Three-dimensional reconstruction of non-Hodgkin's lymphoma in bone marrow trephines.

The objective of this study was to analyse the location in the bone marrow of deposits of low-grade non-Hodgkin's lymphoma. This was achieved using computer-generated three-dimensional reconstruction techniques applied to serial tissue sections of five bone marrow trephines involved by lymphoma. For comparative purposes, previously published three-dimensional models of benign lymphoid aggregates in the bone marrow were need. The images generated by this new study showed that deposits of low-grade non-Hodgkin's lymphoma involving the bone marrow always assumed a paratrabecular pattern of infiltration at some point. This is in direct contrast to the pattern of bone marrow infiltration shown by benign lymphoid aggregates. It is concluded that location within the marrow space is a crucial factor in distinguishing between benign and low-grade malignant lymphoid infiltrates in the bone marrow.

Lymphoid infiltrates in B cell non Hodgkin's lymphoma: comparing nuclear characteristics between lymph node and bone marrow; and evaluating diagnostic features of bone marrow infiltrates in paraffin embedded tissues.

Distinguishing non Hodgkin's lymphoma from benign lymphoid aggregates in bone marrow is well recognised to be difficult. Our objective was to evaluate nuclear morphology, and to perform morphometry on benign and neoplastic lymphoid infiltrates, to establish if objective criteria were of value in the diagnosis of neoplasia. By comparing neoplastic infiltrates in bone marrow with infiltrates in lymph nodes, the validity of grading non Hodgkin's lymphoma on the basis of bone marrow histology alone was assessed. 82 cases of B cell non Hodgkin's lymphoma (44 low grade and 38 high grade), known to have both lymph node and bone marrow involvement at the time of presentation, were compared with bone marrow trephines containing reactive lymphoid infiltrates. The results suggest that in paraffin embedded tissue from bone marrow trephines, nuclear morphology, nuclear area and the nuclear contour index cannot, in most cases, be used to distinguish reactive from neoplastic lymphoid infiltrates although mean nuclear area supports a diagnosis of a neoplastic infiltrate if it can be shown that the nuclei are larger than would be expected in reactive infiltrates. Such differences are subtle and often not appreciable without the use of quantitative techniques. The frequent occurrence of discordant infiltrates in high grade lymphomas means grading of lymphoma on the basis of bone marrow appearances is often unrealistic.

Standardized mitotic counts in breast cancer. Evaluation of the method.

Twenty-one pathologists and technicians participated in a study evaluating the variation present in mitotic counts for prognostication of breast cancer. The participants counted the mitotic figures in 20 breast cancer samples from ten high power fields (mitotic activity index, MAI, giving the results in mitotic figures per 10 fields) and also made a correction for field size and area fraction of the neoplastic epithelium to get the standardized mitotic index (volume fraction corrected mitotic index, or M/VV index, giving the result in mitotic figures per square mm of neoplastic epithelium). The difference in variation between the two methods was not big, but the standardized mitotic index (SMI) showed consistently smaller variation among all participants and different subgroups. Experienced pathologists had the highest variation in mitotic counts, and specially trained technicians, the lowest. The efficiency of the mitotic counts in grading (the grading efficiency) was used to evaluate the mitotic counts. In groups without special training for mitotic counts the mean grading efficiency was lower (experienced and training pathologists both on average had the potential to grade 88% of the cases correctly) than in the group specially trained for the purpose (trained technicians had the potential to grade 95% of the cases correctly). Among the specially trained technicians, the grading efficiency was of the same magnitude as the grading efficiency achieved in determining the S-Phase fraction of cells from paraffin embedded breast cancers by flow cytometry in different laboratories. The results suggest that special training is helpful in making mitotic counts more reproducible, and that in trained hands, the mitotic counts give results comparable to more sophisticated methods of determining proliferative activity in breast cancer.

Three-dimensional reconstruction of benign lymphoid aggregates in bone marrow trephines.

Distinguishing between deposits of low-grade non-Hodgkin's lymphoma and benign lymphoid aggregates in bone marrow trephine sections is a recognized problem in haematopathology. To test the hypothesis that benign lymphoid aggregates do not make contact with a trabecular surface, three-dimensional models were constructed of five serially sectioned bone marrow trephines, containing a total of 19 lymphoid aggregates known to be benign. The computer-generated images showed that benign lymphoid aggregates were located in the central marrow space and did not become paratrabecular. This suggests that a paratrabecular location may help in some cases to distinguish deposits of low-grade non-Hodgkin's lymphoma from benign lymphoid aggregates.

Distribution of alveolar wall per unit volume in the human lung.

Firstly, to assess the reproducibility of a computerised image analysis method of measuring alveolar size in terms of the alveolar wall surface area per unit volume (AWUV) in normal non-smoking adult human lungs fixed in inflation, one lung was fixed by simple manual prebronchial perfusion with formaldehyde (wet-fix), another by prolonged perfusion with formaldehyde followed by air until semi-dried (semi-dry). Blocks from both lungs were taken to assess the relative value of plastic and paraffin wax embedding. There was no statistical difference in AWUV between plastic and paraffin sections from each lung, but the wet-fix method gave statistically significantly (P < 0.05) higher AWUV readings than the semi-dry method. Secondly, when a whole semi-dry lung slice was divided into 66 blocks, the AWUV distribution was not random but largest in the centre and smallest in the periphery anteriorly and superiorly. This is obviously important in sampling of lung tissue for morphometry and other purposes.

Three-dimensional reconstruction of human embryonic notochords: clue to the pathogenesis of chordoma.

Three-dimensional reconstruction experiments performed on serial sections of human embryos showed that the anatomy of the caudal and rostral ends of the notochord was complex. Forking of the ends, with separate fragments of chordal tissue, was demonstrated and these provide a way by which notochordal cell rests could be left behind in the basicranial and sacral regions when the notochord involutes elsewhere. Assuming the histogenesis of chordomas from notochordal cell rests, this would furnish an explanation for the observed skeletal distribution of chordomas.

Three-dimensional reconstruction: methods of improving image registration and interpretation.

(i) Image registration. The use of serial images for computerised three-dimensional reconstruction necessitates the inclusion of three separate sources of information at the stage of data input. These are (i) artificial registration points or fiducials, (ii) a calibration scale and (iii) an outline of each slab of the section to be included in the reconstruction. Most traditional methods rely on the production of drawings of the contours of the structure under investigation which also include both registration points and a calibration scale. We report on a method which considerably reduces the time involved at this labour intensive stage of reconstruction and in addition allows subsequent reconstructions of different structures to be performed without new drawings. Use is made of computerised alignment of tissue sections and the production of composite photomicrographs of both the tissue under investigation and an accurately registered stage micrometer scale. (ii) Improving image interpretation. Images derived from computerised three-dimensional reconstruction can be affected by the number of coordinates used to form the contour of each slice of a structure and by the number of slices that are used to construct the final model. Too little or too much data can considerably reduce the ability of the observer to interpret accurately the image generated by the computer. We report on a feature-based method which enables the experimenter to assess objectively the amount of data required in the two-dimensional plane, i.e. the number of data points per slice, and the three-dimensional plane, i.e. number of slices per structure, so that optimal reconstructions are generated.

Ethanol-induced chronic myopathy in the young rat: a light and electron microscopic study in type I or type II fibre-rich skeletal muscles.

An investigation was made into the characteristics of an experimental chronic alcoholic myopathy in the young rat. Male Wistar rats were fed a diet for 6 weeks in which ethanol comprised 36% of total energy. Controls were pair-fed the same diet except ethanol was substituted by isoenergic glucose. Soleus (type I fibre-rich) and plantaris (type II fibre-rich) muscles were examined by light microscopy, histochemistry and electron microscopy. Muscles from ethanol-fed rats showed a low-grade myopathy and the diameters of individual type II fibres were reduced. Infrequent atrophic fibres were necrotic and undergoing phagocytosis. Ultrastructurally, there were no observable differences in the subcellular organelles of the alcohol-fed and control rats. It was concluded that alcohol causes a specific myopathic process in the rat, selectively affecting type II fibres. These changes correlate well with the abnormalities seen in human chronic alcoholic myopathy.

Nuclear morphometry of primary B cell thyroid lymphoma.

Thyroid lymphoma is usually distinguished from anaplastic thyroid carcinoma and from Hashimoto's thyroiditis by morphological and immunohistochemical assessment of tissue sections. Our objective was to assess the value of nuclear morphometry in the differential diagnosis of these conditions. Nuclear area measurements were performed on 10 cases of thyroid lymphoma using an IBAS 2000 Image Analyser and compared with similar measurements performed on 10 cases of Hashimoto's thyroiditis and 2 of anaplastic thyroid carcinoma. It was found that karyometry demonstrated differences between all three conditions, the cases of thyroiditis being distinguishable from lymphoma on the basis of mean nuclear area alone. Mean nuclear area for lymphomas was greater than for Hashimoto's thyroiditis and lower than for anaplastic carcinomas. The mean nuclear area also reflected the grade of lymphoma, with the exception of one case which had a large reactive T cell population. It is concluded that nuclear morphometry provides valuable information in the diagnosis and assessment of thyroid lymphomas.

Comparison of stains for image segmentation and measurement of nuclear parameters by computerised image analysis using IBAS 2000.

Nuclear measurements using image analysis largely depend upon the quality of the image presented for digitization. To investigate the effects of nuclear stains on image segmentation of nuclei, serial sections of kidney were stained by eleven different methods and presented to an IBAS 2000 interactive image analysis system (Kontron Bildanalyse) via a Zeiss IIIRS microscope at x 800 magnification and a Siemens K30 video camera. Digitised grey level images of each field were processed by an interactive technique and by an automatic segmentation procedure (thresholding). Nuclear areas were measured by each method and the results compared. We conclude that of the stains assessed the uncounterstained haematoxylins offer the best image segmentation for nuclear measurements. Thresholding techniques are suitable for performing measurements using these stains, particularly when additional interactive techniques are used to reject unwanted structures and to separate overlapping nuclei after segmentation. Comparable areas stained with five of the stains were studied to see if the staining techniques themselves affected nuclear area. Our results show that the use of different stains will substantially affect measurements of nuclear dimensions.

Tissue distortion in three-dimensional reconstruction of wax or plastic embedded microscopic structures.

Three-dimensional reconstruction at the light microscopic level depends on obtaining reliable serial sections without "distortion" i.e., expansion and compression during section preparation. We have studied the extent of such distortion in serial sections from paraffin and resin embedded blocks of brain, kidney, liver and lung, using an IBAS 2000 Image Analyser. We found that, taking the uncut block as 100%, the section area, perimeter and minimum diameter varied by no more than 8%, except for the lung sections which varied up to 14%. There was no progressive compression due to knife bluntening. Resin sections also varied up to 8% (16% for lungs) but in addition creasing was a problem. We conclude that, provided the serial sectioning is carefully standardised for block shape and orientation, floating out temperature and time, serial paraffin sections are more suitable for three dimensional reconstruction than resin sections.

A method of image registration for three-dimensional reconstruction of microscopic structures using an IBAS 2000 image analysis system.

Registration of histological tissue sections is a longstanding problem in three-dimensional reconstruction. Our solution is to insert rigid straight narrow birefringent structures namely cactus spines as artificial registration points (ARPs) into the tissue block before embedding in wax or resin. The microscopic image of the tissue section with ARPs in situ is presented to an IBAS 2000 Image Analyser and digitised. Registration is performed in two stages using the ARP nearest to the area to be reconstructed as the principle reference point for alignment. The automatic stage is then used to align the structure under reconstruction. These reconstructions are more accurate than those produced without the use of ARPs.