Quantitative assessment of β-cell apoptosis and cell composition of isolated, undisrupted human islets by laser scanning cytometry.
BACKGROUND
Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes for islet transplantation therapy. Parameters such as percent β-cell apoptosis and cell composition are found to vary markedly between different islet preparations and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry for assessing β-cell apoptosis and islet cell composition on serial sections of intact isolated islets.


METHODS
Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones and acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer.


RESULTS
Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations, we found a mean islet cell composition of 54.5%±1.2% insulin-positive, 33.9%±1.2% glucagon, 12.1%±0.7% somatostatin, and 1.5%±0.2% pancreatic polypeptide-positive cells. The apoptotic β cells were 2.85%±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic β cells correlated well (P<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice.


CONCLUSIONS
The analysis of whole, nondissociated islets for cell composition and β-cell apoptosis using laser scanning cytometry gives reliable and reproducible results and could be performed both before islet transplantation and on preserved cell blocks at any time in future. Thus, they can be a powerful tool for islet quality assessment.
Todorov
Ivan
I
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, CA, USA. itodorov@coh.org
Nair
Indu
I
Avakian-Mansoorian
Alina
A
Rawson
Jeffrey
J
Omori
Keiko
K
Ito
Taihei
T
Valiente
Luis
L
Iglesias-Meza
Itzia
I
Orr
Chris
C
Shiang
Keh-Dong
KD
Ferreri
Kevin
K
Al-Abdullah
Ismail H
IH
Mullen
Yoko
Y
Kandeel
Fouad
F
D017209:Apoptosis; D005934:Glucagon / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology*; D010191:Pancreatic Polypeptide / Q000378:metabolism; D013004:Somatostatin / Q000378:metabolism
D007328:Insulin; D013004:Somatostatin; D010191:Pancreatic Polypeptide; D005934:Glucagon