Morphological detection of X- and Y-chromosomes in smears and paraffin-embedded tissues using a non-isotopic in situ hybridization technique (NISH)

Pharyngeal smears and paraffin-embedded tissue specimens (skeletal muscle, kidney) obtained from 10 male and 10 female individuals were evaluated using non-isotopic in situ hybridization (NISH) with commercial X- and Y-specific biotinylated probes which recognize the pericentromeric regions DXZ1 and DYZ1/DYZ3 of the X- and Y-chromosome, respectively. The results provide evidence that the morphological sex determination of a single cell can be performed by critical application of this staining method leading to one nuclear signal in ldquomalerdquo cells using the Y-specific probe whereas ldquofemalerdquo cells are negative. In situ hybridization of ldquofemalerdquo tissues with an X-specific probe results regularly in 2 signals whereas ldquomalerdquo cells show only one spot in the nucleus

The immunohistochemical analysis of fibronectin, collagen type III, laminin, and cytokeratin 5 in putrified skin

Fibronectin, collagen type III, laminin, and cytokeratin 5 were visualized in normal skin and in skin showing early or advanced signs of autolytic decomposition to prove whether the immunohistochemical analysis of these antigens can provide useful information for an age-estimation of skin wounds obtained from putrified corpses. In cases with early signs of decomposition (visible course of veins, greenish discoloration) and without microscopic alterations like relaxation of the epidermal cell layers or destruction of the blood vessel structures, the staining pattern was identical to that found in normal, non-putrefied skin. In skin already showing microscopic alteration of the tissue structure, fibronectin and collagen type III could not be localized unambiguously. The distribution of laminin and cytokeratin 5, however, was well preserved. In advanced putrefied skin no reliable staining results could be obtained for fibronectin, collagen type III, and laminin. Even though cytokeratin 5 was still detectable in remnants of decomposition-resistant skin appendages, no information useful for an age-estimation of skin wounds can be obtained due to the autolytic detachment of the epidermal layers

The time-dependent expression of keratins 5 and 13 during the reepithelialization of human skin wounds

The time-dependent reepithelialization of 55 human surgical skin wounds with a wound age between 8h and more than 2 months was investigated by the immunohistochemical localization of cytokeratins 5 and 13. A complete, rebuilt epidermal layer over the wound area was first detectable in a 5-day-old wound, while all wounds of more than 18 days duration contained a completely reepithelialized wound area. Between 5 and 18 days the basal layer of keratinocytes showed — in contrast to normal skin — only some cells positive for cytokeratin 5. In some, but not all lesions with a wound age of 13 days or more, a basal cell layer completely staining for cytokeratin 5 was demonstrable. This staining pattern was found in all skin wounds with a wound age of more than 23 days. The immunohistochemical detection of cytokeratin 13 which can be observed regularly in non-cornifying squamous epithelia provides no information for the time-estimation of human skin wounds, since no significant temporary expression of this polypeptide seems to occur during the healing of human skin wounds

The time-dependent rearrangement of the epithelial basement membrane in human skin wounds

In 62 human skin wounds (surgical wounds, stab wounds and lacerations after surgical treatment) we analyzed the immunohistochemical localization of collagen IV in the epithelial basement membrane. In 27 of these wounds the distribution of collagen VII, which represents a specific component of the basement membrane of stratified epithelia, was also analyzed. We were able to demonstrate a virtually identical co-distribution of both collagen IV and VII in the wound area with no significant time-dependent differences in the appearance of both collagen types. Fragments of the epithelial basement membrane could be detected in the wound area from as early as 4 days after wounding and after 8 days a complete restitution of the epithelial basement membrane was observed. In all cases with a wound age of more than 21 days the basement membrane was completely reformed over the former lesional area. The period between 8 and 21 days after wounding was characterized by a wide variability ranging from complete restitution to deposition of basement membrane fragments or total lack of the epidermal basement membrane

Immunohistochemical localization of fibronectin as a tool for the age determination of human skin wounds

We analyzed the distribution of fibronectin in routinely embedded tissue specimens from 53 skin wounds and 6 postmortem wounds. In postmortem wounds a faint but focal positive staining was exclusively found at the margin of the specimens which dit not extend into the adjacent stroma. Vital wounds were classified into 3 groups. The first comprising lesions with wound ages ranging from a few seconds to 30 min, the second comprising those with wound ages upt to 3 weeks, and the third group with lesions more than 3 weeks old. Ten out of 17 lesions with a wound age up to 30 min showed a clear positive reaction within the wound area. Three specimens in this group were completely negative, while in 4 additional cases the result was not significantly different from postmortem lesions. These 7 cases were characterized by acute death with extremely short survival times (only seconds). In wounds up to 3 weeks old fibronectin formed a distinct network containing an increasing number of inflammatory cells corresponding to the wound age. In 2 cases with a survival time of 17 days and in all wounds older than 3 weeks fibronectin was restricted to the surface of fibroblasts and to parallel arranged fibers in the granulation tissue without any network structures. We present evidence that fibronectin is a useful marker for vital wounds with a survival time of more than a few minutes. Fibronectin appears before neutrophilic granulocytes migrate into the wound area. Since a faint positive fibronectin staining is seen in postmortem lesions and bleedings, we propose that only those wounds which show strong positive fibronectin staining also extending into the adjacent stroma should be regarded as vital