Chronic Benign Neutropenia Research Articles

Platelet and neutrophil alloantigen genotyping in clinical practice

Immune responses to platelet and neutrophil alloantigens are involved in the pathogenesis of several clinical syndromes including: neonatal alloimmune thrombocytopenia (NATP), post-transfusion purpura (PTP), refractory responses to platelet transfusion, neonatal alloimmune neutropenia (NAN), transfusion-related acute lung injury (TRALI), and chronic benign autoimmune neutropenia of infancy. Initially, platelet alloantigens were only characterized serologically. Subsequently, they were localized to specific platelet surface glycoprotein structures and ultimately defined to the level of nucleic acid polymorphisms on platelet glycoprotein genes. These advances allowed the tools of molecular biology to be applied to typing for platelet alloantigens. The advantages of such typing methods include: 1) patient platelets are no longer required for the typing assays, and therefore, platelet types can be established on extremely thrombocytopenic samples (by using peripheral blood white blood cells [WBC]); 2) The genotyping methods eliminate the requirement for rare serologic reagents. A number of different genotyping methods have been developed. These include: restriction fragment length polymorphism (RFLP), sequence specific primers (SSP), and Dot-Blot hybridization. Clinical applications of this methodology include: determining the platelet genotype of fetuses at risk for NATP, in the diagnosis of PTP, and identifying causes of refractory responses to platelet transfusions. Analogous to platelet alloantigens, a limited number of neutrophil alloantigens can now be determined by molecular biologic methods. The new methods obviate the need to isolate fresh neutrophils for serologic typing and do not require rare serologic reagents. To date, molecular polymorphisms associated with alloantigens on the neutrophil Fc gamma RIIIb surface glycoprotein have been elucidated. These include the allo-antigens NA1, NA2, and SH.

Determination of neutrophil antigen gene frequencies in five ethnic groups by polymerase chain reaction with sequence-specific primers.

The granulocyte antigens NA1 and NA2 are the two recognized allelic forms of Fc gamma receptor IIIB. These antigens are clinically relevant, because they are the most frequent targets of neutrophil antibodies in alloimmune neonatal neutropenia, transfusion-related acute lung injury, and chronic benign autoimmune neutropenia of infancy. A genotyping assay for NA1 and NA2 using polymerase chain reaction with sequence-specific forward and reverse oligonucleotide primers has been developed and validated. Genomic DNA was isolated from the peripheral blood of 478 unrelated individuals of five ethnic groups and used as template for NA genotyping. A validation study of 22 serologically typed samples (2 NA1/NA1, 10 NA1/NA2, and 10 NA2/NA2) was performed. A concordance rate of 100 percent (22/22 samples) was observed between the genotyping assay and serologic typing. In the genotyping study conducted, the NA1 and NA2 gene frequencies observed were 0.31 and 0.69 for African Americans, 0.30 and 0.70 for Asian Indians, 0.37 and 0.63 for whites, 0.53 and 0.47 for Hispanics, and 0.55 and 0.45 for Native Americans, respectively. Polymerase chain reaction with sequence-specific primers provides a simple and rapid alternative to neutrophil antigen typing by serologic tests. The NA1 and NA2 gene frequencies observed in Asian Indians and African American populations are similar to those observed in white populations, while those observed in Native American and Hispanic populations are more similar to those previously reported for Asian populations.

Effective Twice-Weekly Treatment with Granulocyte Colony-Stimulating Factor in a Patient with Chronic Benign Neutropenia of Childhood

Effective Twice-Weekly Treatment with Granulocyte Colony-Stimulating Factor in a Patient with Chronic Benign Neutropenia of Childhood

Advances in pathophysiology, diagnosis, and treatment of neutrophil defects

Recent literature relevant to neutrophil defects in pediatric patients spans a wide range of interests. Significant new observations on the molecular pathology, pathogenesis, prenatal diagnosis and therapy, as well as thorough clinical reviews of neutrophil defects appeared during the past year. Diverse types of congenital neutropenias are documented to respond to granulocyte-colony stimulating factor and granulocyte-macrophage colony-stimulating factor and differences in responses may yield insight into the pathogenesis of chronic benign neutropenia, Kostmann's agranulocytosis, and the neutropenia of glycogen storage disease 1b. Leukocyte adhesion deficiency can be diagnosed in utero; however, the assays are crude and do not yet take advantage of the rapidly increasing knowledge of transcriptional or translational abnormalities in CD11b expression. In chronic granulomatous disease (CGD), rapidly increasing knowledge of the molecular basis of gene defects in X-linked CGD led to identification of point mutations as well as gross deletions of the gp91phox gene that cause CGD. Furthermore, advances in the therapy of CGD with interferon-7 are now paired with suggestions that increase in nitroblue tetrazolium reduction by monocytes may underlie the efficacy of interferon-y in CGD. Recently improved methods of analyzing cytoskeletal dynamics in the neutrophil has led to the description of a novel neutrophil defect with defective motility and defective actin polymerization, and studies of acquired neutrophil defects point to the importance of transforming growth factor (3 and cytokines as modulators of defective neutrophil function in several immunoregulatory defects associated with increased susceptibility to bacterial infection.

Bone marrow granulocyte reserve in chronic benign idiopathic neutropenia.

Fifteen patients with chronic benign idiopathic neutropenia (CBIN) with neutrophil counts less than 1.0 X 10(9)/1 have been studied. The mean age was 33 years (range 23-50) comprising 11 females and 4 males. Bone marrow cellularity was normal except in two patients who showed slight reduction and one who had a slight increase. Bone marrow differential counts were normal apart from a small increase in the percentage of promyelocytes and reduction in the myeloid/erythroid ratio in some patients. Peripheral blood counts showed no 'compensatory' monocytosis. Epinephrine stimulation tests showed no evidence of excess neutrophil margination. After endotoxin administration there was a one- to two-fold increase in neutrophil counts in three patients, a three-fold increase in three patients and a greater than four-fold increase in the remaining nine patients. The findings suggest that the benign course of CBIN is not due to excess neutrophil margination nor to compensatory monocytosis, but that at least one mechanism includes a functionally adequate release of neutrophils to the peripheral blood under conditions of stress with subnormal delivery of neutrophils under basal conditions. The variability in responses to endotoxin suggests that CBIN is not entirely homogeneous with respect to mechanism. The findings of relatively normal bone marrow cellularity and differential counts and a normal or substantial neutrophil response to endotoxin appear characteristic. They may help predict a benign clinical course in neutropenic patients and assist diagnosis of the CBIN variant of idiopathic neutropenia.

Growth fraction of myelocytes in normal human granulopoiesis.

The labelling index (LI) of myelocytes (M) after flash labelling of normal human bone marrow cells with [3H]-thymidine ([3H]TdR) is always lower that the LI obtained for myeloblasts (MB) and for promyelocytes (PM). This fact can be interpreted in two ways: it may mean that the duration of the G1 phase of the cell cycle is longer in M than in MB or PM, or it may mean that the proportion of cells in cycle, i.e., the growth fraction (GF), is lower in the M population than in MB or PM. The evolution of the LI and of the mean number of grains per cell was monitored in [3H]TdR-labelled normal bone marrow during in vitro incubation for 50 hr. The generation time, measured by the halving time of the mean number of grains per cell after flash labelling, was similar for M to that for MB and PM. During continuous labelling, the LI of MB and PM reached 1 and the LI value for M never rose to more than 50% of the values recorded for MB and PM after 30 hr. These findings give support to the second hypothesis, i.e., a lower GF in the M population. Good correlation was found between the LI of M and the proportion of mature polymorphonuclear cells in the bone marrow of normal subjects and of patients with chronic benign neutropenia or hyperleucocytosis. Variations in the M growth fraction could be a medium-term (2-3 days) regulatory factor in granulocyte production.

Chronic benign idiopathic neutropenia in Jordanians.

Bone marrow granulocyte reserves and the bone marrow storage pool were evaluated in 10 Jordanian adult patients with chronic benign idiopathic neutropenia, by measurement of the peripheral blood granulocyte increment following the administration of hydrocortisone and lithium. The increment in each case was statistically significant, indicating retention of effective neutrophil production in stressful situations and thus the benignity of this disorder.

Intranuclear crystalloids associated with abnormal granules in eosinophilic leukocytes

Ultrastructural evaluation of eosinophilic leukocytes from a 2-yr-old asymptomatic girl with chronic benign neutropenia (CBN) revealed a variety of morphological abnormalities. All eosinophils obtained from blood and marrow specimens contained multipole microcrystalloids in most of the mature cytoplasmic granules. An increase in crystalloid-free, immature granules in late (bilobed nuclei) eosinophils suggested a delay in granule maturation. The eosinophil granules appeared to be of normal size and demonstrated normal acid phosphatase reactivity. Eosinophilic myelocytes contained abnormal cisternae of rough endoplasmic reticulum (RER) and lacked abundant elongated RER cisternae seen in normal cells. A few eosinophilic myelocytes in specimens of bone marrow from the child contained large intranuclear crystalloids measuring up to 3 mu in length. The intranuclear crystalloid contained as cubic lattice of dense material with a periodicity similar to that described for cytoplasmic crystalloids. The ultrastructural morphology of marrow neutrophils was normal, as described in other cases of CBN. Ultrastructural examination of blood eosinophils from the father demonstrated microcrystalloids in cytoplasmic granules identical to those seen in the child. The father was asymptomatic and had normal leukocyte counts. Thus, anomalous crystalloid granule genesis occurred in the father and daughter and was not necessarily associated with neutropenia or clinical symptomatology. This anomaly is associated with the accumulation of intranuclear crystalloid material in eosinophilic myelocytes, which do not appear to be released from the marrow compartment.

Intranuclear Crystalloids Associated With Abnormal Granules in Eosinophilic Leukocytes

Abstract Ultrastructural evaluation of eosinophilic leukocytes from a 2-yr-old asymptomatic girl with chronic benign neutropenia (CBN) revealed a variety of morphological abnormalities. All eosinophils obtained from blood and marrow specimens contained multipole microcrystalloids in most of the mature cytoplasmic granules. An increase in crystalloid- free, immature granules in late (bilobed nuclei) eosinophils suggested a delay in granule maturation. The eosinophil granules appeared to be of normal size and demonstrated normal acid phosphatase reactivity. Eosinophilic myelocytes contained abnormal cisternae of rough endoplasmic reticulum (RER) and lacked abundant elongated RER cisternae seen in normal cells. A few eosinophilic myelocytes in specimens of bone marrow from the child contained large intranuclear crystalloids measuring up to 3 mu in length. The intranuclear crystalloid contained as cubic lattice of dense material with a periodicity similar to that described for cytoplasmic crystalloids. The ultrastructural morphology of marrow neutrophils was normal, as described in other cases of CBN. Ultrastructural examination of blood eosinophils from the father demonstrated microcrystalloids in cytoplasmic granules identical to those seen in the child. The father was asymptomatic and had normal leukocyte counts. Thus, anomalous crystalloid granule genesis occurred in the father and daughter and was not necessarily associated with neutropenia or clinical symptomatology. This anomaly is associated with the accumulation of intranuclear crystalloid material in eosinophilic myelocytes, which do not appear to be released from the marrow compartment.

Phagocytosis of neutrophils by marrow macrophages in childhood chronic benign neutropenia

Chronic benign neutropenia of childhood is a heterogeneous disorder. This study describes two severely neutropenic children with a benign clinical course and the unique bone marrow finding of macrophage engulfment of band and segmented neutrophils. Phagocytic vacuoles in the majority of macrophages contained neutrophils in various stages of digestion at both the light and electron microscope level (with as many as four neutrophils observed in single macrophages). Ultrastructural studies demonstrated that the neutrophils were morphologically normal, and apparently viable at the time of phagocytosis. This type of neutrophil phagocytosis was not observed in five other consecutively studied children with CBN. All of these children with CBN had ultrastructurally normal neutrophils, lacked demonstrable antineutrophil antibodies or serum inhibitors to in vitro myelopoiesis, and had normal colony-forming cells and colony-stimulating activity in vitro. Thus despite many similar clinical and laboratory features in children with CBN, only a unique subgroup of these patients demonstrates abnormal neutrophil-macrophage interaction.

Neutrophil marrow in chronic benign idiopathic neutropenia

Quantitative studies of neutrophil marrow were carried out in 10 patients with chronic neutropenia (60 to 1,970 cells/μl) with no other abnormalities and no serious infections. Neutrophil marrow cellularity was determined from neutrophil-normoblast ratios in marrow sections and ferrokinetic estimation of normoblasts. The results were interpreted in the light of 95 per cent confidence limits previously observed in 13 normal volunteer subjects. Three distinct neutrophil marrow profiles were determined by the numbers of promyelocytes and myelocytes and of segmented marrow cells. Tentative kinetic interpretation was based on the expectation that the physiologic marrow response to removal of neutrophils from circulation would produce an increase in promyelocytes and myelocytes due to influx and proliferation, and a decrease in marrow segmented cells due to accelerated release. In two patients increased segmented marrow cells were consistent with an abnormality of release. Decreased numbers of promyelocytes and myelocytes in three patients was consistent with decreased proliferation. In five patients basal numbers of promyelocytes and myelocytes suggested abnormal proliferation or abnormal regulation of myelopoiesis. The number of metamyelocytes and band forms relative to promyelocytes and myelocytes was normal in all 10 patients: none had evidence of cell loss during postmitotic maturation. The term “chronic benign idiopathic neutropenia” appears to embrace more than one mechanism for neutropenia. All 10 patients had evidence of abnormal neutrophil marrow function.

A Case of Chronic Benign Neutropenia in Childhood associated with Thrombocytopenic Purpura and induced Remission with Glucocorticoid

A Case of Chronic Benign Neutropenia in Childhood associated with Thrombocytopenic Purpura and induced Remission with Glucocorticoid

NE1: a new neutrophil specific antigen.

The sera of three children with chronic benign neutropenia, due to anti-neutrophil antibodies, were studied with respect to their antibody specificity. This was done by screening the sera against a panel of leukocyte donors in the EDTA micro-agglutination test and in the indirect fluorescence test. Two of the sera contained antibodies against the known neutrophil-specific antigen NA2. The third serum was directed against a new neutrophil-specific antigen. Genetic analysis showed no correlation between this antigen and the already known neutrophil-specific antigens: 9A, NA1, NA2, NB1, and NC1. In the Dutch population the frequency of the new antigen, tentatively called NE1, is 23%, which gives a gene frequency of 0.12.

Gingivo‐periodontal manifestations in chronic benign neutropenia

A case of chronic benign neutropenia in a 7-year-old Caucasian male is reported. The predominant clinical symptoms were persistent gingivitis and periodontitis. The clinical, hematological, oral radiographical, histological, and therapeutical features of this uncommon disease are discussed.

Neutrophil kinetics in hereditary and congenital neutropenias.

Neutrophil production and distribution were studied in two families with autosomal dominantly inherited neutropenia to distinguish their illness from other neutropenic disorders. In addition to a reduced post-mitotic pool of bone-marrow neutrophils and neutrophil precursors, mitotic pool size was also reduced, ranging from 1.1 to 2.9 X 10(9) cells per kilogram (normal, 3.8 +/- 0.4 X 10(9) per kilogram). In vitro committed stem cells were reduced as well, ranging from 2 to 12/10(5) marrow cells (normal, 30 to 120/10(5)). In three patients neutrophil counts were observed to return to normal when they were adults. In one such subject studied, however, both the mitotic pool of neutrophil precursors and marrow committed stem cells were reduced. These abnormalities were in contrast to studies of a patient with chronic benign neutropenia of childhood and an infant with Kostmann-type congenital neutropenia, both of whom had increased marrow committed stem cells. These studies demonstrate several different mechanisms for production of neutropenia in these syndromes.

Leucocyte function in a case of chronic benign neutropenia of infancy associated with circulating leucoagglutinins.

A case of chronic benign neutropenia is described in association with circulating leucoagglutinins. IgG and IgM leucoagglutinins demonstrated by Sephadex G-200 chromatography agglutinated neutrophils and monocyte enriched leucocyte preparations. Total IgG, IgA, IgM and IgE, total haemolytic complement, C3 and C4 complement components were all within normal limits. Lymphocyte populations and transformation were normal apart from a relative increase in the number of B lymphocytes. In vitro studies showed that the patient's monocytes responded in chemotaxis, phagocytosis and intracellular killing of S. aureus in a comparable fashion to that of control neutrophils. Although the patient's serum also agglutinated monocytes these findings do not exclude the possibility that leucoagglutinins may have aetiological significance in this disease. The study also emphasizes the versatility of the monocyte in benign neutropenia.

A study of steroid-induced granulocytosis in a patient with chronic benign neutropenia of childhood.

Summary.An infant with chronic benign neutropenia of childhood was found to respond to a sudden, transient, stress‐induced rise in plasma corticoid level with a pronounced rise in peripheral neutrophil count. Neutrophil survival studies were performed following this stress‐induced neutrophilia and during and following hydrocortisone stimulation. Measurement of the decline in total neutrophil‐associated radioactivity showed that neurophils were removed from the circulation at a normal rate while measurement of neutrophil specific activity indicated that the observed steroid‐induced granulocytosis was a result of marrow released of neurtrophils.

Chronic benign neutropenia of childhood with associated oral manifestations

We have presented a case of benign neutropenia of childhood in which the disease was manifested by a destructive process of the supporting structures of the dentition.

Chronic benign neutropenia in infancy and early childhood: Report of a case with a review of the literature

Summary A case is described of “chronic benign neutropenia” in a female infant 8 months of age. The peripheral blood constantly showed a marked, selective neutropenia; the bone marrow was characterized by only a slight degree of increase in young neutrophils, as contrasted with a low normal percentage of polymorphonuclears. Otherwise the bone marrow picture was normal. There was little constitutional involvement after the complaint for which she had been taken to hospital, namely, acute descending laryngotracheobronchitis, had subsided. Spleen, liver, and lymph nodes were never found to be enlarged. The child reacted to infections with a transient rise in circulating neutrophils; injection of ACTH had the same effect. Pentnucleotide did not cure the condition. The cause remained undetermined. The bone marrow picture in this condition is generally considered to show some degree of “maturation arrest” at the stage of the young neutrophils. However, intramuscular injection of ACTH caused the number of circulating neutrophils to rise to normal levels within two hours, a phenomenon difficult to explain on the basis of a “maturation arrest.” In the case reported here it was shown that the polymorphonuclears disappeared from the blood stream with increased rapidity, but there were never found any factors operating in the peripheral blood itself which might lead to increased destruction there. This directs the attention to a selective hyperfunction of the reticuloendothelial system, and of the spleen in particular, resulting in an accelerated removal of the segmented neutrophils from the circulation. This predominance of leukocyte segregation and sequestration over leukocyte production might act as a stimulus to the bone marrow; hence, the increase in young neutrophils. An analogy of this condition might be seen in chronic idiopathic thrombocytopenia in young children, which also shows comparatively little constitutional involvement, a moderate increase in slightly immature “thrombocyte precursors,” i.e., megakaryocytes, and a tendency to spontaneous healing.