premenopausal females was assessed. We found high bone turnover in 36.8xa0% and vitamin D deficiency and insufficiency in 82.8 and 16.1xa0%, respectively, and secondary hyperparathyroidism in 25.9xa0% of the subjects. This is alarming as there is inability to achieve peak bone mass and predisposes to osteoporosis risk.PurposeThis study aimed to assess bone health status in healthy females by using biochemical markers of bone metabolism in blood [N-telopeptide of type I collagen (NTx), 25-hydroxyvitamin D (25OHD), and plasma intact parathyroid hormone (iPTH)].Material and methodsOne hundred and seventy-four healthy premenopausal female volunteers were recruited from an urban residential area in Karachi. Demographic details were collected on a preformed questionnaire. Blood samples for the estimation of serum NTx, 25OHD, and plasma iPTH were taken in a fasting state. Data were analyzed using Statistical Package for Social Sciences 16.0. A p value of <0.05 was considered as significant.ResultsHigh bone turnover, as depicted by NTx, was seen in 36.8xa0% cases. Vitamin D deficiency, insufficiency, and sufficiency were seen in 82.8, 16.1, and 1.1xa0% respectively. Secondary hyperparathyroidism was present in 25.9xa0% of the subjects, while others had blunted PTH response. Significant correlates of bone health were serum 25OHD levels, duration of sun exposure, and the practice of wearing veil (p valueu2009<u20090.001).ConclusionBone turnover is high with high prevalence of vitamin D deficiency in apparently healthy premenopausal females predisposing them to higher risk for development of osteoporosis.

High prevalence of obesity calls for a priority action for non-communicable disease crises in adult women: findings of a community-based study in Karachi, Pakistan.

Pakistan is considered a country in which women and children are facing serious under-nutrition related problems. The National Nutrition Survey of Women in Pakistan (2011) has also shown serious vitamin A, vitamin D, iron, zinc, and calcium deficiency. However, the issue of over-nutrition, as measured by BMI, has received far less attention. Obesity is a recognized risk factor for many noncommunicable diseases, including cardiovascular (CVD), type 2 diabetes, hypertension, sleep apnea, and certain cancers [1]. Furthermore, there is an associated increased risk of morbidity and mortality, as well as reduced life expectancy [2]. Pakistan ranks among the top 10 countries of the world in many of these chronic diseases. An analysis of data from the National Health Survey of Pakistan (1990–1994) showed 25% prevalence of overweight and obesity in the Pakistani adult population [3]. However, prevalence of overweight and obesity was 42.8% in females aged 35–44 years. High prevalence of overweight and obesity in females (36.15%) was also reported previously in a study (n = 834) conducted in employees of universities and health and research institutions in Peshawar in 2003 [4]. In another community-based study (n = 2000) conducted in Multan, high prevalence of overweight and obesity in females (36%) was observed [5]. More recently, in 2011, we conducted a crosssectional community-based study in premenopausal females in 3 randomly-selected towns of Karachi to find out the prevalence of vitamin D3 deficiency and its risk factors. An individual BMI of >25 kg/m2 in our region has been advanced as an Asian cut-off for defining obesity [5–7]. In our survey of 305 subjects, the sample mean was 24 kg/m2. Furthermore, 43.3% and 19.3% of our participants were obese and overweight, respectively (table 1). In countries in which nutrition programs’ emphasis has been on under-nutrition, our findings should have implications for obesity-related, future disease burden due to increasing prevalence of overweight and obesity [8]. This calls for action on behalf of the policy-makers for addressing a very important public health issue in our population. It seems that while we have not addressed the agenda of under-nutrition, we are now facing a dual burden of disease with a growing epidemic of obesity in our population. This high prevalence of obesity is alarming and needs to be addressed immediately. Aysha Habib Khan, Ghazala Naureen, Romaina Iqbal

Ionized Calcium Cannot Predict Vitamin D Deficiency

To the Editor, n nVitamin Dxa0deficiency (VDD) results from inadequate intake, lack of sun exposure, malabsorption, or genetic abnormalities in vitamin D metabolism. Vitamin D deficiency/insufficiency remains undiagnosed, unless 25-hydroxy vitamin D (25OHD) concentrations are measured. Previously VDD was considered to be prevalent in regions where there was not ample sunlight [1]. However, recent literature suggests extensive VDD in many of the sun drenched countries [2, 3]. n nArya et al. [4] reported 78.3% of the healthy hospital staff had 25OHD levels <50xa0mmol/l in Lucknow India despite abundance of sunshine. Reports from Pakistan have similarly shown high prevalence of 25OHD deficiency [5, 6]. n nIn VDD, plasma calcium is maintained at the expense of bone calcium but persistence of deficiency leads to fall in calcium level and secondary hyperparathyroidism. We reported 30% of asymptomatic adults with 25OHD deficiency and secondary hyperparathyroidism but normal calcium levels [6]. In another report on medical clinic patients, calcium was low in patients with severe VDD only and remained normal in patients with mild and moderate deficiency [5]. However, these studies measured total calcium while the true calcaemic status of individuals is best assessed by ionized calcium. n nWe determined whether measurement of iCa could detect VDD in blood samples from 40 apparently healthy laboratory staff aged 27xa0±xa05xa0years and with no known comorbid. None of the individuals were on any medications including calcium and vitamin D supplements. Blood samples were collected according to recommendations by Clinical and Laboratory Standards Institute (CLSI), iCa and pH were simultaneously along with estimation of 25OHD levels by chemiluminescence methodology. n nNinety percent of the group was 25OHD deficient (25OHDxa0<xa050xa0nmol/l). Subjects with insufficient and sufficient 25OHD levels were 5% each. The difference between mean iCa results in 25OHD deficient, insufficient and sufficient groups were non-significant. Negative poor correlation was observed between iCa and 25OHD levels. n nVDD has been widely reported in immigrant Pakistanis to Europe and America. It was suggested that Pakistanis living within homeland are not suffering from VDD as Pakistan is situated between 24° and 37° north latitude and has adequate sunshine. Findings of this study has demonstrated that majority of the healthy population is vitamin D deficient and despite VDD has normal iCa levels, which could be at the expense of mobilization of bone calcium by secondary hyperparathyroidism. These results support the findings of Singh et al. [7] who reported poor correlation between 25OHD insufficiency and other analytes including plasma calcium, alkaline phosphatase or phosphorus levels. Ionized calcium estimation though technically demanding cannot detect VDD/insufficiency. To assess calcium status, total calcium provides enough information. Focus should be on optimization of vitamin D and calcium intake, either by diet or supplementation in our population.