Immature Basophils Research Articles

Accurate white blood cell differential by Alinity hq: A comparison with flow cytometry and manual differential.

White blood cell (WBC) differential by flow cytometry can report a six-part WBC differential and enumerate blasts. Some modern hematology analyzers are also able to provide a six-part WBC differential (including immature granulocytes). Our goal was to compare the WBC differential obtained by the Abbott Alinity hq hematology analyzer to an 8-color single-tube flow cytometry method and to manual WBC differential. Samples from 144 patients were tested with Alinity hq, flow cytometry, and microscopic WBC differential. The WBC count ranged from 1.22 to 359×109 /L, and 34 samples were flagged by the analyzer for abnormal WBC morphology. Strong concordance was demonstrated between Alinity hq and flow cytometry for all six components of the differential, with correlation coefficients ranging from 0.86 (basophils) to 1.00 (lymphocytes). Small, clinically insignificant positive difference was observed between Alinity hq and flow cytometry for mature and total neutrophils (2.51% and 1.85%) and eosinophils (0.14%), and small negative difference for immature granulocytes (-0.65%), lymphocytes (-0.61%), and basophils (-0.21%). No bias was detected between the Alinity hq and flow cytometry monocyte counts. Alinity hq and flow cytometry results agreed with the manual differential, apart from small, clinically insignificant differences. Alinity hq nucleated red blood cell concentrations were equivalent with the manual results (r=0.95, slope=1.16). The percentage of blasts by flow cytometry demonstrated good correlation and agreement with the manual count (r=0.99, slope=1.35). Alinity hq has produced accurate six-part WBC differential in this three-way comparison, equivalent to flow cytometry and morphological classification.

Tryptase: A novel prognostic biomarker in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome. It is treated with tyrosine kinase inhibitor agents targeted against the breakpoint cluster region-Abelson murine leukemia 1 fusion transcript. Disease risk stratification at diagnosis of chronic-phase (CP) CML is done using Sokal, Hasford, and EUTOS scores which use basophilia as a major component. However, basophil counts can be both variable and inaccurate. The serum tryptase level is being studied as a novel biomarker which represents the total basophil compartment. Tryptase, deriving its name from trypsin-like activity commonly expressed by mast cells, and also by immature basophils of patients suffering from various myeloid and leukemic disorders, has a role in tumor proliferation. Patients with seemingly low-normal levels of basophils and raised tryptase levels progress further in disease despite treatment. There is a recent interest in the role of serum tryptase as a prognostic marker in CML-CP.

Abrupt evolution of Philadelphia chromosome‐positive acute myeloid leukemia in myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a clonal disorder arising from an alteration in multipotent stem cells, which lose the ability of normal proliferation and differentiation. Disease progression occurs in approximately 30% MDS cases. Specific chromosomal alterations seem responsible for each step in the evolution of acute myeloid leukemia (AML). Multiple genetic aberrations occur during the clonal evolution of MDS; however, few studies report the presence of the Philadelphia (Ph) chromosome. We report a rare case of Ph-positive AML, which evolved during the course of low-risk MDS. The patient, a 76-year-old man with mild leukocytopenia, was diagnosed with MDS, refractory neutropenia (RN). After 1.5 yr, his peripheral blood and bone marrow were suddenly occupied by immature basophils and myeloblasts, indicating the onset of AML. A bone marrow smear showed multilineage dysplasia, consistent with MDS evolution. Chromosomal analysis showed an additional t(9;22)(q34;q11) translocation. Because progression occurred concurrently with emergence of the Ph chromosome, we diagnosed this case as Ph-positive AML with basophilia arising from the clonal evolution of MDS. The patient was initially treated with nilotinib. A hematological response was soon achieved with disappearance of the Ph chromosome in the bone marrow. Emergence of Ph-positive AML in the course of low-risk MDS has rarely been reported. We report this case as a rare clinical course of MDS.

Serum Tryptase Is a Strong Predictive Biomarker That Improves Prognostication in Ph+ Chronic Myeloid Leukemia.

Abstract 2783Although the prognosis of patients with chronic myeloid leukemia (CML) has improved significantly with the advent and use of novel BCR/ABL tyrosine kinase inhibitors (TKI) in recent years, still a number of patients fail to achieve a durable molecular remission or they relapse. It is of particular importance to define the prognosis in individual patients as early as possible, as timely intervention with novel TKI may improve long term outcome. Basophilia is one of the most predictive and best documented biomarkers in CML at diagnosis. However, in high risk patients, the basophil-committed compartment of the clone may exceed the morphologically identifyable fraction of mature granulated basophils detected in MGG or Giemsa stains. In addition, even granulated basophils may be immature cells and may therefore escape microscopic examination. Tryptase is a serine protease primarily expressed by mast cells but also by immature basophils. In healthy controls, the serum tryptase level ranges between <1 and 15 ng/mL. In freshly diagnosed CML, the serum tryptase level has been reported to be either normal or elevated. In those with elevated serum tryptase, the enzyme is considered to be expressed and released by immature CML basophils. In the current project we asked whether tryptase levels at diagnosis correlate with the phase of disease and/or represent a prognostic marker in CML. To address this question, serum samples from a total number of 69 chronic phase (CP) CML patients (median age: 55, range: 21–84; f:m ratio = 1:1.55) and 10 patients with advanced disease (i.e. 9 patients in accelerated phase = AP and one in blast phase = BP) seen at the Medical University of Vienna between September 1998 and June 2011, were analyzed retrospectively. The median serum tryptase level at diagnosis was significantly lower in CP patients (11.7 ng/mL, range 1.4–65.5 ng/mL) compared to patients in advanced disease, i.e. AP/BP (29.9 ng/mL, range 8.1–67.7 ng/mL; p<0.05). We also found that serum tryptase levels correlate with basophil counts (p<0.05). The median observation time in our CP patients was 3.5 years. All patients received therapy with TKI (imatinib n=66; dasatinib n=2; nilotinib n=1). The median progression-free survival (PFS) in our CP patients was 2.7 years. Overall, 12 CP patients (17%) showed a progression. Eight of these patients (75%) were still alive at the end of the observation period. In the cohort of CP patients with elevated tryptase (>15 ng/mL), 31% showed a progression, whereas in the cohort with normal tryptase (≤15 ng/ml), 9% of the patients were found to progress (p<0.05). Of all peripheral blood parameters analyzed, including white blood cell counts, platelets, eosinophils, basophils, and peripheral blast cells, only basophils and tryptase were found to be significant prognostic variables in univariate analysis. As assessed by multivariate analysis performed with the same paramters as above except basophils, tryptase was found to be an independent prognostic variable. Since basophilia is a key variable used in the recently established EUTOS score, we asked whether basophils can be replaced by serum tryptase levels. Indeed, with regard to PFS, this novel EUTOS-T score was found to be of prognostic significance. In particular, the EUTOS-T score was found to predict the PFS of our patients with high confidence and a p-value of 0.004 compared to a p-value of 0.019 in the original EUTOS score. In addition, the EUTOS-T score was found to be a superior score for predicting event-free survival compared. Together, these data suggest that the serum tryptase level is a useful prognostic biomarker in CML. Moreover, the substitution of basophils by tryptase in established scoring systems may improve the predictive value of these scores. Disclosures:Sperr:Phadia: Research Funding. Valent:Phadia: Research Funding.

Human Cord Blood Derived Immature Basophils Show Dual Characteristics, Expressing Both Basophil and Eosinophil Associated Proteins

Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL)-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95%) and expression of surface markers FcεRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cell-specific transcripts, i.e. β-tryptase, serglycin and FcεRI α-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP), charcot leyden crystal (CLC), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and basophils are closely linked during human hematopoietic development. The dual phenotype also indicates that other cytokines than IL-3 or cell surface interactions are needed to obtain the full basophil specific phenotype in vivo.

Basophilic leukemia in recurrent chronic myelogenous leukemia blast phase

A 31-year-old man with a four-year history of chronic myelogenous leukemia (CML) presents in his second blast phase. The patient’s initial diagnosis was made after routine analysis revealed a white blood cell count (WBC) of 353 10/L. Initial bone marrow biopsy results and cytogenetics were not available for review. The patient responded initially to hydroxyurea followed by imatinib mesylate, at 400 mg daily. A subsequent bone marrow was consistent with morphologic remission; results of cytogenetics and molecular studies were not available. After *3-years, a repeat bone marrow biopsy revealed myeloid blasts comprising 80% of cells consistent with blast phase of CML. Retrospective review of peripheral blood and bone marrow showed only rare (less than 1%) blasts containing granules resembling immature basophils. RT-PCR was positive for BCR-ABL transcript (p210 and p190), and chromosome analysis showed 47, XY, t(2;3)(p23;q29), +8, t(9,22)(q34; q11.2) in 20 of 20 metaphase spreads. Flow cytometric analysis revealed a CD45 positive blast population with moderate side light scatter that strongly expressed CD34, CD117, and CD7 as well as variably expressed CD13, CD33, CD11c, and CD2. Intracellular staining revealed that 1/3 of these cells were myeloperoxidase positive. The imatinib mesylate dose was increased to 800 mg daily but was not tolerated due to nausea. The imatinib mesylate was discontinued, and a second generation ABL kinase inhibitor was initiated. The patient responded to this drug for several months until severe abdominal pain lead to its discontinuation. Less than one week after discontinuation the patient was found to have a WBC of 17 10/L with 30% blasts in peripheral blood, and the tyrosine kinase inhibitor was restarted. Circulating blasts persisted, and the patient underwent induction chemotherapy with an eventual goal of allogeneic transplant. Following this treatment, blasts were no longer detected in peripheral blood, and the patient was thought to be in hematologic remission. After *3-months our patient presented with a WBC of 47 10/L and 76% blasts. These results are consistent with recurrent CML blast phase, though morphologic remission was not established by bone marrow biopsy and persistence of earlier blast crisis remains a possibility. Multiple blast populations were detected morphologically in peripheral blood (Images 1–3), and presence of metachromatic granules after toluidine blue stain indicated a subset (9% of blasts) of basophilic origin (Image 4); maturing basophils were also present (4% of WBC). The patient has since entered a research protocol and had response to a different second generation ABL kinase inhibitor. After 3-weeks of therapy, his WBC has decreased to 1.9 10/L, although occasional blasts (6%) persist. CML is a myeloproliferative disease of bone marrow derived stem cells that contain fusion of BCR on chromosome 22 with c-ABL on chromosome 9 (i.e. Philadelphia chromosome) and which characteristically produces the p210 fusion protein. The initial indolent chronic phase of CML is unstable, and the disease ultimately progresses to accelerated and blast phases. Since the Philadelphia chromosome can be detected in all hematopoietic lineages the blast phase may include neutrophilic, eosinophilic, monocytic, erythroid, megakaryocytic, and basophilic blasts in predominance or in any combination. In about 20–30% of patients the blast phase is due to proliferation of lymphoblasts, and rare patients may have both myeloid and lymphoid blast populations. Acute basophilic leukemia represents <1% of all cases of acute myelogenous leukemia (AML) and must be differentiated from the blast phase of CML, AML with maturation associated with 12p abnormalities or t(6;9), acute eosinophilic leukemia, and acute lymphoblastic leukemia with prominent coarse granules. The persistence of t(9,22), as in the described case, is strong evidence for a predominant basophilic blast population arising from abnormal CML stem cells.

Revealing the Spectrum of Clinical Syndromes in Idiopathic Myelofibrosis by Cluster Analysis: Featuring “Indolent Myelofibrosis”.

To get a clinical classification of MMM, we enquired the database of the Italian Registry of Myelofibrosis (RIMM), a population-based prospective cohort started in 1999, and data of 871 patients at diagnosis were extracted. This population of patients was used as the learning set. The EM-algorithm (WEKA software) conducted a non-supervised data-mining based on 11 variables: age, spleen size, constitutional symptoms, hemoglobin, platelet count, white blood cell count, erythroblasts, immature myeloid cells, myeloid blasts, basophils, and plasma LDH. Differences among clusters were first tested with ANOVA or n-way Chi-square and subsequently investigated with t-test (independent samples) or 2x2 Chi-square. Cluster 1 identified a category of patients whose disease phenotype at diagnosis was of younger age, normal values of hemoglobin, thrombocytosis, and small spleen. We now propose to call this cluster indolent myelofibrosis. Cluster 2 identified patients with mild anemia, no sign of myeloproliferation (neither thrombocytosis nor leukocytosis), and larger splenomegaly. Cluster 3 selected patients which featured severe anemia, large splenomegaly and high number of immature myeloid cells in peripheral blood with the presence of a significant number of blasts, that we propose to call MMM in accelerated phase. Finally, cluster 4 selected patients presenting with trilinear or bilinear cytopenia (mostly thrombocytopenia). By using a decision tree, we tested the best discriminatory parameters to be used for including a new patient in the cluster of indolent myelofibrosis. They resulted: age lower than 55 years, platelet count over 335 x109/L, Hb greater than 12 g/dL, and spleen size smaller than 5 cm from the costal margin. We applied this algorithm to a validation set of 264 patients with idiopathic myelofibrosis who had the follow-up studied (mean follow-up, 67 months, range 1 -315 months). They were 103 females (38.8%), and the mean age was 51.3 years (range 6 to 82 years). Forty-seven (17.8%) of the validation set of patients met the criteria for indolent myelofibrosis. They had a mean age of 39.1 years. The number of WBC was not significantly different in indolent myelofibrosis with respect to that of the remaining patients (9.5 x109/L vs. 9.7 x 109/L). However, the number of circulating immature myeloid cells and erythroblasts, serum LDH, and circulating CD34+ cells were significantly lower in indolent myelofibrosis with respect the remaining patients. Forty-four percent of patients with indolent myelofibrosis had a bone marrow biopsy with a grade 0 fibrosis. Only 1 patient died in the group of indolent myelofibrosis vs. 31 in the group of other patients. The 10-year severe anemia-free survival was 90% (severe anemia = Hb < 10g/dL), in contrast with 30% for other patients; leukopenia-free survival (leukopenia = <4x109/L WBC) was 80% with respect to 40% in other patients; large splenomegaly-free survival (large splenomegaly = spleen margin at more than 10 cm from the left costal arc) was 60% in contrast with 20% in other patients (P<0.001 in all). Only 1 patient had blast transformation with respect to 17 in the remaining population; so, the blast transformation-free 10-year survival was 100% in indolent myelofibrosis and 79% in other patients.

Isolation of transcriptionally active umbilical cord blood‐derived basophils expressing FcɛRI, HLA‐DR and CD203c

Basophils are inflammatory cells associated with allergy and parasite infections. Investigation of their true biological function has long been hampered by the difficulty in obtaining sufficient amounts of pure basophils and by the lack of phenotypic markers. Moreover, it has been very difficult to clone and identify basophil-specific granule proteins, partially because of an almost complete lack of mRNA in mature circulating basophils. To obtain transcriptionally active immature basophils, umbilical cord blood cells were cultured in the presence of interleukin (IL)-3. The cells were analysed by flow cytometry and by histological staining. The continuous presence of IL-3 in cord blood cultures resulted in the expansion of basophil precursors co-expressing FcepsilonRI and the recently described mast cell/basophil marker, 97A6 (CD203c). Several nonbasophil markers (i.e. CD3, CD14, CD15, CD16, CD19 and CD21) were absent on the cultured basophils. However, we show that in early cultures, almost 60% of the CD203c+ cells co-express human leukocyte antigen (HLA)-DR, a marker that is absent on mature circulating basophils. The presence of HLA-DR on basophil precursors may explain the low recovery (24+/-5.2%) obtained after isolation of cultured basophils, when using a conventional basophil isolation kit that removes HLA-DR+ cells. A novel purification method was developed, including a two-step cocktail of antibodies against selected markers, which resulted in both high purity (95+/-0.5%) and recovery (59+/-1.5%) of cultured basophils. We here establish cord blood cultures as a source from which transcriptionally active basophil precursors can be isolated in reasonable quantities for thorough biochemical characterization.

Clinical Classification of Myelofibrosis with Myeloid Metaplasia (MMM): Cluster Analysis of 861 Patients Enrolled into a Nationwide Prospective Registry (RIMM).

BACKGROUND: MMM is a rare chronic myeloproliferative disease with heterogeneous clinical presentation, natural history and prognosis. Knowledge about MMM is burdened by limited sample size and selection bias of the available studies.AIM: To get a clinical classification of MMM for biological and clinical studies.METHODS: The database of the RIMM, a population-based prospective cohort started in 1999, was enquired and data of 861 patients at diagnosis were extracted. The EM-algorithm (WEKA software) was used for a non-supervised data-mining based on 11 variables: age, spleen size, constitutional symptoms, hemoglobin level, platelet and blood cell count, LDH and percentages of erythroblasts, immature myeloid cells, blasts, and basophils. Differences among clusters were first tested with ANOVA or n-way Chi-square and subsequently investigated with t-test or 2x2 Chi-square. Survival differences among clusters were tested through Cox univariate and multivariate regression analysis.RESULTS: Five clusters (C) were identified: C1 identified younger patients with thrombocytosis and scarce myeloproliferation. C2 also identified patients with a high platelet count, but older and anemic. C3 identified patients with intense myeloproliferation (marked leukocytosis and splenomegaly), while C4 (5% of the patients) collected patients with an even more severe myeloproliferation, associated with a high number of circulating immature myeloid cells and blasts. C4 was also characterized by frequent chromosome abnormalities, anemia and thrombocythopenia. Finally, C5 identified patients with bilinear/trilinear cytopenias. The clusters also differed for variables not incorporated by the data-mining process, i.e. CD34+ cell count (p=0.028); i.e. the rate of homozygous V617F mutation of JAK2 gene, which was significantly higher in C3 and C4 (32.4% vs 12.5%, p=0.019). The clusters also significantly differed for overall survival (p<0.0001), independently of patients' age. Finally, referral patterns differed among clusters, with C5 and C1 patients being mostly referred to Internal Medicine and Hematology units (p<0.01), respectively.CONCLUSIONS: Thanks to a large and unbiased repository of cases, we identified 5 classes of MMM patients with specific clinical presentation. The biologic and prognostic value of the clusters deserves further investigations at longer follow-up periods.Clinical parameters in the different clusters (means)CLUSTERAGE (yrs) *HB (g/l) */PLT*10(9)*WBC *10(9) *SPLEEN (cm) *BLASTS*(%)CD34 %/μlSEVERITY SCORE§ *156.713.3 /58311.53.50.210.85 /631.25272.110.6 /48218.32.70.821.12 /2972.04365.110.8 /30318.39.91.471.7 /3132.54467.68.4 /11027.513.45.662.72 /2754.30568.28.4 /1344.26.80.782.92 /523.36All65.510.7 /36213.56.51.181.78 /2002.36* p<0.0001 § The score considers HB, WBC, PLT and SPLEEN (Barosi, Leuk Lymph 2002).

CML Developed in a Japanese Family Transmitting a Novel Point Mutation in the Thrombopoietin Gene(TPO).

Introduction: Four families are reported to have hereditary thrombocythemia (HT) with a mutation in TPO. Their clinical manifestation is essentially thrombocytosis without leukemia. CML is one of myeloproliferative disorders, and shows leukocytosis and thrombocytosis associated with a proliferation of malignant clone originated from a hematopoietic stem cell (HSC). The incidence of CML is about 5 per 100,000 in Japan. Mutations of cytokine receptor including c-kit, flt-3 and G-CSF receptor are reported as a cause of AML. Especially flt-3 abnormalities are found in about 20% of AML. However, abnormality of c-mpl or TPO is not reported as a cause of leukemia. In this paper, we analyzed a CML case with novel point mutation in the TPO who still had thrombocytosis after cytogenetic complete response.Case: Japanese, 35 y.o., male, complained leukocytosis. He had a family history of thrombocytosis in 4 individuals over 3 generations. A physical examination revealed a moderate splenomegaly. Laboratory tests at the time of diagnosis were as follows; WBC 141,000/μl (blast 1.8%, promyelo 2.4%, myelo 20.0%, meta 8.2%, stab 24.2%, seg 22.2%, immature eosinophils 1.8%, eosinophil 3.6%, immature basophils 0.4%, basophils 10.4%, mono 1.0%, lymphocytes 4.0%, erythroblast 3%), PLT 641,000/μl and NAP score 53 (nl; 156–271). Bone marrow showed hypercellularity with the increased megakaryocytes(Meg), bcr-abl fusion mRNA positive, Ph1 chromosome positive. After 5 months treatment with STI571, most of clinical findings including karyotype and fusion mRNA turned to be normal, but thrombocyte(PLT) still showed more than 1,000,000/μl. At this time, serum TPO concentration was 8.14 f mole/ml (nl; 0.40 +/− 0.28 f mole/ml, mean +/− SD). Genetic analysis of TPO revealed novel point mutation at splicing donor site of 3′-end of the exon3. A point mutation at splicing donor site is reported to cause an exon-skipping and intron-retention, which induce a malfunction of a suppressive post-transcriptional and translational regulation, and consequent high-level expression of functional TPO protein.Discussion: TPO was cloned as a c-mpl ligand, which leads to the production of PLTs. Its receptor is a c-mpl proto-oncogene product, which is expressed not only in Meg, but also in HSC. Thus, TPO can stimulate HSC. The c-mpl transgenic mice are reported to have the increased Meg, its committed progenitor and PLT. Knockout mice of TPO presented not only the decreased Meg, but also multi-lineage committed progenitors. Thus, a modulation of c-mpl or its ligand function affects on both Meg and HSC. The c-mpl was cloned as a cellular homolog of a viral oncogene, v-mpl of myeloproliferative leukemia virus (MPL). The MPL causes myeloproliferative leukemia syndrome through v-mpl function in mice. Since v-mpl and c-mpl indicate high homology, it is possible that abnormal c-mpl function causes v-mpl like response. Through continuous stimulation of c-mpl signal, high TPO concentration may have induced a malignant transformation of HSC or supported a survival of an immature malignant clone in the present case. Improvement of thrombocytosis in CML is one of hematological responses to an anti-CML treatment such as STI571. In such a case who had good response other than thrombocytosis, an existence of HT might be considered. On the other hand, during following up HT family, occurrence of CML should be noted.

Pro-Th1 cytokines promote Fas-dependent apoptosis of immature peripheral basophils.

We have previously characterized immature hemopoietic cells of the basophil lineage as a lin(-)c-kit(-) population, which responds to IL-3 by enhancing its histamine synthesis through histidine decarboxylase activation. Herein, we show both in vitro and in vivo that exposure to the pro-Th1 cytokines IL-12 and IL-18 promotes Fas-dependent apoptosis of these cells in the spleen. This conclusion was supported by the following findings: 1) A 24-h treatment with IL-12 plus IL-18 enhanced Fas expression and annexin staining among basophil precursor-enriched lin(-)c-kit(-) splenocytes. 2) Fas or Fas ligand deficiency in mutant mice abolished the inhibitory effect of IL-12 plus IL-18 on IL-3-induced histamine production. 3) The large spectrum inhibitor of the caspase cascade, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone, significantly reduced the effect of IL-12 plus IL-18. The inhibition of histamine production was mediated through NK cells, since it failed to occur upon stimulation of spleen cells from NK cell-deficient mice or after NK cell depletion. IL-12 plus IL-18 rendered NK cells cytotoxic against Fas-transfected target cells and promoted their production of IFN-gamma and TNF-alpha, which are both essential for sensitizing histamine-producing cells to the Fas death pathway. This is the first evidence that pro-Th1 cytokines can promote apoptosis of immature peripheral histamine-producing cells, thus limiting Th2 immune responses. Comparable in vivo data as well as increased histamine production in the spleen of aged Fas-deficient lpr mice support its physiological relevance.

Expression of eosinophil peroxidase in the immature basophil cell line KU812-F

Although peroxidase activity in basophils can be detected by optical and ultrastructural cytochemistry, its characteristics remain to be determined. We have demonstrated the characteristics of peroxidase activity induced in the immature basophil cell line, KU812-F. Ultrastructually, peroxidase activity was detected in granules as well as in the perinuclear space and endoplasmic reticulum. Immunocytochemisty revealed that KU812-F cells were stained by anti-eosinophil peroxidase antibodies, and eosinophil peroxidase mRNA, not myeloperoxidase, was detected in the cells using Northern hybridization and reverse transcription–polymerase chain reaction. Eosinophil peroxidase can be one of the molecules shared with eosinophils and basophils. The biological function of eosinophil peroxidase detected in basophils remains uncertain.

Regulation and possible function of axl expression in immature human mast cells.

The receptor tyrosine kinase Axl which expresses extracellular domains reminiscent of cell adhesion molecules, is involved in homotypic binding as well as in intracellular signaling of myeloid progenitor cells. In order to investigate factors which might influence differentiation pathways through changes of the adhesive properties of cells, we analyzed the expression of axl in immature basophil and mast cell lines and in cultured basophil and mast cell precursors. Axl expression was induced by interferon-alpha in the human leukemic mast cell line HMC-1 and in cultured mast cells derived from CD34+ peripheral blood cells. Axl induction was dose dependent, appeared within 1 h, and was independent of de novo protein synthesis. IFNalpha-treated HMC-1 cells expressing axl formed large cell aggregates within 40 h while untreated cells did not. HMC-1 cells also expressed gas6, the putative ligand of axl, which has been shown to induce axl-mediated homotypic binding. Gas6 expression was independent of interferon treatment in HMC-1 cells. The present results suggest that axl-mediated changes of cellular adhesive properties in mast cells may be important in mast cell differentiation as well as in mast cell-associated inflammation.

Transformation into acute basophilic leukaemia in a patient with myelodysplastic syndrome

We describe a patient with basophilic leukaemia following a 2-year period with myelodysplastic syndrome (refractory anaemia). The marrow showed 59.4% of blasts with 25.0% of mature and immature basophils. The leukaemic blasts contained granules, positively stained with toluidine blue but negative for peroxidase. The basophilic differentiation was confirmed by ultrastructural analysis demonstrating immature basophil granules. In addition, a morphological transition from immature blasts to more mature basophils was observed. Immunophenotypic analysis of blasts and basophils showed positive for CD5, CD7, CD13, CD33 and CD34. Cytogenetic investigation showed an abnormal karyotype, 46,XY,del(5)(q31q35), in 11% of the cells examined when the initial diagnosis of refractory anaemia was made. However, expansion of the same clone up to 100% was observed concomitantly with transformation to basophilic leukaemia.

Development of markers for human basophils and mast cells

Development of markers for human basophils and mast cells

The effects of stem cell factor on the ultrastructure of Fc epsilon RI+ cells developing in IL-3-dependent murine bone marrow-derived cell cultures.

Stem cell factor (SCF) is known to alter the proteoglycans, proteases, and cytokines synthesized by mast cells and to activate basophils. To determine whether SCF could also effect the ultrastructural characteristics of basophils and mast cells, we examined the ultrastructure of these Fc epsilon RI+ cells over 42 days in IL-3-dependent murine bone marrow-derived cell cultures in the presence or absence of SCF. Initial experiments revealed that the addition of SCF to IL-3-dependent cells enhanced their proliferative rate without influencing the percentage of Fc epsilon RI+ or metachromatic cells. We next isolated the Fc epsilon RI+ cells using flow cytometry. Light microscopy of these cells revealed mixed cultures of both immature and mature mast cells and basophils with mature mast cells predominating by 3 wk. One hundred to 150 Fc epsilon RI+ cells were then photographed by electron microscopy at 3, 10, 21, and in some cases, 42 days of culture, and the ultrastructure of each cells was evaluated by morphometry. Each cell was scored as a mast cell or basophil using standard criteria. Analysis of this data revealed that SCF in the presence of IL-3 promoted the development of mast cells, although a significant number of basophils were noted at day 21 but were absent by day 42. When bone marrow cells cultured in IL-3 + SCF were compared with cells cultured in IL-3 alone, a significant decrease in cell and nuclear size and granule number and size was noted in both mast cells and basophils cultured in IL-3 + SCF, and basophils and mast cells under these conditions most resemble their in vivo counterparts. Thus, SCF in the presence of IL-3 increases the ratio of mast cells to basophils and alters the ultrastructural characteristics of mast cells and basophils toward a more mature phenotype.

Infection of Nippostrongylus brasiliensis Induces Normal Increase of Basophils in Mast Cell-Deficient Ws/Ws Rats With a Small Deletion at the Kinase Domain of c-kit

AbstractAll basophils, mucosal-type mast cells (MMC) and connective tissue-type mast cells (CTMC) are derived from the multipotential hematopoietic stem cell. Mutations at the c-kit locus resulted in deficiency of MMC and CTMC in both mice and rats. To investigate the role of the c-kit receptor tyrosine kinase for production of basophils, we used white spotting/white spotting (Ws/Ws) mutant rats that have a small deletion at the tyrosine kinase domain of the c-kit gene. When Ws/Ws, nude athymic, and normal (+ / +) rats were infected with Nippostrongylus brasiliensis (NB), the number of basophils increased greater than 50-fold in the peripheral blood of Ws/Ws and + / + rats but did not increase in that of nude rats. Blood histamine concentration increased significantly in Ws/Ws and + / + rats but did not increase in nude rats. Immature basophils increased greater than 10-fold in the bone marrow of Ws/Ws and + / + rats but did not increase in that of nude rats. Mature and immature basophils that developed after the NB infection were identified by electron microscopy. The present result confirms that T-cell—derived cytokines are indispensable for the augmented production of basophils and suggests that stimulation via the c-kit receptor may not be necessary for the augmented production.

Infection of Nippostrongylus brasiliensis induces normal increase of basophils in mast cell-deficient Ws/Ws rats with a small deletion at the kinase domain of c-kit

All basophils, mucosal-type mast cells (MMC) and connective tissue-type mast cells (CTMC) are derived from the multipotential hematopoietic stem cell. Mutations at the c-kit locus resulted in deficiency of MMC and CTMC in both mice and rats. To investigate the role of the c-kit receptor tyrosine kinase for production of basophils, we used white spotting/white spotting (Ws/Ws) mutant rats that have a small deletion at the tyrosine kinase domain of the c-kit gene. When Ws/Ws, nude athymic, and normal (+/+) rats were infected with Nippostrongylus brasiliensis (NB), the number of basophils increased greater than 50- fold in the peripheral blood of Ws/Ws and +/+ rats but did not increase in that of nude rats. Blood histamine concentration increased significantly in Ws/Ws and +/+ rats but did not increase in nude rats. Immature basophils increased greater than 10-fold in the bone marrow of Ws/Ws and +/+ rats but did not increase in that of nude rats. Mature and immature basophils that developed after the NB infection were identified by electron microscopy. The present result confirms that T- cell-derived cytokines are indispensable for the augmented production of basophils and suggests that stimulation via the c-kit receptor may not be necessary for the augmented production.

Acute Basophilic Leukemia: A Clinical, Morphologic, and Cytogenetic Study of Eight Cases

The authors describe eight cases of acute basophilic leukemia. In six of the eight cases, basophilic involvement was not apparent by light microscopic examination. The cases were identified on the basis of ultrastructural evidence for basophil/mast cell differentiation of the blasts with little or no differentiation into other lineages. Ultrastructural analysis revealed immature basophil granules in blasts in all eight cases and theta granules in blasts in four cases. In three cases, ultrastructural evidence of mast cell differentiation also was present, with rare cells showing evidence for both basophil and mast cell differentiation. No clinical features distinguished this group of patients from others with acute myeloid leukemia. Cytogenetically, the cases were heterogeneous. Three had a Philadelphia chromosome; none had a t(6;9). The authors conclude that ultrastructural analysis usually must be used to diagnose acute basophilic leukemia, that acute basophilic leukemia is associated frequently with the Philadelphia chromosome, and that the ultrastructural findings provide evidence for a common origin of basophils and mast cells.

Production of Interleukin-4 and Other Cytokines following Stimulation of Mast Cell Lines and in vivo Mast Cells/Basophils

Interleukin-3 (IL-3)-dependent mast cell lines, upon stimulation by calcium ionophores or by Fc epsilon RI cross-linking, express mRNA for, and secrete, a distinct pattern of cytokines, similar to those secreted by cloned mouse T cells of the TH2 type. The mast-cell-derived cytokines include IL-3, IL-4, IL-5 and IL-6. Not only in vitro mast cell lines, but also in vivo derived peritoneal mast cells secrete cytokines. An in vivo derived cell, in mouse spleen and bone marrow, secretes IL-4 and other cytokines upon stimulation with calcium ionophores or by Fc epsilon RI cross-linking or Fc gamma RII cross-linking. The IL-4-producing cells are highly enriched in the Fc epsilon R+ subset of spleen and bone marrow cells. These Fc epsilon R+ cells produce large amounts of IL-4, and they have characteristics similar to those of immature mast cells and/or basophils. It is possible that cytokines produced by mast cells and/or basophils participate in allergic inflammatory diseases.