2.50
Hdl Handle:
http://hdl.handle.net/10755/158312
Type:
Presentation
Title:
Natural killer cell cytotoxicity: Methods analysis of two assays
Abstract:
Natural killer cell cytotoxicity: Methods analysis of two assays
Conference Sponsor:Western Institute of Nursing
Conference Year:2003
Author:Motzer, Sandra
P.I. Institution Name:University of Washington, School of Nursing, Department of Biobehavioral Nursing and Health Systems
Title:Assistant Professor
Contact Address:Box 357266, Seattle, WA, 98195-7266, USA
Contact Telephone:206.616.1461
Co-Authors:Tsuji, J.; Hertig, V.; Johnston, S. K.; Scanlan, J.
Purpose and Background: Evaluation of immune function is a central component in the design of many nursing research studies. One important indicator of immune function is natural killer (NK) cell cytotoxicity (NKC). The gold-standard method of assaying NKC is the chromium (51Cr)-release method, despite inherent problems of reliability, sensitivity, radioactivity, and cost. Flow cytometry (FC), a newer method of NKC, may have methodological advantages over the 51Cr-release assay. The purpose of this study was to describe considerations for the use of 51Cr vs. FC NKC assays in research designs. Sample and Methods: The sample was 10 normal women (18-45 yrs) who had participated in an ongoing study of 51Cr NKC. A reserve sample of whole blood collected during the ongoing study was used here for FC NKC. Two additional persons each provided a single assay control (AC) sample (AC-A & AC-B) to be run with every assay. Peripheral blood mononuclear cells (PBMCs) were separated using standard Ficoll-Hypaque centrifugation and then cryopreserved. Standard NKC assay protocols were followed. By batching some assays, we ran 7 FC and 10 51Cr NKC assays. We underestimated the amount of PBMCs required for FC NKC. Thus, both assays used 4 effector-target ratios (E:T) with replicate samples, but FC used lower E:T (75:1-9.38:1 in duplicate) than did our protocol for 51Cr (100:1-12.5:1 in triplicate). Here, to equitably compare NKC results, 2 of 3 51Cr percent specific lysis (PSL) values were randomly selected and then used in calculating lytic units-10 (LU10). Descriptive statistics, coefficients of variation, tests of homogeneity of variance, Wilcoxon signed-ranks tests, and Spearman rho were used in analysis. Results: LU10 coefficients of variation from 10 study subjects were 75% and 98% for 51Cr and FC, respectively. Variances were unequal (p < .01). LU10 51Cr and FC coefficients of variation from AC-A samples were 44% and 86%, respectively; and from AC-B samples were 110% and 42%, respectively. Variances of the two methods were not significantly different for AC-A but were unequal for AC-B (p < .05). Mean 51Cr LU10 was 14.3 + 10.8 and mean FC LU10 was 5.9 + 5.8 (p = .005); they were strongly and positively related (rs = .79, p = .006). Requisite amounts of whole blood to run 1 sample in triplicate at 4 E:T (100:1-12.5:1) was 3.5 ml for 51Cr and 14 ml for FC. After PBMC separation, our cost for supplies/disposal for each 51Cr assay was $67 and for each FC assay was $114. Technician time for running/analyzing each 51Cr and FC NKC was the same: 16 hrs. Conclusions: As expected, both within-subject and between-subject variability was large. Lower mean FC LU10 suggests lower sensitivity. Cost of 51Cr is less. 51Cr has radiation risk, but nuclear stains used in FC likely have carcinogenic risk. More blood is required to perform FC than 51Cr NKC. These considerations about reliability, sensitivity, biohazard risk, cost, and amount of blood may assist in study design.
Repository Posting Date:
26-Oct-2011
Date of Publication:
17-Oct-2011
Sponsors:
Western Institute of Nursing

Full metadata record

DC FieldValue Language
dc.typePresentationen_GB
dc.titleNatural killer cell cytotoxicity: Methods analysis of two assaysen_GB
dc.identifier.urihttp://hdl.handle.net/10755/158312-
dc.description.abstract<table><tr><td colspan="2" class="item-title">Natural killer cell cytotoxicity: Methods analysis of two assays </td></tr><tr class="item-sponsor"><td class="label">Conference Sponsor:</td><td class="value">Western Institute of Nursing</td></tr><tr class="item-year"><td class="label">Conference Year:</td><td class="value">2003</td></tr><tr class="item-author"><td class="label">Author:</td><td class="value">Motzer, Sandra</td></tr><tr class="item-institute"><td class="label">P.I. Institution Name:</td><td class="value">University of Washington, School of Nursing, Department of Biobehavioral Nursing and Health Systems</td></tr><tr class="item-author-title"><td class="label">Title:</td><td class="value">Assistant Professor</td></tr><tr class="item-address"><td class="label">Contact Address:</td><td class="value">Box 357266, Seattle, WA, 98195-7266, USA</td></tr><tr class="item-phone"><td class="label">Contact Telephone:</td><td class="value">206.616.1461</td></tr><tr class="item-email"><td class="label">Email:</td><td class="value">underhil@u.washington.edu</td></tr><tr class="item-co-authors"><td class="label">Co-Authors:</td><td class="value">Tsuji, J.; Hertig, V.; Johnston, S. K.; Scanlan, J. </td></tr><tr><td colspan="2" class="item-abstract">Purpose and Background: Evaluation of immune function is a central component in the design of many nursing research studies. One important indicator of immune function is natural killer (NK) cell cytotoxicity (NKC). The gold-standard method of assaying NKC is the chromium (51Cr)-release method, despite inherent problems of reliability, sensitivity, radioactivity, and cost. Flow cytometry (FC), a newer method of NKC, may have methodological advantages over the 51Cr-release assay. The purpose of this study was to describe considerations for the use of 51Cr vs. FC NKC assays in research designs. Sample and Methods: The sample was 10 normal women (18-45 yrs) who had participated in an ongoing study of 51Cr NKC. A reserve sample of whole blood collected during the ongoing study was used here for FC NKC. Two additional persons each provided a single assay control (AC) sample (AC-A &amp; AC-B) to be run with every assay. Peripheral blood mononuclear cells (PBMCs) were separated using standard Ficoll-Hypaque centrifugation and then cryopreserved. Standard NKC assay protocols were followed. By batching some assays, we ran 7 FC and 10 51Cr NKC assays. We underestimated the amount of PBMCs required for FC NKC. Thus, both assays used 4 effector-target ratios (E:T) with replicate samples, but FC used lower E:T (75:1-9.38:1 in duplicate) than did our protocol for 51Cr (100:1-12.5:1 in triplicate). Here, to equitably compare NKC results, 2 of 3 51Cr percent specific lysis (PSL) values were randomly selected and then used in calculating lytic units-10 (LU10). Descriptive statistics, coefficients of variation, tests of homogeneity of variance, Wilcoxon signed-ranks tests, and Spearman rho were used in analysis. Results: LU10 coefficients of variation from 10 study subjects were 75% and 98% for 51Cr and FC, respectively. Variances were unequal (p &lt; .01). LU10 51Cr and FC coefficients of variation from AC-A samples were 44% and 86%, respectively; and from AC-B samples were 110% and 42%, respectively. Variances of the two methods were not significantly different for AC-A but were unequal for AC-B (p &lt; .05). Mean 51Cr LU10 was 14.3 + 10.8 and mean FC LU10 was 5.9 + 5.8 (p = .005); they were strongly and positively related (rs = .79, p = .006). Requisite amounts of whole blood to run 1 sample in triplicate at 4 E:T (100:1-12.5:1) was 3.5 ml for 51Cr and 14 ml for FC. After PBMC separation, our cost for supplies/disposal for each 51Cr assay was $67 and for each FC assay was $114. Technician time for running/analyzing each 51Cr and FC NKC was the same: 16 hrs. Conclusions: As expected, both within-subject and between-subject variability was large. Lower mean FC LU10 suggests lower sensitivity. Cost of 51Cr is less. 51Cr has radiation risk, but nuclear stains used in FC likely have carcinogenic risk. More blood is required to perform FC than 51Cr NKC. These considerations about reliability, sensitivity, biohazard risk, cost, and amount of blood may assist in study design.</td></tr></table>en_GB
dc.date.available2011-10-26T20:43:15Z-
dc.date.issued2011-10-17en_GB
dc.date.accessioned2011-10-26T20:43:15Z-
dc.description.sponsorshipWestern Institute of Nursingen_GB
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