Tissue fixation with phenol-formaldehyde for routine histopathology.

The addition of 2% phenol had a marked accelerating effect on neutral buffered 4% formaldehyde as a fixative. Histopathological material fixed in buffered phenol-formaldehyde (pH 7.0) and rapidly advanced to paraffin in an enclosed tissue-processor showed improved nuclear and cytoplasmic detail, reduced shrinkage and distortion, and an absence of formalin pigment. Good results were obtained in less time when sequential fixation in phenol-formaldehyde buffered to pH 7.0 and pH 5.5 was carried out at an elevated temperature (40 degrees C) in the enclosed tissue-processor. Standard histological stains and immunoperoxidase methods worked well. In resin-embedded tissue, buffered phenol-formaldehyde (pH 7.0) gave satisfactory ultrastructural results. The penetration rate of buffered phenol-formaldehyde (pH 7.0) in gelatin models did not differ from that of neutral buffered 4% formaldehyde. Polyacrylamide gel electrophoresis showed enhanced protein polymer formation with buffered phenol-formaldehyde (pH 7.0) as compared with neutral buffered 4% formaldehyde. Protein polymer formation increased in response to increased time and temperature. Cells fixed in suspension in buffered phenol-formaldehyde (pH 7.0) and neutral buffered 4% formaldehyde showed similar volume changes.

Buffered phenol formaldehyde (pH 7.0 and pH 5.5): improved fixation in an enclosed tissue processor.

The addition of 2% phenol has a marked adjuvant effect on 4% formaldehyde as a fixative. Sequential use of phenol formaldehyde buffered to pH 7.0 and pH 5.5 results in improved fixation in material rapidly advanced to paraffin in an enclosed tissue processor. Sections stained with alum haematoxylin and eosin show improved nuclear and cytoplasmic detail, reduced tissue shrinkage and an absence of formalin pigment. Excellent results are obtained with conventional special stains and immunoperoxidase methods.

Quantitation of human milk fat globule (HMFG1) expression in breast carcinoma and its association with survival.

Expression of human milk fat globule (HMFG1) in immunohistochemically stained sections of breast carcinomas was assessed subjectively and objectively from 82 women (age range 41-96 years) to determine its prognostic importance. No correlation was observed between the degree of staining and prognosis even when the subcellular distribution of antigen expression was assessed. The total absence of staining with HMFG1 was possibly associated with a favourable outcome, although this did not quite achieve significance with the small numbers involved. The Quantimet 970 was used for objective semiautomated measurement of immunohistochemical reactions in paraffin wax sections and was found to produce better resolution and to eliminate subjective error.

Measurement of the mass of syncytiotrophoblast in a range of human placentae using an image analysing computer.

Placentae from normal term pregnancies, from "normal" preterm pregnancies and from pregnancies complicated by pre-eclampsia have been analysed by computerized histometry. An immunoperoxidase technique was used to stain specifically human placental lactogen in the syncytiotrophoblast. The percentage and mass of syncytiotrophoblast were measured using an image analysing computer. The percentage of syncytiotrophoblast was found to be remarkably constant throughout gestation. Strong evidence was found that a linear relationship exists between syncytiotrophoblast mass and gestation period and that on average pre-eclampsia placentae have a lower percentage and a reduced mass of syncytiotrophoblast.

The effect of advanced old age on the neurone content of the cerebral cortex. Observations with an automatic image analyser point counting method.

The effect of advanced old age on the nerve cell content of the cerebral cortex was examined in 19 non-demented persons aged 69-95 years, using a Quantimet 720 image analysing computer to make area proportion measurements. Neurone loss around 1.0% per annum was found both in the neocortex and in the medial hippocampus. There was also significant shrinkage of neurones in the hippocampus. Macroscopic measurements of the cerebral hemispheres by means of point-counting morphometry showed a corresponding reduction in the volume of white matter amounting to about 0.8% per annum, but only a minor change in the cortex volume. This finding is consistent with the occurrence of dendritic growth of surviving neocortical neurones. By contrast, there appears to be no compensatory dendritic proliferation in the medial hippocampus since tissue atrophy was commensurate with cell loss in this region.

Prognostic value of measurement of elastosis in breast carcinoma.

The extent of elastosis in the stroma within the anatomical "tumour" in 80 patients with breast carcinoma was assessed by observational microscopy using a 4-point grading system, and measured by semiautomated histometry as an area percentage with a Quantimet 720 image-analysing computer. In this retrospective study, measurement of elastosis in tissue removed at time of initial diagnosis was shown to have little value for prediction of duration of survival.

A study of the histological basis of textural analysis.

In an attempt to quantitate histological structure by textural analysis, the characters of a stained microscopical field were recorded as a frequency polygon of the relative areas defined by successive increments of optical density: this is known as the optical density/area (OD/A) profile. In a study of lymph-nodes in Hodgkin's disease and of carcinoma of the female breast, each stained tissue section was found to have an OD/A profile of characteristic shape: moreover, in each of the lesions studied it was shown that the shape of the profile could be replicated by summing the profiles of each constituent cell type and intercellular matrix in proportions determined by differential cell counts and direct measurement of the degree of cell packing in the tissue. In an investigation of the influence of specimen preparation on OD/A profile, it was shown that density of staining and section thickness were important variables. Using the density of staining of tissue lymphocytes as an internal standard, a simple method was developed of compensating for differences in the density of Mayer's haemalum staining greater than those met with in practice. Attempts to compensate for intentionally large differences in section thickness were much less successful, but this was considered to be relatively unimportant since, in practice, the variation in thickness of sections cut on a modern microtome by a competent microtomist had been shown to be slight and to have a negligible effect on the OD/A profile.

Measurement by Quantimet 720 of the proportion of actively growing cells in tissue cultures of human lymphocytes.

The size-distribution of normal human lymphocytes growing in tissue culture was measured with a Quantimet 720 image-analysing computer. The proportion of cells undergoing volumetric growth after phytohaemagglutinin (PHA) stimulation was dervived from a simple mathematical model describing the growth patterns of the cells up to the first mitotic division. The approach may have general application in biology as a method for measuring the proportion of growing cells by microscopy.

Bone marrow cellularity assessed by point-counting.

A simple method for rapid point-counting of bone marrow biopsies is described; the method gave an accurate assessment of cellularity in 100 aspiration biopsy specimens. Measurements of marrow cellularity by the visual point-counting technique correlated well with those obtained with a Quantimet 720 image-analysing computer.

Cytological basis of histological typing of diffuse Hodgkin's disease. Demonstration of an implied misnomer in the terminology of the Rye classification.

Differential cell counts were made on nine lymph nodes whose structure was replaced by diffuse Hodgkin's disease; two of these nodes had the classical histological appearance of the lymphocytic predominance subtype, four of the mixed cellularity subtype, and three of the lymphocytic depletion subtype. Our attempts to achieve valid sampling methods are recorded. The counts, in general, confirm the postulated histological basis of the Rye classification of the subtypes of the diffuse disease. The major discrepancy is that, contrary to the histological descriptions, our direct counts have shown that lymphocytes, are, in general, more numerous in the lymphocytic depletion than in the mixed cellularity subtypes. The cell counts also show that normal mononuclear cells (mainly fibroblasts and macrophage-type cells) are much more numerous in the mixed cellularity subtype than in the other forms of diffuse Hodgkin's disease; this feature has not been emphasised in the Rye classification. On the basis of our differential counts, a hypothesis is proposed that could explain the natural history of the different subtypes of diffuse Hodgkin's disease as the resultant of three processes: (a) tumour aggressiveness, (b) specific cell-mediated immunological reactions, and (c) non-immunological stromal responses.

Grading of astrocytomas using a Quantimet 720 image-analysing computer.

Cerebral astrocytomas and undifferentiated gliomas were graded by (1) Kernohan classification on the basis of visual microscopy, and (2) optical density/area profiles obtained by automated densitometry with a Quantimet 720 image-analysing computer. In a retrospective study of 44 patients the Quantimet measurements seemed to give a more accurate assessment of prognosis than visual grading.

Renal hyalin. A study of amyloidosis and diabetic fibrinous vasculosis with new staining methods.

This describes the sodium sulphate-Alcian Blue (SAB) method for staining amyloid in paraffin sections. Its value lies in the possibility of subsequent counterstaining and thus of revealing the structural relationships of amyloid. In the kidney the topical disposition of amyloid closely resembles the disposition of fibrin in the kidney of diabetics; this suggests that upset in vascular permeability plays a part in determining the site of the amyloid deposits. Furthermore, an aging process in amyloid can now be envisaged resembling the aging of extraluminal fibrin. Both materials proceed to a hyalin material that, staining like collagen, merits the name pseudo-collagen. This term we apply to a hyalin, staining like collagen, for which, we can postulate a specific precursor.