Scanning electron microscope cytochemistry of normal and leukaemic leukocytes

Backscattered Electron Imaging (BEI) is a particular technique which permits to study cytochemical reactions with the Scanning Electron Microscope (SEM). The BEI data pertaining to specific enzymatic activities can be directly correlated to the surface morphology of each individual cell. Leukocytes from 5 normal individuals, 14 patients with acute nonlymphoblastic leukaemia (ANLL), 7 patients with chronic myeloid leukaemia (CML) and 3 patients with acute lymphoblastic leukaemia (ALL) were studied for myeloperoxidase activity, acid phosphatase localization, silver staining of the nuclei and phagocytosis of iron carbonyl in the BEI mode of SEM. Some normal peripheral blood leukocytes which cannot be distinguished by their surface morphology alone were satisfactorily identified with the BEI technique. Leukaemic myeloid cells can be recognized in many cases because of their positive myeloperoxidase reaction, while monocytic elements can be characterized by the presence of surface ruffles, acid phosphatase activity and active phagocytosis. The usefulness of the BEI technique in identifying different blood cell types with the SEM and its possible application to the diagnosis of certain cases of leukaemia are discussed.     

Scanning electron microscope cytochemistry of normal and leukaemic leukocytes

Backscattered Electron Imaging (BEI) is a particular technique which permits to study cytochemical reactions with the Scanning Electron Microscope (SEM). The BEI data pertaining to specific enzymatic activities can be directly correlated to the surface morphology of each individual cell. Leukocytes from 5 normal individuals, 14 patients with acute nonlymphoblastic leukaemia (ANLL), 7 patients with chronic myeloid leukaemia (CML) and 3 patients with acute lymphoblastic leukaemia (ALL) were studied for myeloperoxidase activity, acid phosphatase localization, silver staining of the nuclei and phagocytosis of iron carbonyl in the BEI mode of SEM. Some normal peripheral blood leukocytes which cannot be distinguished by their surface morphology alone were satisfactorily identified with the BEI technique. Leukaemic myeloid cells can be recognized in many cases because of their positive myeloperoxidase reaction, while monocytic elements can be characterized by the presence of surface ruffles, acid phosphatase activity and active phagocytosis. The usefulness of the BEI technique in identifying different blood cell types with the SEM and its possible application to the diagnosis of certain cases of leukaemia are discussed.     

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.     

Simultaneous visualization of myeloperoxidase reactivity and immunogold labeling by backscattered electron imaging with the scanning electron microscope

We describe a method to label circulating human granulocytes with an immunogold marker and then incubate them for demonstration of myeloperoxidase activity. The cells were observed in the backscattered electron imaging (BEI) mode of the scanning electron microscope. This permits simultaneous visualization of cell surface morphology, the immunological surface marker, and the cytochemical reactivity of each individual cell. Enhanced identification of the various cell types and more precise characterization of cell surface features in the different steps of leukocyte differentiation are expected to result from application of this technique.     

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study

The distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.     

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study

Summary The distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.     

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10^–8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10^–8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.     

Mapping gold-labeled IgE receptors on mast cells by scanning electron microscopy: receptor distributions revealed by silver enhancement, backscattered electron imaging, and digital image analysis

Immunogold labeling and silver enhancement techniques are widely used to determine density and distribution of cell membrane receptors by light and transmission electron microscopy. However, these techniques have not been widely used for receptor detection by scanning electron microscopy. We used antigen- or protein A-conjugated colloidal gold particles, together with silver enhancement, sequential secondary and back-scattered electron imaging (SEI and BEI), and digital image processing, to explore cell surface distribution of IgE-receptor complexes on RBL-2H3 cells, a rat leukemia line that provides a model for the study of mucosal mast cells. Cells were first incubated with a monoclonal antidinitrophenol IgE (anti-DNP-IgE) that binds with high affinity to cell surface IgE receptors. The resulting IgE-receptor complexes were cross-linked either with the multivalent antigen, DNP-BSA-gold, or with a polyclonal anti-IgE antibody. Antibody-treated cells were labeled after fixation with protein A-gold. Fixed, gold-labeled cell monolayers were silver enhanced (or not), dehydrated, critical point-dried, and coated with gold-palladium (for SEI analysis) or carbon (for combined SEI/BEI analysis). They were observed in an Hitachi S800 SEM equipped with a field emission tip and a Robinson backscattered electron detector. An image processor (MegaVision 1024XM) digitized images directly from the S800 microscope at 500-1000 line resolution. Silver enhancement significantly improves detection of gold particles in both SEI and BEI modes of SEM. On gold-palladium-coated samples, 20-nm particles are resolved by SEI after enhancement. BEI resolves 15-nm particles without enhancement and 5- or 10-nm particles are resolved by BEI on silver-enhanced, carbon-coated samples. Neither BEI nor SEI alone can yield high resolution topographical maps of receptor distribution (BEI forms images on the basis of atomic number contrast which reveals gold but not surface features). Image analysis techniques were therefore introduced to digitize, enhance, and process BEI and SEI images of the same field of view. The resulting high-contrast, high-resolution images were superimposed, yielding well-resolved maps of the distribution of antigen-IgE-receptor complexes on the surface of RBL-2H3 mast cells. The maps are stored in digital form, as required for computer-based quantitative morphometric analyses. These techniques of silver enhancement, combined BEI/SEI imaging, and digital image analysis can be applied to analyze density and distribution of any gold-labeled ligand on its target cell.     

Colloidal gold labeling of intracellular ligands in dorsal root sensory neurons, visualized by scanning electron microscopy

We report a novel technique that combines high-resolution scanning electron microscopy (SEM) of intracellular structures with backscattered electron imaging (BEI) of colloidal gold-labeled intracellular ligands. Murine dorsal root ganglia were immersion-fixed, freeze-cleaved, labeled with gold complexes, and critical point-dried. Specimens were carbon-coated and viewed by BEI. They were then minimally sputter-coated with gold and previously identified cells relocated by secondary electron imaging (SEI). This permitted increased resolution of intracellular detail while gold particles remained detectable by BEI. Incubation with RNAse-gold and DNAse-gold complexes resulted in specific labeling of cytoplasm and nucleus, respectively. Immunolabeling of neurofilament (NF) and small nuclear ribonucleoproteins (snRNP) resulted in selective labeling of intracellular antigens. Nonspecific binding was abolished by use of 1% skin milk. Specifically, incubation with monoclonal anti-NF68 resulted in labeling of cytoplasm in 66% of neurons, notably of the large cells known to contain large amounts of NF. Satellite cells, which lack NF, showed low levels of background label. Human autoimmune anti-Sm serum recognizes snRNP particles, with the exception of the nucleolar U3 snRNP. Labeling with this serum resulted in specific labeling of 92% of nuclei, with only background labeling over nucleoli and cytoplasm. The results show that it is feasible to employ high-resolution SEM in conjunction with colloidal gold labeling to localize intracellular ligands in situ.     

Programmed cell death in the larval salivary glands of <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera,Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.     

Visualization and rapid quantification of autoradiographic labeling in scanning electron microscopy applied to localization of receptor sites on the surface of whole cells.

The use of backscattered electron imaging (BEI) as a routine procedure for examining autoradiographic reactions in scanning electron microscopy (SEM) is described. This technique allows the determination of the number of receptor sites occupied by 125I-epidermal growth factor (EGF) on whole cells. The effect of 1.25 dihydroxyvitamin D3 (1,25 (OH)2D3) on the number of epidermal growth factor receptors (EGF-R) in the BT 20 human mammary carcinoma cell line (which is known to possess a very high number of EGF-R) has been evaluated with this method. To compare the silver grain density over the cells (controls and 1,25 (OH)2D3-treated cells) we used an image analysis system Quantimet 900. The results were compared with those of a previous study using transmission electron microscopy (TEM). This study confirmed the results obtained with TEM and showed the even distribution of receptors sites on a single cell and a large difference in the number of receptor sites from one cell to another. The use of BEI to visualize the autoradiographic reaction in SEM allowed the examination of a large surface with good contrast and resolution and eliminated artefacts not corresponding to the silver grains. It gave new information not delivered by quantitative TEM autoradiography and was easier and faster to use. The efficient use of SEM autoradiography combined with BEI could facilitate whole area distribution mapping of radioactive labeling.     

A new cytometric method for the immunophenotypic characterization of bone-derived human osteoclasts

BACKGROUND: Osteoclast cell function relates to bone resorption. Isolation and characterization of these cells from in vivo sources remain difficult. The aim of this study was to show the feasibility of using flow cytometry to identify and characterize human mature osteoclasts obtained from bone tissues. METHODS: Bone femoral heads obtained as discarded surgical material were used. To check the nature of 121F(+) (a monoclonal antibody specific for human osteoclasts) cells by flow cytometry, we used laser scanning cytometry to analyze simultaneously the immunophenotype and DNA cell content of osteoclast-like cell-enriched bone samples. RESULTS: Results were compared with conventional morphologic and cytochemical studies. The percentage of cells that showed both cytochemical (tartrate-resistant acid phosphatase [TRAP](+)) and immunophenotypic (121F(+)) osteoclast-associated characteristics was very similar (12.5 +/- 6.2 versus 14.7 +/- 11.7; P = 0.46). Laser scanning cytometry showed that 121F(+) cells were bigger (P = 0.04) and they had a higher DNA cell content (P = 0.04) and more nuclei per cell (P = 0.04) than the 121F(-) cells present in the same sample. DISCUSSION: This study relied on the combined use of the 121F(+) antibody and different cytometry-based techniques to characterize the osteoclast populations from human bone.     

Study on Ultrastructure and Localization of Acid Phosphatase of Gastrodia elata Cortical Cells in Their Digestive Process of Armillaria mellea

In order to study the mechanism of the digestive process of Armillaria mellea in Castrodia data, electron microscopy and cytochemical method for determination of acid phosphatase activity was employed. The provacuoles were formed by means of expanded or convoluted ER under the stimulation of cortical cells and large cells of Gastrodia data by Armillaria mellea. A product of acid phosphatase (lead phosphate deposits) occured on the tonoplast. The papillae were produced in the cell wall of cortex in Gastrodia data when Armillaria mellea penetrated into its cortex. Our results showed that the enzyme was not released from cell of Armillaria mellea. A number of small vacuoles in the cortical cells disappeared. At the same time, lead phosphate deposits on the Armillaria mellea hyphae wall were observed and than Armillaria mellea hyphae wall was going to be digested, and the hyphae lost their structure. The activity of Armillaria mellea hyphae was not observed in the large cell of Gastrodia data. A great deal of small vacuoles and mitochondria were produced, at the same time the renewable nuclei and nuclolar vacuoles etc. appeared in the large cells of Gastrodia data under the stimulation of Armillaria mellea.     

Cytochemical examination of acid phosphatase and beta-glucuronidase enzymes in low-grade B cell non-Hodgkin's lymphomas

The differential diagnostic significance of acid phosphatase and beta-glucuronidase were studied in 77 cases of low-grade B cell non-Hodgkin's lymphomas. In most cases the results of cytochemical enzyme studies performed on malignant cells of the bone marrow were evaluated. B cell chronic lymphocytic leukaemia, centrocytic and centroblastic/centrocytic lymphomas were characterized by a weak or a negative acid phosphatase and beta-glucuronidase activity. Stronger positivity was observed in immunocytoma and in Waldenstr?m's macroglobulinaemia, while the highest activity was found in multiple myeloma. Hairy cell leukaemia of B cell origin showed intensive tartrate-resistant acid phosphatase activity. The cytochemical examination of these lysosomal enzymes may be useful in the diagnosis of low-grade malignant lymphomas of B cell origin by completing other methods.     

The application of the nucleolar organizer region silver staining (AgNOR) to backscattered electron imaging (BEI)

Until recently scanning electron microscopes were mainly used to observe surfaces. However, it has been proved that a backscattered electron detector can give an image (BEI) of the specimen's internal structure after heavy metal staining. In this paper, we report how we have applied the silver staining for NOR-associated proteins to scanning electron microscopy, studying C3H10T1/2 cells in culture. This technique allows to localize, inside the nucleus, the nucleolar arrangement of AgNOR-associated proteins. In BEI imaging, the silver staining shows several intranucleolar silver spot-like deposits sometimes associated in "doublets" as on metaphasic chromosomes. These silver grains probably represent the fibrillar centre location, thought to be the interphasic counterpart of the NORs. However, these silver spot granules are more numerous during interphase.     

Localization of insulin, glucagon and somatostatin in the pancreas of the adult newt, Notophthalmus viridescens

The current study is designed to demonstrate the presence of immunoreactive insulin (IRI), glucagon and somatostatin in the adult pancreas. Methods include aldehyde fuchsin (AF) staining and peroxidase anti-peroxidase (PAP) immunochemical localization for light microscopy as well as protein A gold (PAG) staining for scanning electron microscopy (SEM) in conjunction with backscattered electron imaging (BEI). Our results show the presence of large clusters of AF-positive cells within networks of highly vascularized pancreatic acinar tissue. PAP immunochemistry of pancreas serial sections exhibit positive immunoreactivity to the same AF-positive structure, thus demonstrating the presence of IRI. This immunoreactivity is found in a high percentage of cells in the islet-like structures. These cells tend to be centrally located within the cluster. Antibody specificity controls, including homologous antigen immunoabsorbance, as well as incubation of sections in normal guinea pig serum give negative immunoreactivity. Immunoreactive glucagon-containing cells and somatostatin-containing cells are distributed around the periphery of the central core of IRI-containing cells. SEM in conjunction with BEI confirm the presence of PAG within these cell clusters. We conclude that: (a) newt pancreatic IRI reacts in a specific manner with bovine antibody, suggesting a partial structural similarity to mammalian antigen; (b) IRI is localized within within pancreatic islet-like cell clusters and these IRI-containing cells form a central mass which is surrounded by glucagon and somatostatin-containing cells; this cellular distribution is similar to that found in many mammals. PAG conjugated insulin antibody is detectable by SEM in conjunction with BEI in islet cells of the newt pancreas.     

The application of the nucleolar organizer region silver staining (AgNOR) to backscattered electron imaging (BEI)

Until recently scanning electron microscopes were mainly used to observe surfaces. However, it has been proved that a backscattered electron detector can give an image (BEI) of the specimen's internal structure after heavy metal staining. In this paper, we report how we have applied the silver staining for NOR-associated proteins to scanning electron microscopy, studying C3H10T1/2 cells in culture. This technique allows to localize, inside the nucleus, the nucleolar arrangement of AgNOR-associated proteins. In BEI imaging, the silver staining shows several intranucleolar silver spot-like deposits sometimes associated in “doublets” as on metaphasic chromosomes. These silver grains probably represent the fibrillar centre location, thought to be the interphasic counterpart of the NORs. However, these silver spot granules are more numerous during interphase.     

The lacunar cells and its relationship to interdigitating reticulum cells

Lacunar cells, which are characteristic of the nodular sclerosis type of Hodgkin's disease, were investigated by light and electron microscopy and by enzyme cytochemical and immunohistochemical methods. Characteristic ultrastructural features of the lacunar cells were its size, its multilobated nucleus, and the pale cytoplasm containing only a few organelles. These features distinguish the lacunar cell from typical Sternberg-Reed and Hodgkin cells. Enzyme cytochemically, lacunar cells were weakly positive for acid phosphatase and non-specific esterase. the reaction product was distributed either diffusely or more focally in the cytoplasm. By immunostaining, kappa, lambda, and IgG could be detected in some lacunar cells. The immunostaining pattern was bitypic, which might have resulted from non-specific uptake. All the results of the present study indicate that lacunar cells are non-lymphoid cells. When lacunar cells were compared with cells of normal lymphoid tissue, their ultrastructure was found to be very similar to that of interdigitating reticulum cells. Both cell types showed a bizzarrely shaped nucleus and an electron-transparent cytoplasm with only some vesicles and tubules. Furthermore, lacunar cells and interdigitating reticulum cells exhibited a similar reaction pattern of acid phosphatase and non-specific esterase. Thus, from a cytologic and enzyme cytochemical point of view, a direct relationship between the two cell types is very likely.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

A quantitative cytochemical investigation of osteoclasts and multinucleate giant cells

Summary Quantitative cytochemical, immunocytochemical, autoradiographic and electron cytochemical investigations have been used to compare osteoclasts with multinucleate giant cells that had been freshly obtained from the same animal. The levels of -acid galactosidase activity, the DNA in individual nuclei and the cellular protein content were similar in both cell types. However, osteoclasts generally possessed greater acid phosphatase and NADH dehydrogenase activity but lower levels of fluoride-inhibited non-specific esterase activity than multinucleate giant cells. The acid phosphatase activity in multinucleate giant cells was completely inhibited by 100 mM tartrate, but in osteoclasts only a 20% reduction in activity was observed. Formation of multinucleate giant cells in a bone microenvironment (thin bone slices) did not increase their content of tartrate-resistant acid phosphatase activity. Moreover, in osteoclasts, endogenous peroxidase activity was undetectable but present in several granules within the cytoplasm of multinucleate giant cells. Osteoclasts and multinucleate giant cells displayed a similar microtubular distribution, but calcitonin, which induced rearrangement of microtubules and cellular contraction in osteoclasts, had no effect on multinucleate giant cells. Thus, these investigations reveal both similarities and differences between these two syncytia and support the hypothesis that osteoclasts and multinucleate giant cells are related. Possibly osteoclasts arise from monocyte progenitors before commitment to a macrophage lineage has occurred.