Ultrastructural morphometry of human leucocytes in health and disease

Electron Mierosc. Rev., Vol. 4, pp. 179-195, 1991. ~'rinted in Great Britain. All rights reserved.

0892-0354/91 $0.00 + 0.50 © 1991 Pergamon Press plc.

U L T R A S T R U C T U R A L MORPHOMETRY OF H U M A N LEUCOCYTES IN HEALTH A N D DISEASE R. J. S O K O L * t , G. H U D S O N * , J. W A L E S * a n d N. T. J A M E S +

Departments of *Haematology and of SBiomedical Science, University of Sheffield, Sheffield, $10 2TN, U.K. Abstract--In this review, the literature on ultrastructural morphometry of each of the main types of human blood

leucocytes has been considered, together with the technical and numerical procedures essential for valid analysis. Quantitative data have been reported for these cell types in health and comparisons have been made with those in disease states. In monocytes, and in macrophages developing from them, subtle ultrastructural differences have been detected and quantitated in malignant lymphoma; as the mononuclear phagocytes were not themselves neoplastic, the changes may have related to defects in host defence. Change in the ultrastructural characteristics of leukaemic monoblasts have also been reported. Lymphocytes and malignant lymphoid cells have been extensively investigated: differences between different types and subsets have been shown to be present in both normal lymphocytes and their malignant counterparts in leukaemias and lymphomas. Particular attention has been paid to morphometric assessment of nuclear shape and size in these disorders and to its possible value as a diagnostic tool. Granulocytes have so far been the subject of few morphometric studies, although in hypereosinophilic syndrome, cellular changes have been defined and have thrown light on the abnormal pattern of degranulation. There have also been scattered reports on the cells of acute myelogenous leukaemia. The use of computers and sophisticated statistical packages has greatly facilitated the application of multiple comparison procedures and has permitted discriminant analysis to be carried out where appropriate. This review shows that ultrastructural morphometry of leucocytes will have an increasing application in clinical pathology.

CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

II. Monocytes and macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IIl. Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Technical and numerica[ considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. I N T R O D U C T I O N

179 180 183 187 189 191

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readily available a n d p r e p a r a t i o n artefacts can be kept to a m i n i m u m . A b l o o d sample is t h o u g h t to be representative of the circulating b l o o d p o p u l a tion a n d being already in suspension, the leucocytes are in the r a n d o m o r i e n t a t i o n essential for proper m o r p h o m e t r i c investigation. This also allows the parallel use o f a d d i t i o n a l techniques such as cell s u b t y p i n g , s u s p e n s i o n culture, cytochemistry a n d f u n c t i o n a l assessment. I n this review, the c u r r e n t state o f knowledge relating to the ultrastructural m o r p h o m e t r y of each o f the m a i n types o f leucocyte will be discussed a n d the technical a n d n u m e r i c a l procedures required

U l t r a s t r u c t u r a l m o r p h o m e t r y of h u m a n leucocytes has o p e n e d a p r o m i s i n g field of investigation. In a d d i t i o n to the usual a d v a n t a g e s o f objectivity a n d reproducibility, the q u a n t i t a t i v e i n f o r m a t i o n relating to cellular m o r p h o l o g y of defined leucocyte p o p u l a t i o n s has p e r m i t t e d valid c o m p a r i s o n s in health and disease. Blood leucocytes are particularly suited to such studies. Peripheral b l o o d cells are tCorrespondence to Dr R. J. Sokol, Regional Blood Transfusion Centre, Longley Lane, Sheffield $5 7JN, U.K. 179

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for valid analysis considered. The extent of reported study on the different cell types has varied widely and it is fair to c o m m e n t that the minimal conditions for numerical validity have not always been met. However, the potential value of these techniques in providing information not available from subjective assessment will be apparent. As more of our personal experience in this field has been related to blood monocytes (and macrophages derived from them), this cell line will be considered first.

II. M O N O C Y T E S A N D M A C R O P H A G E S Blood monocytes can provide valuable information about the whole of the mononuclear phagocyte system which has an essential role in host defence (Sokol and Hudson, 1983). In order to provide quantitative data for the three-dimensional features of the normal model cell, we carried out ultrastructural m o r p h o m e t r y on the blood monocytes of 20 healthy individuals (Sokol et al., 1985b; Sokol, 1989). Table 1 shows some of the measurements obtained. These were in general agreement with the results of other smaller studies (James, 1978; Schmid-Sch6nbein et al., 1980; Chien et al., 1984). There has also been a recent report on the m o r p h o m e t r y of monocyte granules (SchmidSch6nbein and Chien, 1988, 1989). Our methodology and analytic procedures will be summarized in Section V. The model of the blood monocyte in health has enabled comparisons to be made with those

Measurement

obtained when similar morphometric studies were carried out in patients with malignant l y m p h o m a (Sokol et al., 1985a; Sokol, 1989). The main differences found in the blood monocytes of 23 patients with Hodgkin's disease related to the mitochondrial and nucleolar contributions to the cellular ultrastructure (significantly smaller mitochondrial and nucleolar volume fractions, volumes and surface areas being found). In 12 patients with nonHodgkin's lymphoma, the main differences from normal related to the nuclear parameters (the nuclear volume, surface area and euchromatin volume being significantly larger and the nucleolar volume fraction, volume and surface area being smaller). Irrespective of the different patterns of change found in these two disease groups, the important point established by this work was that ultrastructural changes can be detected in blood monocytes in malignant lymphoma, which may reflect a disturbance of the mononuclear phagocyte system as a whole. This would be consistent with the m a n y different alterations in functions which have been reported in mononuclear phagocytes in malignant disease (Sokol and Hudson, 1983). One might speculate that the particular changes observed in Hodgkin's disease implied reduced energy potential in the monocytes with less active protein synthesis and this would be consistent with reported reductions in monocyte function: these include chemotaxis (Leb and Merritt, 1978; Lukacs et al., 1980, 1983), phagocytosis (Cruchaud et al., 1977; Estevez et al., 1980, 1985; Urbanitz et al., 1975), antibody-dependent cellular cytotoxicity (Kohl et al., 1980), microbicidal activity (Cruchaud

Table 1. Morphometric Measurements on Normal Leucocytes(means +__SEM) Lymphocytes3 Macrophages2 (T and non-T Neutrophil3 Monocytes~ (6 day culture) combined) granulocytes

Cell Volume (fl) Surface area (pm2) *Membrane excess (#m 2) Nucleus Volume (fl) Suface area (/~m2) *Membrane excess (#m 2)

Eosinophil 4 granulocytes

270 _+_7.1 281 ± 5.9 79 ± 5.9

775 ± 120 843 _+_+83 450 ± 48

123 ± 3.2 174 _+6.4 54 ± 6.4

257 ± 5.1 250 _+_3.7 55 ± 3.7

255 _+_10 228 ± 6.3 34 _+6.3

71 +_2.0 137 ± 3.5 55 ± 3.5

136 ± 17 180 + 15 56 ± 6.8

53 ± 1.7 83 ± 2.2 15 ± 2.2

54 ± 1.7 158 + 4.6 89 + 4.6

51 ± 2.6 116 _+5.1 49 ± 5.1

References: 1, Sokol et al., 1985b; 2, Sokol et al., 1988b; 3, James, 1980; 4, Sokol et al., 1987b. * Difference between actual surface area and surface area of sphere of equivalent volume.

Human LeucocyteMorphometry et al., 1977; Estevez et al., 1980, 1985; Leb and

Merritt, 1978), superoxide anion production (PerezSoler et al., 1985) and cellular acid phosphatase activity (Berenyi et al., 1986). The use of ultrastructural morphometry of blood monocytes to predict subject group was explored by examining the data (Sokol et al., 1985a; Sokol, 1989) with the multivariate statistical technique of discriminant analysis. This technique, to be discussed further in Section V, enables the discriminatory value of all the selected morphometric measurements to be considered simultaneously. In general terms, it has been found to be a particularly appropriate technique for classification problems in biology and pathology where cellular features can be readily assessed (Baak and Oort, 1983); when used in conjunction with morphometry, it can provide an objective as opposed to a subjective basis for classification. In the present context with three subject groups, the overall agreement between predicted and actual group was 64% (Sokol et al., 1989) and whilst this would be of little value for diagnostic purposes, it is noteworthy that the prediction as to whether a subject was healthy or had malignant lymphoma was correct in 80% of cases. Circulating monocytes are non-dividing cells at an intermediate stage of the mononuclear phagocyte life-cycle and are en route to the tissues where they transform into macrophages (van Furth et al., 1979; van Furth and Sluiter, 1985); a similar transformation occurs in tissue culture. The question arose as to whether the differences detected in blood monocytes in lymphoma might be accentuated or become more apparent if the cells were studied during further development. Earlier electron microscopic observations on skin window macrophages supported such a possibility (Sokol et al., 1979, 1980, 1985d). In order to address this question, a method was developed for studying blood monocytes in suspension culture, the cells being in the random orientation necessary for ultrastructural morphometry (Sokol et al., 1985c). Initially, studies were carried out to establish the normal pattern of development of monocytes into macrophages over six days of culture (Sokol et al., 1987a; Sokol, 1988a) and quantitative data were obtained on the sequential morphological changes

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which take place during this period. The increases in cell size and surface complexity (illustrated by comparing monocytes and six-day macrophages in Table 1) had previously been reported as occurring during macrophage maturation (Sutton and Weiss, 1966; Sutton, 1967; Fedorko and Hirsch, 1970; van der Rhee et al., 1979) and similar changes in normal human monocytes cultured in suspension had been shown with electronic sizing (Stevenson et al., 1981). There were also increases in mitochondrial size and number and in the volume and surface area measurements of nucleoli (Sokol et al. 1987a; Sokol, 1988a). These changes were interpreted as reflecting the increasing functional activity of the cells. Using the same culture system, ultrastructural morphometry was employed to compare the developmental patterns of monocytes obtained from healthy subjects with those in untreated patients with malignant lymphoma. Morphometric measurements were made on electron micrographs of cells at five intervals during the six days of suspension culture and the results were subjected to multivariate and univariate analysis of variance (MANOVA and ANOVA). In the first series, cells obtained from 22 patients with Hodgkin's disease were compared with those from 20 healthy subjects (Sokol et al., 1988b; Sokol, 1988b). In the model cell relating to Hodgkin's disease, there were significantly smaller increases in the measurements for the whole cell and in those for mitochondrial and nucleolar volumes and surface areas; mitochondrial numbers failed to increase and nucleolar numbers were actually reduced. These findings were taken as indicating that in the cultures, there was a disruption of the normal pattern of development of monocytes into macrophages in the Hodgkin's group, with impaired growth in size and surface complexity of the cells, and impaired development of mitochondria and nucleoli. Such changes are presumably associated with inadequate functional development and would be consistent with the evidence for a generalized mononuclear phagocyte disturbance in Hodgkin's disease already discussed above. In a second series, we used ultrastructural morphometry to compare the development of cells obtained from 20 patients with non-Hodgkin's

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lymphoma with 20 healthy individuals (Sokol et al., 1988c; Sokol, 1990). The findings were generally similar to those of the first series and again showed a disturbance in the normal pattern of monocytemacrophage development. Such disturbed development would be in keeping with the many reports of mononuclear phagocyte dysfunction in patients with non-Hodgkin's lymphoma [affecting phagocytosis (Ghosh et al., 1973; Urbanitz et al., 1975), antibody-dependent cellular cytotoxity (de Mulder et al., 1984), microbicidal activity (Cline, 1973; King et al., 1975; Estevez et al., 1980; Hamminga et al., 1982), superoxide anion production (Perez-Soler et al., 1985) and glucose metabolism (King et al., 1977)]. The biological significance of such impaired ultrastructural development is still unknown, although it may be related to premature activation (Sokol et al., 1985d). Impairment was present in both Hodgkin's disease and non-Hodgkin's lymphoma at the blood monocyte stage and continued during development in suspension culture. One possibility is that there was an intrinsic change in the cells themselves. This could have been present from the promonocyte or stem cell stage and might, for example, have taken the form of an inadequate expression of receptors for a growth factor. It is of interest in this connection that a disordered development and/or differentiation in cell lineages has been postulated as underlying the immunological deficiency of Hodgkin's disease (Katz, 1981; Katz and Habeshaw, 1985) and that altered functional activity of monocytes in Hodgkin's disease has been corrected with cyclo-oxygenase inhibitors (Estevez et al., 1985). Another possibility is that the mononuclear phagocytes were affected by factors present in the serum of patients with lymphoma. As autologous serum was an integral component of the suspension culture system we used, any such factors would also affect cell development in vitro. There could, for example, have been an increase or a decrease in the concentration or activity of normal factors in the serum. Various serum proteins, including IgG, fibrinogen and fibronectin have been found to have significant activity in promoting the maturation of monocytes into macrophages (Akiyama et al., 1988). Cell surface receptors for normal growth factors (Golde et al., 1990) might

also have been blocked by abnormal factors in the serum. A third possibility is that lymphokines released by circulating lymphocytes or by lymphocytes co-cultured with the mononuclear phagocytes were important: these are known to have profound effects on macrophage development and activities (Adams et al., 1985; McKeever and Spicer, 1980). It would appear that the next step should be to evaluate the role of such factors in mononuclear phagocyte dysmaturation: ultrastructural morphometry with suspension culture of blood monocytes should provide a particularly suitable method for doing this. Discriminant analysis was also used in the study of mononuelear phagocytes in culture (Sokol et al., 1990). All data obtained from blood monocytes grown in suspension culture over the period of six days (i.e. all measurements of whole cell, nucleus, nucleolus and mitochondria for each subject group) were included in the analysis. The combined data were then used to predict the subject group from which an individual specimen had been obtained, and the findings were compared with the known diagnosis. Over 80% of subjects were correctly classified as between the three groups (normal, Hodgkin's disease and non-Hodgkin's lymphoma) and over 90% as to their normality or otherwise. This level of prediction accuracy is much better than that obtained for the blood monocyte study (discussed above) and is comparable with that reported from other classification studies of malignant cells; for example, accuracies of approximately 95% were found in studies of malignant cells in lymph nodes of patients with mycosis fungoides (van der Loo et al., 1980; Meijer et al., 1980), in imprints of biopsy specimens of large cell lymphomas (Ball et al., 1985) and in bone marrow smears from children with acute lymphoblastic leukaemia (Seshadri et al., 1985). In the light of this, a level of prediction accuracy of 90% between normal and patient groups was particularly noteworthy, as it was achieved by studying defence cells, not belonging to the malignant cell line. Caution needs to be exercised in interpreting such changes in mononuclear phagocytes in malignant lymphomas as one would not expect changes in defence cells to be entirely specific in nature, and dysfunctions in these cells have been observed in a

Human LeucocyteMorphometry whole range of other malignant disorders (Sokol and Hudson, 1983). In a test observation on a 43-year-old, apparently normal woman, using the model data, discriminant analysis allocated her developing macrophages to the non-Hodgkin's group; two months later she was found to have a poorly differentiated carcinoma cervix in situ. This observation illustrated that while the changes in mononuclear phagocytes may not be diagnostic, they can be an indication of malignant disease in its early stages. Defence cells have also been studied in a semi-quantitative investigation of cultured monocytes in acute lymphoblastic leukaemia of childhood, an arbitrarily defined cellular differentiation index indicating impaired morphological differentiation (Tsukada et al., 1985). The main conclusion of this section is that detailed examination of mononuclear phagocytes can be of value in assessing the state of the defence mechanisms in individual subjects and ultrastructural morphometry has a particular application in this. Few studies of malignant leucocytes of the mononuclear phagocyte lineage have been carried out using ultrastructural morphometry. Some basic morphometric data has been obtained for leukaemic monoblasts (Dobreva and Meshkov, 1979; Schumacher et al., 1973a, b; Ochiai and Eguchi, 1978) and recently a multivariate morphometric analysis has been carried out on malignant cells from six patients with acute monoblastic leukaemia (James et al., 1988). The latter study showed a highly significant decrease in the surfaceto-volume ratios of both the mitochondria and the nucleus as compared with those of normal monocytes [which were similar to those in our own studies discussed above (Sokol et al., 1985b)]. These changes were taken to reflect a larger total mass of mitochondria and an increase in nuclear size in the malignant cells. Morphometric methods have also been employed to demonstrate the highly irregular shape of the nuclei of histiocytic sarcoma cells (van der Valk et al., 1984). It may be of interest to note that threedimensional reconstructions have provided information about the giant lysosomes of macrophages in the murine counterpart of Chediak-Higashi syndrome but this technique has not so far been

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applied to the human condition (Strausbauch and Sehgal, 1989).

III. LYMPHOCYTES There have been many studies of the ultrastructural morphometry of the blood lymphocyte population of healthy subjects. Quantitative values for the main components of the model lymphocyte (not differentiating between T and non-T types) have been available for a number of years (Konwinski and Kozlowski, 1972; James et al., 1980) and selected measurements are given in Table I. More recently, values have also been obtained for the granules and a new organelle, the nuclear ring. Morphometric studies on granules indicated that there were, on average, 45 granules per cell, the mean volume of each being 0.005/~m 3 (SchmidSch6nbein and Chien, 1988, 1989). The mean outer diameter of the nuclear ring was 0.1/~m, although the measurements varied five-fold (Arechaga et al., 1987). Morphometric changes in blood lymphocytes following mitogen stimulation have also been studied, increases being shown in the volumes of the cell, endoplasmic reticulum, lysosomes, mitochondria and nucleolus (Konwinski and Kozlowski, 1972), as well as in the amount of nuclear interchromatinic material (Dardick et al., 1985b). However, lymphocyte populations are not homogeneous and it would seem important to consider different sub-types separately. Evidence of morphological difference between T and B cell populations in normal human blood were found in studies using automated image processing, with normal distributions being fitted to feature histograms (Harosk6 et al., 1979). Differences were also apparent between sub-types of lymphocyte populations defined by E-rosetting and FAB-peroxidase labelling (Renau-Piqueras et al., 1978), and between nuclear chromatin patterns of small thymocytes and T cells of blood (Renau-Piqueras and Cervera, 1983), but in each case only one subject was studied. Ultrastructural differences between T and non-T lymphocytes were convincingly shown in an in-depth study using cells purified by rosetting techniques with resuspension and centrifugation

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(Boesen and Hokland, 1982a); the T cell had a smaller cell volume and surface area, a lower euchromatin/heterochromatin volume ratio, and a smaller volume and surface area of rough endoplasmic reticulum (RER). A stereological model cell has been described for T cells isolated by passing mononuclear blood cells through a nylonwool column (Petrzilka et al., 1978) and this was used for comparison with PHA-activated T cells, which showed an increase in size and a decrease in their nuclear/cytoplasmic (N:C) ratio (Petrzilka and Schroeder, 1979). There also appear to be morphological differences between different T cell sub-sets. In studies of cells separated by monoclonal antibodies and fluorescent-activated cell sorting (Boesen and Hokland, 1982b), morphometric analysis showed that as compared with T4 + helper/inducer cells, T8 + suppressor/cytotoxic lymphocytes had larger cytoplasmic volumes and surface areas, smaller N : C ratios, and larger Golgi and mitochondrial components. Another study has shown selective differences between these sub-sets in the response to the mixed lymphocyte culture (Balercia et al., 1990). Morphological differences between different NK cell sub-sets have also been shown. Using monoclonal antibodies and immuno-electron microscopy, cells expressing the Leul 1 antigen were compared with the L e u 7 + L e u l l - sub-set; L e u l l + cells had significantly larger areas, smaller nuclear/cell area ratios, and a greater degree of surface villosity (Arancia et al., 1986). These ultrastructural differences are of particular interest in view of other reports of the greater cytotoxic efficiency of L e u l l + cells (Lanier et al., 1983). It may be concluded that ultrastructural morphometry is yielding valuable information about the morphology of different sub-types of lymphocyte in health. It should be borne in mind that the methods of preparation and separation inevitably lead to some inaccuracy in cell selection which may affect the results in absolute terms. There is, for example, evidence that T lymphocytes show morphological differences in relation to their speed of rosetting (Slubowski et al., 1987). Some caution, therefore, needs to be exercised in interpreting the results and in comparing different studies.

Interest in the morphometry of lymphocytes and lymphoid cells in disease states has been stimulated by the possibility that the criteria for diagnosis and the assessment of prognosis may be improved by this means. In view of the large number of studies carried out on nuclear features, these will be reviewed first. Particular attention has been paid to changes in nuclear parameters and to relative dimensions of nucleus and cytoplasm in lymphoid malignancies. For example, the nuclear volumes of the malignant cells in prolymphocytic leukaemia, hairy cell leukaemia and malignant lymphoma were shown to be larger than those in chronic lymphocytic leukaemias of both B and T cell types (Boesen, 1983b) as well as those of normal lymphocytes (Boesen and Hokland, 1982a). The nuclear volume fractions and N : C ratios were also found to be markedly reduced in both hairy cell (James et al., 1980; Boesen, 1983b; Robinson et al., 1989) and prolymphocytic leukaemias (Boesen, 1983b), as might be expected from the large amount of cytoplasm in these conditions. Morphometric assessments of nuclear shape of lymphocytes in disease have yielded much important information, and the nuclear contour index (perimeter/~) and form factor (4n x area/ perimeter2) have been especially useful. For example, in prolymphocytic leukaemia of the T cell type, the significantly larger nuclear contour index has been proposed as a distinguishing feature (Woessner et al., 1978). A highly irregular cerebriform shape is a classical feature of the malignant T lymphocytes in S~zary syndrome and mycosis fungoides and it might therefore be anticipated that measurements of contour index or form factor would be useful in diagnosis of these conditions. This has proved to be the case in studies of the infiltrating lymphocytes in skin biopsies, the nuclear contour index being significantly higher in cutaneous T cell lymphomas than in benign conditions (Meijer et al., 1980; van der Loo et al., 1981) and the index was raised even in early and controversial cases (Willemze et al., 1986). Assessment of nuclear form factor, combined with immunohistochemical examination, was found to be of particular diagnostic value (Payne et al., 1986) and similar studies of T helper cells in the benign inflammatory reaction associated with bot-fly bite did not show

Human LeucocyteMorphometry the features characteristic of mycosis fungoides (Grogan et al., 1987). However, it may be pointed out that another study from the same centre found that even the simple scoring of the number of sharply angled nuclear invaginations gave 100% diagnostic accuracy for mycosis fungoides (Payne et al., 1984). In studies of peripheral blood lymphocytes (Willemze et al., 1983) and of isolated cells from lymph nodes (van der Loo et al., 1980) in patients with erythroderma, those with S6zary syndrome had a significantly higher nuclear contour index than those with unrelated conditions. Other studies have suggested that by considering nuclear contour index together with measurement of the nuclear surface area, aggressive forms of mycosis fungoides can be more readily identified (Simon, 1987). Of interest in this context is the finding of a significant correlation between nuclear contour irregularity (determined by form factor) and the presence of L e u 9 - and L e u 8 - T cells in other benign skin conditions, suggesting that these immunologically identified sub-types may be the normal counterparts of the cells in mycosis fungoides and S6zary syndrome (Payne et al., 1988). However, normal lymphocytes stimulated by mitogens or antigens in vitro did not produce cells with a nuclear contour index or form factor typical of S6zary syndrome (Payne et al., 1985). Measurements of form factor have also been used in B cell malignancies to show an ultrastructural distinction between Burkitt's and Burkitt's-like lymphomas (Payne et al., 1987). There is evidence from studies of B cells in germinal centres that normally the form factor and nuclear contour index are independent of the stage of follicular transformation (Said et al., 1986). Ultrastructural morphometry of nuclear size and shape in lymphocytes of reactive lymph nodes, including study of the frequency distribution of nuclear contour indices and depth of nuclear invaginations, has recently underlined the value of using objective criteria in diagnostic pathology. These studies have thrown doubt on accepted concepts relating to transformation in follicular centre cells and to the presence of a specific subset of cleaved cells---concepts upon which current classifications of non-Hodgkin's lymphoma are based (Dardick et al., 1989a, b).

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In view of the greater convenience of light microscopy, the question arises as to whether morphometric assessment of nuclear size, shape and homogeneity at this level might similarly be of diagnostic value. This has been mainly explored in tissue specimens from patients with lymphomas and the technique has been claimed as useful in a variety of circumstances [which include distinguishing lymphomatous from reactive tissue (Tosi et al., 1984), subclassifying T cell lymphomas (Sigaux et al., 1986), differentiating hairy cell leukaemia (Meijer et al., 1984) and other B cell malignancies (van der Valk et al., 1983) and characterizing other sub-types of lymphoma (van der Valk et al., 1982; Dardick et al., 1985a, 1988; Dawsey et al., 1989; Mihailovici et al., 1989)]. It has also been used in discriminating between benign and malignant lymphoid-rich effusions (Walts and Marchevsky, 1989) and has been proposed as a means of predicting survival using marrow smears in childhood acute lymphoblastic leukaemia (Seshadri et al., 1985). Attempts have been made to improve on the discrimination provided by light microscope measurement of form factor by using more sophisticated shape indicators (Raphael et al., 1985; Lesty et al., 1986; Tosi et al., 1988). Morphometric analysis of nuclear area distribution curves by skewness coefficient has also been put forward as a means of assessing prognosis from lymph node biopsy specimens in chronic lymphocytic leukaemia (Guigui et al., 1986). Optical Fourier transformation has been applied to the analysis of nuclear chromatin patterns of blood lymphocytes and could discriminate between normal and chronic lymphocytic leukaemia (R6zycka et al., 1988). It is apparent that in particular circumstances, morphometric assessment at light microscopy level could be of value. One might expect that more aggressive forms of lymphoid malignancy would have nuclei with a less condensed chromatin pattern. Ultrastructural morphometry provides a means of studying this. A morphometric assessment of the degree of chromatin condensation is given by the euchromatin:heterochromatin ratio and this was significantly greater in both prolymphocytic and hairy cell than in chronic lymphocytic leukaemia (Boesen, 1983b). However, no differences were found in the

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ratio in various immunologically-defined sub-sets in acute lymphoblastic leukaemia (Boesen, 1983a). Using a similar ratio, euchromatin was found to occupy a significantly smaller proportion of the nuclear area of blast cells in acute lymphoblastic leukaemia compared with acute myeloblastic leukaemia (Ochiai and Eguchi, 1987), possibly reflecting a lower rate of DNA synthesis which has been reported in the acute lymphoblastic type (Foadi et al., 1988; Schumacher et al., 1971). Nuclear chromatin compartments have also been studied in lymphocytes of germinal centres in non-Hodgkin's lymphoma (Dardick, 1985). The concept that prominence of nucleoli is an indicator of more aggressive forms of malignancy with a poorer prognosis has similarly received some support from morphometric studies of lymphoid neoplasms. For example, nucleolar measurements were greater in prolymphocytic T cell leukaemia than in normal (Woessner et al., 1978) and in immunoblastic B cell lymphoma compared with the centroblastic type (van der Valk et al., 1983; Ball et al., 1985--in a light microscopy study), although no significant differences were found between chronic lymphocytic leukaemia and normal (Schumacher et al., 1970). Nucleolar volumes in prolymphocytic leukaemia were significantly larger than those in chronic lymphocytic or hairy cell leukaemia or in centrocytic lymphoma (Boesen, 1983b), no significant differences being detected between T and non-T forms of acute lymphoblastic leukaemia (Boesen, 1983a). The percentage of cells with multiple nucleoli was greater in lymphosarcoma cell leukaemia than in chronic lymphocytic leukaemia (Schrek et al., 1971; Schrek, 1972). However, nucleolar frequency has been reported as greater in Burkitt's lymphoma than in Burkitt's-like lymphoma which carries a poorer prognosis (Payne et al., 1987): this suggests that nucleolar prominence may not always be a reliable indicator of the degree of malignancy. Ultrastructural morphometry has also been used to look for changes in whole-cell and cytoplasmic parameters of lymphoid cells in disease states. Larger cell volumes have been demonstrated in hairy cell (James et al., 1980; Boesen, 1983b; Robinson et al., 1989) and prolymphocytic leukaemias (Woessner et al., 1978; Boesen, 1983b;

Robinson et al., 1989). Although the mean volumes of the circulating cells in chronic lymphocytic leukaemia have usually been reported to be within the normal range (Boesen, 1983b), evidence has been found of a bimodal distribution into small and large cell types with mean sizes outside this range (James et al., 1980). The latter observation may be related to the prolymphocytoid transformation of chronic lymphocytic leukaemia where a clone of cells with the morphology of prolymphocytes is also present (Robinson et al., 1989). Mitogen-transformed cells from chronic lymphocytic leukaemia patients have been reported to be smaller than expected (Douglas et al., 1973). Irregularity of cell surface membrane (estimated by relating the cell surface area to that of a sphere of equivalent volume) has been shown to be greater in B cell types as compared with T cell types of chronic lymphocytic leukaemia (Boesen, 1983b), as might be expected from the studies of normal B and T cells as discussed above (Boesen and Hokland, 1982a). The highest measurements of surface irregularity were found in hairy cell leukaemia reflecting the long surface projections characteristic of this disease (Boesen, 1983b; Robinson et al., 1989). Some twodimensional morphometric data for hand-mirror cells (T cells seen in infectious mononucleosis and occasionally in acute lymphoblastic leukaemia) have also been reported (Schumacher et al., 1980). For meaningful ultrastructural morphometry of cytoplasmic organelles, particularly high quality micrographs and meticulous attention to technique are required and there are comparatively few reported studies. Quantitation of the volumes (or volume fractions) of rough endoplasmic reticulum was carried out on circulating cells in chronic lymphocytic leukaemias (Boesen, 1983b; Slubowski et al., 1985) and the B cell types were shown to have larger volumes than the T cell forms (Boesen, 1983b). In B cell non-Hodgkin's lymphomas, the circulating centrocytes had a larger amount of RER than other B cell malignancies (Boesen, 1983b). In acute lymphoblastic leukaemias, the B cell-derived forms also had a large volume of RER, particularly in cells with plasmacytoid differentiation (Boesen, 1983a). Increased length of RER has been shown in the jejunal plasma cells of patients with coeliac disease

Human LeucocyteMorphometry (Guix et al., 1979) while techniques for comprehensive sterological study of RER have been developed and applied to myeloma cells (malignant plasma cells) in different immunological types of myelomatosis (Hughson et al., 1983). An interesting feature of mitochondrial studies has been the apparent uniformity of the mitochondrial volume fractions in the different types of lymphoid malignancy including both acute (Schumacher et al., 1975; Boesen, 1983a) and chronic forms (Boesen, 1983b) although the absolute volumes have varied, and increased numbers of mitochondrial profiles per section have been reported (Woessner et al., 1978). Measurement of size and shape of mitochondria in patients with acute lymphoblastic leukaemia provided no evidence that giant mitochondria (found in only 2 of 28 cases) represented a separate population (Eguchi et al., 1987). The Golgi apparatus was found to be significantly smaller in B cell chronic lymphocytic leukaemia than in the malignant cells of prolymphocytic and hairy cell leukaemias and of centrocytic lymphomas (Boesen, 1983b) but no differences were found between immunologically defined types of acute lymphoblastic leukaemia (Boesen, 1983a). Studies of lysosomal granules have indicated that the volume is greater in the T cell types of acute lymphoblastic leukaemia than in the non-T non-B types (Boesen, 1983a) and in the T cell derived forms of chronic lymphoid malignancy than in the non-T derived forms, with the exception of hairy cell leukaemia which showed the largest volumes of all (Boesen, 1983b). The number of lysosomal granules per section in a case of T cell prolymphocytic leukaemia did not differ from normal circulating lymphocytes (Woessner et al., 1978). It will be apparent from this section that morphological differences can be detected in lymphoid cells in disease and can have an important bearing on diagnosis and prognosis. Ultrastructural morphometry does not yet feature in routine clinical management because its potential contribution is felt to be outweighed by the practical difficulties involved (discussed below under Section V). However, with the rapid advances in modern technology, it seems likely that ultra-

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structural morphometry of lymphocytes and lymphoid cells will soon have an important place in the clinical field.

IV. GRANULOCYTES Quantitative study of granulocyte ultrastructure is greatly facilitated by the ease with which these cells can be identified from their characteristic specific granules, thus permitting the use of all cell sections including those without nuclear profiles; the need to correct for nuclear-biased sampling is thereby avoided. Some basic morphometric data have been obtained for neutrophils in healthy subjects (James, 1980; Schmid-Sch6nbein et al., 1980; Chien et al., 1984) and selected values from one study are shown in Table 1. In another study, the mean number of granules per cell was estimated as 3724 (range 1900-6300) and their radial distribution showed that proportionately more of the granules with light density were in the outer regions of the cytoplasm, proportionately more heavy density granules being placed centrally: there was also an enhancement of granule numbers adjacent to pseudopodia (Schmid-Sch6nbein and Chien, 1988, 1989). Such differences in granule distribution presumably reflect the operation of intracellular mechanisms in relation to functional activity. The actin matrix may be implicated in this, but as yet little is known of the mechanisms involved. Morphometric data has been obtained in our laboratory for the eosinophil granulocytes of 18 healthy individuals, 24 different parameters being studied (Sokol et al., 1987b); some basic data are given in Table 1. This investigation did not reveal any differences between male and female subjects. The apparently smaller nuclear membrane excess in the eosinophils as compared with the neutrophils (Table 1) might be expected, as it is well known that the number of nuclear lobes is smaller in eosinophils. Data in respect of mitochondria and granules are shown in Table 2. Other studies of normal eosinophils have been reported (Schmid-Sch6nbien et al., 1980; Chien et al., 1984; Schmid-Sch6nbien and Chien, 1988, 1989) and in general, the values obtained have been comparable

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R . J . Sokol et al. Table 2. Mitochondrial and Granule Measurements of Blood Eosinophils in Hypereosinophilic Syndrome (after Sokol et al., 1988a)

Measurement Mitochondria Volume (fl) Surface area ~ m 2) Profiles/section Profile area of individual mitochond ria (# m 2) Granules Volumes (fl) Surface area ( # m 2) Profiles/section Profile area of individual granules ( ~ m 2)

N o r m a l subjects (means _+ SEM)

Patient with hypereosinophilic syndrome

4.6 55 4.8 0.12

__+0.4 __+4 _+ 0.3 ___0.01

6.9* 84* 6.3* 0.13

47 437 28 0.21

+ 3 ___21 ___1.1 __+0.01

30* 331" 22* 0.16"

* Asterisks indicate significant difference from corresponding normal value at overall confidence level of < 0.05.

with ours. The total number of granules per cell has been estimated at 418 (range 260-780) (SchmidSch6nbein and Chien, 1988). In rats, ultrastructural morphometry of eosinophil granules in cells at different stages of maturation has provided direct evidence that mature dense granules are derived from earlier vesiculated forms (Elmalek and Hammel, 1987). There have to date been few reports of quantitative electron microscopy of granulocytes in disease states, but these have underlined the potential of such techniques in demonstrating differences which could not have been established by subjective means. In our centre (Sokol et al., 1988a), measurements on the blood eosinophils of a patient with hypereosinophilic syndrome were compared with those of a group of normal individuals. The cells were significantly larger with an increase in surface area and in cell membrane excess; there were also differences in the mitochondrial and granule parameters (Table 2). The increase in volume and surface area of mitochondria (corresponding to an increased number but not size of the individual organelles) presumably indicated increased metabolic activity of the abnormal cells (Carr and Toner, 1982). These findings may be related to the high proportion of light density eosinophils with enhanced function reported in hypereosinophilic syndrome (Winqvist et al., 1982; Prin et al., 1983), some heterogeneity in cell density and function being found in the normal (Pincus et al., 1981; de Simone et al., 1982; Prin et al., 1983, 1984). The granule changes in Table 2 imply that there were

fewer granules and these were of smaller size-a conclusion which has also been reached in a morphometric study of hypodense eosinophils in this syndrome (Peters et al., 1988); the smaller number of granules may in fact explain the hypodensity, as the eosinophil granule crystalloid is known to be composed of major basic protein (Lewis et al., 1978). The reduced number of granules presumably results from inappropriate degranulation, the smaller profile areas perhaps reflecting the selective loss of larger granules, as seen in a morphometric analysis of eosinophil degranulation in vitro (Henderson and Chi, 1985). In another study of hypereosinophilic syndrome, the decreased size of the cytoplasmic granules was confirmed, but the cells were still capable of undergoing degranulation following stimulation by ionophore or zymosan particles (Henderson et al., 1988). It may be noted in passing that the eosinophils present in cerebrospinal fluid of nematode-infected rodents had fewer and smaller granules (Yoshimura et al., 1988). In the acute myelogenous leukaemias, the circulating malignant cells (Dobreva and Meshkov, 1979) showed greater profile areas for whole cell, nucleus and nucleolus in the promyelocytic as compared with the myeloblastic type, together with larger numbers of mitochondria and lysosomal granules. The larger cytoplasmic area ( x 4.5) outweighed the difference in nuclear area ( × 2) and this differential was confirmed in more recent work where the areal fraction of the nucleus within the cell was found to be significantly less in

Human LeucocyteMorphometry promyelocytic (M3) than in myeloblastic (M1 and M2) forms (Ochiai and Eguchi, 1987). Ultrastructural study of leukaemic blasts in the bone marrow indicated that while the myeloblast had significantly more granules per cell profile than other blasts, the mitochondrial numbers and profile areas were approximately the same in all the types studied (Schumacher et al., 1973b) and were also similar to those of the myeloblasts of healthy hospital employees (Schumacher et al., 1974). Changes in the morphological features of mature granulocytes are well-known clinically in a wide variety of acquired and inherited conditions and although ultrastructural morphometry would seem to have obvious applications, no human studies appear to have been reported apart from those mentioned above. In an investigation of irondeficient rabbits, the number of secondary granules per unit area of mature polymorphs was significantly greater than that of normal rabbits (Parmley et al., 1986): this suggests that similar lysosomal changes might be found in human iron deficiency, particularly as reduced bactericidal function has been reported in neutrophil polymorphs (Chandra, 1973; Yetgin et al., 1979). In the circulating neutrophils and eosinophils in a fox with an inherited condition resembling the human Chediak-Higashi syndrome, the volume fraction of the granules was reported to be the same as that of the normal, but there were fewer granules per unit area (Fagerland et al., 1987). It would be interesting to know whether similar findings would be made in the syndrome in humans. Other conditions which would seem to merit morphometric study of mature granulocytes include megaloblastic anaemias, chronic granulocytic leukaemia, toxic states and the Pelger-Huat anomaly. The possible value of examining mature polymorphs in acute myeloid leukaemia was shown in a light microscopy study (BendixHansen, 1986) in which peroxidase-deficient cells were found to be smaller and have bigger nuclei than those with normal myeloperoxidase content, suggesting that they might be the progeny of leukaemic precursors. There would appear to be much scope for further research on the ultrastructural morphometry of myeloid cells in clinical states.

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V. TECHNICAL AND NUMERICAL CONSIDERATIONS Technical and numerical considerations necessary for valid ultrastructural morphometry of human leucocytes have been dealt with in detail in our earlier publications (Sokol, 1988a, b, 1989, 1990; Sokol et al. 1985a, b, 1987a, b, 1988a, b, c, d, 1989, 1990; James et al., 1988) and only the essential points will be covered here. It is obviously important that all blood samples are collected and processed for electron microscopy by similar standard methods. High resolution electron micrographs are a prerequisite and the cells for detailed examination need to be selected in an independent uniform random manner, the magnification of each micrograph being obtained by reference to a test graticule. Measurements on the micrographs can be made by a variety of techniques and automated systems including computerized planimetry are now available. A simple and well-tried method is to carry out point and intersect counting of micrographs covered with a transparent sheet bearing a rectangular lattice of known spacing (Baak and Oort, 1983). This also has the advantage of flexibility and ease of transport as complex apparatus and technical assistance are not required. From the raw data, various morphometric measurements of size may be obtained and the required calculations are now best done by a computer program. Initially, areal fractions (the numerical equivalents of volume fractions) and surface-to-volume ratios are calculated from conventional formulae, compensating for non-equatorial sectioning and, where appropriate, for nuclear-biased sampling (Sokol et al., 1985b); other measurements are then derived. In order to confirm that the size of the sample is adequate, the relative standard error should not exceed the level determined a priori, e.g. 0.05 (Sokol et al., 1987b). For valid analysis of the results, all the statistical test assumptions must be fulfilled. The individual results need therefore to be tested for normality of distribution and homogeneity of variances and when these are not shown, data should be transformed before analysis: one may also have to exclude ratios where both numerator and denominator themselves vary as normal distributions.

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Appropriate steps can be carried out with the SPSS package mentioned below. Only measurements which are considered to be identically and independently drawn should be included, or alternatively, MANOVA or other techniques which can be used even with correlated measures are required. In the analysis, multiple comparison procedures are needed to avoid the false conclusions which would arise from the serial use of single comparison methods. A single comparison method at the 5% significance level (such as the Student t-test) is not valid at that level when two or more morphometric measures are tested simultaneously. The overall significance level (A) for comparing a number of parameters (K) at the individual parameter significance level of ~ is given by the Bonferroni relation: A = 1--(1--~)K. Multiple comparison procedures in common use are the Dunn-gidak procedure and techniques for analysis of variance (ANOVA and MANOVA). In the Dunn-gidak procedure (Sokal and Rohlf, 1981) the overall significance level (A) is set at the desired level, say 0.05, and the individual significance level (~) is then determined from the Bonferroni relation. For example, in our study of the morphometry of blood eosinophils, where 13 independent measurements were being compared in male and female subject groups, an individual significance level (~) of 0.002 was required to give an overall level (A) of 0.05 (Sokol et al., 1987b). Both ANOVA and MANOVA permit the simultaneous analysis of the effects of the multiple comparisons and are normally carried out by means of a computer program. ANOVA may be used alone when a limited number of comparisons are to be considered: for example it was suitable for the analysis of the effects of four culture times on nine independent cell measurements made on developing macrophages from a single group of normal subjects (Sokol et al., 1987a). For more complex studies, MANOVA is required. For example, in a series of studies of the in vitro development of monocytes into macrophages (Sokol, 1988b, 1990; Sokol et al., 1988b, c), the effects of different culture times on independent cell measurements had again to be considered but, in addition, the findings in patients with malignant lymphomas had to be compared with those of

normal subjects and the effects of age, sex, and (in some instances) clinico-pathological status had also to be taken into consideration. For such analysis, a sophisticated statistical package such as the SPSS is required, often run on a mainframe computer (Sokol et al., 1988a, b). SPSS packages are now also available for use with personal computers (Sokol, 1988b, 1990). As already noted in Section II, discriminant analysis may be used to try and identify the subject category of single specimens by reference to an ordered classification from a multivariate population. Discriminant analysis is described in detail in several textbooks (Mardia et al., 1979; Kendall, 1980; Seber, 1980; Manly, 1986; Flury and Riedwyl, 1988; Krazanowski, 1988)and is now included in computer packages. Two types of discriminant analysis are available, linear (such as in the SPSS package which we normally use (Sokol et al., 1990) and non-linear (van der Loo et al., 1980, 1981; Meijer et al. 1980; Ball et al., 1985); in the linear type, the mathematical discriminatory function is a straight line, whereas in the non-linear type it is curved. Two functions may be required for purposes of discrimination where there are three different subject categories, as for example in our ultrastructural study of the development of monocytes into macrophages, where normal subjects, patients with Hodgkin's disease and patients with non-Hodgkin's lymphoma were studied (Sokol et al., 1990). The values of the two functions considered simultaneously are used to assign the morphometric data from an individual subject into the most likely subject category. A stepwise analysis is also included in the discrimination procedure; predictor variables (in the above example, measurements of different morphometric features) are included sequentially in order to ascertain their relative importance, as determined by the minimum value of Wilks' lambda. In this way, it may be possible to reduce the number of variables required for future analysis, while still retaining good discrimination. The main limitation of all types of analysis of leucocyte morphometry in a clinical context is the necessity of producing valid classification models against which subsequent specimens can be tested. This is particularly a problem when the disease

Human Leucocyte Morphometry states b e i n g s t u d i e d h a v e a n u m b e r o f d i s t i n c t i v e c y t o l o g i c a l s u b - g r o u p s as it is n e c e s s a r y to h a v e adequate data for each. However, once valid classification models have been obtained, subseq u e n t a s s e s s m e n t o f i n d i v i d u a l cases is r e l a t i v e l y easy.

Acknowledgements--We thank the Yorkshire Cancer Research Campaign and the Trent Regional Health Authority for financial support, and Mrs. S. M. Coupe for secretarial assistance.

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