Small Skin Biopsies Research Articles

Serum neurofilament light chain levels correlate with small fiber related parameters in patients with hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN)

BackgroundRecent evidence suggests that both serum neurofilament light chain (sNfL) levels and small fiber related diagnostic variables may be valuable disease biomarkers of hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN).Our study aimed to explore the relations between sNfL and small fiber related skin biopsy and quantitative sensory testing (QST) parameters in a cohort of ATTRv-PN patients and pre-symptomatic carriers.MethodsWe retrospectively analyzed data from 13 ATTRv patients and 21 pre-symptomatic carriers who underwent sNfL dosage, skin biopsy, and QST, and analyzed correlations between sNFL, intraepidermal nerve fiber density (IENFD), and cold (CDT) and warm detection thresholds (WDT).ResultsBoth sNfL and small fiber related parameters significantly differed between carriers and patients (sNfL: p < 0.0001; IENFD: p = 0.0008; CDT, WDT: < 0.0001). sNFL levels were normal in all carriers, altered in 85% of patients, negatively correlated with distal IENFD (r = -0.47, p = 0.005), and significantly correlated with CDT (r = -0.68; p < 0.0001) and WDT (r = 0.57; p < 0.0001).ConclusionsOur study showed that sNfL reliably discriminates symptomatic ATTRv-PN patients from pre-symptomatic carriers, and found significant relations between sNfL, skin biopsy, and QST small fiber related parameters, suggesting that sNfL might be a valuable biomarker of peripheral nerve involvement in ATTRv-PN and a supportive criterion for symptomatic disease transition.

Cover Picture and Issue Information

Journal of Animal EcologyVolume 92, Issue 6 p. 1103-1105 COVER PICTURE AND ISSUE INFORMATIONFree Access Cover Picture and Issue Information First published: 07 June 2023 https://doi.org/10.1111/1365-2656.13723AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Cover image: Two killer whales photographed in Vestmannaeyjar, Iceland, after feeding on a school of herring. Despite decades of research, the diets of killer whales remain mysterious, especially in remote parts of the North Atlantic Ocean. But scientists can now infer the precise feeding habits of these apex predators by looking at the lipid (fatty acids) composition of the whales' blubber. All they need is a small skin biopsy (collected from small boats with special darts). Our research team collected biopsies from 200 individuals across the North Atlantic Ocean to reveal large variations in the whales' diets. Photo: Anaïs Remili / The Icelandic Orca Project. Volume92, Issue6June 2023Pages 1103-1105 RelatedInformation

Utility of Prospective Step Sections followed by Reverse Embedding Technique in Increasing Diagnostic Accuracy of Skin Biopsies

Introduction: Small skin biopsies offer a cosmetic advantage to the patient but may provide limited information for making a diagnosis. Non specific and overlapping microscopic features often seen on superficial histopathology sections contribute to this challenge. In such cases, the use of step deeper and reverse embedding (re-embedding) sections has utility in improving diagnostic accuracy in dermatopathology practice. Aim: To examine the use of prospective step sectioning and reverse embedding in skin biopsies to improve diagnosis. Materials and Methods: This prospective, cross-sectional study included 200 consecutive skin biopsies received in the Department of Pathology, BGS Global Institute of Medical Sciences, Bengaluru, Karnataka, India, over an eight-month period from June 2022 to January 2023. Only skin biopsies smaller than 5 mm were included, while large punch biopsies (larger than 5 mm) were excluded. For each sample, a superficial section, step deeper section, and reverse embed section were taken. The pathologist reviewed the microscopic findings and rendered a diagnosis on the first slide. The other two slides were then reviewed, and the information provided by slides 2 and 3 was categorised as either no new information, additional information to make a diagnosis, or a change in diagnosis. Any change in diagnosis based on the information from slides 2 and 3 was noted and analysed. Results: Out of the 200 skin biopsies studied, 32 cases (16%) were non diagnostic on the first slide. Step deeper sections helped in making a diagnosis in 16 (8%) cases, and reverse embedding aided in the diagnosis of 9 (4.5%) cases. For the remaining seven cases where no additional information was obtained even after deeper and reverse embed sectioning, a descriptive report was provided. In eight (4%) cases out of the 200 biopsies where a diagnosis was made on the first slide, deeper/reverse embedding led to a change in diagnosis. Thus, deeper sectioning and reverse embedding improved diagnostic accuracy in 33 cases out of the total 200 skin biopsies studied (16.5%). Conclusion: This study highlights the utility of step deeper and reverse embed (re-embedded) sections in increasing diagnostic accuracy in small skin biopsies. Therefore, implementation of standardised protocol for studying multiple sections of small skin biopsies before rendering a diagnosis can significantly reduce diagnostic errors and aid in providing appropriate treatment to patients.

Small Fiber Functionality in Patients with Diabetic Neuropathic Pain.

Diabetic neuropathic pain is associated with small fiber neuropathy. We aimed to assess the functionality of small fibers in patients with diabetes by using a practical method. Patients with impaired glucose tolerance (IGT), diabetic neuropathic pain (DNP), type II diabetes mellitus without neuropathic pain, and healthy control were included. Axon-reflex flare responses were induced by the intradermal application of capsaicin and histamine at the distal leg. The associated flare characteristics (flare areas and flare intensities) were recorded by using Laser Speckle Contrast Analysis (LASCA). The pain and itch responses were rated while performing LASCA. To verify the structural properties of the small fibers, proximal and distal skin biopsies were performed. DN4, MNSI, NRS, evoked-burning pain scores, and HbA1c levels were the highest in the DNP group. Compatible with length-dependent neuropathy, the distal skin PGP9.5-positive intraepidermal nerve fiber densities (IENFDs) were the lowest, whereas TRPV1-positive IENFDs were the highest in patients with DNP. The distal leg LASCA data showed hypo-functionality in both patients with IGT and DNP and association with disease severity. There is an unmet need to practically assess the functionality of small fibers in patients with pain. In this study, a practical and objective method that does not need special expertise for the measurement of the functional properties of small fibers by using axon-flare responses is presented. The LASCA method could potentially facilitate a practical, quick (within 5 minutes), and very early diagnosis of small fiber hypo-functionality in both patients with IGT and DNP.

Optimized High Quality DNA Extraction from Formalin-Fixed Paraffin-Embedded Human Atherosclerotic Lesions

Formalin-fixed paraffin-embedded (FFPE) tissues represent a valuable source for molecular analyses and clinical genomic studies. These tissues are often poor in cells or difficult to process. Therefore, nucleic acids need to be carefully isolated. In recent years, various methods for DNA isolation have been established for tissues from many diseases, mostly cancer. Unfortunately, genomic DNA extracted from FFPE tissues is highly degraded due to the cross-linking between nucleic acid strands and proteins, as well as random breakings in sequence. Therefore, DNA quality from these samples is markedly reduced, making it a challenge for further molecular downstream analyses. Other problems with difficult tissues are, for example, the lack of cells in calcified human atherosclerotic lesions and fatty tissue, small skin biopsies, and consequently low availability of the desired nucleic acids as it is also the case in old or fixed tissues. In our laboratories, we have established a method for DNA extraction from formalin-fixed atherosclerotic lesions, using a semi-automated isolation system. We compared this method to other commercially available extraction protocols and focused on further downstream analyses. Purity and concentration of the DNA were measured by spectrometry and fluorometry. The degree of fragmentation and overall quality were assessed. The highest DNA quantity and quality was obtained with the modified blood DNA protocol for the automated extraction system, instead of the commercial FFPE protocol. With this step-by-step protocol, DNA yields from FFPE samples were in average four times higher and fewer specimens failed the extraction process, which is critical when dealing with small-vessel biopsies. Amplicon sizes from 200-800 bp could be detected by PCR. This study shows that although DNA obtained from our FFPE tissue is highly fragmented, it can still be used for successful amplification and sequencing of shorter products. In conclusion, in our hands, the automated technology appears to be the best system for DNA extraction, especially for small FFPE tissue specimen.

Development and validation of protein biomarkers of health in grizzly bears.

Large carnivores play critical roles in the maintenance and function of natural ecosystems; however, the populations of many of these species are in decline across the globe. Therefore, there is an urgent need to develop novel techniques that can be used as sensitive conservation tools to detect new threats to the health of individual animals well in advance of population-level effects. Our study aimed to determine the expression of proteins related to energetics, reproduction and stress in the skin of grizzly bears (Ursus arctos) using a liquid chromatography and multiple reaction monitoring mass spectrometry assay. We hypothesized that a suite of target proteins could be measured using this technique and that the expression of these proteins would be associated with biological (sex, age, sample location on body) and environmental (geographic area, season, sample year) variables. Small skin biopsies were collected from free-ranging grizzly bears in Alberta, Canada, from 2013 to 2019 (n = 136 samples from 111 individuals). Over 700 proteins were detected in the skin of grizzly bears, 19 of which were chosen as targets because of their established roles in physiological function. Generalized linear mixed model analysis was used for each target protein. Results indicate that sample year influenced the majority of proteins, suggesting that physiological changes may be driven in part by responses to changes in the environment. Season influenced the expression of proteins related to energetics, reproduction and stress, all of which were lower during fall compared to early spring. The expression of proteins related to energetics and stress varied by geographic area, while the majority of proteins that were affected by biological attributes (age class, sex and age class by sex interaction) were related to reproduction and stress. This study provides a novel method by which scientists and managers can further assess and monitor physiological function in wildlife.

Agar pre-embedding of small skin biopsies: real-life benefits and challenges in high throughput pathology laboratories

Paraffin embedding of small, thin tissue samples requires specific expertise for optimal orientation before tissue sectioning. This study evaluates the real-life utility of the agar pre-embedding technique for small skin...

Characterizing the growth of Sarcoptes scabiei infrapopulations.

During the course of parasitic disease infestations, parasite population sizes change at both individual host (infrapopulation) and host population (metapopulation) levels. However, most studies only report epidemiological values for specific locations and times. In this study we analysed the dynamics of several Sarcoptes scabiei infrapopulations from experimentally infested Iberian ibex, Capra pyrenaica. We obtained mite counts by digesting small skin biopsies, which we compared with indices obtained from histopathological analyses performed on adjacent skin biopsies. We obtained the finite growth rate and the daily growth rate for the mite infrapopulations: mean ± SE = 11.53 ± 10.17 and 0.10 ± 0.08 mites/day, respectively. Mite counts derived from skin sample digestion did not correlate with the histological mite indices obtained from adjacent skin biopsies. At a metapopulational level, both indices of mite abundance were modelled using GLMMs and the factors influencing their variation are analysed and discussed. Our results suggest that mites are not distributed uniformly over the whole area of the skin lesion. Therefore, direct diagnoses of mange and mite counts could be inaccurate if only small skin samples are used.

An evaluation of whale skin differences and its suitability as a tissue for stable isotope analysis

Stable isotope analysis of whale skin has been recurrently used to assess diet and movement patterns. Such studies rely on the untested assumption that the stable isotope ratios in the small skin biopsies analysed are representative of those throughout the skin. In balaenopterids, the ventral skin looks notably different from that of the dorsal region, which is smoother and darker. To investigate possible differences in isotopic ratios throughout the skin, we collected and analysed samples from dorsal and ventral positions in 28 fin whales (Balaenoptera physalus). No significant differences were found between these two skin positions, which might suggest that whale skin is likely a homogeneous tissue. Thus, the isotopic ratios determined at a specific point may be representative of the whole skin in whales.

Clinical efficacy of acquired disorders of pigmentation treated with autologous cell suspension (ReCell® technique)

Objective To evaluate the primarily therapeutic outcomes of a new autologous cell transplantation technique, or namely ReCell® technique, for acquired disorders of pigmentation. Methods Twelve patients with acquired disorders of pigmentation were treated with ReCell® technique in recent 3 years. During the procedure, a small area skin biopsy was harvested from a healthy nearby zone. It was then processed through the ReCell® kit and small amount of cell suspension was obtained. After dermal brasion of the disorder area of pigmentation, the suspension was sprayed to the raw wound and nonadherent dressings were used for temporally coverage. The lesional improvement was finally evaluated with Vancouver scar scale. Results With a mean follow-up of 13±6 months, 11 cases were significant il improved but 1 case was insignificant; there was no major complication occurred except one hematoma. Patient's self-reported data analysis revealed there was statistically significant improvement regarding the appearance of the treated area. The analysis of surgeon's evaluation scale data also reached the same conclusion. Conclusions ReCell® technique can improve the disorders of pigmentaion in Chinese population. It provides an alternative therapeutic choice for plastic surgeon or dermatologist to treat the skin disorders of pigmentation disease. Key words: Epidermic cell transplantation, autologous; Disorders of pigmentation; Surgical treatment

Skin tissue engineering advances in severe burns: review and therapeutic applications

Current advances in basic stem cell research and tissue engineering augur well for the development of improved cultured skin tissue substitutes: a class of products that is still fraught with limitations for clinical use. Although the ability to grow autologous keratinocytes in-vitro from a small skin biopsy into sheets of stratified epithelium (within 3 to 4 weeks) helped alleviate the problem of insufficient donor site for extensive burn, many burn units still have to grapple with insufficient skin allografts which are used as intermediate wound coverage after burn excision. Alternatives offered by tissue-engineered skin dermal replacements to meet emergency demand have been used fairly successfully. Despite the availability of these commercial products, they all suffer from the same problems of extremely high cost, sub-normal skin microstructure and inconsistent engraftment, especially in full thickness burns. Clinical practice for severe burn treatment has since evolved to incorporate these tissue-engineered skin substitutes, usually as an adjunct to speed up epithelization for wound closure and/or to improve quality of life by improving the functional and cosmetic results long-term. This review seeks to bring the reader through the beginnings of skin tissue engineering, the utilization of some of the key products developed for the treatment of severe burns and the hope of harnessing stem cells to improve on current practice.

Evaluation of diagnostic utility of step sections in dermatopathology: A prospective study of 200 consecutive punch biopsies

Background: Obtaining deeper sections or step sections is a common practice for small skin biopsies. Much of the available literature highlights the importance of step sections in neoplastic diseases of skin. However, the routine dermatopathology practice in developing countries shows a predominant burden of nonneoplastic diseases, and the utility of step sections in this context has not been much reported. Objective: The study was aimed to evaluate the utility of prospective step sections in routine dermatopathology practice. Materials and Methods: The present study comprising 200 consecutive skin biopsies was carried out in a prospective manner. Three slides were prepared in each case: Slide 0 was prepared from the ribbon of tissue obtained from untrimmed block, step sections 1 and 2 were obtained at 50 μm and 100 μm depth, respectively. The diagnosis was rendered on slide 0 and subsequently reviewed after examining step section 1 and 2. Results: Of the 200 cases, additional findings on step sections were found in 18 cases (9%) which led to change in diagnosis in 10 (5%) cases. Step section 1 led to correct diagnosis in 6 cases (3%). Step section 2 led to correct diagnosis in 10 cases (5%); however, this was statistically not significant (P ≥ 0.065) when comparing to step section 1. Additional findings which led to diagnosis was most commonly found in the cases of borderline tuberculoid leprosy (5 out of 10 cases) followed by bullous disorders. Conclusion: We therefore believe that step sections improve the diagnostic accuracy in skin biopsies and they are, especially useful in suspected cases of Hansen's disease. There is no statistical advantage of step section 2 versus step section 1; although, step section 2 had shown to include all the additional findings which led to a change in diagnosis.

The importance of cytokeratin 19 expression in the differentiation of Basal cell carcinoma and trichoepithelioma.

Basal cell carcinoma (BCC) is the most common skin neoplasm reported in human. On the other hand, trichoepithelioma (TE) is a rare, benign tumour of skin adnexa. The differentiation of BCC and TE may be difficult since their morphological findings are similar. In a few studies, it has been determined that undifferentiated basaloid cells are highly positively stained with cytokeratin 19. The aim of this study was the comparison of cytokeratin 19 expression in cases of BCC and TE. Sections of skin tissues of 17 TE, 25 BCC and 12 non-neoplastic cases were used for cytokeratin 19 (CK19) immunohistochemical staining. Staining with CK19 of the BCC cases gave 15(60%) diffuse, 7 (28%) focal and 3 (12%) negative staining. On the other hand, among TE cases, 2 (12%) gave diffuse, 5 (29%) focal and 10 (59%) negative staining with CK19. In the non-neoplastic skin tissue samples, while positive staining with cytokeratin 19 in the outer root sheath of hair follicles and sweat glands were observed, there was no staining in basal layers. CK19 expression may be helpful in the differential diagnosis of BCC and TE especially in small skin biopsy samples in which morphologic differentiation is difficult.

Use of Induced Pluripotent Stem Cells in Dermatological Research

Induced pluripotent stem cells (iPSCs) have the potential to differentiate into any cell type of the body. iPSCs are generated through a process termed “reprogramming,” which entails the introduction of a set of transcription factors into somatic cells, such as dermal fibroblasts. iPSC clones, recognizable by their morphology (Figure 1a), arise from these cultures, usually within 14–21 days. In addition to their pluripotency, another important characteristic of iPSCs is that they can be propagated in cell culture indefinitely. Thus, an unlimited supply of iPSCs can be generated from a small skin biopsy. These iPSCs can then be differentiated into different types of somatic cells. For example, iPSCs can be directed to differentiate into epidermal keratinocytes, one of the cell types often affected in skin disorders. iPSC-derived keratinocytes can then be used to generate a stratified epidermis, either in vitro (3D skin equivalent cultures) or in vivo (xenotransplantation of keratinocytes onto immunodeficient mice; see Koch et al., 2014; Koster et al., 2014 for references). The ability to generate unlimited numbers of disease-specific keratinocytes provides an ideal tool for basic scientists to explore the molecular mechanisms underlying different skin disorders. Further, recently developed technologies now enable investigators to correct disease-causing mutations in iPSCs. These cells could then be used to generate gene-corrected, healthy replacement skin for patients affected by genetic skin disorders. A major advantage of this approach is that patients would be treated with cells that are unlikely to be immunologically rejected. Figure 1 Generation of induced pluripotent stem cells (iPSCs) and iPSC-derived keratinocytes

Isolation and Cultivation of Adult Human Keratinocyte Stem Cells for Regeneration of Epidermal Sheets

Background: Keratinocyte stem cell is one of the adult stem cells that inhabits the skin and contributes to skin function and renewal. Adult stem cells are best defined by their capacity to self-renew, and to maintain tissue function for a long period of time. These findings indicate the importance of these cells for clinical applications including regenerative medicine, tissue engineering and gene therapy. In full-thickness damage or injury including burns, the cultured epidermal autografts (CEAs) may be placed directly onto muscle or fascia. Methods: A small split thickness skin biopsy (1 12أ—'> 2 cm) was obtained aseptically to isolate stem cells. The biopsy was cut into thin pieces and treated with trypsin at 4° C overnight (cold trypsin method) to obtain a single-cell suspension. The cells were seeded at a density of 3×104 cells/cm2 onto a preformed mitomycine-C treated 3T3 cell as feeder layer in DMEM medium supplemented with 10% fetal bovine serum (FBS) and other special supplements. Clonogenic keratinocytes divided and colonies quickly expanded and pushed away the 3T3 feeder layer cells, which then detached from the culture vessel and eliminated with medium changes. Primary cultures were usually subcultured when the cells were in exponential growth phase .Colonies of keratinocytes were expanded and after 7-10 days fused and formed a coherent stratified epithelium. Confluent cultured epithelia were detached enzymatically as coherent sheets from the surface of the culture flasks and transferred onto petrolatum- impregnated gauze.Histological studies of cultured epithelium were also carried out. Results: In our experience from 1 cm2 of skin sample, 2,5- 4×106 cells were obtained. It resulted in keratinocytes suspensions which consisted at least 90% single cells. Cultured keratinocytes proliferated and after 8-10 days became confluent. The area of cultured epithelium detached from T-25 and T-75 culture flasks was approximately 12-15 cm2 and 35-40 cm2 respectively. Histological studies showed that 10-day old cultured epithelium had 3-4 cell layers consisting of small basal cells and big scquamous cells with large nucleus. Also in the basal layer few melanocytes with melanin pigments in the cells cytoplasm were found.The 20-day old cultured epithelium had 8-10 layers consisting of small and round basal cells, scquamous cells and 2-3 layers of keratinized cells. Conclusion: Culture of keratinocyte stem cells could result in multilayer epithelium that creates a good cosmetic appearance upon transplantation. This could re-generate an epidermis that is resistant to trauma and infections. It can be considered as an appropriate substitution in skin loss conditions. Keywords: Epidermal Sheets, Skin Adult Stem cells, Keratinocyte.

Utility of prospective step sections in diagnostic skin histopathology for small biopsies

Background: Small skin biopsies have a cosmetic advantage and prospective step sections could potentially improve turnaround time without compromising diagnostic information. The study aimed to examine the use of prospective step sections of small skin biopsies and its effect on histopathological diagnosis and turnaround time. Materials and Methods: This was a hospital based cross-sectional study at Department of Pathology and Department of Dermatology, BPKIHS from June 2011- June 2012. Diagnoses /comment on three levels of prospectively taken 3mm biopsies were compared with those of 5mm biopsies taken from the same/similar lesion from 100 patients. Additional sections from 5mm biopsies were taken retrospectively. Percentage, proportion, mean, standard deviation, diagnostic sensitivity, kappa statistics and paired sample t- test were used as statistical tools. Results: Of 100 cases, 80 were diagnosed using 5mm biopsies while 73 were diagnosed using 3mm biopsies. On additional step sections of 3mm biopsies, 3 more cases were diagnosed. When compared with 5mm biopsies, the sensitivity of the 3mm biopsy rose from 90% to 93.8% after additional sections while the measure of agreement rose from 0.751 to 0.826.Mean turnaround time for prospectively sectioned 3mm biopsies was 2.56 days and that of retrospectively sectioned 5 mm biopsies was 4.64 days with their difference being statistically significant. Conclusion: A statistically significant decrease in turnaround time and an increase in sensitivity and agreement after step sections elucidates the utility of prospective step sectioned 3 mm biopsies in diagnostic skin histopathology. DOI: http://dx.doi.org/10.3126/jpn.v4i7.10298 Journal of Pathology of Nepal (2014) Vol. 4, 552-559

Concise Review: Hurdles in a Successful Example of Limbal Stem Cell-based Regenerative Medicine

Recent breakthroughs in regenerative medicine have generated enthusiasm and many efforts to explore new therapeutic potentials of both somatic and pluripotent stem cells. About 30 years passed since a discovery of a method of producing a great number of human epidermal keratinocytes by cultivation from a small skin biopsy, many possibilities are now envisaged for therapeutic application of different cultured cell types. The importance of stem cell content was proven for many tissues or organs in different pathologies. Ocular burns cause depletion of limbal stem cells, which lead to corneal opacification and visual loss. Most of available treatments are palliative and focused on the relief of the devastating clinical picture. This review is focused on recent developments in cell-based therapy of limbal stem cell deficiency. All findings can provide support for improvement and standardization of the cure for this disabling disease.

Experience of ReCell in Skin Cancer Reconstruction

The ReCell system (Avita Medical) is a cell culture product that allows the immediate processing of a small split-thickness skin biopsy to produce a complete population of cells including keratinocytes, melanocytes, Langerhans cells and fibroblasts. This series is the first to highlight the reconstructive applications of ReCell following ablative skin cancer surgery. The ReCell system was utilized for three patients following skin cancer excision. In two cases, the cells were applied to forehead flap donor sites following nasal reconstruction. In one case, the cells were applied to the calvarial periosteum following wide local excision of a melanoma scar. Assessment of the treated area was performed using the patient and observer scar assessment scale after 1 year. The Patient and Observer Scar Assessment Scale (POSAS) scores for the 2 patients treated with ReCell following forehead flap surgery were 22 and 32. The score for the patient that underwent wide local excision of a melanoma scar was 45. The absence of a donor site, accelerated healing and the satisfactory aesthetic appearance of the mature scars in this series suggest that ReCell may play a useful role in reconstruction following skin cancer excision.

Comparison of intradermal injection of autologous epidermal cell suspension vs. spraying of these cells on dermabraded surface of skin of patients with post-burn hypopigmentation

Introduction:One of the most important complications after burning is hypo/depigmentation. This study was designed to compare two methods of cell spray and intradermal injection of epidermal cell suspension for treatment of burn induced hypopigmentation.Material and Methods:In this study, 28 patients with post burn hypo/depigmentation were selected and divided in 2 groups. A small skin biopsy was taken from normal skin of patients in operation room and epidermal cell suspension was prepared using NaBr 4N and trypsin. In the first group, the epidermal cell suspension was sprayed on the wound surface and then the area was dressed with amniotic membrane and gauze. In the second group, the cell suspension was injected in intradermal manner in the hypopigmented area. The patients were followed up and to evaluate the effect of the cells, photos were taken from the area before operation and also at follow-up. Clinical evaluation was done by the surgeon and a clinical score between “0” to “4” was used to demonstrate the clinical status from poor to excellent pigmentation. Skin biopsies were taken from depigmented area before and after interventions. Melanocytes were stained using anti S100 antibody and were counted in ×400 magnification fields.Results:Eighteen patients were in cell spray and 10 were in cell injection groups. Mean change of pigmentation in two group showed that there was no statistical significant differences in pigmentation between two groups, (P value = 0.52) although a limited improvement in pigmentation status was observed in both groups. Regarding melanocyte numbers per field, there was not a significant difference between two groups and also before and after interventions, but melanocyte number increased after treatment in both groups.Conclusion:We did not find noticeable differences between cell spray and intradermal injection methods. Although both methods showed a limited effect on pigmentation of depigmented skin, the clinical results were not satisfactorily for both patients and clinicians.

Skin explant cultures as a source of keratinocytes for cultivation

Cultivated human keratinocytes can be used successfully in the treatment of burn patients, but efforts to heal burns and other wounds can be hampered by the very small skin biopsies available for cultivation of transplantable keratinocyte sheets. A small biopsy (and correspondingly small number of enzymatically isolated keratinocytes for use in classical cultivation techniques) can lead to a low yield of multilayer sheets for clinical application or unacceptably long cultivation times. One way of addressing this is to make use of skin remnants remaining after enzymatic digestion and culture cells migrating out of these skin explants. Sufficient numbers of explant-derived keratinocytes can be obtained to facilitate additional routine cultivation of these cells. Biopsy remnants can be used to initiate explant cultures repeatedly (we were able to re-use pieces of skin 10 times and still obtain useful numbers of keratinocytes) and this "passaging" yields substantially more cells for classical cultivation than would be available from conventional methodology alone, and in a comparable timeframe. Another advantage of this method is that it does not require additional biopsies to be procured from already-compromised patients and overcomes problems associated with contamination of skin samples with resistant hospital-acquired bacterial infections common during prolonged hospitalization.