Screening human donor corneas during organ culture for the presence of guttae.

AIMS: To detect the presence of guttae by means of light microscopy during organ culture and to evaluate the influence of the presence of guttae in the donor tissue on transplantation outcome. METHODS: Donor corneas were investigated for the presence of guttae by means of light microscopy at the end of organ culture. Recipient corneal buttons from patients with severe Fuchs' dystrophy and donor corneas with advanced guttae were first studied by light microscopy and subsequently by transmission electron microscopy. Lastly, 168 consecutive donor corneas were evaluated for the presence of guttae and issued for transplantation. RESULTS: Corneal specimens with Fuchs' dystrophy displayed numerous round highly reflecting guttae at the level of the corneal endothelium. Donor corneas with advanced guttae showed less numerous guttae. Among 168 organ cultured donor corneas issued for transplantation, low density guttae were found in 43 (25.6%) corneas. The endothelial cell density and figure coefficient were significantly lower and organ culture time was significantly higher in the cornea guttata group than in the control group. The presence of grouped guttae significantly decreased the adjusted graft survival. The incidence of postoperative stage 3 cornea guttata was significantly higher when grouped guttae were found (5/6) than when no guttae or scattered guttae were found (8/101). CONCLUSION: Cornea guttata can be detected during organ culture by means of light microscopy. It is associated with a decrease in endothelial cell figure coefficient and cell density. The presence of grouped guttae is associated with poorer graft survival and more frequent stage 3 cornea guttata in the graft after transplantation.

[Culture of human keratocytes. Influence of culture conditions and ultrastructural aspects]. / Culture des kératocytes humains. Influence des conditions de culture et aspects ultrastructuraux.

BACKGROUND: To investigate the influence of fetal calf serum (FCS) and fibroblast growth factor (FGF) on human keratocyte growth in vitro and cell differentiation, and to describe cultured human keratocyte ultra-structure. METHODS: Human keratocytes were cultured in TC 199/Ham F12 media, supplemented or not with 10% FCS, aFGF, and bFGF. Keratocyte growth was studied. Cultured keratocytes were analyzed by means of immunochemistry and transmission electron microscopy. RESULTS: Without fetal calf serum, cell population doubling occurred after 7 days of culture and no alpha smooth muscle-actin cell expression was observed. With serum, cell population increased by 1 log after 7 days of culture and all of the cells were alpha SM-actin + bFGF or aFGF-addition to the serum-containing medium resulted in a dramatic decrease in this alpha SM-actin expression. Nuclei were found to be oval and regular in cross-sections, and round and indented in frontal sections. Numerous cytoplasmic organelles were observed, as were cell expansions, gap junctions, omega-shaped structures, and fenestrations. Cultured keratocytes synthesized collagen fibers and filaments. CONCLUSION: Fetal calf serum allows human keratocytes to grow with a myofibroblast cell phenotype, whereas addition of FGF results in a fibroblast cell phenotype. Ultrastructure of cultured keratocyte is similar to that observed in situ.

Light microscopic evaluation of human donor corneal stroma during organ culture.

PURPOSE: To try to facilitate evaluation of corneal stroma during organ culture by means of light microscopy. METHODS: Corneal stroma of 53 consecutive organ-cultured corneas was studied by means of light microscopy during endothelial quality control. Out of 9 corneas with bad stromal evaluation, 2 were studied by means of transmission electron microscopy, and 7 were grafted. From the remaining 44 corneas with a normal light microscopic appearance, 35 were grafted. RESULTS: Stromal abnormalities consisted of bright visible structures with a cell-like shape corresponding to keratocyte injuries (i.e. cellular edema, light and dark vacuoles, cell membrane disruption and, finally, internal cytolysis) as observed by TEM. At 3 months postoperatively no clinical differences between the two groups of transplants were observed. CONCLUSION: Corneal stroma can be evaluated qualitatively and easily by means of light microscopy during organ culture. Further studies are needed to investigate whether the presence of lysed keratocytes in the graft's stroma actually influences the outcome of transplantation.

[Performance of Pall filters (RC 100 and PL 100) for the removal of leukocytes from red-cell concentrates and platelets]. / Etude des performances des filtres Pall (RC 100 et PL 100) pour la déleucocytation des concentrés de globules rouges et de plaquettes.

20 red cell concentrates (buffy-coat not removed), 5 pools of 5 and 5 pools of 10 standards plateles concentrates, as well as 5 single donor platelet concentrates obtained through Haemonetics V50-1 cytoplasmapheresis procedure, were filtered, within 48 hours after donation, using RC 100 and PL 100 blood filters developed by Pall Company. Using the RC 100 filter on red cell concentrates, the rate of leukocyte removal exceeded 99% of the initial values. The residual leukocyte count per unit was 5.8 +/- 3.4 10(6). Leukocytes in red cell concentrates filtered after a previous one on the same filter numbered 18.6 +/- 8.9 10(6). The rate of platelet removal exceeded 97% of the initial amount. Red cell loss was 6 +/- 9% or 4 +/- 3% of a single or a subsequent unit were filtered respectively. Using the PL 100 filter on platelet concentrates, the rate of leukocyte removal exceeded 88% of the initial values. Residual leukocyte counts were 1.4 +/- 0.4 10(6), 6.2 +/- 3.8 10(6) and 1.4 +/- 0.8 10(6) respectively in 6-unit pools, 10-unit pools and machine prepared platelet concentrates. The average platelet loss was 15%. There was no evidence of alteration in the qualitative parameters of red cells or platelets. The filters were easy to handle and the time needed for the whole filtration process was remarkably short.

[Pollinosis in the area of the Dalmatian, Istrian and Croatian coast with special emphasis on Parietariae pollen]. / Polinoze na podrucju Dalmacije, Istre i Hrvatskog primorja s posebnim osvrtom na pelud Parietariae.

The distribution of pollen antigens of an area depends on geographic situation, climatic factors and vegetation. Geographic situation of examined area is in latitude from 42.5 degrees (Dubrovnik) to 45.5 degrees north (Rijeka), the climate is Mediterranean and botanically it is Eumediterranean area. In allergologic out-patient departments of Dubrovnik, Split, Sibenik, Zadar, Pula and Rijeka, 300 patients with pollinosis have been tested by the application of the prick method of group allergens of grass, tree and weed pollen, particularly of Parietariae (pellitory) pollen. The object of the investigation was to find out which pollen antigen is actual for the Adriatic area and distribution of Parietariae pollen, regarding the fact that it was almost the only cause of pollinosis in the south part of the Adriatic coast (Dubrovnik), Ninety healthy persons were also examined, 15 in each out-patient department, being the control. The results show that grass pollen is actual allergen in the north and middle Adriatic area, while in the south Adriatic it is without any importance. The number of people oversensitive to trees and weeds pollen is low. The oversensitiveness to Parietariae pollen appears all along the coast, its number decreasing from the south to the north. It is concluded that grass pollen is the main cause of pollinosis, in the area of the Croatian littoral and Istria, important in south and central Dalmatia and almost without importance in south Dalmatia. Parietariae pollen is actual allergen all along the coast, being almost the only cause of pollinosis in south Adriatic area.

[Adaptation of a freezing technic with a low concentration of glycerol for preserving red blood cells resuspended in saline-adenine-glucose]. / Adaptation de la technique de congélation avec faible concentration en glycérol à des concentrés globulaires resuspendus en SAG.

We describe here a freezing method with a final low concentration of 20% glycerol and storage in a aluminium flask at - 150 degrees C. This technique allows the freezing of red blood cells concentrates collected on CPD and resuspended in SAG, without having to eliminate the SAG solution. For this we used an initial solution of 60% glycerol, which we added to the red blood cell concentrate in two steps separated by a resting time. Accordingly to the final concentration of glycerol, this freezing process is similar to the method currently used in our laboratory. This consists in one step addition V/V of 40% glycerol solution to the CPD red blood cells. The two techniques have been compared after thawing, and washing in a IBM Blood Processor 2991. The following parameters have been studied mainly: --haemoglobin level --residual glycerol --leucocytes and platelets --amount of 2-3 DPG and of intra-erythrocyte ATP. The results of the two freezing techniques are similar. They show that the SAG solution does not impair the intracellular penetration of glycerol. The in vitro qualities of the erythrocytes are well preserved.