Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
1997-04-14
2001-11-20
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S001100, C435S001200, C435S003000, C435S040520, C568S420000, C568S422000, C568S449000, C514S694000, C424S466000, C424S474000, C424S477000
Reexamination Certificate
active
06319683
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to the fixation of cell and tissue specimens for biological research or medical testing using a solution of formaldehyde. The present invention particularly relates to a method and a composition for chemically controlling the fixation time of such specimens.
BACKGROUND OF THE INVENTION
Fixation is the first important step in preparing cell and tissue specimens for use in a wide range of analytical tests. Some exemplary tests include immunohistochemistry (IHC), flow immunocytometry, in situ hybridization (ISH) with nucleic acid probes, in situ polymerase chain reaction (PCR), and PCR. These tests are typically used to detect particular DNA or RNA sequences, peptides, proteins, or other kinds of biomolecules, drugs, and general analytes.
Fixation stabilizes microscopic cellular structures and compositions in the specimens to allow them to withstand subsequent processing and to preserve them for retrospective analyses. The fixed cell and tissue specimens can also be used to extract biosynthetic molecules for biochemical or nucleotide sequence analysis. Without fixation, it would be difficult, if not impossible, to sensitively detect, localize, and quantitate biosynthetic or environmental molecules in many kinds of cell and tissue specimens.
A good fixative should harden cell and tissue components to prevent decomposition, putrefaction, and autolysis. The physico-chemical process of tissue modification by a fixative is gradual and complex, involving diffusional penetration into the tissue and a variety of potential chemical reactions. To date, no ideal fixative has been found, i.e., a fixative that perfectly preserves cellular morphology and yet does not modify the specimen composition so as not to change the reactivity of the analyte species therein for subsequent detection. Because of this predicament, the selection of a particular fixative generally entails multiple considerations. Thus, there are many fixatives currently in use.
A fixative with a high content of alcohol or other organic solvent, particularly when acidified with a mild organic acid, hardens tissue specimens by precipitation and coagulation. Such a fixative has several advantages. First, since the fixative does not covalently modify the constituent molecules in the tissue, the reactivity of most antigens in the tissue toward antibodies remains very high. Second and for the same reason, nucleic acids in the tissue may be easily extracted in good condition. Third, the fixative may be completely flushed out of the tissue by rehydrating the tissue in a buffered solution.
Such a fixative, however, has one major drawback. The drawback is that the microscopic morphology of an alcohol- or other solvent-fixed tissue is not as detailed as that of a tissue fixed with a covalent-binding fixative.
In contrast to an alcohol- or other solvent-based fixative, a covalent-binding fixative, such as formaldehyde, provides excellent cellular preservation. Formaldehyde (CH
2
O) was first reported as a tissue preservative by F. Blum in 1893 (10 Z.
Wiss. Mikrosc.
314). It is now the most widely used fixative in histopathology because it is cheap, simple to use, and provides consistent results. A formulation of formaldehyde found in most U.S. and foreign research and clinical laboratories is neutral buffered formalin (NBF). It may also be called “buffered neutral formalin.” See R. Lillie,
Histopathologic Tech.
300 (1948). Other formaldehyde formulations include: 10% formalin; alcoholic formalin; calcium acetate formalin; Bouin's Fluid (containing picric acid or acetic acid, pH 1.6); Cajal's formalin-ammonium bromide; formalin/alcohol/acetic acid; paraformaldehyde (polymerized formaldehyde); and formol-saline (G. Clark,
Staining Procedures
13-16 (1981)).
The commercially available saturated aqueous formaldehyde stock containing 10% methanol stabilizer is called formalin. Its formaldehyde concentration can be denoted in several ways. In particular, it can be denoted as a 100% saturated, a 37% w/w, a 40% w/v, or a 13.3 M solution. A usual working dilution of this stock for fixatives is 1:10 (initial:final) by volume. Such a dilution produces a 10% saturated solution which can also be denoted as 3.7% w/w, 4.0% w/v, or 1.3 M formaldehyde. For example, the standard NBF solution of the U.S. Armed Forces Institute of Pathology Formulation (AFIP) is a 1:10 v:v dilution of formalin in a phosphate buffer at a pH of 6.8 to 7.2 (
Laboratory Methods in Histotechnology
(eds. Edna Prophet et al., 1992)).
The chemistry of aqueous formaldehyde has been thoroughly reviewed, notably in J. Walker's treatise Formaldehyde, ch. 8 (1944). Formaldehyde is a gas that rapidly combines with water (>99.9% according to C. H. Fox et al., 33
J. Histochem. Cytochem.
845-853 (1985)) to form the hydrate, methylene glycol:
CH
2
O+H
2
O→CH
2
(OH)
2
Methylene glycol can be polymerized by commercial processes to form paraformaldehyde (polyoxymethylene glycol):
nCH
2
(OH)
2
→(CH
2
O)
n
.H
2
O+(n−1)H
2
O
which is sometimes used instead of formalin in the formulation of formaldehyde fixatives. In a neutral to alkaline buffered solution, paraformaldehyde depolymerizes to methylene glycol which dehydrates into an equilibrium with active carbonyl formaldehyde (Walker, supra, pp. 74-75). Thus, its effect on tissue is the same as diluted formalin.
Methylene glycol rapidly penetrates into tissue by diffusion at a rate that varies inversely with the tissue specimen temperature. Tissue penetration has been measured at 0.5 cm in 8 hours in rabbit liver (W. T. Dempster, 107
Am. J. Anat.
59-72 (1960)). Dehydration of methylene glycol within the tissue maintains an effective level of reactive carbonyl formaldehyde. Fixation reactions of carbonyl formaldehyde are much slower than the rate of penetration and are temperature dependent. Thus, when
14
C formaldehyde was applied to semi-thin (16 &mgr;m) tissue sections of rat kidney, the binding reaction took over 24 hours to reach equilibrium (C. H. Fox et al., 33
J. Histochem. Cytochem.
845-853 (1985)).
Formaldehyde, which is a very reactive electrophilic species, fixes tissue by combining with proteins and nucleic acids therein (Feldman, 13
Prog. Nucleic Acid Res. Mol. Biol.
1-49 (1973)). Formaldehyde modifications of the tissue proceed in two kinetically distinct stages. Initial reactions modify primary amines (lysine) and thiols (cysteine), and purine but not pyrimidine bases of nucleic acids, forming mono- and di-methylol derivatives, not Schiff bases. Regardless of whether the reaction involves nucleotides, nucleic acid polymers, amino acids, or proteins, this stage reaches equilibrium within 24 to 48 hours. These labile adducts are rapidly reversible if the formaldehyde is removed from the tissue.
Subsequent reactions involve the methylol derivatives that are covalently bound in the tissue. These secondary reactions form methylene crosslinks which are not reversible upon washing. In proteins, the secondary crosslinking reaction occurs via methylene bridges that join the first-modified sites to adjacent, less reactive functional groups including primary amides (glutamine, asparagine), guanidine groups (arginine), and tyrosine ring carbons (H. Fraenkel-Conrat & H. S. Olcott, 70
J. Am. Chem. Soc'y
2673 (1948)). This reaction is very gradual, accumulating over at least 30 days of fixation, and generates relatively stable covalent crosslinkages. In nucleic acids, secondary reactions also result in chain crosslinking. In addition to chain crosslinking, the reaction can produce crosslinkages between nucleic acids and proteins.
All of these secondary reactions produce a lattice of crosslinkages within and between macromolecules in the fixed tissue. The net effect of all covalent modifications is to partially denature the biopolymers in the tissue by interfering with the normal noncovalent bonding patterns of the charged protein side chains, and to lock the conformation into an inflexible configuration, i.e., the secon
Hoag Stephen W.
James William M.
Foley & Lardner
Intergen Company
Lankford , Jr. Leon B.
LandOfFree
Method and composition for controlling formaldehyde fixation... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method and composition for controlling formaldehyde fixation..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method and composition for controlling formaldehyde fixation... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2582101