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Quantification of the Islet Product: Presentation of a Standardized Current Good Manufacturing Practices Compliant System With Minimal Variability
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Forskargrupp Korsgren)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Forskargrupp Korsgren)
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2011 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 91, no 6, 677-683 p.Article in journal (Refereed) Published
Abstract [en]

Background. Accurate islet quantification has proven difficult to standardize in a good manufacturing practices (GMP) approved manner. Methods. The influence of assessment variables from both manual and computer-assisted digital image analysis (DIA) methods were compared using calibrated, standardized microspheres or islets alone. Additionally, a mixture of microspheres and exocrine tissue was used to evaluate the variability of both the current, internationally recognized, manual method and a novel GMP-friendly purity-and volume-based method (PV) evaluated by DIA in a semiclosed, culture bag system. Results. Computer-assisted DIA recorded known microsphere size distribution and quantities accurately. By using DIA to evaluate islets, the interindividual manually evaluated percent coefficients of variation (CV%; n = 14) were reduced by almost half for both islet equivalents (IEs; 31% vs. 17%, P = 0.002) and purity (20% vs. 13%, P = 0.033). The microsphere pool mixed with exocrine tissue did not differ from expected IE with either method. However, manual IE resulted in a total CV% of 44.3% and a range spanning 258 kIE, whereas PV resulted in CV% of 10.7% and range of 60 k IE. Purity CV% for each method were similar approximating 10.5% and differed from expected by +7% for the manual method and +3% for PV. Conclusion. The variability of standard counting methods for islet samples and clinical quantities of microspheres mixed with exocrine tissue were reduced with DIA. They were reduced even further by use of a semiclosed bag system compared with standard manual counting, thereby facilitating the standardization of islet evaluation according to GMP standards.

Place, publisher, year, edition, pages
2011. Vol. 91, no 6, 677-683 p.
Keyword [en]
Islet evaluation, Digital imaging analysis, Purity, Volume, GMP
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-149562DOI: 10.1097/TP.0b013e31820ae48eISI: 000288115800023PubMedID: 21248660OAI: oai:DiVA.org:uu-149562DiVA: diva2:405274
Available from: 2011-03-22 Created: 2011-03-21 Last updated: 2011-07-01Bibliographically approved
In thesis
1. Standardization of Islet Isolation and Transplantation Variables
Open this publication in new window or tab >>Standardization of Islet Isolation and Transplantation Variables
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Currently, the transplantation of islets of Langerhans is a viable means to maintain control of blood sugar levels and reduce the risk of hypoglycemia in defined populations with brittle type I diabetes mellitus or those requiring pancreatectomy. However, the process of islet isolation is highly variable and not all isolations result in islet numbers or quality suitable for transplantation.

This thesis aimed to improve transplantation success through optimization and standardization of the isolation process and to identify pretransplant variables associated with early islet engraftment.

A previously disregarded enzyme activity, tryptic-like activity (TLA), has been identified to influence pancreas digestion efficiency and islet isolation success in both the preclinical and clinical situations. For human pancreases, islet isolation success rates improved from 0% in the lowest TLA group to over 50% in the highest TLA groups without affecting islet quality. These findings should help standardize evaluation of enzymes for clinical islet isolation.

A closed, automated, pump-made gradient system was compared to the open, manual method for islet separation. No differences were observed in expected gradient volumes, islet yields or total purities between the two methods. The pump-made gradient system successfully removed manual influences on density gradient production while fulfilling regulatory requirements for closed system processing.

Islet quantification was evaluated with computer-assisted digital imaging analysis (DIA) and a semi-closed assessment system. By using the DIA system method, which measures islet purity and pellet volume instead of manual counting methods, variation in islet counts and purity reduced by almost half.

By using a transplant outcome measurement of C-peptide adjusted by blood glucose and creatinine, we identified four pretransplant factors that affect early transplant outcome. Of the four factors, one was related to the organ transport time, one to function of the islets, and two to the transplanted tissue volume. When these four factors were put into a predictive model, it accounted for about 40% of the transplant outcome.

The work contained in this thesis identifies and optimizes a number of critical elements related to islet isolation and transplantation protocols.


Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 76 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 669
Islet isolation, standardization, enzyme, gradient separation, digital imaging analysis, DIA, transplantation outcome, islet transplantation, prediction
National Category
Biomedical Laboratory Science/Technology
Research subject
Medical Cell Biology; Computerized Image Processing
urn:nbn:se:uu:diva-150247 (URN)978-91-554-8066-0 (ISBN)
Public defence
2011-05-23, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Available from: 2011-05-02 Created: 2011-03-28 Last updated: 2011-07-01Bibliographically approved

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