Plasma von Willebrand factor multimer quantitative analysis by in-gel immunostaining and infrared fluorescent imaging

Rajiv K. Pruthi, Todd M. Daniels, John A. Heit, Dong Chen, Whyte G. Owen, William L. Nichols

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Introduction: Electrophoretic analysis of plasma von Willebrand factor (VWF) multimer distribution and infrastructure is essential for subtyping von Willebrand disease. To improve the sensitivity, precision and efficiency of this assay, we developed and validated a new in-gel infrared fluorescent VWF multimer imaging method to visualize and quantify VWF multimers directly in the agarose gel, thus eliminating electroblotting or autoradiographic steps. Materials/Methods: VWF multimer analyses of plasma samples from 34 patients with known von Willebrand disease or acquired von Willebrand syndrome, 9 patients with acquired VWF abnormalities, 26 normal volunteer donors and 49 patient samples referred for von Willebrand factor multimer analysis were performed by both traditional autoradiographic and the new infra-red imaging methods and compared. VWF multimer image data were electronically acquired, archived and analyzed. Results: The in-gel infrared method has a sensitivity of detecting VWF antigen as low as approximately 1.6 IU/dL, a reliable fluorescent intensity with intra- and inter-day variability (CV) of 5% and 6% respectively, and provides superior imaging resolution and shortened test turnaround time. Using intermediate resolution agarose gel electrophoresis, the infra-red method sensitively detects subtle loss of highest molecular weight von Willebrand factor multimers in plasmas with acquired VWF abnormalities and in commercial normal reference plasmas, and provides evidence of increased proteolysis of ultralarge multimers in some type 2 VWD plasmas. Conclusions: The in-gel infrared fluorescent VWF multimer imaging method provides a sensitive, reliable, efficient and robust system to improve laboratory testing for von Willebrand disease classification.

Original languageEnglish (US)
Pages (from-to)543-549
Number of pages7
JournalThrombosis Research
Volume126
Issue number6
DOIs
StatePublished - Dec 2010

Fingerprint

von Willebrand Factor
Gels
von Willebrand Diseases
Statistical Factor Analysis
Agar Gel Electrophoresis
Sepharose
Proteolysis
Healthy Volunteers
Molecular Weight
Tissue Donors

Keywords

  • Electrophoresis
  • Infra-red fluorescent antibody
  • Multimer
  • Von Willebrand disease
  • Von Willebrand factor

ASJC Scopus subject areas

  • Hematology

Cite this

Plasma von Willebrand factor multimer quantitative analysis by in-gel immunostaining and infrared fluorescent imaging. / Pruthi, Rajiv K.; Daniels, Todd M.; Heit, John A.; Chen, Dong; Owen, Whyte G.; Nichols, William L.

In: Thrombosis Research, Vol. 126, No. 6, 12.2010, p. 543-549.

Research output: Contribution to journalArticle

Pruthi, Rajiv K. ; Daniels, Todd M. ; Heit, John A. ; Chen, Dong ; Owen, Whyte G. ; Nichols, William L. / Plasma von Willebrand factor multimer quantitative analysis by in-gel immunostaining and infrared fluorescent imaging. In: Thrombosis Research. 2010 ; Vol. 126, No. 6. pp. 543-549.
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abstract = "Introduction: Electrophoretic analysis of plasma von Willebrand factor (VWF) multimer distribution and infrastructure is essential for subtyping von Willebrand disease. To improve the sensitivity, precision and efficiency of this assay, we developed and validated a new in-gel infrared fluorescent VWF multimer imaging method to visualize and quantify VWF multimers directly in the agarose gel, thus eliminating electroblotting or autoradiographic steps. Materials/Methods: VWF multimer analyses of plasma samples from 34 patients with known von Willebrand disease or acquired von Willebrand syndrome, 9 patients with acquired VWF abnormalities, 26 normal volunteer donors and 49 patient samples referred for von Willebrand factor multimer analysis were performed by both traditional autoradiographic and the new infra-red imaging methods and compared. VWF multimer image data were electronically acquired, archived and analyzed. Results: The in-gel infrared method has a sensitivity of detecting VWF antigen as low as approximately 1.6 IU/dL, a reliable fluorescent intensity with intra- and inter-day variability (CV) of 5{\%} and 6{\%} respectively, and provides superior imaging resolution and shortened test turnaround time. Using intermediate resolution agarose gel electrophoresis, the infra-red method sensitively detects subtle loss of highest molecular weight von Willebrand factor multimers in plasmas with acquired VWF abnormalities and in commercial normal reference plasmas, and provides evidence of increased proteolysis of ultralarge multimers in some type 2 VWD plasmas. Conclusions: The in-gel infrared fluorescent VWF multimer imaging method provides a sensitive, reliable, efficient and robust system to improve laboratory testing for von Willebrand disease classification.",
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AB - Introduction: Electrophoretic analysis of plasma von Willebrand factor (VWF) multimer distribution and infrastructure is essential for subtyping von Willebrand disease. To improve the sensitivity, precision and efficiency of this assay, we developed and validated a new in-gel infrared fluorescent VWF multimer imaging method to visualize and quantify VWF multimers directly in the agarose gel, thus eliminating electroblotting or autoradiographic steps. Materials/Methods: VWF multimer analyses of plasma samples from 34 patients with known von Willebrand disease or acquired von Willebrand syndrome, 9 patients with acquired VWF abnormalities, 26 normal volunteer donors and 49 patient samples referred for von Willebrand factor multimer analysis were performed by both traditional autoradiographic and the new infra-red imaging methods and compared. VWF multimer image data were electronically acquired, archived and analyzed. Results: The in-gel infrared method has a sensitivity of detecting VWF antigen as low as approximately 1.6 IU/dL, a reliable fluorescent intensity with intra- and inter-day variability (CV) of 5% and 6% respectively, and provides superior imaging resolution and shortened test turnaround time. Using intermediate resolution agarose gel electrophoresis, the infra-red method sensitively detects subtle loss of highest molecular weight von Willebrand factor multimers in plasmas with acquired VWF abnormalities and in commercial normal reference plasmas, and provides evidence of increased proteolysis of ultralarge multimers in some type 2 VWD plasmas. Conclusions: The in-gel infrared fluorescent VWF multimer imaging method provides a sensitive, reliable, efficient and robust system to improve laboratory testing for von Willebrand disease classification.

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