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Development of a rapid clinical flow cytometric assay of neutrophil cell-surface biomarkers

Weissman, Alexandra (2019) Development of a rapid clinical flow cytometric assay of neutrophil cell-surface biomarkers. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

Rationale: Neutrophils are upregulated in infectious processes. They can be measured as surrogates for the presence of an infection. Flow cytometry is a common tool that uses fluorochrome-conjugated antibodies specific to cell-surface biomarkers to identify cells or biomarkers of interest. Many clinical flow cytometry protocols exist that assess lymphocytes, but fewer protocols exist for rapid clinical evaluation of neutrophils. Expression of neutrophil cell-surface markers integrin α9β1 and CD11b has been shown to increase in elderly patients with bacterial pneumonia. A rapid clinical test for these neutrophil cell-surface markers could help to expedite appropriate antibiotic therapy to patients suffering from complex critical illnesses complicated by bacterial pneumonia for which other diagnostic methods are rendered inadequate.
Methods: We developed a rapid lyse, no-wash clinical protocol for flow cytometric analysis of neutrophil cell-surface biomarkers integrin α9β1 and CD11b from whole blood samples. Assay development included selecting appropriate neutrophil-specific biomarkers and their corresponding fluorochrome profile, determining the most consistent blood acquisition method (BAM) for obtaining samples for analysis, and evaluating the effect of preservative type and time on neutrophil absolute count (ANC), integrin α9β1 mean fluorescence intensity (MFI), and CD11b MFI.
Results: There were significant differences in ANC, CD11b MFI, and integrin α9β1 MFI over time for each BAM and Preservative type. ANC demonstrated the least variability in Citrate, while CD11b MFI and integrin α9β1 MFI were most consistent in Heparin. Therefore, Heparin was selected as the BAM of choice. Streck preservative solution and Streck Blood Collection Tube (BCT) demonstrated the least variability in ANC, integrin α9β1 MFI, and CD11b MFI.
Conclusions: Pneumonia is difficult to detect in patients with critical illness, where the usual diagnostic tools are rendered inadequate secondary to multi-system organ failure. More accurate methods of diagnosis for pneumonia can enhance targeted antibiotic therapy and are of paramount public health importance to improve patient care. Development of a rapid clinical flow cytometric protocol is the first step toward precision methods of pneumonia diagnosis; therefore, a rapid lyse/no-wash clinical flow cytometric assay for whole blood specimens was developed for identification of neutrophil cell-surface biomarkers CD11b and integrin α9β1. Heparin was the least variable BAM for assessing CD11b MFI and integrin α9β1 MFI, while Streck preservative solution and Streck BCT demonstrated the least variability over 24 hours from specimen collection. Heparin is a common type of venipuncture tube in hospitals, it is not as cost effective or feasible to purchase a separate blood collection tube (e.g. Streck BCT) for a single laboratory test. Given these real-world concerns, Streck solution added to samples collected in heparin venipuncture tubes is the most reasonable method by which to perform the rapid lyse/no-wash neutrophil flow cytometric assay.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Weissman, Alexandraajw103@pitt.eduajw103
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairMartinson, Jeremyjmartins@pitt.edu
Committee MemberDonnenberg, Albertdonnenbergad@upmc.edu
Committee MemberCallaway, Cliftoncallawaycw@upmc.edu
Date: 19 June 2019
Date Type: Publication
Defense Date: 17 April 2019
Approval Date: 19 June 2019
Submission Date: 9 April 2019
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 89
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: MPH - Master of Public Health
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Flow cytometry
Date Deposited: 19 Jun 2019 15:59
Last Modified: 19 Jun 2019 15:59
URI: http://d-scholarship.pitt.edu/id/eprint/36401

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