Image analysis of neutrophil nuclear morphology: Learning about phenotypic range and its reliable analysis from patients with pelger-Huët-anomaly and treated with colchicine.

The nuclear morphology of neutrophils depends on different endogenous and exogenous factors, which can lead to hypo- or hypersegmentation of the normally 2-4 segmented nucleus. Hyposegmentation can be due to mutations in the LBR-gene (Pelger-Huët-Anomaly) or can be induced, for example, by colchicine treatment. The range of this phenotypic variation is known as "norm of reaction," which can be of major relevance for clinical diagnosis and therapeutic intervention. In this project, we studied the norm of reaction in 26 subjects with 0-3 wild type LBR alleles. In addition, the phenotypic variation was analyzed in 3 patients with Familial Mediterranean Fever (FMF), before and after colchicine treatment. We measured the phenotype nuclear segmentation of neutrophils based on two conventional qualitative methods, the "rule of threads" and the "rule of thirds." In addition, we tested a morphometric quantitative approach, the "circularity index." The circularity index was superior in cases with hyposegmentation; the rule of thirds with respect to hypersegmentation. Approximately 65% of the observed phenotypic variance was explainable by the number of LBR wild type alleles. The gene-dosage effect followed a non-additive, hysteresis-like characteristic with lower and upper plateaus. Colchicine treatment had a clear, although minor phenotypic effect compared to the number of LBR wild type genes or the mutation type. Thus, the nuclear morphology of granulocytes and its norm of reaction can be regarded as an excellent model both for detailing the interplay between endogenous and exogenous factors and for clinical diagnostic purposes. © 2016 International Clinical Cytometry Society.

Understanding and recognizing the Pelger-Huët anomaly.

The Pelger-Huët anomaly (PHA) is a recognized morphologic variant affecting all granulocytes but is most evident in polymorphonuclear neutrophils (PMNs). PHA is caused by a decreased amount of the lamin B receptor (LBR). Recognition of PHA morphologic features serves as a marker for mutations in the LBR gene. This review summarizes the history of PHA and the current knowledge of the functions of the LBR. Guidance is given for distinguishing PHA from other hematologic disorders in which granulocytes may show similar changes. Recognition of PHA in the laboratory should prompt communication to the patient's physician about the possible clinical significance of this finding and the recommended screening for the anomaly in other family members by CBC and review of a peripheral blood smear.

An in vitro model for Pelger-Huët anomaly: stable knockdown of lamin B receptor in HL-60 cells.

The principal human blood granulocyte (neutrophil) possesses a lobulated and deformable nucleus, important to facilitate rapid egress from blood vessels as these cells migrate to sites of bacterial or fungal infection. This unusual nuclear shape is a product of elevated levels of an integral membrane protein of the nuclear envelope lamin B receptor (LBR) and of decreased amounts of lamin A/C. In humans, a genetic deficiency of LBR produces Pelger-Huët anomaly, resulting in blood neutrophils that exhibit hypolobulated nuclei with redistributed heterochromatin. Structural changes in nuclear architecture occur during granulopoiesis within bone marrow. The exact mechanisms of this nuclear shape change and of heterochromatin redistribution remain largely unknown. As a tool to facilitate analysis of these mechanisms, a stable LBR knockdown subline of HL-60 cells was established. During in vitro granulopoiesis induced with retinoic acid, the LBR knockdown cells retain an ovoid shaped nucleus with reduced levels of lamin A/C; while, the parent cells develop highly lobulated nuclei. In contrast, macrophage forms induced in LBR knockdown cells by in vitro treatment with phorbol ester were indistinguishable from the parent cells, judged by both nuclear shape and attached cell morphology. The capability of differentiation of LBR knockdown HL-60 cells should facilitate a detailed analysis of the molecular relationship between LBR levels, granulocyte nuclear shape and heterochromatin distribution.

The lamin B receptor under transcriptional control of C/EBPepsilon is required for morphological but not functional maturation of neutrophils.

The lamin B receptor (LBR) is an integral nuclear envelope protein that interacts with chromatin and has homology to sterol reductases. Mutations in LBR result in Pelger-Huët anomaly and HEM-Greenberg skeletal dysplasia, whereas in mice Lbr mutations result in ichthyosis. To further understand the function of the LBR and its role in disease, we derived a novel mouse model with a gene-trap insertion into the Lbr locus (Lbr(GT/GT)). Phenotypically, the Lbr(GT/GT) mice are similar to ichthyosis mice. The Lbr(GT/GT) granulocytes lack a mature segmented nucleus and have a block in late maturation. Despite these changes in nuclear morphology, the innate granulocyte immune function in the killing of Staphylococcus aureus bacteria appears to be intact. Granulocyte differentiation requires the transcription factor C/EBPepsilon. We identified C/EBPepsilon binding sites within the Lbr promoter and used EMSAs and luciferase assays to show that Lbr is transcriptionally regulated by C/EBPepsilon. Our findings indicate that the Lbr(GT/GT) mice are a model for Pelger-Huët anomaly and that Lbr, under transcriptional regulation of C/EBPepsilon, is necessary for morphological but not necessarily functional granulocyte maturation.

Impaired heme synthesis in a family with Pelger-Huët anomaly, recurrent abdominal pain attacks and impaired neutrophil motility in vitro.

Heme synthesis was studied by measuring the activity of delta-aminolevulinic acid synthase (AmLev synthase) in granulocytes, the activity of delta-aminolevulinic acid dehydratase (AmLev dehydratase) and of uroporphyrinogen I synthase in erythrocytes as well as the concentrations of coproporphyrin and protoporphyrin in erythrocytes of 6 patients with Pelger-Huët anomaly. 3 of these patients from the same kindred had a syndrome of recurrent attacks of fever and abdominal pains, a tendency to skin infections, delayed wound healing and impaired neutrophil motility. The other 3 patients were asymptomatic. The activity of AmLev synthase was depressed in all 3 symptomatic patients and normal in the asymptomatic patients. 1 symptomatic patient had a decreased erythrocyte protoporphyrin concentration. These findings indicate a derangement of heme synthesis in the symptomatic patients. Their abdominal pain attacks could be due to heme depletion. The findings suggest that the abdominal pains of patients with porphyria could be due to decreased heme synthesis rather than due to accumulation of porphyrin precursors in tissues. The cause of the impaired neutrophil motility may be a defect in energy metabolism due to decreased supply of heme for oxidative metabolism.