How to Use Image Analysis for Islet Counting

Abstract

■ AbstractAIM: Assessment of islet mass before islet transplantation requires a reliable technique to enable exact analysis of islet volume. This study aimed to test the applicability of digital image analysis (DIA) for evaluation of samples of purified and non-purified islets. METHODS: Pancreatic islets were isolated from 10 Lewis rats. Samples of purified (n = 10) and non-purified islets (n = 30) were counted conventionally and by using a computerized method. The equipment for the computerized counting consisted of a digital camera installed on a stereomicroscope and connected to a personal computer. Images of 2272x1704 pixels were processed using a previously described non-commercial program originally developed for this purpose. Islets were converted to equivalents using globe and ellipsoid models. The insulin content of purified islets was assessed using radioimmunoassay and was correlated to the absolute and standardized islet number. RESULTS: Mean absolute numbers of purified islets ± SD were 908 ± 130 and 1049 ± 230 (manually and DIA respectively). Mean insulin content ± SD obtained from purified islets was 161 ± 45 mU. The mean equivalents of purified islets (1589 ± 555 for globe and 1219 ± 452 for ellipsoid) significantly correlated with insulin content. However, this correlation was not significant when absolute islet numbers were used, counted using either method. There was no significant difference in absolute non-purified islet numbers assessed by manual and computerized methods (average ± SD in 50 µl samples; 12.6 ± 4.1 and 13.3 ± 5.3 respectively; p = 0.22). The manual method showed a significantly higher yield of islet equivalents (IE; p < 0.001 for both globe and ellipsoid). CONCLUSION: The computer-based system for islet counting correlated better to insulin content than conventional islet estimation and prevented overestimation. Reproducibility and ease of assessment make it potentially applicable to clinical islet transplantation.

Validation of methodologies for quantifying isolated human islets: an islet cell resources study

“…Strict adherence to the international conventions for image analysis assessment of the IEQ in our study ensured comparability to the expert standard. Other reports using image analysis for evaluating islet isolation of mostly animal preparations also lacked the thorough approach of this study (15, 16).…”

“…Instead of losing any record of the original islet sample as is inherent in the practice using standard microscopy assessment of islet preparations, the digital nature of the new imaging technique lends itself to storage of the original images of islet samples that can be analyzed and a permanent record kept in an electronic archive. Moreover, more advanced applications of image analysis have been already proposed, i.e., to apply an ellipsoid formula instead of a sphere, which might better reflect the islet shape and thus provide a better estimate of the islet volume (16).…”

The Flaws and Future of Islet Volume Measurements

“…One of the most consistent criticisms of the IEQ has been its mathematical basis on the ideally spherical 150 μm diameter islet equaling one IEQ. In fact, most islets are not spherical, but are irregularly shaped, both in situ and in culture 20 , 49 , 59 – 61 . Fig.…”

Reduced Expression of PDX-1 Is Associated With Decreased Beta Cell Function in Chronic Pancreatitis

“…The resultant dispersed pancreatic islets were washed 3 times with RPMI 1640 medium, centrifuged, and resuspended in 5 mL of CMRL 1066 medium (invitrogen, Grand Island, NY/Gibco, Carlsbad, Calif ). Islet cells were stained with dithizone, counted under an inverted microscope (Olympus CX 41; Olympus Corporation, Tokyo, Japan), 25 and their yield was expressed in islet equivalents (IEq) per gram of tissue. The size of freshly isolated islets was measured with an inverted microscope (Olympus IX41; Olympus Corporation) using a commercial software (ProgRes Capture pro 2.5; Jenoptik GmbH, Jena, Germany) and expressed in micrometers.…”