Trimester-specific reference ranges for thyroid hormones of pregnant females at tertiary care hospitals in Lahore, Pakistan.

BACKGROUND: The significance of investigation for diagnosing and managing thyroid dysfunction in pregnant females has been extensively documented in the medical literature. This study aimed to determine trimester-specific reference ranges for thyroid-stimulating hormones (TSH), free T3 (FT3), and free T4 (FT4) in apparently healthy pregnant women attending tertiary care hospitals in Lahore. METHODS: This cross-sectional study was conducted at two tertiary care Hospitals in Lahore, Pakistan. In this multi-centric study, 500 pregnant females were initially enrolled from September 2019 to December 2019 who fulfilled the inclusion criteria. For measurement of serum FT3, FT4, thyroid stimulating hormone (TSH), anti-thyroid peroxidase (anti-TPO), and thyroglobulin antibodies, 5 ml of the blood sample was drawn, under aseptic conditions, from each subject using Maglumi 800 chemiluminescence immunoassay (CLIA) system. RESULTS: Out of 500 subjects, 23 subjects with positive anti-TPO, 19 subjects with anti-TG antibodies, and 12 subjects due to less volume of serum yielded from whole blood (serum less than 3 ml) were excluded from the analysis. Ten samples were hemolyzed and not included in the analysis. A total of 436 samples were examined for analysis. Of the remaining 436 subjects, 133 (30.5%) were from 1st trimester, 153 (35.1%) from 2nd trimester, and 150 (34.4%) from 3rd trimester. As the data were non-normal, the 2.5th, 50th, and 97.5th percentiles were calculated to express each group's results. Trimester specific range of TSH 0.168-4.294, 0.258-4.584 and 0.341-4.625 mIU/mL, FT31.857-4.408, 1.958-4.621 and 2.025-4.821 pmol/L and FT4 8.815-18.006, 8.306-17.341 and 7.402-17.292 pmol/L. CONCLUSION: In this study, we established a trimester-specific reference range for our local population's thyroid function test. The results of this study have complemented the results of previous studies.

Frequency of lipoprotein(a) testing and its levels in Pakistani population.

BACKGROUND: Lipoprotein(a) [Lp(a)] is a highly atherogenic particle identified as an independent risk factor for the development of atherosclerotic cardiovascular disease (ASCVD). This study aimed to investigate the frequency of Lp(a) testing and the incidence of elevated Lp(a) levels in the Pakistani population. METHODS: For this observational study, Lp(a) and lipid profile data from five years (June 2015 to October 2020) were acquired from the electronic patient records of a diagnostic laboratory with a countrywide network. The association of age and total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), non-HDL, and triglyceride (TG) levels with two thresholds for Lp(a), that is, <30 mg/dL and ≥30 mg/dL, was calculated using the Kruskal-Wallis test, while the association between Lp(a) levels and lipid variables was calculated using Spearman correlation. RESULTS: For five years, 1060 tests were conducted, averaging 212 tests per year. Of these, 37.2% showed Lp(a) levels above 30 mg/dL. No significant differences were observed in the results between males and females. However, younger individuals displayed significantly higher Lp(a) levels. Additionally, there was only a weak correlation between the Lp(a) levels and other lipid variables. CONCLUSION: Despite being recognized as a risk factor for ASCVD in the Pakistani population, only a small proportion of the large population underwent Lp(a) testing. Moreover, a significant proportion of the population exceeded this threshold.

Fingerprinting hyperglycemia using predictive modelling approach based on low-cost routine CBC and CRP diagnostics.

Hyperglycemia is an outcome of dysregulated glucose homeostasis in the human body and may induce chronic elevation of blood glucose levels. Lifestyle factors such as overnutrition, physical inactivity, and psychosocials coupled with systemic low-grade inflammation have a strong negative impact on glucose homeostasis, in particular, insulin sensitivity. Together, these factors contribute to the pathophysiology of diabetes (DM) and expanding landscape of its prevalence regionally and globally. The rapid rise in the prevalence of type 2 diabetes, therefore, underscores the need for its early diagnosis and treatment. In this work, we have evaluated the discriminatory capacity of different diagnostic markers including inflammatory biomolecules and RBC (Red Blood Cell) indices in predicting the risk of hyperglycemia and borderline hyperglycemia. For that, 208,137 clinical diagnostic entries obtained over five years from Chugtai Labs, Pakistan, were retrospectively evaluated. The dataset included HbA1c (n = 142,011), complete blood count (CBC, n = 84,263), fasting blood glucose (FBG, n = 35,363), and C-reactive protein (CRP, n = 9035) tests. Our results provide four glycemic predictive models for two cohorts HbA1c and FBG) each having an overall predictive accuracy of more than 80% (p-value < 0.0001). Next, multivariate analysis (MANOVA) followed by univariate analysis (ANOVA) was employed to identify predictors with significant discriminatory capacity for different levels of glycemia. We show that the interplay between inflammation, hyperglycemic-induced derangements in RBC indices, and altered glucose homeostasis could be employed for prognosticating hyperglycemic outcomes. Our results then conclude a glycemic predictor with high sensitivity and specificity, employing inflammatory markers coupled with RBC indices, to predict glycemic outcomes (ROC p-value < 0.0001). Taken together, this study outlines a predictor of glycemic outcomes which could assist as a prophylactic intervention in predicting the early onset of hyperglycemia and borderline hyperglycemia.

Reference Interval of Serum Gastrin 17 (G-17) for Healthy Population: A Non-Invasive Screening Biomarker for Gastric Disorders.

OBJECTIVE: To analyse fasting serum concentrations of G-17 in healthy individuals to establish the reference intervals (RIs) in the Pakistani population. STUDY DESIGN: Cross-sectional, observational study. Place and Duration of the Study: Department of Clinical Chemistry and Immunology, Chughtai Institute of Pathology, Lahore, Pakistan, from October to December 2022. METHODOLOGY: Fasting serum samples from one hundred and twenty healthy individuals between the age of 18-65 years were collected according to the CLSI recommendations after taking written informed consent. Samples were analysed on the auto-analyser for the quantitative measurement of serum G-17 by sandwich chemiluminescence immunoassay. Kolmogorov-Smirnov test was applied to check normality. A p-value of <0.05 was considered significant; 2.5th and 97.5th percentiles were computed using the formula 0.025 (n+1) and 0.0975 (n+1), respectively. RESULTS: Of the 120 samples, 74 were obtained from male patients and 46 from females. The mean age was 30.2 ±10.36 years. The histogram revealed a non-parametric distribution of the data. The established reference intervals by the rank-based method were 2.31 pg/mL and 49.36 pg/mL which corresponds to 2.5th and 97.5th percentiles, respectively. These were markedly different from the Chinese reference ranges. CONCLUSION: Ethnic and geographic variations affect the trends of RIs of Serum G-17. There is a need to establish its population-specific RIs for G-17, so it can be used as a non-invasive option in identifying patients requiring invasive endoscopic intervention. KEY WORDS: Gastrin, Atrophic Gastritis, Biomarker, Reference values.

Genetic and clinical characteristics of patients with lipoprotein lipase deficiency from Slovenia and Pakistan: case series and systematic literature review.

Introduction: Hypertriglyceridemia (HTG) is a complex disorder caused by genetic and environmental factors that frequently results from loss-of-function variants in the gene encoding lipoprotein lipase (LPL). Heterozygous patients have a range of symptoms, while homozygous LPL deficiency presents with severe symptoms including acute pancreatitis, xanthomas, and lipemia retinalis. Methods: We described the clinical characteristics of three Slovenian patients (an 8-year-old female, an 18-year-old man, and a 57-year-old female) and one Pakistani patient (a 59-year-old male) with LPL deficiency. We performed next-generation sequencing (NGS) targeting all coding exons and intron-exon boundaries of the LPL gene, and Sanger sequencing for variant confirmation. In addition, we performed a systematic literature review of all cases with three identified variants and described their clinical characteristics. Results: Two Slovenian patients with a heterozygous pathogenic variant NM_000237.3:c.984G>T (p.Met328Ile) were diagnosed within the first three years of life and had triglyceride (TG) values of 16 and 20 mmol/L. An asymptomatic Pakistani patient with TG values of 36.8 mmol/L until the age of 44 years, was identified as heterozygous for a pathogenic variant NM_000237.3:c.724G>A (p.Asp242Asn). His TG levels dropped to 12.7 mmol/L on dietary modifications and by using fibrates. A Slovenian patient who first suffered from pancreatitis at the age of 18 years with a TG value of 34 mmol/L was found to be homozygous for NM_000237.3:c.337T>C (p.Trp113Arg). Conclusions: Patients with LPL deficiency had high TG levels at diagnosis. Homozygous patients had worse outcomes. Good diet and medication compliance can reduce severity.

Highly Sensitive and Ultrafast Liquid Chromatography-tandem Mass Spectrometry Internal Standard Method for Determination of Alpha-Tocopherol in Serum and Application of Validated Method on Patient Serum Samples in Pakistan.

OBJECTIVE: To optimize and validate a specific, sensitive and fast liquid Chromatography coupled to triple quadrupole Mass Spectrometric (LC-MS / MS) technique for accurate detection of serum α-tocopherol (Vitamin E) levels. STUDY DESIGN: An experimental based study. PLACE AND DURATION OF STUDY: The Clinical and Forensic Toxicology section of Chughtai Lab, Jail Road Lahore, from April to September 2022. METHODOLOGY: Methanol was used to deproteinize serum samples. The chromatographic separation was achieved using an Agilent Infinity-Lab Poroshell 120EC-C18 column, Agilent 6470 LC-MS/MS (equipped with an Electron Spray Ionization source) in gradient elution mode using 0.1% LCMS grade formic acid in water and LCMS-grade methanol as mobile phases. Hexa-deuterated α-tocopherol was employed as internal standard to minimise matrix interferences. RESULTS: The retention time of α-tocopherol was 3.0 ± 0.1 minutes. The linear concentrations obtained were ranged from 0.05-2 mg/dL with ≥0.985% coefficient of linearity. Detection and lower quantification limits determined were 0.025mg/dL and 0.05mg/dL, respectively. Recovery ranged from 96.5 to 99.8% and ionization suppression was -15.2% and -15.9% at high and low concentrations of α-tocopherol in serum. Intra-day and inter-day coefficient variation values were 4.2-4.9% and 5.0-5.9%, respectively. CONCLUSION: An efficient and reliable tandem mass spectrometric technique for vitamin E analysis in serum was optimized, validated, and applied to 80 patient samples. This method has usefulness in clinical application for the accurate determination of vitamin E without potential matrix interferences. KEY WORDS: Vitamin E, LC-MS/MS, Tocopherol, Internal standard, Validation.

Reference Interval of Soluble FMS­like Tyrosine Kinase­1 in Non-Pregnant and Pregnant Females: A Novel Biomarker for Pre-eclampsia.

OBJECTIVE: To determine the reference interval of soluble FMS-like tyrosine kinase-1 (sFIt-1) in healthy, non-pregnant and pregnant females. STUDY DESIGN: Observational study. Place and Duration of the Study: Department of Chemical Pathology, Chughtai Institute of Pathology, Lahore, from January to May 2023. METHODOLOGY: Blood samples were collected from 120 disease-free non-pregnant females of reproductive age group and 120 disease-free pregnant females with singleton fetuses from 15 to 28 weeks of gestational age. Healthy reference individuals were selected by correlating history with medical disorders like diabetes mellitus, hypertension, autoimmune diseases, inherited disorders, and by excluding any other drug history. All findings were recorded on health screening questionnaire. Levels of sFlt-1 were measured by a fully automated immunoassay analyser Cobas e601. Kolmogorov-Smirnov test was applied. The value of p <0.05 was considered significant. The 2.5th and 97.5th percentiles were computed at 90% CI by using the formula 0.025x (n+1) and 0.975x (n+1) which corresponded to rank number 1 and 7, respectively. The reference interval was calculated by the Rank-based method. RESULTS: Reference interval of sFlt-1 in non-pregnant and pregnant females were determined on the basis of 2.5th and 97.5th percentiles which were 57.7 to 118.5 pg/mL and 563.5 to 3288.0 pg/mL, respectively. CONCLUSION: The present study determined reference interval of sFlt-1 in healthy, non-pregnant and pregnant females in Lahore. KEY WORDS: Reference interval, Soluble FMS-like tyrosine kinase-1, Pre-eclampsia, Rank-based method.

Prediction Of Renal Flare In Adult Patients With Lupus Induced Nephritis.

BACKGROUND: The most common complication of SLE is lupus nephritis (LN) causing high morbidity and mortality. The routine biomarkers used for the diagnosis of LN do not have the ability to predict the worsening in renal disease activity. Thus, there is need of a new biomarker leading to detection of flare in LN. The objective of this study was to assess the role of urinary neutrophil gelatinase associated lipocalin (uNGAL) as a predictor of renal flare in patients with lupus nephritis. METHODS: Including a total of 84 subjects, 42 cases were lupus patients without renal involvement and 42 cases were lupus patients with nephritis (24 active nephritis and 18 inactive nephritis). The diagnosis of lupus nephritis was established on the basis of renal biopsy. uNGAL was estimated in both groups. RESULTS: This study revealed that the nephritis group had increased levels of uNGAL as compared to systemic erythematosus patients without having lupus nephritis (p-value <0.05). Patients with active nephritis had increased uNGAL levels as compared to patients with inactive nephritis. CONCLUSIONS: From the findings in our study, it can be stated that uNGAL can prove to be a noninvasive, reliable and sensitive biomarker to predict flare in cases of lupus nephritis.

Seroprevalence of COVID-19 IgG antibodies among healthcare workers of Pakistan: a cross-sectional study assessing exposure to COVID-19 and identification of high-risk subgroups.

OBJECTIVE: In this study, we aimed to find the seroprevalence of healthcare workers (HCWs) of Pakistan involved in the treatment and care of patients with COVID-19. SETTING: This was a cross-sectional study and total of 15 000 HCWs involved in providing services and care to the patients with COVID-19 were randomly selected from all over Pakistan. PARTICIPANTS: Informed consent was taken from all participants and were included according to inclusion and exclusion criteria. All testing was done on serum samples for the qualitative detection of SARS-CoV-2 IgG antibodies using Abbott Chemiluminescent microparticle immunoassay. An index of 1.4 was used as a cut-off to mark reactive and non-reactive cases. SPSS V.23.0 was used for data analysis. OUTCOME: Immune status of the study population depicting seroprevalence among HCWs. RESULTS: Out of all the candidates, majority of the HCWs were men (61.9%) and were doctors (62.4%). The mean age of participants was 32.8 years (SD 8.7) and majority were asymptomatic (51.8%). In this study, 33% of the HCWs were reactive for SARS-CoV-2 IgG antibody. Around 44% of the reactive cases were asymptomatic. The symptoms more significantly associated with seropositivity were: fever (OR 1.31; 95% CI 1.16 to 1.48), headache (OR 2.43; 95% CI 2.16 to 2.73), cough and shortness of breath (OR 2.10; 95% CI 1.91 to 2.31), loss of sense of smell or taste (OR 3.70; 95% CI 3.29 to 4.17) (p<0.001). Factors which showed significant association with the presence of antibodies were professional category (absolute risk (AR) 0.09; OR 1.46; 95% CI 1.36 to 1.56), availability of protective masks (AR 0.02; OR 0.90; 95% CI 0.84 to 0.96), safety goggles (AR 0.02; OR 0.90; 95% CI 0.84 to 0.97) and living arrangements (AR 0.03; OR 1.12; 95% CI 1.04 to 1.20) (p<0.05). CONCLUSION: Our study showed a high seropositivity of HCWs dealing with patients with COVID-19 in Pakistan revealing significant association with professional category, nature of work place and precautions taken while performing duties.

Seroconversion in Newly Diagnosed Cases of Coronavirus Disease.

OBJECTIVE: To determine the percentage of seroconverted real time reverse transcriptase polymerase chain reaction (RT-PCR) positive coronavirus disease (COVID-19) cases at different days post-symptom onset; and also find the agreement of chemiluminescence assay used for total antibody detection using RT-PCR as a reference method. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Chughtai Institute of Pathology from April to May 2020. METHODOLOGY: Fifty pre-pandemic samples (healthy population) and 75 COVID-19 patients were included in the study. RT-PCR confirmed COVID-19 patients were divided into 3 equal groups (25 each), according to the days of symptom onset. The samples were analysed using electro-chemiluminescence as assay principle. Positive and negative agreement of COVID-19 antibodies was calculated using EP evaluator to find out the sensitivity of chemiluminescence assay for total antibody detection. The results were analysed using SPSS version 23.0. RESULTS: All the pre-pandemic samples tested were negative for antibodies with a negative agreement of 100%. Total agreement at day 7 post-symptom onset was 84%; whereas, it was 94% at day 14 and increased rapidly to 100% at day 21 post-symptom onset. At day 7 post-symptom onset, 68% of patients were seroconverted; and this percentage was 88% and 100% at day 14 and 21 post-symptom onset, respectively. CONCLUSION: Pre-pandemic samples were non-reactive for COVID-19 antibodies and seroconversion started within the first week post-virus exposure. There was 100% concordance between RT-PCR result and antibody positivity 21 days post-symptom onset. Key Words: COVID-19, SARS CoV-2, Seroconversion, Chemiluminescence.

Frequency of COVID-19 IgG Antibodies among Special Police Squad Lahore, Pakistan.

OBJECTIVE: To find out the sero-prevalence of COVID-19 IgG antibodies among policemen performing duties at high risk areas of Lahore, Pakistan. STUDY DESIGN: Cross-sectional, observational study. PLACE AND DURATION OF STUDY: Chughtai Lab Lahore from 20th to 30th May 2020. METHODOLOGY: One hundred and fifty-four young policemen (mean age 27.1 ±3.8 years) were included in the study after written consent. Squad officers who had any sign of acute respiratory infection were excluded from the study. The participants were asked to fill a questionnaire regarding nature of exposure with the infected cases, kind of personal protective equipment they are provided with and living arrangements. Three ml of blood samples were collected from the participants and immediately sent to the lab for analysis. COVID-19 IgG antibodies were analysed using chemiluminescence immunoassay method on Architect Ci8200 (Abbott). SPSS 23.0 was used for data analysis. RESULTS: All participants were males and mean age was 27.1 ±3.8 years. Out of 154 squad members, 24 were reactive for COVID-19 IgG antibodies and performing duties at the areas of high virus exposure. None of COVID-19 IgG reactive cases exhibited symptoms and most of them performed duties at high risk areas with only masks provided as personal protective equipment. Only 10% of the participants who were exposed to confirmed cases of COVID-19 were found to have anti-COVID IgG. CONCLUSION: COVID-19 IgG antibody reactive cases may not show any symptoms. Police force is at high risk of exposure. Serosurveys can help to find the spread of infection in the community and aid in planning healthcare strategies. Key Words: COVID-19, SARS CoV-2, Seroprevalence, Pandemic, Asymptomatic cases.