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PubMed1981

Brain lactic acidosis and ischemic cell damage: 2. Histopathology.

The influence of severe tissue lactic acidosis during incomplete brain ischemia (30 min) on cortex morphology was studied in fasted rats. Production of lactate in the ischemic tissue was varied by preischemic infusions (i.v.) of either a saline or a glucose solution. The brains were fixed by perfusion with glutaraldehyde at 0, 5, or 90 min of recirculation. In saline-infused animals (tissue lactate about 15 mumol g-1), changes observed at 0 and 5 min of recirculation were strikingly discrete: slight condensation of nuclear chromatin, mild to moderate mitochondrial swelling, and only slight astrocyte edema. These changes had virtually disappeared after 90 min recirculation and, at this time, only discrete ribosomal changes were observed. In contrast, glucose-infused rats (tissue lactate about 35 mumol g-1) showed severe changes: marked clumping of nuclear chromatin and cell sap in all cells was already evident at 0 and 5 min recirculation, while mitochondrial swelling was mild to moderate. Although tissue fixation was inadequate at 90 min, the ultrastructural appearance indicated extensive damage. It is concluded that excessive tissue lactic acidosis during brain ischemia exaggerates structural alterations and leads to irreversible cellular damage. A tentative explanation is offered for the paucity (less than 0.2%) of condensed neurons with grossly swollen mitochondria, previously considered a hallmark of ischemic cell injury.

Acidosis↗

PubMed1983

Diagnosis of human lymphoma with monoclonal antileukocyte antibodies.

Two monoclonal antibodies have been produced that react with antigens present on human white cells. These reagents differ from other monoclonal antibodies of similar specificity in that the antigens they recognize are resistant to conventional tissue-fixation and embedding procedures. These reagents can therefore be used in immunocytochemical staining of paraffin-embedded tissue sections. We assessed the practical usefulness of this technique in the histopathological diagnosis of human lymphoid neoplasms by staining a wide range of routine surgical biopsy specimens of normal and neoplastic tissue (gathered from five institutions), using an indirect immunoperoxidase technique. In all 40 cases of non-Hodgkin's lymphoma, positive labeling of neoplastic cells was obtained with one or both antibodies. In contrast, no staining of neoplastic cells was observed in 60 samples of nonlymphoid neoplasms. We conclude that many of the difficulties encountered by histopathologists in distinguishing between lymphoid and nonlymphoid neoplasms may be overcome by immunohistologic labeling with monoclonal antibodies such as the ones we have studied.

Animals↗

PubMed2002

Optimizing gene expression analysis in archival brain tissue.

Analysis of gene expression in the brain is a valuable tool to study the function of the brain under normal and pathological conditions. Although there are many techniques used to measure gene expression the validity of any such experiment is directly related to the quality of the RNA in the samples. The most readily available source of human brain tissue is post-mortem and while frozen tissue is sometimes available, most archived tissue is fixed and paraffin-embedded. The use of fixed tissue for expression analysis introduces variables, which must be considered in the experimental design. In addition, factors associated with clinical variability of the patient and with tissue procurement can affect RNA transcript levels. In order to illustrate the effects of two common tissue fixatives, formalin and ethanol, on the quality of RNA for expression analysis we compare RNA extracted from these fixed tissues to the gold standard, flash-frozen tissue. We describe RNA extraction from fixed tissue and ways to assess the quality or intactness of the RNA using reverse transcription combined with polymerase chain reaction amplification. An advantage of using archived tissue is the ease with which single cells or subpopulations of cells can be obtained by laser microdissection. The successful isolation of RNA from microdissected cells is also presented. From our results and a review of the literature we conclude that RNA from fixed tissues is a viable source of RNA for expression analysis which should enable new experimental approaches and discoveries as long as attention is given to variables that can affect RNA at all levels of analysis.

Archives↗

PubMed2003

Role of soft tissues in metacarpal fracture fixation.

The contribution of soft tissues in stabilizing fracture fixation in metacarpals is appreciated clinically, but no quantitative biomechanical study of their role has been done. All previous studies of fracture fixation in vitro have been done on metacarpals denuded of soft tissues. To quantify the role of soft tissues in metacarpal fracture fixation, the biomechanical effectiveness of four fixation devices was examined in human cadaver metacarpals with and without soft tissues. Values were compared for three nonrigid methods (expandable intramedullary fixation devices, crossed Kirschner wires, and single half-pin frames) and one rigid method (dorsal plates) in 45 disarticulated metacarpals stripped of soft tissues (denuded) and in 46 metacarpals in whole hands with all soft tissues remaining (intact). Mechanical testing to complete failure in three-point apex dorsal bending was done in all specimens. Ultimate moment (strength) of each of the four fixation methods was significantly greater in intact specimens than in denuded specimens. Crossed Kirschner wires were most stable in intact specimens, and dorsal plates were more stable in denuded specimens. The results show that soft tissues contribute to the strength of fracture fixation. Clinically, surgeons may be able to use a less invasive fixation method than plating without compromising the strength of metacarpal fixation in patients whose soft tissues are not severely disrupted and the fracture configuration allows. Plating may offer optimum stability in patients whose soft tissues are damaged severely and provide less strengthening of the fracture construct.

Biomechanical Phenomena↗

PubMed2005

Analysis of outcomes of anterior cruciate ligament repair with 5-year follow-up: allograft versus autograft.

PURPOSE: To prospectively compare outcomes of primary anterior cruciate ligament (ACL) reconstruction with either Achilles tendon allograft with soft-tissue fixation or standard bone-patellar tendon-bone autograft with interference screw fixation. TYPE OF STUDY: Prospective comparative case series. METHODS: A group of 41 patients who underwent soft-tissue allograft reconstruction and a group of 118 patients who underwent autograft bone-patellar tendon-bone reconstruction were included in the final results. Patients were evaluated preoperatively and postoperatively at 1 to 2 weeks, 6 weeks, 3 months, 6 months, and then annually for 5 years. Objective measures of outcome included KT-1000 measurements, range of motion, ligamentous integrity, thigh atrophy, and International Knee Documentation Committee score. Subjective evaluations included patient completion of 5 questionnaires documenting functional status, pain, and health-related quality of life: (1) the short-form McGill Pain Questionnaire, (2) a patient subjective assessment of knee function and symptoms, (3) a patient subjective assessment follow-up, (4) a knee pain scale, and (5) the RAND 36-Item Health Survey. Mixed models analysis of variance was used to compare the outcomes of the treatment groups using baseline values of the study variables as a covariate. RESULTS: Autograft patients reported significantly more pain on the bodily pain subscale of the RAND-36 than the allograft group at 1 week (P = .0006), 6 weeks (P = .0007), and 3 months (P = .0270). Autograft patients reported more pain than allograft patients on the McGill Pain Scale visual analog scale at 1 to 2 weeks (P < .0001) and 6 weeks (P = .0147). Patient assessment of function and symptoms showed that a higher proportion of patients reported normal or nearly normal knee function in the allograft group than in the autograft group at 3 months (33% v 14%, P = .0558, respectively). Fewer activity limitations were reported by allograft patients than autograft patients at 6 weeks (P = .0501), 3 months (P = .0431), and 6 months (P = .0014). After reconstruction, the allograft group displayed significantly more laxity in KT-1000 measurements at all time points than the autograft group (P = .0520). These measurements decreased over time for both groups (P < .0001). CONCLUSIONS: Five-year follow-up of patients undergoing ACL reconstruction with allograft versus autograft were compared objectively and subjectively. Both groups of patients achieved similar long-term outcomes. Overall, the allograft patients reported less pain at 1 and 6 weeks after surgery, better function at 1 week, 3 months, and 1 year, and fewer activity limitations throughout the follow-up period. LEVEL OF EVIDENCE: Level II, prospective cohort study.

Adult↗

PubMed1991

A monoclonal antibody to human leukocyte common antigen, SHL-1, and its use for formalin-fixed, paraffin-embedded tissues.

The authors describe a newly characterized murine monoclonal antibody to the human leukocyte surface antigen, SHL-1. The antigen belongs to the leukocyte common antigen (LCA) family, and its molecular weight is about 180,000 daltons, which is similar to that of some previously characterized LCAs. The SHL-1 antigen is resistant to conventional tissue-fixation and embedding procedures. This antibody can therefore be used in the immunohistochemical staining of paraffin-embedded tissue sections. Wide screening with a sufficient number of both fresh and routinely processed paraffin-embedded tissues was done with indirect immunoperoxidase technique. With this procedure, SHL-1 labeled the majority of normal leukocytes and hematopoietic malignancies. Some B-cell malignancies were not stained with this antibody. The non-hematologic malignancies posing diagnostic problems of differentiation from lymphomas or leukemias were completely negative to SHL-1. The immunoreactivity to SHL-1 of samples from 24 leukemic patients and 15 human tumor cell lines was determined by the immunofluorescence method. Of 24 leukemic preparations, 23 were strongly reactive to this antibody. One case of B-cell leukemia did not react with SHL-1. No immunoreactivity was demonstrated in non-hematopoietic tumor cell lines. The overall reaction pattern of SHL-1 proved its usefulness in both diagnostic and research practice in hematological disorders. This antibody detected cell surface antigens of the T cell series more effectively than those of the B-cell series in terms of the positive number of cells and mean fluorescence intensity.

Antibodies, Monoclonal↗

PubMed2002

Biomechanical comparison of hamstring and patellar tendon graft anterior cruciate ligament reconstruction techniques: The impact of fixation level and fixation method under cyclic loading.

PURPOSE: To mechanically test different reconstruction techniques of the anterior cruciate ligament (ACL) under incremental cyclic loading and to evaluate the impact of the level and method of graft fixation on tensile properties of each technique. TYPE OF STUDY: In vitro biomechanical study. METHODS: Four hamstring and 1 patellar tendon reconstruction techniques were performed on 40 young to middle-aged human cadaveric knees (average age, 39 years). An anterior drawer with increasing loads of 20 N increments was applied at 30 degree of knee flexion. Anatomic, direct interference screw fixation was tested in 2 hamstring and in the patellar tendon groups. Nonanatomic (extracortical) graft anchorage was tested in the remaining 2 hamstring groups with indirect graft fixations on both sides and the combination of indirect tibial and direct femoral fixation. Structural properties were determined throughout the cyclic loading test. RESULTS: The more anatomic reconstruction techniques provided significantly higher structural properties and smaller loss of fixation compared with nonanatomic, extracortical fixation, with indirect repair on both fixation sites resulting in the lowest structural properties. The tibial fixation site was the weakest link in all of the anatomic reconstructions. Patellar tendon fixation with attached bone blocks in both bone tunnels significantly improved construct stiffness and decreased graft slippage. CONCLUSIONS: The results of this study suggest that anatomic fixation should be preferred for anchorage of hamstring tendons and linkage materials should be avoided. Direct soft-tissue fixation with interference screws still allows considerable graft slippage, which can be limited by using a bone block or application of a backup or hybrid fixation, especially on the tibial fixation site.

Absorbable Implants↗

PubMed1983

[Submicroscopic study of brown adipose tissue in young rats by the freeze-fracturing method].

The authors report the results of their ultrastructural study on the rat brown adipose tissue using the freeze-fracturing techniques. It is well known from transmission electron microscopy that lipid vacuoles of the brown adipose tissue cells are bound by a dense line corresponding to a structure as yet totally unknown. The results of the study using freeze-fracturing technics show that the morphology of lipid vacuoles surface is different from that of the typical unit membrane. The possibility is discussed that the particular aspect of the surface is due to the lypolysis induced by tissue fixation.

Adipose Tissue, Brown↗

PubMed2000

Alkaline fixation drastically improves the signal of in situ hybridization.

In situ hybridization (ISH) is widely used to detect DNA and RNA sequences within the cell and tissue sections. The important step in performing this technique is tissue fixation. We investigated the influence of the pH of the fixative on the outcome of ISH. Our studies indicate that alkaline formaldehyde dramatically increases the ISH signal with RNA probes. The increase in signal was observed for detection of low as well as for high abundance messages. The sensitivity of the method was increased 5- to 6-fold.

Cell Cycle Proteins↗

PubMed1978

Immunocytochemical localization studies of myelin basic protein.

The location of myelin encephalitogenic or basic protein (BP) in peripheral nervous system (PNS) and central nervous system (CNS) was investigated by immunofluorescence and horseradish peroxidase (HRP) immunocytochemistry. BP or cross-reacting material could be clearly localized to myelin by immunofluorescence and light microscope HRP immunocytochemistry. Fine structural studies proved to be much more difficult, especially in the CNS, due to problems in tissue fixation and penetration of reagents. Sequential fixation in aldehyde followed by ethanol or methanol provided the best conditions for ultrastructural indirect immunocytochemical studies. In PNS tissue, anti-BP was localized exclusively to the intraperiod line of myelin. Because of limitations in technique, the localization of BP in CNS myelin could not be unequivocally determined. In both PNS and CNS tissue, no anti-BP binding to nonmyelin cellular or membranous elements was detected.

Animals↗

PubMed1984

Surgical management of doxorubicin (Adriamycin) extravasation.

The advent of potent chemotherapeutic agents has been helpful in the medical management of a variety of malignancies. Unfortunately, the side effects and complications have been significant. Our recent experience with a patient who had the avoidable complication of extravasation with doxorubicin (Adriamycin) into the upper extremity is described. Frequently, the medical management of this problem has been observation alone followed by late surgical referral. This tissue fixation of the chemotherapeutic agent is such that it stays bound locally and causes an ever enlarging ulceration of tissue. This slough can occur not only in a circumferential fashion, but also in the superficial to deep manner, resulting in full-thickness lesions. Early surgical intervention is important to shorten the morbidity and to return the patients to their home environment.

Adolescent↗

PubMed1987

The effect of diffusion on the immunolocalization of antigen.

Diffusion prior to tissue fixation may result in immunohistochemical localization of antigen at sites in which it does not occur in vivo. The extent and speed with which this may occur have been investigated with several antigens of different molecular weights in tissues which were left unfixed for various lengths of time. There was clear evidence of diffusion when fixation was delayed for as little as 1 h, resulting in focal labelling of cells which did not contain antigen in rapidly fixed tissues. Falsely localized antigen was particularly evident adjacent to sites containing high antigen concentrations and might be expected in areas permeated by antigen-containing tissue fluid. These findings substantiate the view that diffusion of antigen should be considered as a cause of false positivity in any immunolocalization study.

Animals↗

PubMed1991

PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies.

The development of polymerase chain reaction (PCR) DNA amplification methods has afforded molecular studies of fixed paraffin-embedded tissue samples and other archival material. Some fixation methods damage DNA, and thus deleteriously affect subsequent PCR analysis. This study addressed the effect of short- and long-term storage (2 hr to 30 days) in a variety of fixatives (10% buffered-neutral formalin [BNF], 95% ethanol, acetone, and OmniFix) before paraffin embedding. We tested the ability of prepared tissue sections to yield DNA amplification products ranging from 268 to 1327 bp. Results indicated that tissues fixed for 8 days in BNF were able to amplify 536-bp but very little 989-bp DNA fragments; after 30 days of BNF fixation only a 268-bp fragment was amplifiable. Samples fixed in OmniFix and acetone yielded products of 989 and 1327 bp, respectively, after 8 days of fixation; both fixatives yielded 989-bp amplification products after 30 days of fixation. Tissues fixed in 95% ethanol for up to 30 days efficiently produced DNA amplification fragments of up to 1327 bp in length. The results provide important information for prospective studies that involve PCR analysis from archival material. Furthermore, fixation and long-term storage in ethanol should prove particularly useful in remote areas where refrigeration or immediate sample-processing is unavailable.

DNA↗

PubMed1976

Reinterpretation of the ultrastructure of cartilage matrix.

The epiphyseal cartilage from new-born mouse was treated with collagenase in two ways: either before fixation or after glutaraldehyde fixation. The electron dense granules of the matrix were not seen in the micrographs of cartilage treated with collagenase before fixation. It is concluded that collagen plays a definite role in the formation of the granules at the time of tissue fixation and that the granules are fixation artifacts.

Animals↗

PubMed1989

Histochemical blockade of the antigen-antibody reaction using immunoperoxidase demonstration of lysozyme in paneth cells and lamina propria mononucleocytes of human small intestine as a model system.

Nitrosation and acetylation, two histochemical blocking procedures for amino groups, were used to establish the extent to which these groups intervene in the antigen-antibody reaction in immunohistochemistry. We used the peroxidase-antiperoxidase method (PAP) to demonstrate lysozyme in Paneth cells and in lamina propria mononucleocytes of human small intestine as a model system. We studied the relationship of these groups to fixation, concentration of the primary antiserum, and length of blockade, as well as the possibility of reversing blockade as proof of specificity. Our findings support the contention that amino groups are also an important factor in antigen-antibody binding, even in fixed tissue. Fixatives influence the binding process in many ways, with acetylation producing a more successful result than nitrosation in tissue fixed in Bouin without acetic acid, whereas the reverse is true in formaldehyde-fixed tissue.

Acetates↗

PubMed1989

Postfixation of cryostat sections improves tumor definition in Mohs surgery.

Precise identification and localization of tumor is the key to success in Mohs micrographic surgery. In a pilot study, we demonstrated improvement in tumor definition and staining characteristics after formalin fixation of cryostat sections (postfixation) when compared with unfixed specimens. We further investigated the benefits of postfixation with a series of tissue fixatives and with varying fixation times. In all cases, postfixation was found to be beneficial. No improvement was noted by extending the postfixation time beyond 1 minute.

Acetates↗

PubMed1975

Electron microscopical investigations on the glycocalyx of cultivated cells after incubation with extracts from rapidly growing normal and tumour tissues.

Electron microscopical investigations by means of the ruthenium red method on Huggin-tumour cells cultivated in vitro resulted in a different thickness of the glycocalyx depending on the fixation of the cells in monolayer or, after mechanical detachment, in suspension. Cells fixed in suspension display a thicker glycocalyx, which is supposed to be due to a contraction of the cells before fixation. Tissue extracts from rapidly growing tissues (mammary gland of pregnant cows, tumour tissue) cause a significant reduction of the glycocalyx in transformed hamster fibroblasts and Huggin-tumour cells after an 18-hour period of action. These findings are in accordance with previous findings on the glycolytic effect of tissue extracts on sections of cartilaginous tissue. It is discussed whether the action of the tissue extracts on the glycocalyx is due to its detachment or to an inhibition of its renewal.

Animals↗

PubMed1990

Potential value of estrogen receptor immunocytochemical assay in formalin-fixed breast tumors.

Sixty-two primary breast carcinomas were analyzed for estrogen receptor (ER) by both the dextran-coated charcoal (DCC) technique and estrogen receptor immunocytochemical assay (ER-ICA) on cryostat and permanent sections. Paraffin sections of formalin-fixed breast tissue underwent DNase pretreatment to expose the nuclear antigenic site as described by P. Shintaku and J. H. Said (Am J Clin Pathol 87:161, 1987). The results of immunocytochemical staining agreed with those of the DCC biochemical assay in 89% of paraffin-sectioned tissue and in 94% of the cryostat sections. Comparison of the results of ER-ICA on permanent and frozen sections showed 85% agreement (kappa statistic = 0.704). This study suggests that ER can be demonstrated immunocytochemically on paraffin-sectioned breast tissue. However, although highly specific, immunoperoxidase determination on paraffin-embedded tissue is less sensitive than that on frozen tissue. The commercial source of DNase, length of incubation, and tissue fixation are important factors in the demonstration of ER immunoreactivity. The assay may offer an alternative for assessment of ER when tissue is not suitable or available for biochemical assay or conventional cytochemical analysis.

Breast Neoplasms↗