Development and Performance Evaluation of a Quantitative Reverse Transcription PCR Kit for the Determination of prohibitin Gene Expression

Background@#Prohibitin (PHB) regulates intracellular signal pathways, transcription, and cell cycles. Aberrant expression of the PHB gene is known to be related totumorigenesis, tumor progression, and chronic metabolic and inflammatory diseases. The present study aimed to develop a one-step quantitative reverse transcription PCR (RT-qPCR) kit for quantifying PHB mRNA levels and evaluate its performance in the laboratory. @*Methods@#TaqMan chemistry was used to develop the one-step PHB1 and PHB2 RT-qPCR kit. Normal peripheral blood cells from healthy individuals (N=20) and leukemia cells from patients initially diagnosed with acute myeloid leukemia (AML, N=20), chronic myeloid leukemia (CML, N=13), and acute lymphoid leukemia (ALL, N=7) were enrolled to evaluate the laboratory performance of the kit using commercially available total human RNA controls. @*Results@#The intra-assay and inter-assay precision of the kit developed in this study was less than 2%. The distribution of PHB1 mRNA expression of AML, CML, and ALL was 0.898-0.993 (median: 0.936), 0.817-0.976 (0.918), and 0.844-1.074 (0.973), respectively. The distribution of PHB2 mRNA expression of AML, CML, and ALL was 0.957-1.024 (median: 0.985), 0.988-1.047 (1.002), and 0.937-1.059 (1.004), respectively. The sensitivity, specificity, positive and negative predictive value, and test effectiveness of the developed PHB1 and PHB2 kit were greater than 50% for each parameter. @*Conclusions@#Our developed kit would be useful for diagnosing leukemia as well as detecting residual disease. Additionally, this kit could be used for monitoring and conducting molecular pathophysiological studies of obesity, metabolic, and inflammatory diseases.

Analytical Performance and Reference Interval of a Microfluidic Viscometer, Viscore-300 for the Measurement of Whole Blood Viscosity

BACKGROUND: Whole blood viscosity (WBV) refers to the internal resistance that occurs when blood flows through blood vessels. WBV is known to be related to many diseases including cardiovascular and neurovascular diseases. We have investigated the analytical performance and established reference intervals for a newly developed microfluidic viscometer, Viscore-300 (NanoBiz, Korea), used for the measurement of WBV. METHODS: We performed a precision test of 240 measurements over 20 days using three control materials. For evaluation of repeatability, a total of 60 WBV measurements were made in 3 whole blood samples 20 times a day. A total of 100 whole blood samples were used to evaluate the accuracy of the Viscore-300 in comparison to a rotating viscometer, DV3T (Brookfield, USA), in accordance with the the Clinical and Laboratory Standards Institute's guidelines. To establish the reference intervals, 122 healthy individuals were enrolled in this study. RESULTS: The precision and repeatability results showed that the CV was less than 5% for three samples and two shear rates. In the accuracy test, the mean differences between two viscometers were 0.09 cP (0.9%) and −0.07 cP (−1.4%) at shear rates of 10 s−1 and 300 s−1, respectively. The reference intervals of WBV for men were 6.88–13.52 cP at 10 s−1 and 4.32–6.43 cP at 300 s−1; those of women were 5.74–13.29 cP at 10 s−1 and 3.60–6.12 cP at 300 s−1. CONCLUSIONS: Viscore-300 showed excellent precision and accuracy and it might be a good instrument for reporting WBV quickly and accurately.

Comparison between V-Tubes and BD Vacutainer Tubes for Use in Laboratory Tests

BACKGROUND: Vacuum tubes are widely used in clinical laboratories for routine tests. We compared a newly developed V-tube (AB Medical, Korea) and BD tubes (BD, USA) in common clinical assays, i.e., hematological, chemical, and immunological tests. METHODS: In total, 100 volunteers comprising 79 patients and 21 healthy volunteers were recruited and peripheral blood samples were collected with 2 brands of EDTA tubes and serum-separating tubes (SSTs). EDTA-tube samples were evaluated using 16 routine hematological tests. The SST samples were analyzed for 32 routine chemical parameters and 3 thyroid hormones. The results were statistically analyzed using the paired t-test and Bland-Altman plot. In addition, the stability of each analyte in 2 brands of vacutainers was evaluated. The results of the hematological tests at t=0 hr were compared with those at t=72+/-2 hr, and the results of the chemical parameters and thyroid hormones at t=0 hr were compared with those at t=72+/-2 hr and t=168+/-2 hr for each tube. RESULTS: Paired t-test analysis revealed that the test results of 16 routine hematological parameters, 32 routine chemical parameters, and 3 thyroid hormones showed clinically allowable differences between the 2 brands of vacuum tubes (t=0 hr). The results obtained when using V-tubes showed a statistically significant correlation with those obtained when using BD tubes. The stability of each analyte was similar in both vacuum tubes. Except for 10 parameters (white blood cell count, mean corpuscular volume, basophils [%], mean corpuscular hemoglobin concentration, monocytes [%], phospholipid, sodium, potassium, chloride, and free T4), all parameters showed significant but clinically allowable differences with regard to storage duration. CONCLUSIONS: The newly developed V-tube vacutainers provide a suitable alternative to BD tubes in common clinical laboratories.