Mehdi Nassiri

A Tissue Fixative that Protects Macromolecules (DNA, RNA, and Protein) and Histomorphology in Clinical Samples

Preservation of macromolecules (DNA, RNA, and proteins) in tissue is traditionally achieved by immediate freezing of the sample. Although isolation of PCR-able RNA has been reported from formalin-fixed, paraffin-embedded tissues, the process has not been shown to be reproducible because high molecular weight RNA is usually degraded. We investigated the potential value of a new universal molecular fixative (UMFIX, Sakura Finetek USA, Inc., Torrance, California) in preservation of macromolecules in paraffin-embedded tissue. Mouse and human tissues were fixed in UMFIX from 1 hour to 8 weeks. They were then processed by a rapid tissue processing (RTP) system, embedded in paraffin, and evaluated for routine histology as well as for the quality and quantity of DNA, RNA, and proteins. Formalin-fixed tissues were processed by RTP and evaluated in a similar manner. Fresh-frozen samples were used as controls. The morphology of UMFIX-exposed tissue was comparable to that fixed in formalin. High molecular weight RNA was preserved in tissue that was immediately fixed in UMFIX and stored from 1 hour to 8 weeks at room temperature. There were no significant differences between UMFIX-exposed and frozen tissues on PCR, RT-PCR, real-time PCR, and expression microarrays. Similarly, physical and antigenic preservation of proteins in UMFIX tissue was similar to fresh state. Both RNA and proteins were substantially degraded in formalin-fixed and similarly processed specimens. We concluded that it is now possible to preserve histomorphology and intact macromolecules in the same archival paraffin-embedded tissue through the use of a novel fixative and a rapid processing system.

Simultaneous measurement of multiple Th1 and Th2 serum cytokines in psoriasis and correlation with disease severity.

BACKGROUND: Psoriatic plaques have been shown to contain increased levels of proinflammatory cytokines. Serum levels of interleukin (IL)-6, IL-7, IL-8, and interferon (IFN)-gamma have been reported elevated in psoriatic patients. AIM: To evaluate serum cytokine profiles in psoriasis patients by improved enzyme-linked immunosorbent assay (ELISA) technique and to correlate these levels with disease severity. METHODS: We analyzed single serum samples from 10 patients with active untreated psoriasis, two patients with active treated psoriasis, and five healthy volunteers for major T helper type 1 and T helper type 2 cytokines using the LINCOplex ELISA multi-analyte detection system that permits simultaneous detection of multiple cytokines from a single sample. The disease severity, including erythema, induration, scale, and surface area, was assessed. RESULTS: IFN-gamma was markedly elevated in all sera from psoriasis patients, 33.8 +/- 1.3 pg/ml (mean +/- standard error) versus 8 +/- 1.5 pg/ml for normal controls (p < 0.01), and positively correlated with all indices of disease severity (Spearman r > 0.6). IL-8 was also increased in psoriasis patients (24.4 +/- 1.8 pg/ml) versus normal controls (3.6 +/- 1.2 pg/ml) (p < 0.05) and positively correlated with the degree of erythema (Spearman r > 0.6). Mean IL-12 levels were decreased in sera from psoriasis patients (8.5 +/- 1.2 pg/ml) compared with normal controls (42.2 +/- 5.3 pg/ml) (p < 0.01). Also, serum IL-10 levels were below detection levels in psoriatics compared with controls (6.4 +/- 1.3 pg/ml). CONCLUSIONS: This new ELISA system allowed rapid and reliable detection of numerous cytokines in single serum samples from patients with psoriasis. We observed that IFN-gamma and IL-8 cytokines were elevated in psoriatics and correlated with parameters of disease severity while IL-10 and IL-12 were decreased.

A gene signature of nonhealing venous ulcers: Potential diagnostic markers

BACKGROUND Venous leg ulcers are responsible for more than half of all lower extremity ulcerations. Significant interest has been focused on understanding the physiologic basis on which patients fail to heal with standard therapy. OBJECTIVE This study uses complementary DNA microarray analysis of tissue samples from healing and nonhealing venous leg ulcers to identify the genetic expression profiles from these dichotomous populations. METHODS Ulcer size and chronicity, factors that have been identified as prognostic indicators for healing, were used to distribute venous leg ulcers as healing versus nonhealing. Punch biopsy samples were obtained from the wound edge and wound bed of all venous leg ulcers. The top 15 genes with differential expression greater than 2-fold between the two populations of wounds (P < .05) were reported. RESULTS Significant differences were demonstrated in the expression of a diverse collection of genes, with particular differences demonstrated by genes coding for structural epidermal proteins, genes associated with hyperproliferation and tissue injury, and transcription factors. LIMITATIONS Small sample size may mitigate potential clinical implications of findings. CONCLUSIONS The genetic expression profiles displayed here may have implications for the development of novel therapies for chronic venous leg ulcers, and may also serve as prognostic indicators for wound healing.

Preservation of biomolecules in breast cancer tissue by a formalin-free histology system

BackgroundThe potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer – estrogen receptor and HER2 – at the RNA and protein levels.MethodsParallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR.ResultsThe immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p < 0.05). Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05). The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative. Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens.ConclusionThe formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.

Immunohistochemically Determined Estrogen Receptor Phenotype Remains Stable in Recurrent and Metastatic Breast Cancer

We evaluated the estrogen receptor (ER) phenotype of recurrent and/or metastatic breast cancers and compared it with the ER status of the primary tumor in 278 cases. All patients had undergone surgical excision of the primary tumor, followed by observation only or treatment modalities that included radiation and/or chemotherapy or hormonal therapy. The immunohistochemical expression of ER was evaluated by using monoclonal antibody ER-1D5 and heat-induced antigen retrieval. At diagnosis, 165 patients had locoregional disease and 7 had distant metastases. Local recurrences and/or distant metastases occurred from 2 months to 21 years after the diagnosis of the primary tumor. Overall, 159 primary tumors (57.2%) were positive for ER. In 269 cases (96.8%), the ER status of the primary and metastatic tumors was the same. In 9 patients with ER+ primary tumors, the metastases were ER-. There were no ER- primary tumors with ER+ metastases. The time to distant metastasis was significantly longer for ER+ tumors. The immunohistochemically determined ER phenotype of primary breast cancers remains stable in most recurrent and metastatic disease.

Topical application of imiquimod 5% cream to keloids alters expression genes associated with apoptosis

Keloids are benign mesenchymal tumours, usually present at and extending beyond the margins of sites of previous injury. It is reported that keloids display aberrant expression of apoptotic genes: TGFB1 is activated, whereas caspase 8 and 3 are not, thus indicating a block upstream in the apoptosis cascade in keloids. Interferon‐α2b normalizes the excessive synthesis of collagen, glycosaminoglycans and collagenase by keloidal fibroblasts, reduces recurrences following keloid excision, and enhances the expression of native p53 and apoptosis. Imiquimod, a rapid and potent inducer of interferons locally at the site of application to the skin, reduces recurrences following keloid excision and alters gene expression of markers of apoptosis in basal cell carcinoma cells. We investigated the effects with respect to the expression of apoptotic genes in keloidal tissue compared with nontreated controls of imiquimod 5% cream applied topically to keloids. Total RNA was extracted from excised keloidal tissue, cDNA probes synthesized and then hybridized to gene‐specific cDNA fragments spotted on membranes. The expression levels of 96 genes involved in apoptosis, relative to cyclophilin expression, were compared in the imiquimod‐treated and untreated groups. The mean ratio of expression, relative to cyclophilin of caspase 3 and DFFA were significantly enhanced. Caspase 3 was significantly downregulated and DFFA was significantly upregulated in the group of imiquimod‐treated keloids (P < 0·05) compared with the untreated group of keloids. Although imiquimod is capable of altering the expression of these markers of apoptosis in keloids, their role, if any, in the therapeutic response of keloids to imiquimod requires further investigation.

The Pathology of full-thickness cadaver skin transplant for large abdominal defects: a proposed grading system for skin allograft acute rejection.

Closure of large abdominal defects after extensive abdominal surgery is a major technical surgical problem. Failure to close the abdomen leaves the patient at risk for grave complications. Full-thickness abdominal wall skin transplantation appears to solve this problem. This is the first time that detailed histopathologic features of skin abdominal wall transplantation from cadaver donors are described. Five adults and four children underwent 10 transplants because of large abdominal wall defects. Twenty-two posttransplantation skin specimens were evaluated during a mean follow-up of 23.5 weeks, and the findings were compared with the clinical appearance of the skin. Rejection was manifested as a maculopapular rash. The histologic features were categorized as perivascular infiltrates, epidermal changes, and stromal changes. A grading system is proposed based on the number of cases encountered: No rejection, grade 0 (n = 9): No perivascular infiltrates. Indeterminate for rejection, grade 1 (n = 2): Up to 10% of vessels show infiltrates of small lymphocytes. No eosinophils, large lymphocytes, spongiosis, epidermal, or stromal inflammation are seen. Mild rejection, grade 2 (n = 5): 11% to 50% of vessels are infiltrated by small lymphocytes. Eosinophils and mild spongiosis may or may not be present. No epidermal infiltrates, stromal infiltrates, or large lymphocytes are seen. Moderate rejection, grade 3 (n = 4): Greater than 50% of vessels show lymphocytic infiltrates that may be accompanied by epidermal and stromal inflammation. Spongiosis is absent or mild. Endothelial plumping, eosinophils, and large lymphocytes may be seen. Severe rejection, grade 4 (n = 2): Greater than 50% of vessels show infiltrates, but different from moderate rejection, there is dyskeratosis and the epidermis shows heavier lymphocytic infiltrates and moderate to severe spongiosis. The stroma shows infiltrates extending into the base of the epidermis. Endothelial plumping, eosinophils, and large lymphocytes are present. The mean number of weeks after transplantation for the development of clearcut rejection (grades 2–4) was 8.36. Among the 9 nonrejection cases, 4 specimens from 3 patients had thrombosis of the vessels feeding the graft. A grading system serves to better assess skin allograft rejection.

IMMUNOHISTOCHEMISTRY OF TISSUE PREPARED BY A MOLECULAR-FRIENDLY FIXATION AND PROCESSING SYSTEM

Abstract:A recently introduced histologic fixative (Universal Molecular Fixative [UMFIX]) has been shown to preserve macromolecules in tissue at ambient temperature. When UMFIX-exposed tissues are processed by a formalin-free, microwave-assisted rapid processing system, the resulting paraffin blocks retain good histomorphology and intact nucleic acids suitable for expression microarray analysis. Because UMFIX may be used as an alternative to formalin, the authors set out to study the effect of this new fixation and processing system on immunohistochemistry (IHC) by analyzing a range of human neoplastic and non-neoplastic specimens. Parallel slices from surgically removed specimens were fixed in formalin and UMFIX and processed in a rapid microwave-assisted tissue processor. IHC was performed following routine procedures. The staining for those antibodies that normally required antigen retrieval was carried out with and without that step. The intensity and pattern of reactions were compared in 144 tissue samples fixed by the two methods using 70 monoclonal and polyclonal antibodies. The intensity of IHC reactions for most cytoplasmic antigens was generally equal or stronger in UMFIX tissues. This was particularly true with intermediate filaments and HercepTest, where the antigen retrieval step became unnecessary. Conversely, there was a decrease in the intensity of reactions for HepPar1, bcl-2, and three nuclear antigens (Ki-67, TTF-1, and estrogen receptor). Increasing their exposure times optimized the sensitivity of the latter four antibodies. The study shows that IHC staining results of tissues fixed in UMFIX and processed by the microwave-assisted system are comparable to those obtained on formalin-fixed, similarly processed specimens. There is an enhancement of the sensitivity of few antibodies in UMFIX-exposed tissue, rendering antigen retrieval unnecessary. This increased sensitivity may be due to the effect of eliminating formalin from fixation and processing or the microwave energy.

Vaginal smooth muscle cell apoptosis is increased in women with pelvic organ prolapse

To compare the smooth muscle content and apoptosis of the vagina in women with and without anterior vaginal wall prolapse. Vaginal tissues were sampled in women with (n = 6) or without (n = 6) anterior vaginal wall prolapse undergoing hysterectomy. Smooth muscle of the vagina was studied by immunohistochemistry. Digital image analysis was used to determine the fractional area of smooth muscle in the histologic cross-sections. Apoptosis was assessed by TUNEL assay. The fractional area of non-vascular smooth muscle in the vagina of women with anterior vaginal wall prolapse was significantly decreased compared to women without prolapse (0.36 ± 0.12 vs. 0.16 ± 0.12 P = 0.021) and the apoptotic index was significantly higher compared to women without prolapse (0.04 ± 0.01 vs. 0.02 ± 0.03, P = 0.041). The fraction of smooth muscle in the vagina is significantly decreased and the rate of apoptosis is higher in women with anterior vaginal wall prolapse compared to women without prolapse.

Methodology for preservation of high molecular-weight RNA in paraffin-embedded tissue: application for laser-capture microdissection.

Laser-capture microdissection techniques have enhanced the ability to perform molecular studies of pure-cell populations. Although many technical factors affect the outcome of the procedure, none is more critical than the appropriate handling of the tissue. Because extraction of intact RNA from paraffin-embedded tissue is a difficult and inconsistent process, frozen sections with their attendant problems are used for this purpose. The major limitation of frozen section is its inferior morphologic quality compared with paraffin-embedded sections that may complicate accurate identification of cells during microdissection. We have developed a procedure that provides both high-quality histomorphology and RNA preservation in paraffin-embedded tissue. It is based on the use of a methanol-based fixative coupled with microwave-assisted rapid tissue processing. This technology in conjunction with a modified hematoxylin-eosin stain and a RNA extraction method allows isolation of high molecular-weight RNA from laser-capture microdissected, hematoxylin and eosin-stained paraffin sections. The high quality of the extracted RNA was confirmed by capillary electrophoresis and RT-PCR. The combination of a methanol-based fixative, rapid microwave tissue processing, and a modified hematoxylin and eosin stain produces paraffin sections that yield high molecular-weight RNA upon microdissection. This methodology opens the door for a wide range of gene expression analyses using paraffin-embedded tissue.