Chronic childhood neutropenia: studies on the in vitro clonogenicity of bone marrow myeloid progenitors

Bone marrow mononuclear cells (MNC) from 6 pediatric patients with chronic neutropenia were tested for myeloid colony formation upon stimulation with the supernatant of the 5637 cell line or with recombinant granulocyte-macrophage colony-stimulating factor or interleukin 3. Heterogeneous patterns of response of myeloid progenitors were observed in the individual patients, with no colony growth in 2 cases and abnormalities of colony formation or composition in two additional cases. Morphologic and surface marker analyses showed that the bone marrow of some patients contained an excess of lymphocytes with an altered subset distribution. In order to investigate whether or not there was a relationship between the latter abnormality and the observed clonogenic defects, marrow MNC were tested for myeloid colony formation before and after lymphocyte depletion. No evidence for a cell-mediated suppression of colony growth was obtained; likewise, patient sera failed to inhibit colony formation by normal bone marrow myeloid progenitors. Taken together, these data make it unlikely that, in our cases, immunologic mechanisms were responsible for the pathogenesis of chronic neutropenia.     

Response of neutropenia and anaemia to immunosuppressive therapy: report and bone marrow culture studies

Soft agar culture of bone marrow cells was used to investigate abnormalities of granulopoiesis in a patient with acquired anaemia and neutropenia which responded to immunosuppressive therapy. Results showed that the patient's peripheral blood leucocytes inhibited granulopoiesis in vitro. Variation in the response of different target marrows suggested that inhibition was not due to direct action on granulocyte-monocyte committed stem cells but was directed against or mediated through the cells in target marrow samples which produce granulopoietic stimulators. Cell separation showed that the inhibitory activity was associated with the patient's mononuclear cells rather than neutrophils. A tentative explanation of the mechanism of production of the anaemia is also offered.     

Response of neutropenia and anaemia to immunosuppressive therapy: report and bone marrow culture studies.

Soft agar culture of bone marrow cells was used to investigate abnormalities of granulopoiesis in a patient with acquired anaemia and neutropenia which responded to immunosuppressive therapy. Results showed that the patient's peripheral blood leucocytes inhibited granulopoiesis in vitro. Variation in the response of different target marrows suggested that inhibition was not due to direct action on granulocyte-monocyte committed stem cells but was directed against or mediated through the cells in target marrow samples which produce granulopoietic stimulators. Cell separation showed that the inhibitory activity was associated with the patient's mononuclear cells rather than neutrophils. A tentative explanation of the mechanism of production of the anaemia is also offered.     

Myelokathexis: Chronic neutropenia with hyperplastic bone marrow and hypersegmented neutrophils in two siblings

Summary The author describes the clinical and pathological characteristics of a chronic neutropenia in two siblings. The disease was probably inherited as an autosomal recessive trait. The clinical course and the results of the laboratory investigations corresponded to the rare cases of myelokathexis reported so far.     

Chronic idiopathic neutropenia: Granulopoietic assessment by both marrow culture and granulocyte kinetics

Granulopoietic assessment was made in 14 patients with chronic idiopathic neutropenia (CIN) whose neutrophils were consistently less than 1.5 × 10⁹/ liter and in whom there was absence of splenomegaly and recent drug ingestion. Granulopoiesis was studied using a combination of bone marrow culture in nutrient agar and granulocyte kinetics. Agar colony growth assessed bone marrow concentration of granulocyte progenitor cells (GPC) and the proportion of GPC in DNA synthesis by in vitro ³HTdR suicide. Granulocyte kinetics with in vitro DF³²P labelling of patient granulocytes measured granulocyte half-life (T^1/2), turnover rate (GTR), and the circulating, marginated, and total blood granulocyte pools. The results indicated that either GPC concentration or the proportion of GPC in DNA synthesis was outside the normal range in all but one patient and decreased in ten out of 14 patients. CIN was also characterized by reduced total, circulating, and marginated blood granulocyte pools, reduced GTR, and normal granulocyte half-life. The neutropenia appeared to be due to a variety of intra-marrow causes, including either a reduction in the GPC compartment, a reduction in GPC proliferation, a maturation arrest, or a reduced amplication during granulopoiesis. Increased granulocyte utilization, intra-vascular destruction or excessive margination could be excluded as possible causes of CIN in this series. Although GPC parameters tended to be reduced, suggesting a production defect, there were signs in nine patients that the bone marrow was attempting to compensate for the peripheral neutropenia. It is suggested that for a complete assessment of granulopoiesis in man, granulocyte kinetic studies need to be combined with quantitative studies of the bone marrow granulocyte progenitor compartment using the agar colony system.     

Characterization of the bone marrow immunofluorescence test in childhood autoimmune neutropenia

The bone marrow immunofluorescenece test (BMIFT) demonstrates autoantibodies to granulocytes and their precursors on fresh-frozen bone marrow slides. It may be used to differentiate childhood autoimmune neutropenia (AIN) from other causes of childhood neutropenia, even when circulating neutrophil counts are low. We sought to characterize the diagnostic utility of the BMIFT in childhood AIN. All BMIFT requests for investigation of children with neutropenia between January 1998 and May 2007 were reviewed. Patients were classified as AIN or nonautoimmune causes. Baseline demographic data, results of BMIFT, granulocyte immunofluorescence testing and bone marrow findings were collected from clinical records and the institutional laboratory database. Seventy-six children had BMIFT performed for investigation of neutropenia. There were 45 patients diagnosed with AIN, 28 with nonimmune neutropenia and three failed tests. The median age of children with AIN was 1.2 years (range 0.3-15.3), compared with 3.6 years (range 0.1-15.7) in the nonautoimmune group. The median neutrophil count in AIN was 0.3 x 10(9)/l (0.9 x 10(9)/l in nonautoimmune). BMIFT was positive in 24 of 45 patients with AIN and 0 of 28 with nonautoimmune neutropenia (sensitivity 53%, specificity 100%, positive predictive value (PPV) 100%, negative predictive value 57%). Ten patients had other autoimmune diatheses at diagnosis. The BMIFT is a simple, highly specific test with excellent PPV and thus is a clinically useful test to confirm AIN in children.     

Lung and bone marrow granulomas associated with human parvovirus B19 infection]

A 56-year-old man was admitted with flu-like symptoms, and his platelet count abruptly decreased. A chest X-ray film showed granular shadows, and lung and bone marrow specimens disclosed non-caseating epithelioid cell granulomas. The patient's serum IgM titer for human parvovirus (HPV) B19 was elevated. Our conclusion was that HPV B19 must be kept in mind as a possible pathogenic agent of granuloma formation.     

Human parvovirus B19 infection in bone marrow transplantation patients

We report the results of a survey of parvovirus B19 infection carried out with the aim to evaluate the frequency and the role of this infection in bone marrow transplant (BMT) recipients, as it is known that B19 virus can persist in clinical circumstances of immunodeficiency. Fifty-one patients subjected to BMT in the Bone Marrow Transplantation Center of Florence were enrolled in this study. Immunological and virological indications of B19 infection were tested weekly during the stay in hospital. A high rate of seroconversion or B19 antibody rise was observed, but, in absence of B19 IgM or B19 DNA presence, this result seems to be attributable to a passive immunization, rather than to a recent viral infection. In these 51 patients, as well as in 59 others not included in this study, clinical manifestations imputable to B19 infection have never been observed. It is possible that the isolation measures and the intravenous immunoglobulins (IVIG) administration may contribute in preventing B19 infection in the BMT recipients at least until the hospital discharge. © 1993 Wiley-Liss, Inc.     

Chediak-Higashi syndrome: studies in long-term bone marrow culture

The granulocyte defects of Chediak-Higashi syndrome (CHS) include neutropenia, characteristic giant lysosomal granule morphology, and functionally abnormal cell motility, degranulation, and bacterial killing. Findings of elevated levels of adenosine 3', 5' cyclic monophosphate nucleotide (cAMP) and of concanavalin A (Con A) capping have suggested a pathogenic role of a cyclic-nucleotide-related defect in microtubule polymerization, but not all patients exhibit these abnormalities. In order to test which defects derive from the cells' genetic program and which from the host environment, we examined granulocytes produced by CHS bone marrow progenitors in long-term in vitro bone marrow cultures. These cells exhibited the characteristic giant-granule morphology and defective cell motility of CHS. However, culture-derived CHS granulocytes had normal cAMP contents and normal spontaneous capping of Con A. Granulopoiesis diminished dramatically after five weeks in culture, with accompanying autophagocytosis by mononuclear phagocytes. In mixing experiments, the phenotype of the mature granulocytes corresponded to the genotype of the hematopoietic component of the culture rather than the stroma. These results indicate that the hallmark giant-granule morphology and cell motility defect of CHS are expressions of the genetic program of the hematopoietic cells. However the abnormalities in resting cyclic nucleotide levels and in Con-A capping may be secondary manifestations of the disease and are not essential to the pathogenesis of the chemotactic defect.     

Aplastic crisis in sickle cell disorders: bone marrow necrosis and human parvovirus infection

Aplastic crisis in patients with sickle cell disease who develop a parvovirus infection may be associated with extensive bone marrow necrosis as well as acute selective erythroblastopenia. This illness may be manifested by pyrexia, lymphadenopathy, bone tenderness and significant hypoxemia with minimal roentgenographic findings in the lungs. It is uncertain whether the hypoxemia is caused by the effects of the viral infection on the lungs or is secondary to sickling of red blood cells in the pulmonary vasculature or both. The hypoxia may be sufficiently severe to require treatment with both oxygen and transfusion. The physical damage to the bone marrow associated with bone marrow necrosis may be more important than selective acute erythroblastopenia in inducing aplastic crisis in patients with sickle cell disorders. Studies of bone marrow biopsy specimens collected during parvovirus-associated aplastic crisis in patients with nonsickle cell hemolytic disorders would be helpful in determining the pathophysiology of parvovirus-associated disorders.     

Acute parvovirus B19 infection mimicking myelodysplastic syndrome of the bone marrow

Summary A 36-year-old, previously healthy woman was referred to our institution with pancytopenia and splenomegaly for suspected acute leukemia. Bone marrow aspiration showed marked dysplastic changes, excess of blasts, and only spurious red blood cell precursors. Action was taken to prepare allogeneic bone marrow transplantation from an HLA identical sibling for myelodysplastic syndrome. Repeat cytological examination of the bone marrow revealed striking hyperplasia of the red cell line with presence of abnormal giant proerythroblasts. Acute parvovirus B19 infection was suspected and confirmed by detection of anti-B19 IgM and B19 DNA. The underlying disease for this transient aplastic crisis was a formerly unknown hereditary spherocytosis.     

Cytomegalovirus infection after bone marrow transplantation: an association with acute graft-v-host disease

Among 181 patients undergoing allogeneic bone marrow transplantation over a five-year period (1978 through 1982), cytomegalovirus (CMV) infection was a frequent and often lethal complication. Recipient pretransplant serology was the most important predictor of posttransplant CMV infection. CMV infection occurred in 26/137 seronegative recipients and in 28/44 seropositive recipients (P less than .001). Among patients who developed CMV infection, the time to infection was identical in seronegative and seropositive patients (median, 71 days post transplant). Bone marrow donor CMV serology did not significantly influence CMV infection rate. CMV infection was strongly associated with acute graft-v-host disease (AGVHD), occurring in 34/81 patients with AGVHD and 20/100 without GVHD (P less than .001). AGVHD preceded CMV infection by 33.7 days (mean) in patients developing both complications. Patients who developed CMV infections had also received more cellular blood products post transplant. These data suggest that CMV infection may occur through reactivation of latent virus (in seropositive recipients) or through exogenous exposure, possibly through transfused blood products, but that duration of immunoincompetence may be more critical than route of exposure in timing of clinically evident CMV infection. Prophylaxis tailored to the likely infectious source and more effective GVHD prevention both may be critical in preventing CMV infection after bone marrow transplantation.     

Chronic hepatitis C infection in a patient with bone marrow hypoplasia

Chronic hepatitis C virus （HCV） infection is associated with multifarious extra-hepatic manifestations; the most described and discussed being mixed cryoglob-ulinemia which is strongly related to B-cell lympho- proliferative disorders （LPDs）. We present a case of chronic HCV infection and mixed cryoglobulinemia, with minimal liver involvement. The case is a 53-year- old patient who was diagnosed as having bone marrow hypoplasia at the age of three. She received several blood transfusions to normalize her haemoglobin. At the age of 31, she was diagnosed with rheumatoid arthritis on account of her diffuse joint pain and inflam- mation, elevated rheumatoid factor （RF） and Raynaud＇s phenomenon. Twenty years later, monoclonal gam- mopathy of IgG Lambda （one year later, changed to IgM Kappa） was detected during a routine examination. A bone marrow biopsy showed hypoplasia, Kappa positive B-lymphocytes and low-grade malignant lymphoma cells. PCR of the bone marrow aspirate was not contributory. No treatment was initiated owing to her poor bone marrow function and she is under regular follow-up.     

Productive infection by B19 parvovirus of human erythroid bone marrow cells in vitro

B19 parvovirus, the cause of fifth disease and transient aplastic crisis, has been successfully propagated in suspension cultures of human erythroid bone marrow cells obtained from patients with sickle cell disease and stimulated by erythropoietin. B19 inoculation in vitro resulted in a marked decline in identifiable erythroid cells over seven to nine days of incubation. Characteristic giant early erythroid cells were seen on Wright's-Giemsa stain of infected cultures. By in situ hybridization, 30% to 40% of erythroblasts were infected at 48 hours; a similar proportion of cells showed B19 capsid protein by immunofluorescence. B19 DNA was present in erythroblasts but not in the leukocyte fraction of bone marrow. B19 replication, as determined by Southern analysis, and B19 encapsidation, as determined by sensitivity of isolated cell fractions to DNase I, were restricted to the nuclei. B19 DNA was detectable in the nuclei from infected cultures beginning at 18 hours and in the supernatant at 32 hours; B19 genome copy number was estimated at about 25,000 to 30,000/infected cell at 48 hours. Recovery of virus depended on the multiplicity of infection (moi); at low moi, approximately 200x input virus was recovered from total cultures and 50x from the culture supernatants. Virus released into the supernatant was as infectious or more infectious than virus obtained from sera of infected patients. Human erythroid bone marrow culture represents a safe in vitro system for the elucidation of the cellular and molecular biology of the pathogenic B19 parvovirus.     

Ganciclovir-related neutropenia after preemptive therapy for cytomegalovirus infection: comparison between cord blood and bone marrow transplantation

We studied ganciclovir (GCV)-related neutropenia after preemptive therapy for cytomegalovirus infection: 9 of 17 (53%) cord blood transplantation (CBT) patients and 18 of 20 (90%) bone marrow transplantation (BMT) patients developed GCV-related neutropenia with an absolute neutrophil count (ANC) of less than 1000/l. Among the patients who did not receive granulocyte colony-stimulating factor, 2 (13%) and 1 (7%) CBT patients, and 10 (56%) and 8 (44%) BMT patients, developed neutropenia with an ANC of less than 500 and 250/l, respectively. The incidences of neutropenia in patients with an ANC of less than 1000, 500, and 250/l were significantly lower after CBT in comparison with BMT. Two BMT patients, but no CBT patients, developed neutropenic fever, and both patients recovered after antibiotic therapy. In CBT patients, a creatinine clearance rate of less than 50 ml/min and an absence of steroid therapy were associated with a greater incidence of GCV-related neutropenia. No risk factors for GCV-related neutropenia were found in BMT patients. These results suggest that GCV may be less toxic to myeloid progenitor cells from cord blood than those from bone marrow.     

Haemophagocytic lymphohistiocytosis in association with granular lymphocyte proliferative disorders in early childhood: characteristic bone marrow morphology

Five paediatric cases of haemophagocytic lymphohistiocytosis (HLH) which showed proliferation of granular atypical lymphocytoid cells in bone marrow are reported. All cases were girls aged 8 months to 4 years who had marked hepatosplenomegaly. Marker analysis on peripheral blood mononuclear cells revealed an increase in the CD3⁺HLADR⁺ subset in three cases and the CD³⁻CD56⁺ subset in one case. An Epstein-Barr virus genome was detected in three cases, and monoclonality was confirmed in two cases. A characteristic morphology of large granular lymphocytes (LGL) was identified, with elongated bizarre features that resembled horsetail-, tadpole-, cucumber- or shooting star-type configurations on the bone marrow smear. Serum concentrations of soluble interleukin-2 receptor and interferon-gamma were elevated in all cases. All five cases required multi-agent chemotherapy which resulted in two complete remissions, two partial remissions and one no response. Refinement of treatment is required for these paediatric GLPD cases which probably comprise a specific high-risk subgroup among secondary HLH patients which had previously escaped notice.