Evaluation of noninvasive biospecimens for transcriptome studies.

Transcriptome studies disentangle functional mechanisms of gene expression regulation and may elucidate the underlying biology of disease processes. However, the types of tissues currently collected typically assay a single post-mortem timepoint or are limited to investigating cell types found in blood. Noninvasive tissues may improve disease-relevant discovery by enabling more complex longitudinal study designs, by capturing different and potentially more applicable cell types, and by increasing sample sizes due to reduced collection costs and possible higher enrollment from vulnerable populations. Here, we develop methods for sampling noninvasive biospecimens, investigate their performance across commercial and in-house library preparations, characterize their biology, and assess the feasibility of using noninvasive tissues in a multitude of transcriptomic applications. We collected buccal swabs, hair follicles, saliva, and urine cell pellets from 19 individuals over three to four timepoints, for a total of 300 unique biological samples, which we then prepared with replicates across three library preparations, for a final tally of 472 transcriptomes. Of the four tissues we studied, we found hair follicles and urine cell pellets to be most promising due to the consistency of sample quality, the cell types and expression profiles we observed, and their performance in disease-relevant applications. This is the first study to thoroughly delineate biological and technical features of noninvasive samples and demonstrate their use in a wide array of transcriptomic and clinical analyses. We anticipate future use of these biospecimens will facilitate discovery and development of clinical applications.

Automated Urine Cell Image Classification Model Using Chaotic Mixer Deep Feature Extraction.

Microscopic examination of urinary sediments is a common laboratory procedure. Automated image-based classification of urinary sediments can reduce analysis time and costs. Inspired by cryptographic mixing protocols and computer vision, we developed an image classification model that combines a novel Arnold Cat Map (ACM)- and fixed-size patch-based mixer algorithm with transfer learning for deep feature extraction. Our study dataset comprised 6,687 urinary sediment images belonging to seven classes: Cast, Crystal, Epithelia, Epithelial nuclei, Erythrocyte, Leukocyte, and Mycete. The developed model consists of four layers: (1) an ACM-based mixer to generate mixed images from resized 224 × 224 input images using fixed-size 16 × 16 patches; (2) DenseNet201 pre-trained on ImageNet1K to extract 1,920 features from each raw input image, and its six corresponding mixed images were concatenated to form a final feature vector of length 13,440; (3) iterative neighborhood component analysis to select the most discriminative feature vector of optimal length 342, determined using a k-nearest neighbor (kNN)-based loss function calculator; and (4) shallow kNN-based classification with ten-fold cross-validation. Our model achieved 98.52% overall accuracy for seven-class classification, outperforming published models for urinary cell and sediment analysis. We demonstrated the feasibility and accuracy of deep feature engineering using an ACM-based mixer algorithm for image preprocessing combined with pre-trained DenseNet201 for feature extraction. The classification model was both demonstrably accurate and computationally lightweight, making it ready for implementation in real-world image-based urine sediment analysis applications.

Persistently Elevated HBV Viral-Host Junction DNA in Urine as a Biomarker for Hepatocellular Carcinoma Minimum Residual Disease and Recurrence: A Pilot Study.

Hepatitis B virus (HBV)-host junction sequences (HBV-JSs) has been detected in the urine of patients with HBV infection. This study evaluated HBV-JSs as a marker of minimum residual disease (MRD) and tumor recurrence after treatment in HBV-hepatocellular carcinoma (HCC) patients. Archived serial urine DNA from two HBV-HCC with recurrence as confirmed by MRI and four HBV-related cirrhosis (LC) patients were used. Urinary HBV-JSs were identified by an HBV-targeted NGS assay. Quantitative junction-specific PCR assays were developed to investigate dynamic changes of the most abundant urinary HBV-JS. Abundant urinary HBV-JSs were identified in two cases of tumor recurrence. In case 1, a 78-year-old female with HBV- HCC underwent a follow-up MRI following microwave ablation. While MRI results were variable, the unique HBV-JS DNA, HBV-Chr17, steadily increased from initial diagnosis to HCC recurrence. In case 2, a 74-year-old male with HBV-HCC contained two HBV-JS DNA, HBV-Chr11 and HBV-TERT, that steadily increased after initial HCC diagnosis till recurrence. One LC examined had HBV-TERT DNA detected, but transiently in 3.5 years during HCC surveillance. HBV-JS DNA was persistently elevated prior to the diagnosis of recurrent HCC, suggesting the potential of urinary HBV-JS DNA to detect MRD and HCC recurrence after treatment.

Phenotype of Urine Sediment Cells in Patients with Bladder Cancer.

Phenotype of urine sediment cells were studied in patients with bladder cancer depending on the cancer stage and recurrence prognosis. In T1N0M0 stage, the number of lymphocytes decreased, in T2N0M0 stage, the most pronounced shift was an increase in the number of erythrocytes. Irrespectively of the disease stage, we observed increased number of innate immunity cells and cells that inhibit antitumor immunity in the composition of the leukocyte fraction of urine sediment cells. At T1N0M0 stage, the epithelial-endothelial fraction was characterized by increased content of cells expressing CD13 marker (responsible for tumor growth and metastasis) and reduced number of cells expressing CD15 marker (responsible for intercellular adhesion). In patients developing relapse of bladder cancer, the number of lymphocytes was decreased in urine sediment cells and the number of epithelial and endothelial cells expressing CD13 marker was increased.

[Detection and identification of urinary crystals using an automated urine particle analyzer coupled with a digital particle imaging device]. / Détection et identification de cristaux dans les urines à l'aide d'un analyseur de cytologie urinaire couplé à un dispositif d'imagerie numérique.

Polarized light microscopy (POM) remains the gold standard for crystalluria analysis. However, such method is time consuming and requires well-trained staff. Here, to address this issue, we tested the Sysmex UF-4000 analyzer coupled to a UD10 module as an automated flow cytometry-digital particle imaging workflow to assess (i) the ability of the system to detect and identify the crystals species and (ii) the quality of the images provided by the UD-10 module (n = 40) for each urine sample analyzed. First, systematic analysis of 76 samples by POM and the UF-4000/UD-10 analyzer showed that only attentive examination of the 40 photos was able to confidently detect crystalluria-positive samples with no misses and thus serve to discriminate positive-test crystalluria from negative-test crystalluria. These first results were confirmed by sensitivity analysis and the negative predictive value calculated on 200 samples for the results provided by the UF-4000 (39% and 46%) and after examination of the 40 UD-10 photos (100% for the both values). Digital images can therefore serve to screen crystalluria without missing crystals. A part of samples were treated by POM whereas it was not necessary (positive predictive value: 78%). Finally, we compared the crystal identification performances of the Sysmex UF4000/UD10 workflow and the 'gold standard' POM method on 131 urine samples containing crystals. Only calcium oxalate dihydrate crystals were identified by the Sysmex UF-4000. A close examination of the digital photographs enabled exact identification of crystals in 84.7% of the samples, suggesting however that POM is still require as soon as crystals are observed on the photographs. We conclude that a SYSMEX UF-4000 coupled with a UD-10 module can be used in practice with close examination of the photographs to discriminate positive crystalluria from negative crystalluria.

Cutoff values of PD-L1 expression in urinary cytology samples for predicting response to immune checkpoint inhibitor therapy in upper urinary tract urothelial carcinoma.

BACKGROUND: The objective of this study was to determine the cutoff value of PD-L1 expression that can predict response to immune checkpoint inhibitor (ICI) immunotherapy for upper tract urothelial carcinoma (UTUC). METHODS: The concordance of PD-L1 expression between paired surgical resection specimens (SRSs) and urine cell blocks (UCBs) (cohort 1) was studied in a retrospective set of 58 UTUC patients to determine its suitability as a predictor of ICI immunotherapy efficacy. PD-L1 expression in UCBs obtained before neoadjuvant ICI immunotherapy was verified in a prospective set of 12 UTUC patients (cohort 2). PD-L1 (SP263 clone) expression was assessed for percentage (tumor proportional score) of tumor cell (TC) showing PD-L1 staining. RESULTS: The authors found an overall agreement of 94.4% (51 of 54) between UCBs and SRSs in cohort 1 (positive percent agreement = 100%, negative percent agreement = 93.8%, r value = 0.63). PD-L1 expression in <10% and ≥10% of tumor cells (TCs) of UCBs were the best predictors of negative (<25%) and positive (≥25%) expression in TCs of SRSs, respectively (concordance = 98.1%, r value = 0.93). These findings were verified in cohort 2: at the 10% cutoff for PD-L1 expression, the best response predictive value was 83.3% (5 of 6) in PD-L1-positive patients, and the nonresponse predictive value was 50% (3 of 6) in PD-L1-negative patients. The sensitivity, specificity, and area under the receiver operating characteristic curve values for predicting ICI immunotherapy efficacy based on PD-L1-expressing TCs in UCBs were 62.5%, 75%, and 0.688, respectively. CONCLUSIONS: Immunocytochemistry of UCBs is reliable for determining PD-L1 expression, which can predict the efficacy of ICI immunotherapy at a cutoff of 10%.

The application value and research progress of urine free DNA in tumor molecular diagnosis / 中华检验医学杂志

Urine cell-free DNA (ucfDNA) contains DNA fragments released from the lysis of cells in the urinary system, and circulating cell-free DNA (ccfDNA) can also enter the urine after glomerular filtration, and the mutation information carried by tumor DNA contained in ucfDNA and ccfDNA is consistent. Therefore, as a biomarker for molecular diagnosis of urological and non-urinary tumors, ucfDNA, has become a research hotspot in the field of liquid biopsy in recent years. UcfDNA has potential application value in individualized treatment, early diagnosis, dynamic monitoring of therapeutic efficacy, and prognosis assessment, etc. However, in order to realize the clinical application of urine ucfDNA, the extraction and accurate detection of ucfDNA still need to be solved.

Concentration and integrity indexes of urine cell-free DNA as promising biomarkers for early lung cancer diagnosis.

Aim: To explore the role of urine cell-free DNA (ucfDNA) concentration and integrity indexes as potential biomarkers for lung cancer diagnosis. Materials & methods: Quantitative real-time PCR targeting Arthrobacter luteus (ALU) repeats at three size fragments (ALU-60, 115 and 247 bp) was performed in 55 lung cancer patients and 35 healthy individuals. Results: ucfDNA concentration and integrity indexes were significantly higher in lung cancer patients than in healthy controls. The area under the receiver operating characteristic curve for differentiating patients with stage I/II from healthy controls by ALU fragments concentration were 0.856, 0.909 and 0.932, respectively. In addition, the ucfDNA integrity indexes in patients with lymph node metastasis were significantly higher than in patients with non-metastatic. Conclusion: ucfDNA concentration and integrity indexes could serve as promising biomarkers for lung cancer diagnosis.

Improved efficiency of urine cell image segmentation using droplet microfluidics technology.

Recent advances in the recognition of biological samples using machine vision have made this technology increasingly important in research and detection. Image segmentation is an important step in this process. This study focuses on how to reduce the interference factors such as the overlap between different types (or within the same type) of urine cells according to microfluidics and improve the machine vision segmentation accuracy for cell images. In this study, we demonstrate that the platform can realize this hypothesis using urine cell image segmentation as an example application. We first discuss the reported urine cell droplet microfluidic chip system, which can realize the test conditions in which urine cells are encapsulated in the droplet and isolated from salt crystallization and/or bacteria and other urine-formed elements. Then, based on the analysis conditions set in the aforementioned experiment, the proportions of red blood cells, white blood cells, and squamous epithelial cells covered by various formed elements in the total urine cells in the same urine sample are measured. We simultaneously analyze the percentage of urine cells covered by salt crystallization and the incidence of overlapping between urine cells. Finally, the Otsu algorithm is used to segment the urine cell images encapsulated by the droplet and the urine cell images not encapsulated by the droplet, and the Dice, Jaccard, precision, and recall values are calculated. The results suggest that the method of encapsulating single cells based on droplets can improve the image segmentation effect without optimizing the algorithm.

Urine cell-free DNA as a promising biomarker for early detection of non-small cell lung cancer.

BACKGROUND: While blood-derived cell-free DNA has been shown to be a candidate biomarker able to provide diagnostic and prognostic insight in cancer patients, little is known regarding the potential application of urine cell-free DNA (ucfDNA) in diagnosis of cancer. Thus, the aim of this study was to investigate ucfDNA concentration and integrity index as potential biomarkers for early detection of non-small-cell lung cancer (NSCLC). METHODS: Urine samples were collected from 35 healthy controls and 55 NSCLC patients at various tumor node metastasis (TNM) stages. Two long interspersed nuclear element 1 (LINE1) fragments (LINE1-97 and 266 bp) were quantified via quantitative real-time PCR (qPCR). DNA integrity index was calculated as the ratio of LINE1-266/LINE-97. RESULTS: LINE1 fragments concentrations of ucfDNA (LINE1-97, 266 bp) were significantly higher in NSCLC patients with stage III/IV than in stage I/II and in healthy controls. The receiver operating characteristic (ROC) curves for discriminating patients with stage III/IV from healthy controls had areas under the curves (AUC) of 0.84 and 0.886, respectively. Moreover, ucfDNA integrity LINE1-266/97 was significantly higher in patients with stage III/IV than in stage I/II and in healthy controls. The AUC of ROC curve for discriminating patients with stage III/IV from healthy controls was 0.800. Furthermore, LINE1-266 fragment concentration was significantly higher in lymph node metastasis (LNM)-positive patients relative to LNM-negative patients. The ROC curve for discriminating LNM-positive from LNM-negative patients had an AUC of 0.822. CONCLUSION: UcfDNA could serve as a promising biomarker for early detection of NSCLC.

Generation of sepsis encephalopathy patient-specific inducible pluripotent stem cells with urine cells / 中华危重病急救医学

Objective To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients. Methods Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCATM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway. Results The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t =-10.688; REX1 mRNA (2-ΔΔCt):1.419±0.206 vs. 0.103±0.066, t =-14.245; OCT4 mRNA (2-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t =-9.028; Sox2 mRNA (2-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t =-12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC. Conclusion The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.

Generation of sepsis encephalopathy patient-specific inducible pluripotent stem cells with urine cells / 中华危重病急救医学

Objective@#To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients.@*Methods@#Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCATM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway.@*Results@#The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t = -10.688; REX1 mRNA (2-ΔΔCt): 1.419±0.206 vs. 0.103±0.066, t = -14.245; OCT4 mRNA (2-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t = -9.028; Sox2 mRNA (2-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t = -12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC.@*Conclusion@#The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.

Generation of Urine Cell-Derived Non-integrative Human iPSCs and iNSCs: A Step-by-Step Optimized Protocol.

Objective: Establishing a practical procedure to generate induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) from human urine cells (UCs). In this report, we optimized a non-integrative protocol to generate patient-specific iPSC and iNSC lines with high reprogramming efficiency. Methods: UCs were electroporated with the pEP4-EO2S-ET2K and pEP4-M2L plasmids containing the OCT4, SOX2, KLF4, SV40LT, c-MYC, and LIN28 genes, and then cultured with N2B27 medium plus four small molecule compounds (A83-01, PD0325901, Thiazovivin, and CHIR99021). When iPSC or iNSC clones emerged, the medium was replaced with mTeSR1 or neural growth medium. Morphological changes were seen at day 4-7. After day 10, the clones were picked up when the clone diameter exceeded 1 mm. Results: iPSCs and iNSCs were successfully derived from UCs with up to 80 clones/well. These iPSCs and iNSCs showed typical hESC or NSC morphology and were self-renewable. The iPSCs had pluripotency to differentiate into the three germinal layers and displayed high levels of expression of pluripotency markers SOX2, NANOG, OCT4, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase (AP). They maintained normal karyotype and had no transgene expression or genomic integration. The iNSCs were positive for NSC markers NESTIN, PAX6, SOX2, and OLIG2. Conclusion: The optimized protocol is an easy and fast procedure to yield both iPSC and iNSC lines from a convenient source of human urine in a single experiment.

Urine cell-free DNA integrity as a marker for early bladder cancer diagnosis: preliminary data.

OBJECTIVES: Urine cell-free (UCF) DNA has recently been proposed as a potential marker for early bladder cancer diagnosis. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. Therefore, we verified the potential role of UCF DNA integrity in early bladder cancer diagnosis. MATERIALS AND METHODS: UCF DNA was isolated from 51 bladder cancer patients, 46 symptomatic patients, and 32 healthy volunteers. To verify UCF DNA integrity, sequences longer than 250 bp, c-Myc, BCAS1, and HER2, were quantified by real time PCR. RESULTS: At the best cutoff value of 0.1 ng/µl, UCF DNA integrity analysis showed a sensitivity of 0.73 (95% CI 0.61-0.85), and a specificity of 0.84 (95% CI 0.71-0.97) in healthy individuals and 0.83 (95% CI 0.72-0.94) in symptomatic patients. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.834 (95% CI 0.739-0.930) for healthy individuals and 0.796 (95% CI 0.707-0.885) for symptomatic patients. CONCLUSIONS: These preliminary data suggest that UCF DNA integrity is a potentially good marker for early noninvasive diagnosis of bladder cancer. Its diagnostic performance does not seem to vary significantly, even in an "at risk" population of symptomatic individuals.

Evaluation and Establishment of Reference Range of Automated Urine Cell Analyzer UF-100 / 임상검사와정도관리

BACKGROUND: The automated urine cell analyzer UF-100 (Syxmex co., Japan), flow cytometer-based instrument, has enabled to perform rapid and efficient work. We evaluated the UF-100 by comparing performance in urine sediment testing with counting chamber, standardized method, and traditional manual microscopy widely used in laboratories, and established reference ranges in our hospital. METHODS: Urine samples were obtained from patients in their 20s to 60s who visited hospital for regular check-up between March and April 2007 at Ewha Womans University Mokdong Hospital. We selected randomly a total of 261 samples (male 130, female 131) and evaluated correlations of red blood cell (RBC) and white blood cell (WBC) counts of UF-100 with counting chamber, and manual microscopy. Moreover, we established reference ranges of UF-100 and counting chamber according to CLSI guideline, using 156 urine samples (male 93, female 63) with normal dipstick (strip) test results. RESULTS: The RBC correlation coefficients between UF-100 and counting chamber, UF-100 and manual microscopy, counting chamber and manual microscopy were 0.538, 0.873, and 0.619, respectively. The WBC correlation coefficients between UF-100 and counting chamber, UF-100 and manual microscopy, counting chamber and manual microscopy were 0.992, 0.902, and 0.893, respectively and showed good correlations. The results of UF-100 were higher than counting chamber and manual microscopy. The RBC reference ranges of UF-100 nd counting chamber were 0.5-24.9/microliter (male 0.4-12.2/microliter, female 0.9-38.8/microliter) and 0-4/microliter (male 0-4/microliter, female 0-5/microliter), and the WBC reference ranges of those were 0.9-21.8/microliter (male 0.8-12.6/microliter, female 2.0-23.4/microliter) and 0-7/microliter (male 0-7/microliter, female 0-9/microliter). CONCLUSIONS: The fully automated analyzer UF-100 could be useful to enhance efficiency by labor-saving, turnaround time reduction and improving throughput and to enable standardization. But it is needed for further study including clinical evaluation, because the results and reference ranges between UF-100 and counting chamber or manual microscopy showed considerable differences.

Evaluation and Establishment of Reference Range of Automated Urine Cell Analyzer UF-100 / 임상검사와정도관리

BACKGROUND: The automated urine cell analyzer UF-100 (Syxmex co., Japan), flow cytometer-based instrument, has enabled to perform rapid and efficient work. We evaluated the UF-100 by comparing performance in urine sediment testing with counting chamber, standardized method, and traditional manual microscopy widely used in laboratories, and established reference ranges in our hospital. METHODS: Urine samples were obtained from patients in their 20s to 60s who visited hospital for regular check-up between March and April 2007 at Ewha Womans University Mokdong Hospital. We selected randomly a total of 261 samples (male 130, female 131) and evaluated correlations of red blood cell (RBC) and white blood cell (WBC) counts of UF-100 with counting chamber, and manual microscopy. Moreover, we established reference ranges of UF-100 and counting chamber according to CLSI guideline, using 156 urine samples (male 93, female 63) with normal dipstick (strip) test results. RESULTS: The RBC correlation coefficients between UF-100 and counting chamber, UF-100 and manual microscopy, counting chamber and manual microscopy were 0.538, 0.873, and 0.619, respectively. The WBC correlation coefficients between UF-100 and counting chamber, UF-100 and manual microscopy, counting chamber and manual microscopy were 0.992, 0.902, and 0.893, respectively and showed good correlations. The results of UF-100 were higher than counting chamber and manual microscopy. The RBC reference ranges of UF-100 nd counting chamber were 0.5-24.9/microliter (male 0.4-12.2/microliter, female 0.9-38.8/microliter) and 0-4/microliter (male 0-4/microliter, female 0-5/microliter), and the WBC reference ranges of those were 0.9-21.8/microliter (male 0.8-12.6/microliter, female 2.0-23.4/microliter) and 0-7/microliter (male 0-7/microliter, female 0-9/microliter). CONCLUSIONS: The fully automated analyzer UF-100 could be useful to enhance efficiency by labor-saving, turnaround time reduction and improving throughput and to enable standardization. But it is needed for further study including clinical evaluation, because the results and reference ranges between UF-100 and counting chamber or manual microscopy showed considerable differences.