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Population-adjusted cut-off: A new approach for enhancing the diagnostic efficacy of hematological discrimination formulae for screening β-Thalassemia trait

2026-09-01T02:28:09Z

Title: Population-adjusted cut-off: A new approach for enhancing the diagnostic efficacy of hematological discrimination formulae for screening β-Thalassemia trait Authors: Karunaithas, R.; Aravinda, I.; Dharmasiri, S.; Sewwandi, C.; Karunaithas, S.; Goonetilleke, S. Abstract: Screening for β-thalassemia trait (βTT) is crucial for preventing β-thalassemia major in offspring. Although hematological discrimination formulae (HDF), developed using complete blood count parameters, are cost-effective tools for screening βTT, their performance varies across different populations. This study evaluated the performance of 32 HDF for screening βTT in the Sri Lankan population. Data were retrieved from laboratory databases and categorized into confirmed βTT and non-βTT groups based on high-performance liquid chromatography results. The βTT screening performance of the HDF was assessed using accuracy measurements, the receiver operating characteristic (ROC) curve, and Youden’s index (YI). Furthermore, a population-adjusted cut-off was determined using the Index of Union (IU) method to optimize the predictive accuracy of HDF in screening βTT. Bordbar demonstrated excellent predictive performance in males (AUC = 0.908; YI = 0.815), while Shine & Lal, Kerman-I, Nishad, Sehgal, Bordbar, and Roth demonstrated high discriminative ability in females (AUC > 0.833; YI > 0.666). Applying a population-adjusted cut-off improved the βTT screening potential of Shine & Lal, Kerman-I, Nishad, Bordbar, and Roth in males (AUC > 0.911; YI > 0.822) and enhanced the performance of Kerman-II in females (AUC > 0.861; YI > 0.722). Notably, Shine & Lal (AUC = 0.937; YI > 0.873) and Nishad (AUC = 0.897; YI > 0.794) demonstrated the best performance for males and females, respectively, when a population-adjusted cut-off was applied for screening βTT. In conclusion, determining a population-adjusted cut-off is a new initiative to enhance the βTT screening performance of HDF across different populations.

Exploring urinary biomarkers for the diagnosis of diabetic and hypertensive chronic kidney disease: A promising pilot study

2025-04-21T03:10:51Z

Title: Exploring urinary biomarkers for the diagnosis of diabetic and hypertensive chronic kidney disease: A promising pilot study Authors: Saseevan, S.; Nishanthi, N.; Sanath, R.; Magana-Arachchi, D. Abstract: In the current clinical setting, conventional serum biomarkers such as serum creatinine (Scr) and estimated glomerular filtration rate (eGFR) have several lapses in chronic kidney disease (CKD) diagnosis. Diagnosing CKD using non-invasive methods is crucial for implementing prompt therapeutic interventions and preventing disease progression. This study aims to identify the potential diagnostic urinary biomarkers and their correlation with existing renal markers, Scr, eGFR, and proteinuria in diabetic and hypertensive CKD. RNA was extracted from eighty-two urine samples of CKD patients and healthy controls (HC) and reverse transcribed for gene expression analysis using quantitative polymerase chain reactions. The expression of NGAL, MMP9, ANXA3, OLFM4, PI3, and PRMT3 genes was analyzed relative to the reference gene, B2M. Fold changes (FC) in gene expression in diabetic nephropathy (DN), and hypertensive nephropathy (HT) were calculated against HC. Log2 normalized FC was used to determine significance levels and correlation with existing serum markers. NGAL, ANXA3, and OLFM4 exhibited the highest upregulations in DN with mean Log2FC 1.42, 2.66, and 5.87, respectively. A two-fold increase in NGAL FC was observed in early DN than in late DN, suggesting its potential as an early urinary biomarker for DN. PI3 and MMP9 were upregulated in HT patients with higher FC values. PRMT3 showed a significant negative correlation (P<0.05) in HT patients with Scr (r=−0.738) and proteinuria (r=−0.906). The gene panels including ANXA3, OLFM4, and NGAL, and PI3, PRMT3, and MMP9, could have potential diagnostic value in DN and HT, respectively.

RNA extraction from urine sediment: A cost-effective protocol for gene expression analysis in renal pathology.

2025-04-03T07:29:55Z

Title: RNA extraction from urine sediment: A cost-effective protocol for gene expression analysis in renal pathology. Authors: Saseevan, S.; Rajapakse, S.; Magana-Arachchi, D.N. Abstract: Urine is an appropriate choice of specimen to study the biomarkers for metabolic and renal disorders because it is readily available with less harm to the patients. However, RNA extraction from voided urine is challenging due to the presence of RNases and cell scarcity. This study aims to optimize a protocol for RNA extraction from urine samples for gene expression studies in renal pathology. Hundred and two urine samples were collected from both healthy controls (HC) (𝑛 = 15; 54 ± 11 years) and chronic kidney disease (CKD) patients (𝑛 = 87; 56 ± 10 years) and centrifuged at 6,500 ɡ for 20 min at 4 °C to obtain sediment. RNA was extracted from urine sediments using a phenol-based technique. The extracted RNA was quantified and reverse-transcribed into complementary DNA (cDNA). Reverse transcriptase quantitative polymerase chain reactions (RT- qPCR) were carried out using 2 ng of template cDNA to amplify the housekeeping gene, β2- microglobulin (B2M). The total yield of RNA from CKD and HC samples were 718 ± 164 ng and 790 ± 231 ng, respectively, and a statistically significant difference was not observed between the two study groups (p > 0.05). The urinary RNA recovery was significantly increased with CKD progression (p < 0.05). Further, the results show that urine volume, gender, and serum creatinine level significantly influence the RNA yield in only disease groups (p < 0.05). The mean threshold cycle (Ct) values for B2M amplification of CKD and HC were 27.36 ± 3.09 and 20.97 ± 3.90, respectively. This modified phenol-chloroformbased urinary RNA extraction method is less expensive and does not require pre- and post-purification steps. It provides a higher yield of RNA with less inhibition to qPCR and is sufficient for downstream applications than column-based techniques.

Culturable bacterial pathogens in midstream urine of chronic kidney disease patients in Vavuniya, Sri Lanka: A preliminary study

2025-04-03T07:10:31Z

Title: Culturable bacterial pathogens in midstream urine of chronic kidney disease patients in Vavuniya, Sri Lanka: A preliminary study Authors: Jayalath, J.M.S.D.; Saseevan, S.; Perera, W.A.K.; Magana-Arachchi, D.N. Abstract: Background: Contribution of the midstream urine microbiome to chronic kidney disease (CKD) is clinically relevant, yet it’s understudied. Objectives: To identify bacteria from midstream urine of CKD patients with comorbidities of diabetes mellitus (DM), hypertension (HT) and other causes, in Vavuniya. Methods: A total of seventeen (n = 17) midstream clean-catch urine samples were collected from CKD patients (56.59 ± 12.91 years) at the District General Hospital, Vavuniya, belonging to CKD + HT (n = 8), CKD + DM (n = 2), CKD + HT + DM (n = 3) and causes of CKD other than DM and HT (n = 4). Non-CKD healthy controls (n = 8; 56 ± 11 years) were included for comparison. Samples were transported on ice and immediately refrigerated at 4°C until processed. 10μl per sample was inoculated onto Luria-Bertani (LB) agar plates in duplicates and incubated aerobically at 37°C for 24 hours. Morphologically different bacterial colonies were subcultured in LB broth to obtain pure isolates, which underwent gram staining, and biochemical analyses for preliminary identification. Results: Mean colony count for CKD subjects was 2,500 ± 967 CFU/mL and 286 ± 181 CFU/mL for controls. The CKD + HT + DM group had the highest mean colony count. In total, 24 bacterial isolates were obtained from the patients’ urine of which 45.8% were gram positive cocci, 37.5% were gram negative rods and bacterial genera Staphylococcus (33%), Streptococcus (12.5%), Pseudomonas (4%), Klebsiella (4%) and Proteus (4%) were identified. Staphylococcus, Corynebacteria, Proteus, Escherichia, and Citrobacter were found in controls. Conclusion: Various viable uropathogens in the midstream urine of CKD patients were identified using culture-based tests. This preliminary study is currently ongoing to further identify the midstream urine microbiome in CKD with molecular techniques too, as culture alone is insufficient.