Immature Basophils Research Articles

An Unusual Form of Chronic Myeloproliferative Disorder Aleukemic Basophilic Leukemia

A 52-year-old Japanese man manifested various clinical signs and symptoms such as vomiting, high fever, dyspnea, cough, sweating, palpitation, eosinophilic leukocytosis and hepatosplenomegaly. These histamine-related clinical manifestations showed a dramatic response to steroid therapy. After 10 months of hospitalization, he suddenly succumbed to candidal septicemia at the end of the third cycle of steroid therapy. Autopsy revealed neoplastic proliferation of immature basophils in various internal organs without involvement of the skin. The neoplastic cells, positive immunohistochemically for leukocyte common antigen, possessed lobulated nuclei and weakly metachromatic cytoplasmic granules, predominantly of the basophil type, which exhibited weak naphthol ASD-chloroacetate esterase activity. Mast cell-type granules were also observed ultrastructurally. The neoplastic infiltration was associated with fibrosis in the liver, spleen and bone marrow and with extramedullary hematopoiesis in the liver, spleen, lymph nodes and perihypophyseal tissue. The bone marrow showed uneven and multifocal involvement. Despite the lack of leukemic manifestations and the results of chromosomal analysis, the most suitable diagnosis was aleukemic basophilic leukemia within the category of chronic myeloproliferative disorder. Kinship of this neoplasia to systemic mastocytosis is discussed.

The purification of human basophils: their immunophenotype and cytochemistry.

An IgM class monoclonal antibody Bsp-1 that selectively reacts with human basophils was used to label basophils in normal and leukaemic haemopoietic samples. The immunophenotype of Bsp-1+ basophils was determined using a panel of 21 IgG subclass monoclonal antibodies in two-colour immunofluorescence assays. Basophils expressed the leucocyte common antigen, HLA-ABC antigens and antigens defined by CD11 and CD13 monoclonal antibodies. Other myeloid cell (granulocyte-monocyte) associated anti-antigens, lymphoid cell surface determinants and HLA-DR antigens were not detected. Basophil preparations of 95-98% purity were obtained from the peripheral blood of patients with CML and umbilical cords using fluorescence activated cell sorting techniques. Purified basophils exhibited metachromatic staining with toluidine blue, alcain blue and astra blue. PAS staining was observed in 7% of cord Bsp-1+ cells and of 55% CML Bsp-1+ cells. Between 5% and 10% of basophils were chloroacetate esterase-positive which suggests that some Bsp-1+ cells are immature basophils.

Immunophenotyping of Acute Myeloid Leukemia Using Monoclonal Antibodies and the Alkaline Phosphatase-Antialkaline Phosphatase Technique

The leukemic cells from 41 cases of acute myeloid leukemia (AML) and 17 cases of acute lymphocytic leukemia (ALL) were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technique utilizing eight monoclonal antibodies (MoAb) reactive with cells of myeloid origin and seven MoAb reactive with lymphoid antigens. Ninety percent of the cases of AML reacted with one or more of the pan-myeloid MoAb, My7, My9, or 20.3. Reactivity of the myeloid panel of MoAb showed some correlation with the French-American-British (FAB) classification of AML. Five of six cases of acute promyelocytic leukemia (APL) were HLA-DR negative; the one HLA-DR-positive APL had a minor population of HLA-DR-negative promyelocytes. OKM5 and/or My4 reacted with 16 of 16 monocytic leukemias. No specific marker of early erythroid development was identified. AP3, a MoAb reactive with platelet glycoprotein (GPIIIa), was specific for acute megakaryoblastic leukemia. Immunocytochemistry was also helpful in classifying seven cases of AML with equivocal or negative routine cytochemistry. Two cases of AML had minor populations of blasts detected by the APAAP technique that were immunologically distinct from the major blast population; these minor populations emerged as the predominant cell type at relapse. Two cases of ALL expressed multiple myeloid and lymphoid antigens. Two other cases that morphologically were ALL reacted with only myeloid MoAb; one consisted entirely of immature basophils on ultrastructural examination. Immunophenotyping results using the APAAP technique were comparable with those obtained with flow cytometry. The APAAP technique is a reliable method for immunophenotyping leukemia that complements other methods of immunologic evaluation. The primary advantages of this method include its use with routinely prepared blood and bone marrow smears and the ability to correlate immunocytochemical reactions with morphology.

Immunophenotyping of acute myeloid leukemia using monoclonal antibodies and the alkaline phosphatase-antialkaline phosphatase technique

The leukemic cells from 41 cases of acute myeloid leukemia (AML) and 17 cases of acute lymphocytic leukemia (ALL) were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technique utilizing eight monoclonal antibodies (MoAb) reactive with cells of myeloid origin and seven MoAb reactive with lymphoid antigens. Ninety percent of the cases of AML reacted with one or more of the pan-myeloid MoAb, My7, My9, or 20.3. Reactivity of the myeloid panel of MoAb showed some correlation with the French- American-British (FAB) classification of AML. Five of six cases of acute promyelocytic leukemia (APL) were HLA-DR negative; the one HLA-DR- positive APL had a minor population of HLA-DR-negative promyelocytes. OKM5 and/or My4 reacted with 16 of 16 monocytic leukemias. No specific marker of early erythroid development was identified. AP3, a MoAb reactive with platelet glycoprotein (GPIIIa), was specific for acute megakaryoblastic leukemia. Immunocytochemistry was also helpful in classifying seven cases of AML with equivocal or negative routine cytochemistry. Two cases of AML had minor populations of blasts detected by the APAAP technique that were immunologically distinct from the major blast population; these minor populations emerged as the predominant cell type at relapse. Two cases of ALL expressed multiple myeloid and lymphoid antigens. Two other cases that morphologically were ALL reacted with only myeloid MoAb; one consisted entirely of immature basophils on ultrastructural examination. Immunophenotyping results using the APAAP technique were comparable with those obtained with flow cytometry. The APAAP technique is a reliable method for immunophenotyping leukemia that complements other methods of immunologic evaluation. The primary advantages of this method include its use with routinely prepared blood and bone marrow smears and the ability to correlate immunocytochemical reactions with morphology.

Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils.

We used ultrastructural autoradiographic and cytochemical methods to localize esterase activities in unstimulated guinea pig basophils and in basophils undergoing degranulation or recovery from degranulation. We used tritium-labeled diisopropylfluorophosphate (DFP) as a probe for serine enzymes and localized this probe by ultrastructural autoradiography to cytoplasmic granules of immature or mature unstimulated basophils, as well as to granules released by degranulating basophils. Ultrastructural cytochemistry using alpha naphthyl acetate (ANA) as substrate localized nonspecific esterase activity to extruded granules, either within the interiors of degranulation sacs or within granules completely separated from degranulating basophils. Extruded granules retained their esterase activity for as long as 24 hr after antigen-induced degranulation. The plasma membranes of unstimulated or degranulating basophils, as well as of basophils recovering from degranulation, displayed prominent cell surface ANA esterase ectoenzyme activity. Lipid bodies, organelles present in the cytoplasm of both control and recovering basophils, were also alpha naphthyl acetate esterase (ANAE)-positive. Thus, cytochemical and autoradiographic techniques localized esterase and/or [3H]-DFP-binding activities to cytoplasmic granules, lipid bodies, and cell surface of basophils, and these enzyme activities persisted during both degranulation and recovery from degranulation.

Ultrastructural Cytochemistry of Congenital Basophilic Leukemia

A newborn infant was diagnosed as having congenital basophilic leukemia, an extremely rare disorder, by examination of cord blood. An ultrastructural cytochemical study was conducted on the blasts of the infant. Granules of basophil blasts showed ultrastructural characteristics of immature basophil granules and were similar in size to those of normal basophil granules but were fewer in number in each profile. Histochemical studies showed weak reactions for acid mucopolysaccharides and neutral glycoconjugates in the granules, but they were negative for peroxidase. Acid phosphatase was markedly positive. The findings of congenital basophilic leukemia are different from those of basophils in chronic myelocytic leukemia. The patient underwent induction therapy with vincristine and prednisone and has been in complete remission for 21 months.

A monoclonal antibody reactive with human eosinophils

EO-1, an IgGl murine monoclonal antibody raised against human eosinophilic leukemia cells, reacts with eosinophils, basophils, platelets, and a few (2%) mononuclear cells but not with neutrophils. In the bone marrow, mature and immature eosinophils and basophils express the EO-1 antigen, whereas immature myeloid cells do not. The distribution of EO-1 antigen on leukemic cells is concordant with this finding, ie, typical myeloid lines (HL-60, KG-1, and ML-1) and fresh acute myelogenous leukemia cells are all unreactive with EO-1. Immunoprecipitation of an extract from surface-131I-labeled platelets with EO-1 and sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing or nonreducing conditions, yielded a specific band of molecular weight 23,000. Previously described monoclonal antibodies reacting with eosinophils also recognize neutrophils. EO-1 is a unique antibody with specificity restricted to eosinophils, basophils, and platelets and might therefore be a valuable reagent for the study of their function and differentiation.

A Monoclonal Antibody Reactive With Human Eosinophils

EO-1, an IgGl murine monoclonal antibody raised against human eosinophilic leukemia cells, reacts with eosinophils, basophils, platelets, and a few (2%) mononuclear cells but not with neutrophils. In the bone marrow, mature and immature eosinophils and basophils express the EO-1 antigen, whereas immature myeloid cells do not. The distribution of EO-1 antigen on leukemic cells is concordant with this finding, ie, typical myeloid lines (HL-60, KG-1, and ML-1) and fresh acute myelogenous leukemia cells are all unreactive with EO-1. Immunoprecipitation of an extract from surface-131I-labeled platelets with EO-1 and sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing or nonreducing conditions, yielded a specific band of molecular weight 23,000. Previously described monoclonal antibodies reacting with eosinophils also recognize neutrophils. EO-1 is a unique antibody with specificity restricted to eosinophils, basophils, and platelets and might therefore be a valuable reagent for the study of their function and differentiation.

Dual expression of lymphoid/basophil markers on single blast cells transformed from chronic myeloid leukemia

A 27-yr-old man developed blastic crisis after the chronic phase of Philadelphia chromosome positive chronic myeloid leukemia (CML). The blast cells expressed terminal deoxynucleotidyl transferase (TdT) +/common acute lymphoblastic leukemia antigen (CALLA) + phenotypes, corresponding to common ALL type. A vincristine plus prednisolone regimen initially suppressed the blastic proliferation, but the blasts soon reappeared as lymphoblasts, and 65% of them possessed basophil-like granules. Immunologic markers were not altered. The blasts were negative for myeloperoxidase, Sudan black B and periodic acid-Schiff reactions, but were positive for toluidine blue (TB) stain and supravital peroxidase (PO) stain using diaminobenzidine (DAB). These blasts were considered to have immature basophil granules. The supravital staining, for TB or PO in combination with fluorescinated-CALLA staining, directly revealed that single blasts expressed both basophil and lymphoid markers. This biphenotypic blast population was found to be a distinct clone from the initial crisis clone by cytogenetic examination. These findings suggest that the CML clone is derived from a multipotent stem cell common to lymphoid and myeloid lineages, or that dual markers may be expressed on transformed lymphoid or basophil clone as the result of differentiation infidelity probably determined by the genetic derangement in acute crisis.

Ultrastructural cytochemistry of glycoconjugates in basophils from humans and animals

The distribution of glycoconjugates in the basophil granules of humans, guinea pigs, and rabbits was compared. The observation of acid mucopolysaccharides using the dialyzed iron method and of sulfated glycoconjugates using the high iron diamine method revealed three types of reactions in the basophil granules of all three species: granules showing a strong overall reaction, granules showing reaction only at their periphery, and granules showing no reaction. With regard to the relationship between maturation and the types of basophil granules, it appeared that, in general, there were many type-1 granules among immature basophils, but that these granules decreased in mature basophils as type-3 granules increased. The reaction patterns of periodate-reactive neutral glycoconjugates, as shown by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method, were different from those of acid mucopolysaccharides: the reaction of basophil granules was diffusely positive, and localization at the periphery was rarely observed. Therefore, unlike the acid mucopolysaccharides, it was difficult to classify the glycoconjugates into three types. However, as with acid mucopolysaccharides, there was a tendency for periodate-reactive glycoconjugates to decrease as maturation progressed. In terms of different species of animals, the reaction of periodate-reactive glycoconjugates with PA-TCH-SP was stronger in humans and rabbits than in guinea pigs.

A new leukemia cell line with philadelphia chromosome characterized as basophil precursors

A new myeloid cell line (KU812) was established from a patient with blastic crisis of chronic myelogenous leukemia. His blasts were morphologically characteristic of immature basophils and basophil colonies were grown in agar culture of the blood mononuclear cells. Suspension culture of his blood cells was continued for more than 2.5 years. The KU812 cells morphologically showed a fine reticular nuclei with nucleoli, and some of them contained metachromatic granules with toluidine blue (TB) staining. These granules were positive for astra blue (AB) staining. Immunological marker studies revealed that there were no lymphoid characters except Fc receptors. The KU812 cells grew colonies in in vitro agar cultures, which were proved to be composed of basophils by TB staining and AB staining. Cytogenetical analysis showed marked aneuploidy and was positive for the Philadelphia chromosome (Ph 1). The cell lysate was proved to contain histamine. These data suggest that KU812 is a cell line from leukemic basophil precursors. This is the first human basophil cell line, and KU812 will be useful in clarifying the mechanism of basophilic differentiation of the stem cells.

Secretory cycle of the pituitary basophils and its morphological evidence.

The pituitary basophils of rats were examined electronmicroscopically at various time courses of the following experiments: Acute, subacute and chronic administration or perfusion of TRH and LRH separately or in combination to normal male and ovariectomized female rats as well as thyroidectomy on male rats. The findings showed a sequential, ultrastructural transformation (type I to V) of the basophil which might perform its secretory cycle. The I-type cells represent immature basophils. The II-type cells (correspond to classical TSH-cells) are angular cells in a synthesizing and/or storing phase showing an abundance of secretory granules 100-150nm in diameter. The II/III-type cells come into two categories of cell type, presenting a peripheral vesiculation of the cytoplasm. The III-type cells (correspond to classical LH-cells) are round in shape and possess a number of vesicular structures and secretory granules of about 200nm in diameter throughout the cell body. The III/IV-type cells are characterized by numerous round large cisternae which are filled with fine granulated particles. Secretory granules in these cells are 200-250nm in diameter. The enlarged spherical IV-type cells (correspond to classical FSH-cells) are packed with irregularly expanded endoplasmic reticula. Secretory granules are about 250nm in size. The IV-type cells are presumably at the end stage of granular storage or at the initiation of granular secretion, since there is a transitional cell type (IV/II-type) in which secretory granules are reduced in size (100-150 nm in diameter) and in electron density. Vesicular endoplasmic reticula are infrequently found in the IV/II-type cells, which may be either at the end of the secreting phase or at the subsequent resting phase. The V-type cells appear even in the normal and numerously after repeated or prolonged administrations of TRH or LRH. They may be exhausted basophils. It was not, however, concluded that II-, III-and IVtype cells might correspond to TSH-, LH-and FSH-cells, respectively.Based on a sequential appearance of the cells mentioned above, we propose a hypothesis: The II-type may transform into the III-type, and subsequently into the IV-type which may come back to the II-type.

Development of pituitary acidophils and their granulation in pre- and postnatal rats.

Development of acidophils was investigated electron microscopically in pituitaries from the rat embryos on the 15th, 17th, 18th and 20th day of gestation and from the neonatal and postnatal rats on the 1st, 3rd, 4th, 6th, 12th and 18th day of birth. Pituitary agranular primordial cells are the stem cells from which acidophils develop. During embryonic life, the male is more advanced in differentiation of primordial cells than the female. Initial granulation begins to take place in “ambiguous cells” (Yoshimura et al., 1970) on the 17th day of gestation as a row of minute granules about 50 mμ along the plasmamembrane. Incidence of ambiguous cells with various shapes is higher in the male than in the female. During embryonic period, only a small number of immature acidophils have developed from ambiguous cells despite spherical enlargement of cell-bodies where dense granules maintain their size within the proximity of 250 mμ. Most of ambiguous cells tend to transform into immature basophils, and even gonadotroph-like basophils have accumulated during the latest pregnancy. On the other hand many immature acidophils emerge directly from primordial cells without the mediation of ambiguous cells. In a certain primordial cells, a row of dense granules as large as 130-200 mμ in diameter suddenly appear along the plasmamembrane on the 18th day of gestation. In equilibrium with the prompt increase in the cell number, their granules rapidly increase in dimension and their maximal size reaches 250 mμ towards the end of fetal period. Postnatal development of acidophils is quite quick as to the population and granulation in both sexes. Numerous immature acidophils, not always spherical but somewhat elongated, develop appositionally from primordial cells. Appearance of a row of large granules of 200-250 mμ along the plasmamebrane of primordial cells seems to be related to morphological representation for the main developmental process of acidophils. Granules in immature acidophils increase in number, their size being persistently less than 200 mμ up to the 6th postnatal day. Enlarged spherical premature acidophils containing numerous granules of 200-300mμ in diameter begin to appear on the 12th day. So-called somatotrophs identical with the fully developed acidophils filled with large granules of about 350 mμ start to appear on the 18th day of age.

Degranulated acidophils as a possible original source of "thyroidectomy cells" in the rat hypophysis.

The original source of thyroidectomy cells (TX-cells) in rats was investigated electron microscopically. Thyrotrophs did not respond so definitely to thyroidectomy as gonadotrophs did to castration. In thyrotrophs, changes in terms of hyperplasia, hypertrophy and de-or hypergranulation were not manifested. There was no evidence that TX-cells belonged to hyperactive thyrotrophs. The course of transformation from the degranulated acidophils to TX-cells was clearly demonstrated in sequence in this study. Hypertrophy, deformation of the cell, expansion of cisternae, occurrence of intracisternal granules and enlargement of the Golgi-ring, these were manifestations of the developmental process of degranulated acidophils into TX-cells. The other process of transformation of acidophils into TX-cells seemed related to the gradual dilation of ER without discharging their large granules completely. During the chronic phase of thyroidectomy, the anterior pituitaries were filled with TX-cells and their precursors. High vesiculation could be considered as an intrinsic biological reaction which is commonly seen in most of hypophyseal cells during thyroxine deficiency. It was, therefore, suggested that degranulated or slightly granulated acidophils might be predominantly the original sources of TX-cells. Thus, the acidophil theory has been firmly established as to the genesis of TX-cells. We, at the same time, wanted to open up a possibility that some TX-cells might arise from immature basophils, but the valid evidence was not produced in this study. It was verified electron microscopically that the fully developed round TX-cells seemingly like signet-ring-typed castration cells were quite different from genuine castration cells, although both cells had the same staining properties and light microscopical characteristics. However, a number of immature gonadotrophs came into being during the chronic phase of thyroidectomy.

Fine structure of pituitary acidophils in thyroidectomized rats with or without the treatment with thyroxine and dexamethasone.

Electron microscopic study was made on rat anterior pituitaries in an attempt to clarify the causal mechanism of “loss of acidophils” which could be detected with the light microscope following thyroidectomy. In the advancing days after thyroidectomy, the acidophils (somatotrophs) were gradually deprived of their large granules whose diameter was about 350mμ. There was evidence in vivo for the first time that there were some acidophils containing only the small granules, about 130mμ in diameter, after the 7th postoperative day. This clearly showed the simultaneous existence of two different sizes of granules in the same acidophils. As time elapsed, the large granules dispersed from the acidophils. After the 30th day these acidophils began to adjust their shape into the polygonal cells. Eventually, the acidophils completely lacking granules of either size occassionally appeared on the 90th and 200th days. Thus thyroidectomy had a strong influence to release the large granules, whereas an influence to release the small ones was not remarkable. The replacement injection of thyroxine into the thyroidectomized rats efficaciously reproduced the large granules which were generally considered to be responsible for GH storage. The repeated administrations of dexamethasone to the thyroidectomized rats resulted in the increase in number of small granules that were thought by us to be the storage carriers of ACTH. On the other hand, this compound alleviated loss of large granules following thyroidectomy. The final fate of acidophils after thyro cectomy was, however, still vague because of the equivocal distinguishment of these degranulated acidophils from the immature basophils. Thus, the “loss of acidophils” may not mean the intrinsic disappearance of the cells themselves, but represent a transient loss of the large dense granules in the remaining acidophils.

Electrom microscopic study of the anencephalic adenohypophysis with reference to the adrenocorticotrophs and their correlation with the functional differentiation of the hypothalamus during the foetal life.

The anterior pituitaries of three anencephalic embryos were observed by light and electron microscope. Normal embryos during terminal and mid-foetal life were served as controls. Many degenerating cells appeared in the pituitaries of the anencephalic embryos. Despite a shrinkage, these cells retained numerous so-called a-granules (300-400mμ in diameter) in their cytoplasm. The cells may be related to the corticotrophs which are destined to involute after mid-foetal life in the hypothalamusless state. The profound degeneration of the acidiopils was closely related to the involution of the adrenal cortex of the anencephalic foetal. The thyroid showed advanced development in the anencephalic embryos. Electron microscopically, the polygonal or sometimes round cells containing small granules (100-200mμ) were often observed. These cells may be related to immature basophils, but gonadotrophs were not yet recognized. Basophils were not degenerated in the anencephalics. It was suggested from the present observations that ACTH might be secreted from the adenohypophyseal cells during the early half of foetal life even in the absence of hypothalamic stimulation, but thereafter its secretion might be reduced or cease in parallel with the atrophic change of the adrenocorticotrophs. On the contrary, TSH was produced through the entire course of gestation independently of lack of hypothalamic mediation. These facts may indicate that functional development of the hypothalamus-pituitary-adrenal axis is established after mid-foetal life, whereas that of the other axes are done after birth.

Electron microscopy of the anterior lobe of pituitary in normal and castrated rats.

The ultrastructures of cells of the anterior lobe in normal and castrated male rats, 50, 128 and 300 days after the removals were investigated.1. Chromophobe cell is a maternal or immature cell from which acidophil and basophil may develop. The transformation axis from chromophobe cell to basophils was exclusively stimulated in degree by the castration. Following the castration, granulation was progressive in the enlarged cytoplasm of chromophobe cells, which may turn into polygonal or round basophils.2. The size-difference in dense beta-granules in basophils is not indispensable for the identification of gonadotroph and thyrotroph, because beta-granules, 100 to 150mμ in diameter, in immature basophil (thyrotroph-type) of castrated rats may grow up to large one, 200 to 250mμ in diameter, which are common with those in chronic castration cells. Thus the castration made the number and the size of granules in basophils increase. Polygonal thyrotroph may correspond to a cell preceding to round gonadotroph in a series of life history of the basophile cell.3. Beta-and alpha-granules may be produced together in Golgi area. Evidences were presented that vesicles were gradually transformed into granules of variable density. The former, therefore, assumed to be a precursor of the latter. The present observation indicated that granules were stable vehicles for the longterm preservation in the cytoplasm, and vesicles were seemingly important carriages which might work as the active principle.4. Following the chronic castration, most of basophils turned into the cast ration cells which were thereafter converted into signet-ring cells, in which huge colloid lumen seems to be originated from the coalescence of smooth endoplasmic reticulums. Within 128 and 300 days, signet-ring cells were provided with a degenerating sign on account of prolonged preservation. Big granules (700 mμ) yielded numerously.5. In addition to alpha-granules, so-called prolactin granules with ellipsoid contour are rarely released out of cell-body by adhesion of the limiting membrane of the granules to the plasma membrane even in male rats. They may not be pushed out but mostly dissolved by enzymatic activity. On the other hand, the discharge of beta-granules presumably takes place in normal and castrated rats by diffusion of the granular content due to the vanishment of the limiting membrane. Diffused material might be eliminated through the membrane of endoplasmic reticulum and stored in it.6. The above ultrastructures would reinforce the previous doubt of the authors about the subdivision of gonadotroph and thyrotroph.