Molecular pathology curriculum for medical laboratory scientists: A report of the association for molecular pathology training and education committee.

Molecular diagnostics is a rapidly growing specialty in the clinical laboratory assessment of pathology. Educational programs in medical laboratory science and specialized programs in molecular diagnostics must address the training of clinical scientists in molecular diagnostics, but the educational curriculum for this field is not well defined. Moreover, our understanding of underlying genetic contributions to specific diseases and the technologies used in molecular diagnostics laboratories change rapidly, challenging providers of training programs in molecular diagnostics to keep their curriculum current and relevant. In this article, we provide curriculum recommendations to molecular diagnostics training providers at both the baccalaureate and master's level of education. We base our recommendations on several factors. First, we considered National Accrediting Agency for Clinical Laboratory Sciences guidelines for accreditation of molecular diagnostics programs, because educational programs in clinical laboratory science should obtain its accreditation. Second, the guidelines of several of the best known certifying agencies for clinical laboratory scientists were incorporated into our recommendations. Finally, we relied on feedback from current employers of molecular diagnostics scientists, regarding the skills and knowledge that they believe are essential for clinical scientists who will be performing molecular testing in their laboratories. We have compiled these data into recommendations for a molecular diagnostics curriculum at both the baccalaureate and master's level of education.

Circulating neutrophil CD14 expression and the inverse association of ambient particulate matter on lung function in asthmatic children.

BACKGROUND: Identifying baseline inflammatory biomarkers that predict susceptibility to size-specific particulate matter (PM) independent of gaseous pollutants could help us better identify asthmatic subpopulations at increased risk for the adverse health effects of PM. OBJECTIVE: To evaluate whether the association between lung function and exposure to ambient levels of PM less than 2.5 microm in diameter (PM2.5) (fine) and 10 to 2.5 microm in diameter (PM(10-2.5)) (coarse) in children with persistent asthma differed across baseline measures of inflammation and innate immune activation. METHODS: We performed a panel study on a local population of 16 children with persistent asthma and evaluated daily pulmonary function (percentage of predicted peak expiratory flow and forced expiratory volume in 1 second) while concurrently measuring daily PM2.5 and PM(10-2.5) exposure from a central site in Chapel Hill, North Carolina. The children underwent a baseline medical evaluation that included assessment of several immunoinflammatory biomarkers in peripheral blood. RESULTS: Children without measurable CD14 expression on circulating neutrophils had significantly reduced pulmonary function (forced expiratory volume in 1 second and peak expiratory flow) with each interquartile range (IQR) increase in PM2.5 (IQR = 8.5 microg/m3) and PM(10-2.5) (IQR = 4.1 microg/m3) concentration, unlike children with measurable CD14 expression (P < .001 for interaction). CONCLUSIONS: Asthmatic children with muted surface expression of CD14 on circulating neutrophils may have a decreased capacity to respond to bacterial components of PM.

A novel gene from Brugia sp. that encodes a cytotoxic fatty acid binding protein allergen recognized by canine monoclonal IgE and serum IgE from infected dogs.

Brugia pahangi infection of dogs is a well characterized model of human lymphatic filariasis in which sera consistently show IgG or IgE reactivity to a 35-kDa antigen. Using dog lymph node B cells, we previously established a heterohybridoma cell line producing canine monoclonal IgE (cmAb 2.39) that activates and degranulates canine mast cells, and specifically recognizes a 35-kDa B. pahangi antigen. By affinity purification and sequencing of the native protein from B. pahangi adults, a 19-amino acid sequence was obtained; the derived nucleotide sequence showed homology to a Brugia malayi and 2 related Onchocerca volvulus expressed sequence tag (EST) clones from the Filarial Genome Project database. Consensus primers amplified a 244-bp product from adult and infective larval stage cDNA libraries of B. malayi, O. volvulus, and Wuchereria bancrofti, but not from those of nonfilarial nematodes. The B. malayi EST clone only showed nucleotide sequence homology to O. volvulus EST sequences. A 684-bp region from the open reading frame was expressed as a glutathione S-transferase fusion protein designated BmAl-1. CmAb 2.39, as well as serum IgE from dogs infected with B. pahangi and canine filarial heartworm, Dirofilaria immitis, recognized BmAl-1 on enzyme-linked immunosorbent assay and Western blots. BmAl-1 showed high binding affinity for a fatty acid; however, a search for sequence homology with known fatty acid binding proteins indicated that BmAl-1 is a unique fatty acid binding protein. This 35-kDa protein seems to be highly conserved in different stages and species of filarids, and it represents a previously unknown allergen that is possibly involved in the pathogenesis of filarial disease.

Relative contributions of enzyme cytochemistry and flow cytometric immunophenotyping to the evaluation of acute myeloid leukemias with a monocytic component and of flow cytometric immunophenotyping to the evaluation of absolute monocytoses.

We evaluated the contributions of enzyme cytochemical stains and flow cytometric immunophenotyping (FCI) data to detection of monocytic cells (MCs) in acute myelomonocytic and acute monocytic leukemias (AMMLs and AMoLs) and compared FCI findings in AMoL, chronic myelomonocytic leukemia (CMML), and normal peripheral blood (PB) and bone marrow (BM) monocytes to classify and evaluate absolute monocytoses (AMs). We reviewed 10 AMMLs and 6 AMoLs with a-naphthyl-acetate esterase (ANAE) and a-naphthyl-butyrate esterase stains and a complete FCI profile and compared FCI data for 6 AMoLs, 7 CMMLs, 2 AMs, and normal monocytes. We confirmed increased sensitivity of ANAE staining to FCI data in detecting MCs in AMML and AMoL. CD14 was insensitive for confirming MCs; other characteristic markers of MCs were absent or partially lost in AMML and AMoL. Aberrant expression of CD56 (detected in 50% of AMMLs and AMoLs), CD34, and CD117 indicated malignancy. The mature MCs of the CMMLs revealed variable FCI abnormalities (partial loss of CD13, CD14, and CD15; expression of CD56), as in the monoblasts of AMoL. These FCI abnormalities in morphologically mature MCs might indicate markers for CMML. AMs revealed FCI abnormalities, indicating clues to their correct classification as CMML.

Practical evaluation of methods for detection and specificity of autoantibodies to extractable nuclear antigens.

Detection and specificity of autoantibodies against extractable nuclear antigens (ENA) play a critical role in the diagnosis and management of autoimmune disease. Historically, the detection of these antibodies has employed double immunodiffusion (DID). Autoantibody specificity was correlated with diagnoses by this technique. Enzyme immunoassays have been developed by multiple manufacturers to detect and identify the specificity ENA autoantibodies. To address the relationship of ENA detection by DID and enzyme immunoassay, the performances of five immunoassays were compared. These included two DID and three enzyme-linked immunoassays (ELISA) (both screening and individual antigen profile kits). The sample set included 83 ENA-positive, antinuclear-antibody (ANA)-positive specimens, 77 ENA-negative, ANA-positive specimens, and 20 ENA- and ANA-negative specimens. Sensitivity and specificity were calculated by two methods: first, by using the in-house DID result as the reference standard, and second, by using latent class analysis, which evaluates each kit result independently. Overall, the results showed that the ELISA methods were more sensitive for detection of ENA autoantibodies than DID techniques, but presence and/or specific type of ENA autoantibody did not always correlate with the patient's clinical presentation. Regardless of the testing strategy an individual laboratory uses, clear communication with the clinical staff regarding the significance of a positive result is imperative. The laboratory and the clinician must both be aware of the sensitivity and specificity of each testing method in use in the clinical laboratory.

Minimizing red blood cell contamination while isolating mononuclear cells from whole blood: the next step for the treatment of severe hemolytic disease of the fetus/newborn.

OBJECTIVE: We examined whether mononuclear cell purification of human blood could be done while minimizing contamination with RhD-positive red blood cells to treat hemolytic disease of the fetus/newborn that was caused by rhesus disease. STUDY DESIGN: Whole blood from 16 individuals who tested rhesus positive was diluted and centrifuged over a Ficoll gradient. The cell pellet was incubated with red blood cell lysis buffer, divided into three samples, and analyzed for cell count, mononuclear cell yield, and RhD-positive red cell contamination by flow cytometry. RESULTS: Mean RhD-positive red cell contamination was 0.24% (range, 0%-1.9%). The average yield of mononuclear cells was 11.5% (range, 1.8%-23.6%). Through regression analysis, 34 to 180 mL of paternal whole blood would be necessary to achieve an antigen load that is sufficient for an HLA antibody response. CONCLUSION: Purification of human blood is possible to produce reasonable mononuclear cell yields with minimal rhesus activity, which makes paternal leukocyte therapy a plausible treatment for severe rhesus alloimmunization.

Differential expression of TcR-CD3 zeta as evidence for altered immunoregulation in preeclamptic versus normotensive women.

OBJECTIVE: The study was undertaken to exhibit and quantify the difference in modulation of CD3-zeta protein (an integral component of the T-cell receptor) in preeclamptic and normotensive women. STUDY DESIGN: Serum was collected from 10 preeclamptic and 10 normotensive women at >or=37 weeks' gestation on admission. Jurkat E-61 cells were incubated with the sera (20% volume to volume) and analyzed with Western immunoblot using mouse monoclonal CD3-zeta antibody. Enhanced chemiluminescence and densitometry were used to qualitatively measure zeta expression of the cells. A de novo flow cytometry assay was developed to quantify the difference in CD3-zeta expression of these cells. Comparisons were performed by t test (P<.05 was significant). RESULTS: Preeclamptic patient sera produced a 2.4-fold increase in CD3-zeta expression than normotensive patients on Western blot (P<.01). Flow cytometry showed that preeclamptic sera had a 1.4-fold higher expression of CD3-zeta compared with normotensive patients (P<.0003). CONCLUSION: TcR/CD3-zeta expression is normally suppressed in pregnancy. Loss of this suppression occurs in preeclamptic patients, implying increased T-cell function.