Both Granulocytic and Non-Granulocytic Blood Cells Are Affected in Patients with Severe Congenital Neutropenia and Their Non-Neutropenic Family Members: An Evaluation of Morphology, Function, and Cell Death

Objective: To examine granulocytic and non-granulocytic cells in children with severe congenital neutropenia (SCN) and their non-neutropenic parents. Materials and Methods: Fifteen patients with SCN and 21 non-neutropenic parents were evaluated for a) CD95, CD95 ligand, annexin V, propidium iodide, cell cycle, and lymphocyte subsets by flow cytometry; b) rapid cell senescence (of leukocytes) by senescence-associated ß-galactosidase stain; c) aggregation tests by aggregometer; d) in vitro bleeding time by PFA-100 instrument; e) mepacrine-labeled dense granule number of thrombocytes by fluorescence microscope; and f) hematomorphology by light and electron microscope. HAX1, ELANE, G6PC3, CSF3R, and JAGN1 mutations associated with SCN were studied in patients and several parents. Results: Significant increase in apoptosis and secondary necrosis in monocytes, lymphocytes, and granulocytes of the patients and parents was detected, irrespective of the mutation type. CD95 and CD95 ligand results implied that apoptosis was non-CD95-mediated. Leukocytes of 25%, 12.5%, and 0% of patients, parents, and controls showed rapid cell senescence. The cell cycle analysis testable in four cases showed G1 arrest and apoptosis in lymphocytes of three. The patients had HAX1 (n=6), ELANE (n=2), G6PC3 (n=2), and unidentified (n=5) mutations. The CD3, CD4, and NK lymphocytes were below normal levels in 16.6%, 8.3%, and 36.4% of the patients and in 0%, 0%, and 15.4% of the parents (controls: 0%, 0%, 5.6%). The thrombocytes aggregated at low rates, dense granule number/thrombocyte ratio was low, and in vitro bleeding time was prolonged in 37.5%-66.6% of patients and 33.3%-63.2% of parents (vs. 0% in controls). Under electron and/or light microscope, the neutrophils, monocytes, lymphocytes, and thrombocytes in the peripheral blood of both patients and parents were dysplastic and the bone marrow of patients revealed increased phagocytic activity, dysmegakaryopoiesis, and necrotic and apoptotic cells. Ultrastructurally, thrombocyte adhesion, aggregation, and release were inadequate. Conclusion: In cases of SCN, patients' pluripotent hematopoietic stem cells and their non-neutropenic parents are both affected irrespective of the genetic defect.

Transcobalamin II Deficiency in Four Cases with Novel Mutations.

OBJECTIVE: Transcobalamin II deficiency is one of the rare causes of inherited vitamin B12 disorders in which the patients have characteristically normal or high vitamin B12 levels related to the transport defect of vitamin B12 into the cell, ending up with intracellular cobalamin depletion and high homocysteine and methylmalonic acid levels. MATERIALS AND METHODS: Herein, we describe the findings at presentation of four patients who were diagnosed to have transcobalamin II deficiency with novel mutations. RESULTS: These patients with transcobalamin II deficiency were found to have novel mutations, of whom 2 had the same large deletion (homozygous c.1106+1516-1222+1231del). CONCLUSION: Transcobalamin II deficiency should be considered in differential diagnosis of any infant with pancytopenia, failure to thrive, diarrhea, and vomiting.

Fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin (FLAG-IDA) for the treatment of children with poor-prognosis acute leukemia: the Hacettepe experience.

Fludarabine, cytarabine, granulocyte colony-stimulating factor (G-CSF), and idarubicin (FLAG-IDA) regimen has been proven to be a potentially useful chemotherapy regimen for relapsed or poor-prognosis childhood leukemia. The aim of the study was to evaluate complete remission (CR) rate, toxicity, and overall survival of children with poor-prognosis acute leukemia who received the FLAG-IDA regimen. Furthermore, the authors investigated the children who achieved CR following FLAG-IDA treatment regarding their eligibility for allogeneic hematopoietic stem cell transplantation (HSCT). Between January 2002 and April 2007, 25 children with poor-prognosis acute leukemia were treated with FLAG-IDA regimen in our center. Of the 25 children (16 AML, 9 ALL) with poor-prognosis acute leukemia, 7 (28.0%) received 1 cycle, 17 (68.0%) received 2 cycles, and 1 (4%) received 3 cycles of FLAG or FLAG-IDA regimen. After 44 cycles of FLAG-IDA or FLAG regimen, 10/25 (40%) children were nonresponders, 15/25 (60.0%) showed CR. Five (20%) of these patients in CR who underwent allogeneic HSCT are still in remission. The remaining 20 (80.0%) children were lost due to infection or relapse of the primary diseases. The overall survival of patients who are still alive and underwent allogeneic HSCT (mean: 40.6 ± 4.7, median: 40, range: 34-46 months) was longer than that of patients (mean: 5.5 ± 4.3, median: 4, range: 1-15 months) who did not undergo allogeneic HSCT. The CR rate was quite high in the present study using the FLAG-IDA regimen, and the authors believe this regimen is a possible option prior to allogeneic HSCT in children with poor-prognosis acute leukemia.

Risks and outcome of fungal infection in neutropenic children with hematologic diseases.

In this retrospective study, we report the results of antifungal treatments (AFTs) in febrile neutropenic episodes in patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and aplastic anemia (AA) in our center. From January 2004 to December 2005, a total of 52 patients and 221 febrile neutropenic episodes were evaluated. AFT was started in 96 (43%) of the 221 episodes. Amphotericin B and fluconazole were used in 44 (46%) and 52 (54%) febrile neutropenic episodes, respectively. Microbiologically or histopathologically evident fungal infections were detected in 35 of 96 febrile neutropenic episodes. The mortality rate due to fungal infection was higher in patients with AA (7/8 patients) and AML (7/12 patients) than in ALL patients (1/32). Mortality for the whole group was 28%. When the mortality rate was compared between the two treatment groups (amphotericin B vs fluconazole), mortality was significantly higher in patients receiving amphotericin B [n = 14 (93%) and n = 1 (7%), respectively].

Evaluation of the effects of and earliest response rate to anti-D treatment in children with chronic idiopathic thrombocytopenic purpura: a pilot study.

In this pilot study, 30 (14 male, 16 female; median age: 8 years, range: 2-18) chronic non-splenectomized idiopathic thrombocytopenic purpura (ITP) patients with Rh+ blood group and their 49 attacks were evaluated after intravenous (i.v.) anti-D (WinRho SDF, Cangene Corporation, Winnipeg, MB, Canada) treatment at a dose of 50 microg/kg x 3 days (n = 21 cases; 35 attacks) or a single dose of 75 microg/kg (n = 9 cases; 14 attacks) to define the hemostatic dose of anti-D. Five of 30 patients (22/49 attacks) were resistant to steroid, intravenous immunoglobulin (IVIG) and vincristine treatment. Hemoglobin (Hb), white blood cells (WBC), platelets (plt) and reticulocytes (ret) were evaluated before and after treatment during the follow-up in sequences on the 1st, 7th, 14th and 21st days after anti-D treatment if the patients had no symptom. All patients, even the resistant ones, experienced an increase in plt count to provide protection from bleeding (> or = 20 x 10(9)/L in patients with symptoms, > or = 10 x 10(9)/L in patients without symptoms). The plt responses of one resistant and five non-resistant patients treated with a single 75 microg/kg dose of i.v. anti-D in 8 attacks were monitored at the 2nd, 4th, 8th, 24th and 48th hours of the treatment. A protective plt level was attained within 2 hours in 6 attacks of five non-resistant cases and in 24 hours in the remaining 2 attacks of one resistant case. This pilot study suggests that anti-D treatment in ITP patients is effective and can increase plt to a level adequate enough to protect from hemorrhage within 2 hours, when given in a 75 microg/kg dose. A few adverse events (i.e. chills, hemolysis and hemoglobinuria) resolved without intervention.

Acral erythema with bullous formation: a side effect of chemotherapy in a child with acute lymphoblastic leukemia.

Chemotherapy-induced acral erythema (CIAE) with bullous formation is an uncommon complication in children. We describe herein a child who developed CIAE with bullous formation following high-dose methotrexate and cytarabine for relapsed acute lymphoblastic leukemia. The rash completely resolved within two weeks of the appearance without any specific treatment. CIAE may develop in children with hematological malignancies as a complication of pediatric chemotherapeutic regimens, and pediatricians should be aware of this phenomenon despite its rarity.

Measles, mumps, and rubella antibody status and response to immunization in children after therapy for acute lymphoblastic leukemia.

Seventy-seven patients with acute lymphoblastic leukemia (ALL) who were in complete remission and whose therapies had been stopped for at least 6 months before enrollment in this study were retrospectively analyzed regarding their antibody status for measles, mumps, and rubella, with the aim to demonstrate the seropositivity rate after treatment in the authors' group. Each patient's serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA) method to determine the antibody titers before and after immunization. Measles serology was available in 77 children; 45 (58%) were seronegative. Initial ages of measle-seronegative patients were statistically lower than those of seropositive cases (median 3.29 versus 4.91 years, respectively). Mumps serology was available in 76 children; 36 (47%) were seronegative. Mumps-seropositive cases tended to have more frequent previous history of infection than seronegative cases (55.0% versus 28.6%, respectively, P = .05). Rubella serology was available in 76 children, and 20 (26.3%) were seronegative. It was determined that initial ages of rubella-seronegative patients were statistically lower than those of seropositive cases (median 3.03 versus 4.32 years, respectively). The authors concluded based on the results of their study that at a median of 3.31 years after completion of chemotherapy for ALL, the majority of cases had antibody levels lower than protective values for measles (58.4%); however, these values were 47.3% for mumps and 26.3% for rubella. Seroconversion rates after measles (55%) and mumps vaccination (57.1%) were still low. However, in the available cases, relatively adequate response to rubella vaccination (92.3%) was observed.

Chronic recurrent multifocal osteomyelitis as the first presentation of acute lymphoblastic leukemia in a 2-year-old boy.

SUMMARY: We present herein a 2-year-old boy who suffered from chronic recurrent multifocal osteomyelitis for 6 months and was later diagnosed as acute lymphoblastic leukemia. In view of the rarity of bilateral symmetric and multifocal lesions in osteomyelitis in children, we suggest that leukemia should be investigated with bone marrow aspiration in such patients, even if complete blood count parameters are normal, and there is no hepatosplenomegaly.

Parvovirus-B19 and hematologic disorders.

Parvovirus-B19 (PV-B19) is a member of Parvoviridae, which is one of the smallest DNA viruses. PV-B19-associated diseases usually serve as a good representation of the balance of virus, host response and the immune system. The diseases manifested with PV-B19 are erythema infectiosum, which is common in children, hydrops fetalis, transient pure red cell aplasia in patients with chronic hemolytic anemia, arthralgia - mostly observed in women, and chronic pure red cell aplasia in immunocompromised individuals. Cytopenia (bicytopenia, monocytopenia or pancytopenia) may also accompany the diseases mentioned above. On the other hand, there are many diseases, including neurologic, vasculitic, hepatic, rheumatoid, nephritic, autoimmune, myocardial, and others in which the mechanisms of the diseases are not clear, which may be associated with PV-B19. The virus may manifest with unexpected and unexplained clinical pictures and lead to misdiagnosis. Therefore, hematologic disorders in any unestablished clinical diagnosis should be investigated for PV-B19 infection. However, serologic examination for PV-B19 diagnosis is not sufficient in immunocompromised status. The virus can be determined with polymerase chain reaction (PCR) in the serum or tissue samples. Supportive therapy, blood transfusion and immunoglobulin are the conventional therapeutic interventions for PV-B19 today. Vaccination studies are under examination.

Alternative algorithm for L-asparaginase allergy in children with acute lymphoblastic leukemia.

BACKGROUND: L-asparaginase is a crucial chemotherapeutic agent for the treatment of acute lymphoblastic leukemia. The alternatives to L-asparaginase are not available in many parts of the world, including Turkey. OBJECTIVE: We sought to evaluate the utility of premedication with or without a desensitization protocol in children with acute lymphoblastic leukemia and systemic hypersensitivity reactions to Escherichia coli-asparaginase. METHODS: In this prospective study patients with systemic hypersensitivity reactions to E coli-asparaginase for whom we were unable to ascertain/provide other alternatives to asparaginase were either premedicated, desensitized, or both to receive their chemotherapy as E coli-asparaginase according to the severity of the hypersensitivity reaction. RESULTS: Nineteen patients (13 male patients) with a mean age of 7.4 +/- 4.7 years experienced a systemic hypersensitivity reaction to E coli-asparaginase during a 4-year period. Polyethylene glycol-asparaginase could be used for 3 patients. Eight of the remaining 16 children, who had experienced anaphylaxis, were premedicated and desensitized with E coli-asparaginase, and in 7 patients treatment was tolerated. The other 8 patients, with acute allergic reactions to E coli-asparaginase, were premedicated first, and 5 of them showed no reaction subsequently. Three of them demonstrated systemic hypersensitivity reactions again (anaphylaxis, n = 3), and premedication and desensitization with E coli-asparaginase resulted in anaphylaxis. Polyethylene glycol-asparaginase was administered uneventfully to the patients who could be provided it. CONCLUSION: E coli-asparaginase could be administered to more than half of the patients who had a hypersensitivity reaction, and all of these patients were able to receive their planned doses of asparaginase. In countries with shortages of alternative asparaginase preparations, our approach might be a suitable option.

The evaluation of acquired aplastic anemia in children and unexpected frequency of varicella-zoster virus association: a single-center study.

In this study, 32 patients under the age of 17 years with acquired aplastic anemia (AAA) were evaluated. Nine patients developed AAA associatedwith viral infection in which viral hepatitis and varicella infection were nearly equal. Four of the patients were administered drugs before developing AAA. Patients were treated as follows: combined immunosuppressive therapy (CIST) including anti-thymocyte or anti-lymphocyte globulin plus high-dose methylprednisolone (HDMP) and cyclosporin A and granulocyte colony-stimulating factor (G-CSF) (14 patients); mega-dose (30 mg/kg) methylprednisolone (8 patients); and HDMP combined with cyclosporin A or anapolon or cyclophosphamide (6 patients). Complete remission was seen in 10 patients and partial remission in 2 patients. The response rate was similar in the CIST and MDMP groups. The most striking findings of this study were the frequent association of AAA withvaricella infection and the low cure rate, which was due to patient non-compliance with the treatment and inadequate isolation conditions in the hospital.

Dysplasia and disorder of cell membrane entirety in iron-deficiency anemia.

Peripheral blood smears of 43 patients (26 males, median age 18 months, range: 6-180 months) with nutritional iron-deficiency anemia (IDA) were examined for the presence of trilineage hematological dysplasia. Twelve patients were reexamined for dysplastic findings after achieving a normal Hb and hematocrit level for age by the end of 2-3 months of iron treatment. A control group of 17 age-matched healthy children were also included. Neutrophils with loss of membrane entirety and protrusions were remarkable in 34/43 (79%) in the IDA group versus 1/12 (8%) after iron treatment and none of the control group. Microspherocytes were seen in 9/43 (21%) of IDA patients. Additionally, trilienage dysplasia was observed in the bone marrow samples available in 3 of the patients. It has been shown that iron-deficiency results in cellular DNA and RNA alterations, cell-cycle G1/S phase arrest, and apoptosis. Rac GTPases have been shown to control actin cytoskeleton, influencing cell polarity, microtubule dynamics, and the cytoskeletal organization of hematopoietic cells. Thus, the findings described above in neutrophils and red cells suggest a plausible link between iron and the Rac GTPase gene family. It may be a new avenue for iron waiting for proof.

Intravenous immunoglobulin in the treatment of severe methotrexate-induced acral erythema.

Chemotherapy-induced acral erythema is an uncommon and dramatic reaction to high-dose chemotherapy. It is characterized by painful erythema of both palms and soles with symmetrically well-defined borders, which may progress to bullae formation and desquamation. The bullous variant of this reaction has been reported with methotrexate and more frequently cytosine arabinoside. Rapid differential diagnosis and discrimination from more serious conditions such as graft versus host disease or toxic epidermal necrolysis is essential. In this case report, we present a 13-year-old boy who developed severe and prolonged chemotherapy-induced acral erythema after high-dose methotrexate treatment and successfully responded to intravenous immunoglobulin.

Weekly long-term intravenous immunoglobulin for refractory parvovirus B19 and Epstein-Barr virus-induced immune thrombocytopenic purpura.

In pediatric acute immune thrombocytopenic purpura (ITP) cases, it is usually possible to determine an underlying pathology; among them, viral infections are the leading causative agents. In this report, we describe two cases of acute ITP complicated secondary to parvovirus B19 or Epstein-Barr virus (EBV) infections who were unresponsive to initial therapeutic measures, but who responded to long-term intravenous immunoglobulin (IVIG) treatment, given weekly for five to eight weeks.

Novel HAX1 mutations in patients with severe congenital neutropenia reveal isoform-dependent genotype-phenotype associations.

Homozygous mutations in HAX1 cause an autosomal recessive form of severe congenital neutropenia (CN). By screening 88 patients with CN, we identified 6 additional patients with HAX1 mutations carrying 4 novel mutations. Of these, 2 affect both published transcript variants of HAX1; the other 2 mutations affect only transcript variant 1. Analysis of the patients' genotypes and phenotypes revealed a striking correlation: Mutations affecting transcript variant 1 only were associated with CN (23 of 23 patients), whereas mutations affecting both transcript variants caused CN and neurologic symptoms, including epilepsy and neurodevelopmental delay (6 of 6 patients). In contrast to peripheral blood, transcript variant 2 was markedly expressed in human brain tissue. The clinical phenotype of HAX1 deficiency appears to depend on the localization of the mutation and their influence on the transcript variants. Therefore, our findings suggest that HAX1 isoforms may play a distinctive role in the neuronal system.

The prognostic impact of myeloid antigen expression in pediatric acute lymphoblastic leukemia patients.

The incidence of mixed-lineage leukemias in the pediatric age group was previously reported as 13.8% for myeloid antigen-positive ALL and 11.1% for lymphoid antigen-positive acute myeloid leukemia (AML). Recent studies showed that extensive chemotherapy protocols overcome the risk of myeloid lineage. Our study also supports most of the previous data and we postulate that myeloid antigen expression in pediatric ALL cases has insignificant effect on clinical presentation, relapse rates and survival. Importantly, 54% of myeloid antigen-expressing ALL patients received high-risk treatment protocols for some other reasons and this may also have contributed to similar outcome in these patients to that observed in myeloid antigen-negative ALL patients.

The absence of peripheral blood blasts at diagnosis may predict CNS involvement or CNS relapse in pediatric acute lymphoblastic leukemia patients.

A high tumor burden at the time of diagnosis of childhood acute lymphoblastic leukemia has an unfavorable outcome. Peripheral white blood cell count is commonly used to reflect the leukemic burden and is used as one of the most important factors during determination of the risk-based treatment. However, peripheral blood blast count may not always reflect the tumor burden if leukocytes are not in blast nature. In the present study, we observed no central nervous system involvement at the time of diagnosis in patients with no peripheral blood blasts at the beginning, and furthermore, none of the patients with no peripheral blasts at the diagnosis had central nervous system relapse.

Rapid cell senescence and apoptosis in lymphocytes and granulocytes and absence of GM-CSF receptor in congenital dysgranulopoietic neutropenia.

A girl with congenital dysgranulopoietic neutropenia (CDN) and her non-neutropenic mother with aphthae (A) were investigated. Apoptosis in lymphocytes and granulocytes of both patients (mother A+) were documented by high annexin and electron microscopic morphology. CD11b/CD18 of the daughter's granulocytes ranged between low to normal while that of the mother changed between very low to high levels through A(-) to A(+) periods. In both patients, CD11b/CD18 on lymphocytes were high; GM-CSF receptor was negative; CD4-/CD8- lymphocytes were high and the leukocytes which showed abnormal cell cycle were stained by senescence associated beta-galactosidase. We think that increased apoptosis and rapid cell senescence of leukocytes underlies the pathophysiology of CDN.

Congenital dysgranulopoietic neutropenia.

We investigated a 15-year-old female with congenital dysgranulopoietic neutropenia (CDN) and her non-neutropenic mother who had recurrent stomatitis. In both patients, cells of the neutrophilic, eosinophilic, monocytic, megakaryocytic, and basophilic series were dysmorphic. Plasmacytoid lymphocytes and mild megaloblastic erythroid precursors were present. Bleeding times of both patients were prolonged. The mother had a secondary aggregation defect; the number of the plasmacytoid lymphocytes, dense granules of platelets, and dysmorphic neutrophils, neutrophil chemotaxis, and myeloperoxidase content fluctuated according to the presence or not of aphthae. The daughter's karyotype revealed 46,XX/46,XX, t(1;8). No ELA2 or G-CSFR mutation was detected. These findings support stem cell involvement in CDN.