Is manual counting of corneal endothelial cell density in eye banks still acceptable? The French experience

Abstract

Aim: To examine the differences in manual endothelial cell counting methods in French eye banks and to analyse whether these differences could explain some substantial discrepancies observed in endothelial cell density (ECD) for corneas made available for transplant.

Methods: A questionnaire was sent to the 22 eye banks asking for details of the technical features of the light microscopes used, the microscope calibration, strategy for cell counting, the technical staff, and the method of presenting endothelial data.

Results: All eye banks responded and 91% (20/22) used only manual counting methods, in real time, directly through a microscope, and 62 different technicians, with varying experience, were involved in such counting. Counting of cells within the borders of a grid that were in contact with two adjacent borders was the most common method (17/22, 77%). Of the eight banks (8/22, 36%) that did not calibrate their microscopes, six reported the highest ECD values. Of the 14 others (64%), six applied a "magnification correcting factor" to the initial cell counts. In five of these cases, the corrected ECD was lower than estimated on initial count. Most of the banks (12/22, 55%) counted 100 cells or less in one to six non-adjacent zones of the mosaic. 14 of the banks (14/22, 64%) also graded cell polymegethism while seven (7/22, 32%) also graded pleomorphism ("hexagonality").

Conclusions: Lack of microscope calibration appears to be the leading cause of variance in ECD estimates in French eye banks. Other factors such as differences in counting strategy, the evaluation of smaller numbers of cells, and the different extent of experience of the technicians may also contribute to intraobserver and interobserver variability. Further comparative studies, including cross checking and the outcome of repeated counts from manual methods, are clearly needed with cross calibration to a computer based image archiving and analysis system.

Figure 1

Example of the procedure to calibrate a microscope fitted with an objective of ×10 theoretical magnification. The reticule (a square grid of 100 mm² real surface area) was placed in the eyepiece, with the engraved side in the plane of the diaphragm. A strip bearing a micrometric slide (arrowed) of 1 mm certified length was placed on the microscope’s viewing stage, to measure the length of one side of the reticule border image. If the micrometer image did not coincide with the reticule border image, a magnification correcting factor was necessary. In this example, the length of the projected image of the 1 mm micrometer was not 10 mm as it should have been, but only 8 mm. A real endothelial area of 1 mm² thus projected in a reticule area of 8×8  =  64 mm² (and not 100 mm²). It was consequently necessary to multiply the obtained cell count by 64/100 to obtain the real ECD in cell/mm². The magnification factor was thus 0.64 in this example. If the micrometer image coincided with the reticule border image, no correcting factor was necessary.

Figure 2

Mean number of endothelial cells taken into account for the endothelial assessment (n = 22 eye banks). More than half the eye banks (12/22, 55%) took into account only 50 or 100 cells.

Figure 3

Illustration of the two endothelial counting strategies on a theoretical mosaic with uniform endothelial cell density (ECD), using a reticule with a 0.01 mm² square surface area. The cells taken into account are marked. (A) “Zone strategy.” Cells within the square unit are counted, and those touching two adjacent borders (in this case, the right hand side border and the bottom border) are excluded. This procedure is performed 1–10 times, depending on the eye bank. In this example, ECD  =  20×100  =  2000/mm². (B) “Border strategy.” Cells touching two adjacent borders, two square units long. In this example, ECD  =  9×9×100/4  =  2025 cell/mm². This example highlights a 1.25% difference between the two strategies.

Figure 4

Proposed counting exercise (A). The endothelial cells usually taken into account had to be ticked. With the “zone strategy,” the most common method, there were four acceptable answers depending on which borders were chosen. In the case of this non-uniform mosaic, the theoretical results were either 10 (B and E) or 11 (C and D) cells. This slight variation is an intrinsic drawback of the fixed frame method on a non-uniform mosaic. However, it is minimised by taking account of several zones.