Unexplained Hematocrit Increase after Therapeutic Phlebotomy in a Patient with Marked Erythrocytosis.

We report a patient with hereditary erythrocytosis who underwent a therapeutic phlebotomy and had a post-phlebotomy hematocrit that was higher than the pre-phlebotomy hematocrit. We could not discern a reason for this hematocrit increase after phlebotomy. Instead of performing another phlebotomy, we performed an automated red cell depletion via an apheresis instrument. This procedure is essentially a red cell exchange, but 5% albumin is used as the replacement fluid instead of red blood cells. The patient's hematocrit decreased from 80% to 39% after three consecutive daily red cell depletion procedures. We share our experience to report the unusual finding of a patient's hematocrit that increased with phlebotomy and to raise awareness of the red cell depletion procedure.

Human rhinovirus 1B exposure induces phosphatidylinositol 3-kinase-dependent airway inflammation in mice.

RATIONALE: Infection with rhinovirus (RV) triggers exacerbations of asthma and chronic obstructive lung disease. OBJECTIVES: We sought to develop a mouse model of RV employing RV1B, a minor group serotype that binds to the low-density lipoprotein receptor. METHODS: C57BL/6 mice were inoculated intranasally with RV1B, replication-deficient ultraviolet (UV)-irradiated RV1B, or RV39, a major group virus. MEASUREMENTS AND MAIN RESULTS: Viral RNA was present in the lungs of RV1B-treated mice, but not in those exposed to UV-irradiated RV1B or RV39. Lung homogenates of RV-treated mice contained infectious RV 4 days after inoculation. RV1B exposure induced neutrophilic and lymphocytic airway inflammation, as well as increased lung expression of KC, macrophage-inflammatory protein-2, and IFN-alpha and IFN-beta. RV1B-exposed mice showed airway hyperresponsiveness 1 and 4 days after inoculation. UV-irradiated RV1B induced modest neutrophilic airway inflammation and hyperresponsiveness 1 day after exposure. Both RV1B and UV-irradiated RV1B, but not RV39, increased lung phosphorylation of Akt. Confocal immunofluorescence showed colocalization of RV1B and phospho-Akt in the airway epithelium. Finally, pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 attenuated chemokine production and neutrophil infiltration. CONCLUSIONS: We conclude that RV1B induces airway inflammation in vivo. Evidence is presented that viral replication occurs in vivo and is required for maximal responses. On the other hand, viral replication was not required for a subset of RV-induced responses, including neutrophilic inflammation, airway hyperresponsiveness, and Akt phosphorylation. Finally, phosphatidylinositol 3-kinase/Akt signaling is required for maximal RV1B-induced airway neutrophilic inflammation, likely via its essential role in virus internalization.

A Rare Presentation of Biclonal Gammopathy in Multiple Myeloma with Simultaneous Extramedullary Involvement: A Case Report.

Multiple myeloma is characterized by an abnormal clone of plasma cell infiltration in the bone marrow and presence of a high level monoclonal immunoglobulin (M-protein) in the serum or urine in most cases. The monoclonal protein is usually detected as a discrete band in the gamma globulin region by serum protein electrophoresis. Rarely, it may show a simultaneous presence of two distinct bands, which could be either from a single clone, or two separate clones. We report a very unusual presentation of biclonality with lambda restricted IgG predominant cells from cervical lymph node and kappa restricted IgA cells from the bone marrow simultaneously.

Assessing p16 Status of Oropharyngeal Squamous Cell Carcinoma by Combined Assessment of the Number of Cells Stained and the Confluence of p16 Staining: A Validation by Clinical Outcomes.

Human papillomavirus-related oropharyngeal squamous cell carcinoma (OPSCC) has favorable prognosis relative to other head and neck squamous cell carcinomas. Criteria for predicting human papillomavirus status based upon p16 staining, including difficult cases with partial staining patterns, have been developed; however, clinical validation of these criteria and the clinical significance of partial p16 staining have not been reported. Eighty-one archival OPSCC cases were initially stained for p16 by immunohistochemistry with clone G175-405. The percentage of p16 cells and percentage of confluence of p16 cells were categorized as 25%, 26% to 75%, or >75%. Of all cases, 16 (20%) had partial p16 expression, with 26% to 75% p16 cells. Applying previously developed criteria of >75% p16 cells or >50% positive cells with >25% confluence, 48 (59%) patients were categorized p16 and demonstrated expected clinical characteristics and superior disease-free survival and overall survival (P<0.001) compared with p16 patients. By themselves, the partial staining patients had intermediate outcomes; however, separating the partial staining cases by degree of confluence showed that those with >75% confluence had superior disease-free survival (P=0.042). When the 16 original partial staining cases were re-stained with the alternative anti-p16 E6H4 clone, p16 status remained concordant for all cases, but only 3 of the 16 were interpreted as demonstrating partial staining. This report shows that the prevalence of partial p16 staining varies with the antibody utilized and clinically validates the application of a graded evaluation of both the number as well as confluence of positive cells for risk stratification of patients with OPSCC.

A colorful future of quantitative pathology: validation of Vectra technology using chromogenic multiplexed immunohistochemistry and prostate tissue microarrays.

The Vectra platform (Caliper Life Sciences, Hopkinton, MA) is an advanced multispectral imaging system for biomarker quantitation in tissue microarray or intact tissue sections. This is the first study to validate its reliability for quantitating spatially overlapping biomarkers using chromogenic multiplexed immunohistochemistry on prostate tissue microarrays. Two tissue microarray cohorts (an outcome tissue microarray and a progression tissue microarray) were used. The outcome tissue microarray cohort consists of 462 duplicate cores with more than 5-year outcome information. The progression tissue microarray cohort consists of 384 duplicate cores from different disease (stage) groups. The tissue microarray slides were stained with different combinations of antibodies (anti-androgen receptor, anti-E-cadherin, anti-erythroblastosis virus E26 oncogene-related gene product, and anti-α-methylacyl-CoA racemase). Three outcome tissue microarrays were stained with androgen receptor + erythroblastosis virus E26 oncogene-related gene + E-cadherin (outcome tissue microarray 1), androgen receptor + E-cadherin (outcome tissue microarray 2), and erythroblastosis virus E26 oncogene-related gene + E-cadherin (outcome tissue microarray 3), respectively. One progression tissue microarray section was stained with E-cadherin and α-methylacyl-CoA racemase; tissue microarray slides were then scanned with the Vectra platform. Biomarker expression analysis was performed with Vectra software-Nuance 3.0.0, and inForm 1.2. IBM SPSS Statistics 19 was used for statistical and correlation analysis (SPSS, Chicago, IL). Close concordance was found between the triple- and double-immunostaining assays used for quantitating spatially overlapping biomarkers androgen receptor and erythroblastosis virus E26 oncogene-related gene using outcome tissue microarrays (r = 0.897 for androgen receptor and 0.613 for erythroblastosis virus E26 oncogene-related gene, respectively). α-Methylacyl-CoA racemase and E-cadherin expression levels measured in progression tissue microarray were consistent with previously published data by other groups. In conclusion, Vectra technology is reliable for objective and high-throughput biomarker quantitation and colocalization study using chromogenic multiplexed immunohistochemistry.

Lung cells from neonates show a mesenchymal stem cell phenotype.

RATIONALE: Mesenchymal stem cells have been isolated from adult bone marrow, peripheral blood, adipose tissue, trabecular bone, articular synovium, and bronchial submucosa. OBJECTIVES: We hypothesized that the lungs of premature infants undergoing mechanical ventilation contain fibroblast-like cells with features of mesenchymal stem cells. METHODS: Tracheal aspirate fluid from mechanically ventilated, premature (< 30 wk gestation) infants 7 days old or younger was obtained from routine suctioning and plated on plastic culture dishes. MEASUREMENTS AND MAIN RESULTS: A total of 11 of 20 patients studied demonstrated fibroblast-like cells, which were identified as early as 6 hours after plating. Cells were found to express the mesenchymal stem cell markers STRO-1, CD73, CD90, CD105, and CD166, as well as CCR2b, CD13, prolyl 4-hydroxylase, and alpha-smooth muscle actin. Cells were negative for the hematopoietic and endothelial cell markers CD11b, CD31, CD34, or CD45. Tracheal aspirate monocyte chemoattractant protein-1/CCL2 levels were ninefold higher in aspirates in which fibroblast-like cells were found, and cells demonstrated chemotaxis in response to monocyte chemoattractant protein. Placement of cells into appropriate media resulted in adipogenic, osteogenic, and myofibroblastic differentiation. Patients from whom mesenchymal stem cells were isolated tended to require more days of mechanical ventilation and supplemental oxygen. CONCLUSIONS: Together, these data demonstrate that tracheal aspirate fluid from premature, mechanically ventilated infants contains fibroblasts with cell markers and differentiation potential typically found in mesenchymal stem cells.