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PubMed2004

Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections.

The epidermal growth factor receptor (EGFR) is highly expressed in a variety of solid malignant tumors and its expression has been correlated with disease progression and poor survival. With the advent of targeted therapies, especially IMC-C225 (Cetuximab), a monoclonal antibody (MAb) directed against the EGFR, there is an increasing interest in immunohistochemistry (IHC)-based EGFR screening methods using paraffin-embedded tumor specimens to select cancer patients eligible for treatment with Cetuximab. With the EGFRpharmDX kit, a complete assay for demonstration of EGFR is now available. Because no information about the preservation of the EGFR under various conditions of fixation is available, we performed a prospective study on a panel of commonly used fixatives to determine optimal tissue preservation protocols. The stability of the epitope on cut tissue sections stored for a period up to 24 month was also tested using material originating from patients with head and neck cancer, non-small-cell lung carcinomas, and colorectal adenocarcinomas. Depending on the fixative used and the time of storage of cut tissue sections, a variation in the determined level of EGFR expression was demonstrated compared with the most optimal fixation procedure.

Adenocarcinoma↗
PubMed1978

Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity.

As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.

Cell Line↗
PubMedDate not supplied

Effect of dynamic glutaraldehyde fixation on the viscoelastic properties of bovine pericardial tissue.

We have previously proposed dynamic fixation as an alternative method to fix a porcine aortic heart valve xenograft with better tissue fixation and better preservation of its natural biomechanical properties. Bovine pericardium was fixed under dynamic conditions, low pressures (< 4 mmHg) and low vibration rate (1.2 Hz) in a 0.5% glutaraldehyde phosphate buffer (pH 7.4, 0.2 M). After fixation, tensile testing (i.e. relaxation and stress-strain curves) was performed at low and high extension rates (3 and 30 mm s(-1)) and tissue denaturation temperatures were determined by the hydrothermal isometric tension method. Conventional fresh and statically fixed pericardium were used as controls. In this instance, we found no significant biomechanical differences between the dynamically and statically fixed pericardial tissue (e.g. moduli and stress relaxation). However, differences in tissue extensibility were delineated, since the extensibility of the dynamically fixed tissue was closer to that of the fresh tissue compared to that of the statically fixed tissue. The final relaxation rate of the dynamically fixed tissue (-3.5 +/- 1.0% of stress remaining per log(second)) was similar to that of the statically fixed tissue (-3.2 +/- 0.60% log(s(-1))) and significantly lower than the fresh tissue(-9.5 +/- 1.2% log(s(-1))). The denaturation temperatures of the dynamically fixed pericardial tissue (mean +/- SD) (86.0 +/- 1.2 degrees C) and the statically fixed (85.2 +/- 1.6 degrees C) were similar but significantly higher than that of the untreated (fresh) valves (69.3 +/- 0.4 degrees C). The results suggest a similar degree of internal cross-linking for both statically and dynamically fixed pericardium. Although fundamental structural differences exist between both porcine and bovine xenograft tissue, how these differences contribute to biomechanical differences in the effects of dynamic versus static fixation remain to be explained.

Animals↗
PubMed1996

Is the frequency of mitoses and PCNA positive cells in tissues affected by delay in fixation? Lessons for accurate tumor grading.

Using uniform systematic random sampling, we tested whether a drop in mitoses and PCNA (proliferation cell nuclear antigen) positive cells following a delay in tissue fixation of up to five hours, would be unequivocally discernible at the well perfused periphery from 11 aggressive rat adenocarcinomas. No statistically significant difference (0.05 < P < 0.10; 95% Cl (-13.3, 1.3)) was found. We attributed this finding to the high proliferative heterogeneity of rapidly growing solid tumors because: (1) vascular perfusion is heterogeneous as rapidly proliferating epithelial tumors tend to outgrow their blood supply; (2) the ratio of perfused/ non-perfused tumor volumes may change as a solid tumor grows, altering to the same extent the ratio of proliferative vs. non proliferative regions in the tumor; and (3) the duration of mitoses is not constant. The consequences for accurate tumor grading are discussed.

Adenocarcinoma↗
PubMed1985

Changes of relaxation times T1 and T2 in rat tissues after biopsy and fixation.

NMR spectroscopical measurements of relaxation times were conducted on muscle, intestine, fatty tissue and cerebral cortex and white matter of the rat at various time intervals following removal of the tissue. It appeared that most tissues can be stored at 4 degrees C up to 24 hours without noticeable effects on NMR relaxation parameters. Exceptions are the T2 of muscle and the T1 and T2 of intestine, which tended to change in the first hour after biopsy. Relaxation parameters change considerably after fixation of the tissues. Therefore the effects of fixation have to be taken into account when carrying out NMR measurements on fixed tissues.

Adipose Tissue↗
PubMed2005

[The effect of anterior or posterior tibial tunnel placement of a soft tissue graft with a soft tissue interference screw on fixation biomechanics].

OBJECTIVES: We investigated the biomechanical characteristics of anterior or posterior tibial tunnel placement of the soft tissue graft with a soft tissue interference screw in anterior cruciate ligament (ACL) reconstruction. METHODS: Twelve bovine tibiae and digital extensor tendons were divided into two homogeneously equal groups after they were stripped of all soft tissues. Tibial tunnels were prepared and digital extensor tendons were fixed at nonanatomic (apart from the joint) anterior (n=6, group I) or posterior (n=6, group II) tibial tunnel positions with a soft tissue metal interference screw, 9x30 mm in size. All the specimens were cycled 500 times from 50 to 250 N at 1 Hz frequency in a servo hydraulic test device, after which ultimate load-at-failure testing was performed at a rate of 20 mm/min. RESULTS: The mean screw insertion torque values were 8.2+/-2.4 Nm and 8.4+/-2.8 Nm in groups I and II, respectively (p=0.88). No significant differences were found between the two groups with respect to graft displacement (1.9+/-0.8 mm vs 2.3+/-0.4 mm; p=0.38) and stiffness (132.7+/-10.9 N/mm vs 126.4+/-8.5 N/mm, p=0.98) at the end of cyclic loading. CONCLUSION: Our results show that the site of nonanatomic soft tissue graft fixation in the tibial tunnel (anterior or posterior) with a soft tissue interference screw do not affect the biomechanical parameters in ACL reconstruction.

Animals↗
PubMed1997

Degradation potential of biological tissues fixed with various fixatives: an in vitro study.

The purpose of this study was to investigate the in vitro degradation potential of porcine pericardia fixed with various aldehyde or epoxy compound (EC) fixatives, using bacterial collagenase and pronase. The fixatives investigated were formaldehyde (FA), glutaraldehyde (GA), monofunctional EC (EX-131), and multifunctional ECs (EX-810, EX-313, and EX-512). Fresh porcine pericardium was used as a control. The test samples were well immersed in a 20-U/mL collagenase solution or a 10-U/mL pronase solution and incubated at 37 degrees C at pH 7.5 for 24 h. The extent of degradation of each test sample was determined by measuring its increment in free amino group content and changes in collagen structure, denaturation temperature, and tensile stress after degradation. In general, the extent of tissue degradation with pronase was more notable than with collagenase. As observed with fresh tissue, the EX-131 EC fixed tissue radically disintegrated after either collagenase or pronase degradation, whereas the other test samples remained intact. The reason for this may reside in the more random molecular packing of the EX-131 EC-fixed tissue, which led to some loss in its helical integrity. This made penetration of enzymes into biological tissue easier. Of the multifunctional EC test groups, tissues fixed with tetrafunctional EC (EX-521) or trifunctional EC (EX-313) had relatively better resistance to degradation than those fixed with bifunctional EC (EX-810). The extent of degradation for the EX-313 or EX-512 EC fixed tissues was similar to that observed for the FA- or GA-fixed tissues. The results of this study indicated that the biological tissue fixed with monofunctional EC (EX-131) cannot resist bacterial collagenase or pronase degradation. However, resistance to degradation of the multifunctional EC (EX-313 or EX-152)-fixed tissues was comparable to that of the aldehyde (FA or GA)-fixed tissues. Therefore, of various EC fixatives, the EC with a greater number of functional groups should be chosen for tissue fixation to increase its resistance to enzymatic degradation.

Amino Acids↗
PubMed2004

Interference screw fixation of soft tissue grafts in anterior cruciate ligament reconstruction: part 2: effect of preconditioning on graft tension during and after screw insertion.

BACKGROUND: Preconditioning of tendon grafts is believed to eliminate natural viscoelasticity of the tendons and prevent knee laxity after anterior cruciate ligament (ACL) reconstruction. HYPOTHESIS: Preconditioned ACL grafts maintain their initially set tension. STUDY DESIGN: Randomized experimental study. METHODS: Forty-two human anterior tibialis (AT) tendon grafts were subjected to either no preconditioning (group 1), cyclic preconditioning (group 2), or isometric preconditioning (group 3). The residual graft tension was then recorded immediately after the application of an initial graft tension of 80 N and fixation into tibia with an interference screw, as well as 10 minutes later. In another experiment, the residual graft tension was recorded 1, 10, and 60 minutes after 10 AT and quadrupled hamstring tendon (HT) grafts alone (no fixation) had been subjected to isometric preconditioning (80 N). RESULTS: Immediately after screw insertion, the residual (AT) graft tensions were 79 +/- 19 N, 100 +/- 17 N, and 102 +/- 15 N in groups 1 through 3, respectively. Ten minutes later, the corresponding values were 49 +/- 16 N, 60 +/- 11 N, and 64 +/- 12 N. For the AT and HT grafts alone, the residual graft tensions were 67 +/- 2 N and 67 +/- 2 N, 45 +/- 2 N and 46 +/- 4 N, and 29 +/- 3 N and 34 +/- 5 N at 1, 10, and 60 minutes, respectively. CONCLUSIONS: A steady decrease (-60% within 60 minutes after initial tensioning) occurs in the initially set tension of the soft tissue ACL grafts. CLINICAL RELEVANCE: Clinically applicable preconditioning protocols cannot eliminate the intrinsic viscoelasticity from ACL soft tissue grafts, and thus, the reasonableness of preconditioning per se is questioned in ACL reconstruction.

Adolescent↗
PubMed1997

Comparison of formalin, ethanol, and Histochoice fixation on the PCR amplification from paraffin-embedded breast cancer tissue.

The polymerase chain reaction (PCR) has been successfully employed for the laboratory analyses of genetic and infectious disorders using DNA extracted from paraffin-embedded tissues. However, fixative type and fixation time influenced PCR reactions and in some circumstances amplification fragments could not be efficiently generated. In this study, we determined the effects of three commonly used fixatives including ethanol, formalin and Histochoice, on the PCR amplification of DNA from paraffin-embedded breast cancer tissue. The effect of fixatives and fixation times was measured by the ability of the extracted DNA to serve as a template for the amplification of 280 and 530 base pair DNA fragments. On amplifying DNA, positive reactions were uniformly seen in the ethanol specimens. The next best fixative was Histochoice with positive results almost constantly observed in the PCR reactions performed. Formalin fixation sometimes compromised DNA amplification. Our results are consistent with previous reports investigating the effect of ethanol and formalin fixation on DNA amplification by PCR. Moreover, this is the first study showing that paraffin-embedded tissues fixed with Histochoice can be efficiently used for PCR gene amplification.

Breast Neoplasms↗
PubMed1994

Fixation of bioprosthetic tissues with monofunctional and multifunctional polyepoxy compounds.

Collagen from a native tissue is fixed with a polyepoxy compound (PC) for use as a new biologic prosthetic material. Prior studies have shown that this biomaterial has comparable properties with collagen fixed with glutaraldehyde (GA), and thus has great promise for biomedical applications. A prior kinetic study indicated that the reaction between the functional groups of collagen and the multifunctional epoxy EX-313 is a 2.5th-order reaction. The purpose of this study was to understand the mechanism of the amino acid-PC reactions in a fixation process. Bovine arteries were fixed with a monofunctional PC (EX-131) and a multifunctional PC (EX-313) as a function of fixation time. A sequential fixation with a second fixative was used to identify the available remaining reactive sites from a prior fixation. The denaturation temperature (Td) was measured on each sample. Because the denaturation temperature is a direct indication of crosslinking of individual amino acids with the fixative, the increase in Td of a subsequent fixation may be indicative of the available remaining amino acids. The fixation index was measured on each sample to reflect the increase of fixation completion in a sequential fixation process. The fixation index and crosslink data also revealed that the reactive amino acids for EX-131 and EX-313 may not be exactly the same. The data in this study suggest that a monofunctional fixative can pre-react with the amino acids of collagen to effectively block further fixation of collagen with a second fixative. This amino acid masking may be associated with collagen branching. Collagen branching and its effect on denaturation temperature are described.

Animals↗
PubMed1999

Crosslinking characteristics and mechanical properties of a bovine pericardium fixed with a naturally occurring crosslinking agent.

Currently available crosslinking agents used in fixing bioprostheses are all highly (or relatively highly) cytotoxic, which may induce an adverse inflammatory reaction in vivo. It is therefore desirable to provide a crosslinking agent that is of low cytotoxicty and may form stable and biocompatible crosslinked products. To achieve this goal, a naturally occurring crosslinking agent-genipin-was used by our group to fix biological tissues. Genipin may be obtained from its parent compound, geniposide, which may be isolated from the fruits of Gardenia jasminoides Ellis. In our previous studies, it was found that the cytotoxicity of genipin is significantly lower than both glutaraldehyde and an epoxy compound. Also, it was shown that genipin can form stable and biocompatible crosslinked products. The present study further investigates the crosslinking characteristics and mechanical properties of a genipin-fixed bovine pericardium. Fresh and glutaraldehyde- and epoxy-fixed counterparts were used as controls. It was found that the denaturation temperatures of the glutaraldehyde- and genipin-fixed tissues were significantly greater than the epoxy-fixed tissue, although their fixation indices were comparable. The mechanical properties of fresh bovine pericardium are anisotropic. However, fixation tended to eliminate tissue anisotropy. The tendency in the elimination of tissue anisotropy for the genipin-fixed tissue was more remarkable than for the glutaraldehyde- and epoxy-fixed tissues. In addition, the genipin-fixed tissue had the greatest ultimate tensile strength and toughness among all the fixed tissues. Distinct patterns in rupture were observed in the study: The torn collagen fibers of the genipin- and glutaraldehyde-fixed tissues appeared to be bound together, while those of fresh and the epoxy-fixed tissues stayed loose. The results obtained in the study suggests that tissue fixation in glutaraldehyde, epoxy compound, and genipin may produce distinct crosslinking structures. The differences in crosslinking structure may affect the crosslinking characteristics and mechanical properties of the fixed tissues.

Animals↗
PubMed2003

Early internal fixation and soft tissue cover of severe open tibial pilon fractures.

We studied 32 consecutive patients with open distal tibial pilon fractures. All patients had radical debridement with immediate skeletal stabilisation and early soft-tissue cover with a vascularized muscle flap. The minimum follow-up was 1 (range 1-8) year. There were four superficial infections, two deep infections and two amputations. There were no long-term problems with union and no patient required an ankle fusion. Patients were assessed using the SF-36 questionnaire. There were significant differences from the US norm in physical function score ( p<0.01), role physical score ( p<0.05) and physical component score ( p<0.01). Physical component score of 38.5 was significantly better ( p<0.01) when compared with amputees from severe lower-extremity trauma. Our protocol for management resulted in a good functional outcome with low infection and amputation rates.

Amputation, Surgical↗