An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these “low density granulocytes” (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I interferons. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared to those of autologous normal density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize extracellular traps (NETs) which display increased externalization of bactericidal, immunostimulatory proteins and autoantigens, including LL-37, IL-17, and double-stranded DNA (dsDNA). Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-α synthesis by pDCs. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and ds-DNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role.

BACKGROUND:The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method. RESULTS: The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (P = 0.86) and superior precision with a CV c15 percent for blood samples with >0.1 percent fetal cells.Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. CONCLUSIONS The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies. he detection of circulating fetal cells in maternal blood represents an important area of laboratory support to the obstetrical management of ABBREVIATIONS: ELISA = enzyme-linked immunosorbent assay; FMH = fetomaternal hemorrhage; HAT = hypoxanthine aminopterin thymidine; Hb = hemoglobin; HbA = hemoglobin A; HbF = hemoglobin F; PBS = phosphate-buffered saline; PBS-BSA = PBS with bovine serum albumin; RBC(s) = red cell(s); RhIG = Rh immune globulin; WBC(s) =white cell(s). From the Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan.

As a result of overlapping morphologic and immunohistochemical features, it can be difficult to distinguish synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma/primitive neuroectodermal tumor in core biopsies. To analyze and compare immunohistochemical profiles, we stained tissue microarrays of 23 synovial sarcomas, 23 malignant peripheral nerve sheath tumors, and 27 Ewing sarcomas with 22 antibodies potentially useful in the differential diagnosis, and analyzed the data with cluster analysis. Stain intensity was scored as none, weak, or strong. For CD99, tumors with membranous accentuation were independently categorized. Cluster analysis sorted five groups, with like tumors clustering together. Synovial sarcoma clustered into two groups: one cytokeratin and EMA positive (n ¼ 11), the other mostly cytokeratin negative, EMA positive, bcl-2 positive and mostly CD56 positive (n ¼ 9). Malignant peripheral nerve sheath tumor clustered into two groups: one S100 positive, with nestin and NGFR positivity in most (n ¼ 10), the other mostly S100 negative, and variably but mostly weakly positive for nestin and NGFR (n ¼ 11). Ewing sarcomas clustered into a single group driven by membranous CD99 staining. Thirteen cases failed to cluster (outliers), while three Ewing sarcomas clustered into groups of other tumor types. Paired antibodies for each tumor type determined by visual assessment of cluster analysis data and statistical calculations of specificity, sensitivity, and predictive values showed that EMA/CK7 for synovial sarcoma, nestin/S100 for malignant peripheral nerve sheath tumor, and membranous CD99/Fli-1 for Ewing sarcoma yielded high specificity and positive predictive values. Cluster analysis also highlighted aberrant staining reactions and diagnostic pitfalls in these tumors. Hierarchical cluster analysis is an effective method for analyzing high-volume immunohistochemical data.

Graft-versus-host-disease (GVHD), the major complication of allogeneic bone marrow transplantation (BMT), affects the skin, liver and gastrointestinal (GI) tract. There are no plasma biomarkers specific for any acute GVHD target organ. We used a large scale, quantitative proteomic discovery procedure to identify biomarker candidates of skin GVHD and validated the lead candidate, elafin, by ELISA in samples from 492 patients. Elafin was overexpressed in GVHD skin biopsies. Plasma levels of elafin were significantly higher at the onset of skin GVHD, correlated with the eventual maximum grade of GVHD, and were associated with a greater risk of death relative to other known risk factors (hazard ratio of 1.78). We conclude that elafin has significant diagnostic and prognostic value as a biomarker of skin GVHD.

Our experience confirms the utility of current staging systems in predicting outcomes of mucosal melanoma of the head and neck and stresses the importance of achieving negative surgical margins. Pathologically, fewer mitoses and the absence of ulceration predict better outcomes and should be reported as part of routine histological profiles of mucosal melanoma. Further studies are necessary to change the paradigm of care for this rare and deadly disease.

CD163 is a useful adjunct in distinguishing AFX from other malignant cutaneous spindle cell tumors and offers improved specificity in identifying cutaneous histiocytic/dendritic lesions.

Eccrine porocarcinoma (EP), although rare, is widely recognized as the most common malignant sweat gland tumor. EP typically grows slowly and usually is cured by surgical excision with clear margins. An elevated mortality rate, however, is observed when regional lymph nodes are involved. We herein describe cytohistologic findings in a case of metastatic EP. An 86-year-old man with a history of EP of the left lateral ankle and squamous cell carcinoma in situ (Bowen's disease) of the penis presented with enlarged left inguinal lymph nodes. A superficial fine-needle aspiration (FNA) was performed and demonstrated a hypercellular sample with discohesive clusters and/or individual tumor cells. The tumor cells were round or oval with most of the cells showing dense, refractile cytoplasm. Intracytoplasmic vacuoles were readily appreciated in some of the cells. Nuclear enlargement, high N/C ratio, nuclear hyperchromasia, bi- and multinucleation, and prominent nucleoli were seen. A diagnosis of metastatic eccrine porocarcinoma was rendered. Enlarged retroperitoneal lymph nodes were detected and CT-guided left retroperitoneal core biopsy was performed 1 week later. The biopsy revealed features consistent with metastatic eccrine porocarcinoma.

Context.—Sepsis, affecting millions of individuals annually with an associated high mortality rate, is among the top 10 causes of death. In addition, improvements in diagnostic tests for detecting and monitoring sepsis and infection have been limited in the last 25 years. Neutrophil CD64 expression has been proposed as an improved diagnostic test for the evaluation of infection and sepsis. Objective.—To evaluate the diagnostic performance of a quantitative flow cytometric assay for leukocyte CD64 expression in comparison with the standard tests for infection/sepsis in an ambulatory care setting. Design.—Prospective analysis of 100 blood samples from patients from an emergency department setting in a 965-bed tertiary care suburban community hospital was performed for neutrophil CD64 expression, C-reactive protein, erythrocyte sedimentation rate, and complete blood count. The laboratory findings were compared with a clinical score for the likelihood of infection/sepsis, which was obtained by a blinded retrospective chart review. Results.—The diagnostic performance, as gauged by the clinical score, varied with neutrophil CD64 (sensitivity 87.9%, specificity 71.2%, efficiency 76.8%) and outperformed C-reactive protein (sensitivity 88.2%, specificity 59.4%, efficiency 69.4%), absolute neutrophil count (sensitivity 60.0%, specificity 50.8%, efficiency 53.8%), myeloid left shift (sensitivity 68.2%, specificity 76.3%, efficiency 73.3%), and sedimentation rate (sensitivity 50.0%, specificity 65.5%, efficiency 61.0%). Conclusion.—Neutrophil CD64 expression quantitation provides improved diagnostic detection of infection/sepsis compared with the standard diagnostic tests used in current medical practice.