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PubMed1991

Proteinuria after renal transplantation: diagnosis with highly sensitive silver stain in sodium dodecylsulphate-polyacrylamide gradient gel electrophoresis.

Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of urinary proteins is becoming increasingly significant in monitoring renal allograft recipients. After conventional Coomassie Blue staining, changes in renal proteinuric patterns during acute renal transplant rejection could not be seen in many cases. Discrimination between rejection and nephrotoxic side effects of cyclosporine overdosage was also not possible. We therefore examined the possible advantages of a highly sensitive silver staining technique in this study. A total of 734 urine samples obtained from 38 patients after allogenic kidney transplantation were examined by SDS-PAGE with consecutive Coomassie Blue and silver staining. Twenty-two histologically proven rejections and 20 cyclosporine overdosage episodes were diagnosed in these patients within a time period of up to 524 days after transplantation. No changes in proteinuric patterns were seen in 9 of 22 patients after Coomassie stain during rejection, and only 12 cases showed a rise of glomerular protein bands, whereas silver stain revealed an increase in 19 of 22 cases. Discrimination between cyclosporine overdosage and rejection was possible with a probability of p less than 0.001 after silver stain when using the changes in the number of glomerular protein bands as a criterion. These findings suggest that application of a highly sensitive silver stain instead of the conventional Coomassie stain after SDS-PAGE reflects considerable progress in monitoring renal allograft recipients.

Adult↗

PubMed1982

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX.

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.

Alcian Blue↗

PubMed2002

Fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counter ion-dyes, Coomassie brilliant blue R-250 and neutral red.

A fast and sensitive protein staining method in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using both an acidic dye, Coomassie Brilliant Blue R-250 (CBBR) and a basic dye, Neutral Red (NR) is described. It is based on a counter ion-dye staining technique that employs oppositely charged two dyes to form an ion-pair complex. The selective binding of the free dye molecules to proteins in an acidic solution enhances the staining effect of CBBR on protein bands, and also reduces gel background. It is a rapid staining procedure, involving fixing and staining steps with short destaining that are completed in about 1 h. As the result, it showed two to fourfold increase in sensitivity comparing with CBBR staining. The stained protein bands can be visualized at the same time of staining.

Benzenesulfonates↗

PubMed2001

Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids.

The objective of the present study was to develop a stain permitting automated quantification of myocellular lipid depositions in skeletal muscle sections together with immunolocalisation of other myocellular constituents by fluorescence microscopy. Lipid droplets were detected in skeletal muscle by oil red O (ORO). Conventional ORO was modified to diminish background staining, prevent crystallisation of ORO and to optimise lipid retention in cryosections. These modifications resulted in a punctate staining of lipid droplets, rather than the somewhat diffuse staining by conventional ORO. Small cavities in muscle sections (like the lumen of small blood vessels) lack ORO when using the protocol presented here. In addition a staining protocol is presented combining ORO with immunofluorescence. This combination permits multiple staining studies in the same section. Thus, lipid droplets can be studied together with immunolabelling of proteins involved in lipid handling and metabolism. This will extend our knowledge on the subcellular localisation of lipid handling proteins (i.e. enzymes and fatty acid transporting proteins) in relation to the localisation of lipid depositions. In conclusion, the protocol presented here permits examination of ORO-stained lipid droplets in skeletal muscle sections together with multiple staining of other immunodetectable proteins present in skeletal muscle by quantitative fluorescence microscopy.

Animals↗

PubMed2003

Use of oolong tea extract (OTE) for elastin staining and enhancement in ultrathin sections.

To assess the usefulness of oolong tea extract (OTE) staining for connective tissue observation, we examined the visceral pleural mesothelium of rat lung by transmission electron microscope. Four kinds of electron microscopic staining methods (routine, tannic acid, OTE in distilled water, and OTE in 0.1 M phosphate buffer) were compared to determine the most suitable method for electron microscopic observation of ultrathin sections. Elastin (elastic laminae) was selectively stained by tannic acid and both water and buffered OTE. Except for elastin, connective tissue and cell ultrastructures were also electron enhanced by tannic acid and both water and buffered OTE staining. However, using water OTE, the electron-dense filaments (10-12 nm in diameter) were obscured. In tannic acid staining, the unit membranes of the visceral pleural mesothelial cells were weaker as compared with routine and buffered OTE stains. Thus, the buffered OTE staining method is a highly useful technique for connective tissue observation and electron-enhanced staining in transmission electron microscopic preparations.

Animals↗

PubMed2005

Photodynamic effect of argon and diode laser on cholesteatoma cell cultures after intravital staining with absorption enhancers.

Chronic epitympanic otitis media, or chronic suppurative osteitis, is a destructive form of chronic middle-ear inflammation. The therapy of choice is complete surgical removal of the squamous epithelium from the middle ear. It is often impossible to inspect all areas of the middle ear with the posterior canal wall intact. Not all recesses can be reliably monitored with the microscope, particularly in the area of the antrum and hypotympanum. Residual squamous epithelium here causes frequent recurrences following cholesteatoma surgery. This study examines the effect of argon and diode lasers on cholesteatoma tissue. The aim is to develop a laser treatment selectively directed against cholesteatoma cells that can be performed after cholesteatoma surgery to eliminate any residual squamous epithelium. Intraoperatively harvested monolayer-cultured cholesteatoma cells stained in vivo with various absorption enhancers served as the in vitro examination model. Argon (499 nm) and diode lasers (810 nm) were applied since their irradiation has an appropriate tissue penetration depth and is absorbed by various chromophores such as neutral red (475-500 nm), fluorescein (488 nm), and indocyanine green (790-810). Intracellular staining of cultured cells increased the optical density at the wavelength corresponding to the dye. Neutral red damaged 50-60% of cultured cells merely by intracellular accumulation at high concentrations. An additive cell destruction of about 30% was achieved by also applying argon laser irradiation. Fluorescein diacetate caused no appreciable stain-induced damage to cultured cholesteatoma cells. Argon laser irradiation destroyed up to 60% of the cultures. Indocyanine green resulted in only minor damage to cultured cells. The diode laser destroyed up to 60% of the irradiated cells. Selective staining of cholesteatoma cells was not achieved with any of the dyes examined. Thus, other stained tissue could be damaged. Staining and subsequent laser irradiation destroys up to 60% of cultured cholesteatoma cells. Unstained irradiated cells are not affected. Indocyanine green and fluorescein are nontoxic and may thus be used as absorption enhancers. The diode and argon lasers appear to be basically suitable. Cell staining is not selective, i.e., other tissues would also be stained and damaged. To avoid such unwanted damage, it would be desirable to couple the chromophore to a specific antibody that binds only to cholesteatoma cells.

Argon↗

PubMedDate not supplied

Microstaging of breast cancer patients using cytokeratin staining of the sentinel lymph node.

BACKGROUND: Sentinel lymph node (SLN) mapping is an effective and accurate method of axillary nodal evaluation for metastatic disease. Cytokeratin (CK) immunohistochemical (IHC) staining of the SLN has found micrometastatic disease previously undetected by routine hematoxylin and eosin (H&E) stains. The purpose of this study is to determine the number of patients who were upstaged or microstaged, i.e., detected to have micrometastatic disease only by combined lymphatic mapping with CK IHC. METHODS: Two hundred and ten patients with newly diagnosed breast cancer underwent intraoperative lymphatic mapping using a combination of vital blue dye and technetium-labeled sulfur colloid. The excised sentinel lymph nodes were examined grossly, by imprint cytology, by standard H&E histology, and by IHC stains for CK. SLNs that were only CK positive were confirmed to be malignant by histologic examination. RESULTS: CK IHC staining was performed on 381 SLNs in 210 breast cancer patients. Forty-seven of 210 patients (22.4%) had positive nodes. Thirty of these 47 patients (63.8%) had both H&E- and CK-positive SLNs, and an additional 17 of the 47 positive patients (36.2%) had only CK-positive SLNs. Seventeen of the 180 patients (9.4%) who were negative on H&E staining were upstaged by CK IHC staining of malignant cells in the SLN. Comparison of tumor size with the total number of node-positive patients demonstrated that 16 of 30 node-positive T0 and T1 patients (53.5%) and 22 of 39 nodes (56.4%) were upstaged by CK IHC staining. T2 and T3 patients were less frequently upstaged by cytokeratin analysis of lymph nodes. Only one of 17 node-positive patients (5.9%) and seven of 34 nodes (20.6%) in patients with T2 and T3 tumors were upstaged. CONCLUSION: CK IHC staining of SLNs shifted 9.4% of patients from stage I to stage II. There was a significant upstaging influence noted in patients with tumor sizes under 2 cm. This microstaging shift or upstaging may account for the significant proportion of stage I breast cancer treatment failures. Microstaging of the SLNs using more sensitive assays may help identify a subgroup of patients with invasive breast cancer who would benefit from systemic adjuvant treatment, while sparing a disease-free subset of patients the additional risks of toxic adjuvant chemotherapy.

Biopsy, Needle↗

PubMed2006

Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat.

We used cytochrome oxidase (CytOx) staining intensity, which is correlated with neuronal functional activity, to evaluate maturity and functionality of newborn rat olfactory epithelium (OE) and olfactory receptor neurons (ORNs). Nasal olfactory tissue of neonatal rats was stained with CytOx and analyzed qualitatively and quantitatively. Results revealed that newborn OE shows six differentially stained horizontal bands. Bands run parallel to the OE surface and were categorized as very light, medium or darkly stained. A narrow and pale Band 1 overlapped with horizontal basal cells. Next, a wide and lightly stained Band 2 was observed that coincides with the globose basal cell layer and immature ORNs, deep in OE. Next apically, a medium-staining Band 3 overlapped with ORN perikarya. Closer to the surface, a medium to light Band 4 was discerned where dendrites of mature ORNs normally occur. This band was interrupted with lighter areas due to the presence of supporting cells nuclei. Next, a superficial but dark Band 5 occurred, populated by the apical portions of ORN dendrites and their ciliated knobs and by supporting cell apices; mitochondria in apices of supporting cells contribute predominantly to dense staining of this Band 5. Apical to Band 5, a thin and fairly light Band 6 was observed which overlaps with the mucus layer that contains part of the ORN knobs, their cilia and supporting cell microvilli. Along the length of ORN dendrites, apical segments just below the ORN knobs, and wide basal segments showed a darker staining than the middle segments implying "microzones" of higher neural activity within the most apical and basal regions of dendrites. Our findings agree with ultrastructural studies showing a presence of mitochondria in knobs, basal portions of ORN dendrites and in OE supporting cell apices, suggesting that apical regions of both olfactory and supporting cells near the surfaces are metabolically most active, in odorant detection, signal processing, and detoxification, the latter for supporting cells.

Animals↗

PubMed1990

Staining properties of bovine low molecular weight hydrophobic surfactant proteins after polyacrylamide gel electrophoresis.

Reports describing polyacrylamide gel electrophoresis patterns of bovine hydrophobic surfactant proteins are not consistent. In this study, we found unusual staining characteristics of these proteins that may explain some of these inconsistencies. Low molecular weight surfactant proteins extracted from bronchoalveolar lavage with organic solvent are partially delipidated with Sephadex LH-20 chromatography using chloroform and methanol. Fractions from the first protein peak are dried under nitrogen then subjected to SDS electrophoresis on 20% polyacrylamide gels. Under nonreducing conditions, silver staining identifies 5- and 26-kDa bands, and Coomassie blue identifies 6-, 12-, and 26-kDa bands. When gels are stained with Coomassie blue then silver, the 5- and 26-kDa bands stain with silver and 6- and 12-kDa bands remain stained with Coomassie blue. If gels are first stained with silver then Coomassie blue, similar results occur. We modified the silver staining protocol by treating gels with dithiothreitol or 2-mercaptoethanol after electrophoresis. With this modification, 5-, 6-, 12-, 26-, and also 17-kDa bands are identifiable. Using the modified protocol and restaining gels previously stained with silver, 6-, 12-, and 17-kDa bands that were not identified previously all became visible. In further experiments, protein bands of 6-, 12-, and 26-kDa that were identified by Coomassie blue were electroeluted under nonreducing conditions. After electrophoresis of the eluted 26-kDa protein, bands of 17-, and 26-kDa under nonreducing, and 8-kDa only under reducing conditions, were apparent by using the modified silver protocol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

Silver staining of histone-depleted metaphase chromosomes.

To investigate a possible relationship between the core-like structures seen in silver-stained chromosomes (prepared by standard cytogenetic methods) and the scaffolds observed in histone-depleted chromosomes, the ability of the scaffold to stain with silver has been examined. Isolated chromosomes were histone-depleted by washing in ammonium acetate or by spreading the chromosomes on an ammonium acetate hypophase. The residual chromosome structures were carbon-platinum shadowed or stained with silver, and then examined by electron microscopy. The results provide clear evidence that the scaffold structure has a high affinity for silver and is therefore similar in its silver-staining potential to the core structure in standard chromosomes. This suggests that the silver core in standard chromosomes may represent the scaffold visualized by histone depletion. The peripherally dispersed DNA radiating from the scaffold also proved to be silver-reactive, and additional experiments demonstrated that purified DNA is capable of binding silver. This result indicates that cytological silver staining is not simply a matter of staining protein, as has previously been thought, but may also involve the staining of chromosomal DNA. In the ammonium acetate-treated and carbon-platinum-shadowed preparations, the scaffold structure was highly variable in its morphology and appeared to be composed of undispersed or incompletely dehistonized chromatin fibers. The silver-stained scaffold reflected this variability. Taken together with other evidence, these findings lead to a questioning of the reality of chromosome core structures.

Acetates↗

PubMed1983

A modification of the Papanicolaou exfoliative cytology stain to demonstrate keratin in paraffin-block tissue sections.

A method of staining for keratin by application of a modified Papanicolaou staining procedure to formalin-fixed paraffin-embedded tissues is described. The modification is achieved by increasing the staining time for the three basic Papanicolaou stains and incorporating phloxine B as an additional staining component. Since the Papanicolaou staining components are common to most histopathology laboratories and phloxine B is not a rare stain component, this procedure can be instituted with a modicum of effort. The procedure is less time consuming than the usual staining methods for demonstrating keratin and does so with a high degree of reliability.

Humans↗

PubMed1982

The correlation between bismuth and uranyl staining and phosphorus content of intracellular structures as determined by electron spectroscopic imaging.

Four groups of intracellular structures can be recognized according to bismuth and uranyl staining and phosphorus content. (1) Those which contain phosphorus and stain strongly with uranyl acetate but not with bismuth (ribosomes, heterochromatin and mature ribosomal precursor granules), presumably because of their nucleic acid content. (2) Those which contain phosphorus and stain with uranyl acetate and bismuth (interchromatin granules, immature ribosomal precursor granules and mitochondrial granules), presumably because at least some of their phosphate is available to react with bismuth. (3) Those which contain little phosphorus but which stain strongly with bismuth and weakly with uranyl acetate (Golgi complex beads), perhaps because some ligand in addition to phosphate reacts with bismuth, and (4) those which do not contain phosphorus and stain with neither uranyl acetate nor bismuth (portasomes). Uranyl staining correlates strongly with the phosphorus content of nucleic acids, proteins and inorganic deposits. Bismuth will stain some phosphorylated molecules but not all. Thus only some phosphates stain with bismuth.

Animals↗

PubMed1994

Simultaneous visualization of Helicobacter pylori and gastric morphology: a new stain.

Although the histopathologic patterns associated with Helicobacter pylori infection have been described, details of the interaction between bacteria, epithelial cells, and inflammatory cells remain elusive. One of the limiting factors has been the lack of a staining technique that allows the simultaneous visualization of H pylori and tissue morphology. By combining three commonly available stains (Steiner, hematoxylin-eosin, and alcian blue at pH 2.5) into a single procedure we have developed a stain that permits the optimal detection of H pylori in tissue sections while simultaneously allowing the histopathologic evaluation of all salient characteristics of the gastric mucosa. This procedure is inexpensive, as easy to perform as any silver stain, and provides consistent results. This stain is as sensitive as the Warthin-Starry stain for the detection of H pylori and significantly more sensitive than hematoxylin-eosin alone (> 99% v 85% when compared with the hematoxylin-eosin stain in a series of 332 positive biopsy specimens; > 99% v 61% in 49 biopsy specimens with rare bacteria). This stain is particularly useful for the visualization of small numbers of bacteria, such as in the evaluation of posttreatment gastric biopsy specimens, in specimens with abundant mucus or debris, and in specimens from the corpus, where H pylori usually does not elicit strong inflammatory responses and, therefore, pathologists' index of suspicion tends to be low.

Biopsy↗

PubMed1993

The interference of cysteine thiols in the detection of glycated and non-glycated proteins by modified silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

In the previous study a staining intensification of in vitro glycated collagen type I versus a non-glycated one after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under a modified silver staining procedure was observed (Hodny, Z., Struzinsky, R. and Deyl, Z. (1992) J. Chromatogr. 578, 53-62). While investigating the specificity of this stain to glycation product(s) on protein we have observed that a great number of proteins (e.g. bovine serum albumin) was sensitive to this stain even in a non-glycated state. It is proposed from results of the analysis of amino acid composition of these proteins that their better stainability correlates with the amount of cysteine present in the protein. Modification of SH groups by iodoacetamide (or N-ethylmaleimide) had an inhibitory effect on the staining of bovine serum albumin (and some other proteins) in its 'native' state but had no visible inhibitory effect on their staining in the glycated state. However, the positive staining response of a great number of components from cellular lysates even after iodoacetamide treatment indicates the existence of further chemical groups (either of protein or nucleic acid origin) participating in this silver staining method.

Collagen↗

PubMed1986

Monoclonal antibodies to human hypoglossal nucleus which stain neurons and astrocytes in normal brains and brains from cases of Alzheimer-type dementia.

Monoclonal antibodies were raised to membranes of hypoglossal nuclei from normal human post-mortem brain. Two of these clones were recloned to yield antibodies ES.18 and ES.19. Antibody ES.18 stained some, but not all, neuronal perikarya in the medulla oblongata and other brain areas. Neurons stained by this antibody did not have a common neurotransmitter or physiological function, although they tended to be large. Perikarya in the basal forebrain nucleus from a case of Alzheimer-type dementia were stained much more intensely by ES.18 than were these perikarya in a control brain. Antibody ES.19 did not stain neuronal perikarya but stained glial fibrillary acidic protein-positive processes below the pia, in the subependymal layer and in the molecular layer of the cerebellum of control and Alzheimer brains. This antibody also stained the numerous glial fibrillary acidic protein-positive astrocytes in Alzheimer cerebral cortex, but did not stain glial fibrillary acidic protein-positive astrocytes in the white matter of brains from controls or cases of Alzheimer-type dementia. The staining pattern of ES.19 suggests that fibrous astrocytes in Alzheimer cerebral cortex are antigenically different from fibrous astrocytes in white matter.

Alzheimer Disease↗

PubMed1996

Distribution of terminal fields stained for zinc in the neocortex of the rat.

Staining for zinc in terminal fields of the rat neocortex was studied by applying the sulphide/silver histochemical method. The stain was arranged in a distinct layered pattern. Two bands of heavy reaction were found in deep layer 1 plus layers 2-3 and in upper layer 5. A band of moderate-to-heavy reaction was found in layer 6. Three bands of lighter staining were found in upper layer 1, layer 4 and deep layer 5. The layers of reaction showed variations in width and intensity of staining from area to area. In the frontal and cingulate cortices and in the association cortices, the heavily stained bands were dominant over the narrow, inconspicuous, lightly stained bands. In contrast, in primary sensory regions (Par1, Oc1 and Te1), the lightly stained bands were wide and prominent. The sulphide/silver method gives a straightforward delimitation of many cortical areas and reveals a clear distinction between (A) allocortical and isocortical areas, and (B) primary sensory areas, secondary or association areas, and prefrontal plus motor areas. The presence of vivid bands of staining for zinc appears to be linked to areas with prominent pyramidal layers.

Animals↗

PubMed2006

Effect of trypan blue staining on the density and viability of lens epithelial cells in white cataract.

PURPOSE: To assess the effect of anterior capsule staining with trypan blue 0.0125% on the density and viability of the lens epithelial cells (LECs). SETTING: Iladevi Cataract and IOL Research Center, Ahmedabad, India. METHODS: This randomized prospective study comprised 40 eyes having phacoemulsification for white mature cataract. Patients were randomized to have the anterior capsule stained with 0.1 mL trypan blue 0.0125% solution before the anterior capsulorhexis (Group 1, n = 20) or to have no anterior capsule staining before the anterior capsulorhexis (Group 2, n = 20). The density and viability of LECs were analyzed. The first 20 samples (10 from Group 1, 10 from Group 2) were stained with hematoxylin-eosin for cell density analysis using image-analysis software. The remaining 20 samples (10 from each group) were stained with a solution containing 0.5 microM calcein AM and 5 microM propidium iodide for 10 minutes at 37 degrees C for viability assay. RESULTS: The mean cell density in Group 1 (with staining) was 3533.15 cells/mm(2) +/- 664.01 (SD). This was significantly lower than the mean of 4235.59 +/- 414.93 cells/mm(2) in Group 2 (no staining) (P = .01). The mean capsule area covered with LECs (80.75% +/- 8.04% versus 94.63% +/- 3.78%) and the mean live LECs in the covered area (51.69% +/- 11.54% versus 68.67% +/- 9.15%) were also significantly lower in Group 1 (P = .002). CONCLUSION: Staining the anterior capsule with trypan blue affected the density and viability of LECs.

Capsulorhexis↗

PubMed2005

Prevention of indocyanine green toxicity on retinal pigment epithelium with whole blood in stain-assisted macular hole surgery.

PURPOSE: To examine whether whole blood prevents indocyanine green (ICG) toxicity on in vitro retinal pigment epithelium (RPE) and prevents RPE staining in ICG-assisted macular hole (MH) surgery. DESIGN: In vitro study and prospective case series. PARTICIPANTS: In vitro study and 20 patients who underwent ICG-assisted MH surgery (20 eyes). METHODS: In the in vitro study, cultured human RPE cells were covered with balanced saline solution (BSS), heparinized whole blood, plasma, or packed red blood cells, then exposed to various concentrations of ICG. One cohort was incubated in the dark; the other cohort was exposed to light. Indocyanine green toxicity was evaluated by mitochondrial dehydrogenase assay. In the clinical study, a prospective noncomparative review of 20 consecutive patients (20 eyes) with stage 3 to stage 4 MH who underwent surgery with ICG to stain the internal limiting membrane (ILM) was performed. Indocyanine green solution (0.5 mg/ml) was used to stain the ILM after autologous whole blood covered the macular hole area. Postoperative staining of ICG on RPE was detected by an infrared-sensitive camera. MAIN OUTCOME MEASURES: Cultured human RPE cell viability, macular hole closure rate, median visual acuity preoperatively and postoperatively, postoperative ICG staining, and retinal changes. RESULTS: Cultured human RPE cells covered by whole blood or plasma showed no decrease of mitochondrial dehydrogenase even in 5.0 mg/ml concentration of ICG for 20 minutes with or without light exposure. Conversely, those exposed to ICG and covered with BSS demonstrated a significant decrease of mitochondrial dehydrogenase after incubation in 5, 2.5, and 1.0 mg/ml for 20 minutes in the dark and in 5 to 0.05 mg/ml with light. Clinically, no postoperative staining on RPE was detected. No atrophic RPE changes or other retinal changes were observed in the previous MH area that was covered by autologous whole blood in all 20 eyes on average follow-up of 10.6 months. The hole closed in 19 eyes (95%) on first surgery. Visual acuity improved in 17 eyes (85%) on most recent follow-up. CONCLUSIONS: Whole blood prevents ICG toxicity in RPE cell culture and prevents staining of RPE in surgery of MH when the ILM is stained with ICG.

Adult↗