Effects of Platelet Lysate Preparations on the Proliferation of HaCaT Cells

백새윤,임영애,강선주,안선현,이위교,김철호 대한진단검사의학회 2014 Annals of Laboratory Medicine Vol.34 No.4

Background Standard protocols are lacking for the preparation of platelet lysates (PL) as an alternative to using fetal bovine serum as a cell culture supplement. This study aimed to establish optimum conditions for preparing PL for use in cell cultures. Methods Cell density in three pooled platelet concentrates (PC) were adjusted to 1×1012/L and 2×1012/L. PL was prepared from PC by 1 to 3 freeze-thaw (FT) cycles. HaCaT cells were cultured in media supplemented with 5% or 10% PL. Cell numbers were estimated using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Growth factors were quantified by using the Luminex 200 system (Luminex Corporation, USA). Results Cell proliferation rates in the presence of PLs were similar when prepared from PCs of both cell densities. The rates were higher in media containing 5% PL than 10% PL when prepared by two FT cycles. Concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor-AB/BB (PDGF-AB/BB), PDGF-AA, and epidermal growth factor (EGF) were significantly higher in PL prepared from PC with a cell density of 2×1012/L than 1×1012/L PC. However, only VEGF and PDGF-AA concentrations in PLs were correlated with HaCaT cell counts. Conclusions The 5% PL from PC with a cell density of 1×1012/L prepared by two FT cycles treatment was the most effective condition that supported steady HaCaT cell proliferation. Our finding may be useful for preparing PL-supplemented cell culture media.

Effects of Platelet Lysate Preparations on the Proliferation of HaCaT Cells

백새윤,임영애,강선주,안선현,이위교,김철호 대한진단검사의학회 2014 Annals of Laboratory Medicine Vol.34 No.1

Background: Standard protocols are lacking for the preparation of platelet lysates (PL) as an alternative to using fetal bovine serum as a cell culture supplement. This study aimed to establish optimum conditions for preparing PL for use in cell cultures. Methods: Cell density in three pooled platelet concentrates (PC) were adjusted to 1×10 12 / L and 2×10 12 /L. PL was prepared from PC by 1 to 3 freeze-thaw (FT) cycles. HaCaT cells were cultured in media supplemented with 5% or 10% PL. Cell numbers were estimated using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Growth factors were quantified by using the Luminex 200 system (Luminex Corporation, USA). Results: Cell proliferation rates in the presence of PLs were similar when prepared from PCs of both cell densities. The rates were higher in media containing 5% PL than 10% PL when prepared by two FT cycles. Concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor-AB/BB (PDGF-AB/BB), PDGF-AA, and epidermal growth factor (EGF) were significantly higher in PL prepared from PC with a cell density of 2×10 12 /L than 1×10 12 /L PC. However, only VEGF and PDGF-AA concentrations in PLs were correlated with HaCaT cell counts. Conclusions: The 5% PL from PC with a cell density of 1×10 12 /L prepared by two FT cy- cles treatment was the most effective condition that supported steady HaCaT cell prolifer- ation. Our finding may be useful for preparing PL-supplemented cell culture media.

Comparison of ABO Antibody Titers on the Basis of the Antibody Detection Method Used

강선주,임영애,백새윤 대한진단검사의학회 2014 Annals of Laboratory Medicine Vol.34 No.4

Background: Detection methods for ABO antibody (Ab) titers vary across laboratories, and the results are different depending on the method used. We aimed to compare titer values using different detection methods for the measurement of ABO Ab titers. Methods: For ABO Ab detection, pooled group A or B red blood cells (RBCs) were reacted with each of 20 sera from blood groups A, B, or O without dithiothreitol treatment. The room-temperature (RT) incubation technique and the indirect antiglobulin test (IAT) were used in the tube test and gel card test. Flow cytometry (FCM) was performed by using anti-IgM and anti-IgG Abs. Results: Regardless of the blood groups tested, the FCM assay with anti-IgM showed the highest titer compared to the tube test and gel card test with RT incubation in both. The tube test with IAT showed a higher titer than the gel card test with IAT (Gel-IAT) or FCM with anti-IgG in blood group A and B, while Gel-IAT showed the highest titer relative to the other tests, only for the anti-A Ab in blood group O. Conclusions: There were significant differences in the titers depending on the detection method used, and each method showed a different detection capacity for each ABO Ab depending on the ABO blood group tested. Therefore, caution should be exercised in in- terpreting ABO Ab titer results, taking into consideration the detection method used and the blood group.

Anti-Rods and Rings Autoantibodies in a Patient With Hepatitis C Virus Infection

최경호,임영애,김신규,전래희,백새윤,조성원,정은주 대한진단검사의학회 2015 Annals of Laboratory Medicine Vol.35 No.6

Progress of in vitro factor VIII coagulant activity from 0 to 8 hours after reconstitution

심예지,이건수,김욱현,서진경,백새윤,현신영 대한혈액학회 2014 Blood Research Vol.49 No.4

Background Continuous infusion of factor VIII (FVIII) is a more cost-effective method for treating hemophilia A than intermittent bolus injection. However, there is currently no specific data in Korea about the progress of in vitro FVIII coagulant activity (FVIII:C) after reconstitution from its lyophilized form. Methods Three commercial FVIII concentrate products (two recombinant FVIII and one plasma-derived) were used. In vitro FVIII:C was measured at 0, 2, 4, 6, and 8 hours following reconstitution in both the indoor light-exposed and light-shielded groups. Results For the three drugs, in vitro FVIII:C decreased over the 8 hours following reconstitution (P<0.001). The decline of FVIII:C was linear (P<0.001). In vitro FVIII:C for the indoor light-exposed groups was 95.3±1.9% and 90.6±2.5% after 4 and 8 hours following reconstitution, respectively, compared to baseline activity. In the light-shielded group, FVIII:C was 95.4±1.1% and 90.9±1.7% of the baseline activity after 4 and 8 hours, respectively. There was no statistical difference between FVIII:C in the indoor light-exposed and light-shielded groups (P=0.849). Conclusion In vitro FVIII:C decreased after reconstitution, but activity was maintained at over 90% of the baseline value during 8 hours. Exposure to indoor light did not accelerate the loss of FVIII:C over the experimental time. This result indicates that CI with FVIII is available in 8-hour intervals, with no indoor light-exposure precautions needed.

Progress of in vitro factor VIII coagulant activity from 0 to 8 hours after reconstitution

심예지,이건수,김욱현,서진경,백새윤,현신영 대한혈액학회 2014 Blood Research Vol.49 No.4

Background Continuous infusion of factor VIII (FVIII) is a more cost-effective method for treating hemophilia A than intermittent bolus injection. However, there is currently no specific data in Korea about the progress of in vitro FVIII coagulant activity (FVIII:C) after reconstitution from its lyophilized form. Methods Three commercial FVIII concentrate products (two recombinant FVIII and one plasma-derived) were used. In vitro FVIII:C was measured at 0, 2, 4, 6, and 8 hours following reconstitution in both the indoor light-exposed and light-shielded groups. Results For the three drugs, in vitro FVIII:C decreased over the 8 hours following reconstitution (P<0.001). The decline of FVIII:C was linear (P<0.001). In vitro FVIII:C for the indoor light-exposed groups was 95.3±1.9% and 90.6±2.5% after 4 and 8 hours following reconstitution, respectively, compared to baseline activity. In the light-shielded group, FVIII:C was 95.4±1.1% and 90.9±1.7% of the baseline activity after 4 and 8 hours, respectively. There was no statistical difference between FVIII:C in the indoor light-exposed and light-shielded groups (P=0.849). Conclusion In vitro FVIII:C decreased after reconstitution, but activity was maintained at over 90% of the baseline value during 8 hours. Exposure to indoor light did not accelerate the loss of FVIII:C over the experimental time. This result indicates that CI with FVIII is available in 8-hour intervals, with no indoor light-exposure precautions needed.