Immunoferritin electron microscopic studies with antibasophil serum of guinea pig basophil degranulation and regranulation in vitro.

We have previously shown that degranulated guinea pig basophils in vitro do not die. Rather, they recover, and these mature cells rebuild granules in the absence of nuclear changes of immaturity or of mitoses. This ability to survive the explosive release of cytoplasmic granules and to rebuild granules was the first such demonstration for a mature granulocyte. We utilized this model of antigen-induced basophil degranulation and recovery in vitro, in conjunction with a specific antibasophil serum (ABS) and indirect immunoferritin electron microscopy, in order to localize the anatomic sites of expression of the antigen(s) recognizing ABS during the secretory and recovery events. Basophil surface membranes were labeled prior to degranulation, during degranulation, after degranulation, and as mature, polynuclear, granule-free cells began to rebuild new granules. In addition, the plasma membranes of non-granule-containing, glycogen positive, small mononuclear cells were labeled. These were few in number and were thought to be precursor cells in the basophil lineage. Controls, either nondegranulated basophils at a variety of culture intervals, or basophils following degranulation and during recovery, were done using normal rabbit serum (NRS) and an indirect immunoferritin electron microscopic technique. These were all negative. Moreover, contaminating eosinophils, neutrophils, and lymphocytes were also negative. Extruded granules were labeled with ferritin (ABS) on their membrane-free, finely granular exteriors, but not within the lamellar array of their matrixes. Extruded granules were not labeled in control experiments. When tested with ABS, membrane-bound granules in the cytoplasm prior to release and membrane-free, released granules contained within degranulation sacs, were negative, as was the membrane of sacs. Immature, membrane-bound granules were also negative. Ferritin binding to membranes was seen in narrow channels, vesicles, and larger vacuoles, as well as in multivesicular bodies, present in the peripheral cytoplasm. This distribution of labeled sites was not observed in neutrophils, eosinophils, and lymphocytes which were also routinely present in these preparations. These sites were not labeled in basophils or other cells in control studies using ferritin and NRS. This indicates the necessity for specific binding to basophils in order to visualize this process. Whether most such sites represent residual endocytosis at 4 degrees C or binding to open invaginations near surfaces of cells does not detract from their specificity and thus expression of specific antigen(s)-binding sites in basophils.

Vesicular uptake of eosinophil peroxidase by guinea pig basophils and by cloned mouse mast cells and granule-containing lymphoid cells.

Guinea pig basophils, cloned mouse mast cells, and cloned mouse granule-containing lymphoid cells were found to utilize a vesicular transport system to internalize eosinophil peroxidase (EPO) added in vitro. Kinetic analysis indicated that EPO internalization involved the binding of EPO to the plasma membrane, the formation of complex surface invaginations, and the movement of EPO-laden vesicles, tubules, and vacuoles toward the center of the cells. EPO became associated with multivesicular bodies in granule-containing lymphoid cells and mast cells, with immature granules in mast cells, and with mature granules in basophils. In other cells, the endogenous production of granule peroxidases (neutrophils and eosinophils) or the prior uptake of exogenous peroxidatic substances (some basophils) precluded cytochemical analysis of granules for EPO. Vesicular transport of EPO provides a possible explanation for the variable detection of peroxidase activity in mast cells or basophils. It also provides a mechanism for sequestration of this potentially toxic material or for its storage for possible future use.

One-micron plastic sections of human peripheral blood and bone marrow specimens and peroxidase cytochemistry: use in the differential diagnosis of acute leukemias.

Peripheral blood or bone marrow samples from 142 patients with hematologic disorders were prepared for light microscopic study using 1-micron plastic sections. The same methodology provided excellent material for ultrastructural study of the same samples. Sixty-one of these cases were studied by electron microscopy and have been reported elsewhere. The techniques employed allowed the study of peripheral blood, a specimen generated by a relatively noninvasive procedure. The methods used provided light or electron microscopic identification of glycogen or peroxidase. These methods provided rapid results. Both light microscopic results and electron microscopic data were available within the same time frame as were routine paraffin sections. Preparation of plastic sections for light microscopy makes use of simple, well-known techniques and uses inexpensive manual microtomes. These methods are, therefore, accessible to any routine histopathology laboratory. The same material (blocks) can then be submitted to any diagnostic electron microscopic laboratory for further analysis if necessary. The extra resolution afforded by use of thin plastic sections for light microscopy allows easy identification of cell types. We have found these methods useful in the accurate classification of many acute leukemias and suggest that such improvement in diagnosis should be made available to physicians concerned with the diagnosis and care of patients with hematologic disorders.

Guinea pig bone marrow basophilopoiesis.

Basophilopoiesis was produced in guinea pigs with daily injections of sheep blood. Bone marrow preparations from these animals and controls were studied by electron microscopy at frequent intervals during 14 days of injections. We found that the composition of normal bone marrow consisted primarily of neutrophils and their precursors, small lymphocytes, plasma cells, macrophages, red blood cell precursors, megakaryocytes, and myeloblasts. The basophil lineage and blasts of any type were seen infrequently prior to injections. Serial injections of sheep blood produced a marked, early expansion of blasts and young basophilic myelocytes, followed later by the presence of numerous mature basophils. This proliferation was accompanied by a generalized granulocytic proliferation. Megakaryocytes also increased in number and were often surrounded by basophils. An important finding in this model of basophilopoiesis was the marked early expansion of blasts which were not distinguishable morphologically from a proliferation of large lymphoid cells and lymphoblasts. These actively dividing, ribosome-filled cells underwent Golgi expansion and accumulated vesicle-containing vacuoles. Later, single and then multiple, typical, immature basophil granules appeared. The presence of these granules allowed classification of these cells as young basophilic myelocytes. The origin of immature granules from the Golgi apparatus was evident. The association of small vesicles in the cytoplasm immediately adjacent to immature granules as well as within these evolving structures suggests a role for these vesicles in the construction of new granules. These in vivo observations of granulogenesis are identical to a much accelerated process we have described in mature basophils as they rebuild granules after degranulation in an in vitro model. Moreover, the presence of multivesicular structures and vesicle-containing immature granules in granulogenesis in in vivo studies of guinea pig basophilic myelocytes has been helpful for the identification of mouse basophilic myelocytes, both in vitro and in vivo.

Cloned mouse cells with natural killer function and cloned suppressor T cells express ultrastructural and biochemical features not shared by cloned inducer T cells.

We have examined the morphology, cytochemistry, and biochemistry of mouse leukocyte subsets by analyzing cloned leukocyte populations specialized to perform different immunologic functions. Cloned cells expressing high-affinity plasma membrane receptors for IgE and mediating natural killer (NK) lysis and cloned antigen-specific suppressor T cells contained prominent osmiophilic cytoplasmic granules similar by ultrastructure to those of mouse basophils. Both clones also incorporated 35SO4 into granule-associated sulfated glycosaminoglycans, expressed a characteristic ultrastructural pattern of nonspecific esterase activity, incorporated exogenous [3H]5-hydroxytryptamine, and contained cytoplasmic deposits of particulate glycogen. By contrast, cloned inducer T cells lacked cytoplasmic granules and glycogen, incorporated neither 35SO4 nor [3H]5-hydroxytryptamine, and differed from the other clones in pattern of nonspecific esterase activity. These findings establish that certain cloned cells with NK activity and cloned suppressor T cells express morphologic and biochemical characteristics heretofore associated with basophilic granulocytes. However, these clones differ in surface glycoprotein expression and immunologic function, and the full extent of the similarities and differences among these populations and basophils remains to be determined.

Unusual inclusions in plasmacytoid cells. Their occurrence in a patient with Waldenström's macroglobulinemia.

The bone marrow from a patient with Waldenström's macroglobulinemia was studied, and histologic, immunopathologic, and ultrastructural features correlated. The infiltrating plasmacytoid lymphocytes contained PAS-negative cytoplasmic inclusions, which were shown to contain a specific immunoglobulin that matched the patients's circulating paraprotein. Electron microscopic studies revealed abundant dense fibrillar and globular material within dilated cisternae of rough endoplasmic reticulum. This material was morphologically similar to, but quantitatively different from, an endoplasmic reticulum-associated structure previously described in several patients with chronic lymphocytic leukemia. The differences between these fibrillar inclusions and Russell bodies are stressed.

Crohn's disease. Ultrastructural studies showing basophil leukocyte granule changes and lymphocyte parallel tubular arrays in peripheral blood.

Buffy coat preparations from the peripheral blood of a 40-year-old woman with a six-year history of Crohn's disease were studied by electron microscopy. This patient did not receive treatment with prednisone, sulfasalazine, or azathioprine, and the blood samples for this study were taken prior to ileocolectomy. Four patients without Crohn's disease were studied as controls. In the circulating blood from the patient with Crohn's disease, there were several significant findings: lymphocytes that contained parallel tubular arrays were prominent, and degranulation was observed in the circulating basophilic leukocytes. These observations may be related to defective immunologic inflammatory processes in this disease, which is of unknown origin, and may lead to an understanding of some of the extraintestinal symptoms experienced by patients with Crohn's disease.

Metastatic adenocarcinoma of unknown primary site: diagnostic electron microscopy to determine the site of tumor origin.

Poorly differentiated malignant tumors and metastatic adenocarcinomas have been studied by electron microscopy. Rapid ultrastructural analysis allows the accurate diagnosis of the cell of tumor origin to be reported in conjunction with the routine surgical signout of paraffin sections. The identification of specialized intracellular structures is useful for determining the site of origin of metastatic tumors. Particularly useful in this material was the presence of lamellar (surfactant) bodies, typical of alveolar cell carcinoma of the lung, as well as the presence of apical terminal webs, diagnostic of gut epithelial cells.

Ultrastructural localization of nonspecific esterase activity in guinea pig and human monocytes, macrophages, and lymphocytes.

Using either hexazotized pararosaniline or new fuchsin as coupling agents, we investigated the ultrastructural localization of alpha-naphthyl acetate esterase (ANAE) activity in guinea pig bone marrow and peritoneal exudates, and in human peripheral blood cells. DFP-inhibitable ANAE activity was present on the cell surface of lymphocytes, monocytes, macrophages, neutrophils, eosinophils, basophils, megakaryocytes, platelets, and blasts. Demarcation lines in megakaryocytes and the perinuclear cisternae in normoblasts were also positive. In addition, lymphocytes, monocytes, and macrophages displayed ANAE activity associated with cytoplasmic-vesicle clusters (CVC). Reaction product was always present on the cytoplasmic surfaces of these vesicles and in the adjacent cytoplasm; vesicle interiors were invariably ANAE-negative. Small lymphocytes generally had a single large paranuclear ANAE-positive CVC, whereas mononuclear phagocytes had multiple discrete foci of similar appearing ANAE-positive CVC that sometime became confluent. ANAE activity was also found in the Gall bodies of human lymphocytes and in coated vesicles of macrophages. Cytoplasmic ANAE activity was increased in oil-induced guinea pig peritoneal macrophages. Both surface and cytoplasmic esterase activities had a neutral pH optimum. An identical distribution of reaction product was observed when alpha-naphthyl butyrate was employed as substrate. The function of these esterases, and their relation to known surface and cytoplasmic neutral proteases, awaits further investigation.

Diagnostic electron microscopy. I. Hematology: differential diagnosis of acute lymphoblastic and acute myeloblastic leukemia. Use of ultrastructural peroxidase cytochemistry and routine electron microscopic technology.

We have described in detail a number of ultrastructural methods which we have found to be useful for the evaluation of hematologic cases submitted to our diagnostic electron microscopic unit. The techniques include the preparation of peripheral blood for study as both buffy coat and cell suspension specimens and the preparation of bone marrow spicules. Ultrastructural methods for the demonstration of glycogen and peroxidase are detailed. The study of such material includes light microscopic study of plastic-embedded, alkaline-Giemsa-stained one micron sections as well as ultrastructural studies. All hematological cases submitted for ultrastructural analysis in a two-year period were reviewed and are presented here. The identification of individual mature cells was relatively simple using light microscopy. Populations of blasts could also easily be recognized. Further differentiation of blasts, primarily lymphoblasts or myeloblasts, was done using ultrastructural cytochemistry where needed. These techniques can easily be done in electron microscopy units concerned with diagnostic work. We submit that pathologists and hematologists should have access to the diagnostic tools described here in order to manage patients with acute leukemia.

Crohn's disease: transmission electron microscopic studies. III. Target tissues. Proliferation of and injury to smooth muscle and the autonomic nervous system.

Transmission electron microscopy was done on surgical specimens from 12 patients with Crohn's disease and three control subjects. Nonulcerated involved areas of ileum as well as proximal, grossly uninvolved resection margins were chosen for study. Specimens for transmission electron microscopy were prepared by a variety of techniques and 112 blocks were examined by electron microscopy. The study was concentrated on two target tissues of the gut: the autonomic nervous system and the smooth muscle. Proliferative and injurious changes were found in each. Proliferation, myofibroblastic transformation, hypercontraction, and necrosis characterized the smooth muscle changes seen in Crohn's disease of the ileum. Autonomic nervous system changes included proliferation of axons containing dense core granules (catecholamines) and axonal necrosis. The possible pathogenetic significance of these changes is discussed here and in the accompanying article beginning on page 606 of this issue.

Crohn's disease: transmission electron microscopic studies. II. Immunologic inflammatory response. Alterations of mast cells, basophils, eosinophils, and the microvasculature.

Transmission electron microscopy was done using surgically resected specimens from 12 patients with Crohn's disease and three control subjects. Nonulcerated involved areas of ileum as well as proximal, grossly uninvolved resection margins were chosen for study. Specimens for transmission electron microscopy were prepared for viewing by a variety of techniques. Six hundred five Epon embedded blocks were studied by light microscopy, and 112 of these were viewed in the transmission electron microscope. Study of the immunologic inflammatory response revealed a number of changes of interest. The number of mast cells was markedly increased, and they were found predominantly in edematous submucosa and between smooth muscle cells in the muscular coats of the involved gut. Evidence of focal and complete degranulation of mast cells was frequently seen. Basophilic leukocytes, some of which had degranulated, were also identified. Another frequently encountered cell, the eosinophil, showed changes in the granules, which might reflect the release of eosinophil granule materials. Eosinophils also contained an increased number of small dense granules of the arylsulfatase containing type. Eosinophils, basophils, and mast cells formed close associations. Evidence of acute and chronic damage to vascular endothelia was present. Small lymphocytes, plasma cells, macrophages, and epithelioid cells were present. Multinucleated giant cells were infrequently encountered. Lymphoblasts, however, were rarely seen.