A review of guidelines for evaluating a minor modification to a validated assay.

Any modification to a validated assay must be evaluated in terms of the impact on the assay's performance characteristics and whether the assay remains fit for the intended purpose. The comparison is referred to as a 'method comparison', 'method comparability', 'method change', or 'comparative validation'. This review presents recommendations and examples of studies found in the current literature as a means of assessing minor modifications. In addition, the authors discuss common statistical approaches used for these comparisons.

Toute modification apportée à un essai validé doit être évaluée afin de mesurer l'impact de cette modification sur les paramètres de performances de l'essai et déterminer si l'aptitude à l'emploi qui lui a été assigné demeure valable suite à la modification en question. Cette comparaison est désignée sous les termes de « comparaison de méthodes d'essai ¼, « comparabilité de méthodes ¼, « changement de méthode d'essai ¼ ou « validation comparative ¼. Les auteurs font part de leurs recommandations et donnent des exemples d'études émanant de la littérature récente concernant l'évaluation de modifications mineures. En outre, ils examinent les approches statistiques couramment utilisées pour ces comparaisons.

Toda modificación que se introduzca en un ensayo validado debe ser objeto de evaluación para determinar la influencia del cambio en las características de funcionamiento del ensayo y saber si este sigue estando adaptado a su función. Para referirse a la comparación, los autores emplean expresiones como 'comparación de métodos', 'comparabilidad de métodos', 'cambio de método' o 'validación comparativa'. Los autores presentan aquí recomendaciones y ejemplos de estudios extraídos de la bibliografía actual como medio de evaluar modificaciones de importancia menor. Además, los autores examinan las lógicas estadísticas comunes utilizadas para estas comparaciones.

Hematopoiesis in the equine fetal liver suggests immune preparedness.

We investigated how the equine fetus prepares its pre-immune humoral repertoire for an imminent exposure to pathogens in the neonatal period, particularly how the primary hematopoietic organs are equipped to support B cell hematopoiesis and immunoglobulin (Ig) diversity. We demonstrated that the liver and the bone marrow at approximately 100 days of gestation (DG) are active sites of hematopoiesis based on the expression of signature messenger RNA (mRNA) (c-KIT, CD34, IL7R, CXCL12, IRF8, PU.1, PAX5, NOTCH1, GATA1, CEBPA) and protein markers (CD34, CD19, IgM, CD3, CD4, CD5, CD8, CD11b, CD172A) of hematopoietic development and leukocyte differentiation molecules, respectively. To verify Ig diversity achieved during the production of B cells, V(D)J segments were sequenced in primary lymphoid organs of the equine fetus and adult horse, revealing that similar heavy chain VDJ segments and CDR3 lengths were most frequently used independent of life stage. In contrast, different lambda light chain segments were predominant in equine fetal compared to adult stage, and surprisingly, the fetus had less restricted use of variable gene segments to construct the lambda chain. Fetal Igs also contained elements of sequence diversity, albeit to a smaller degree than that of the adult horse. Our data suggest that the B cells produced in the liver and bone marrow of the equine fetus generate a wide repertoire of pre-immune Igs for protection, and the more diverse use of different lambda variable gene segments in fetal life may provide the neonate an opportunity to respond to a wider range of antigens at birth.

Developmental expression of B cell molecules in equine lymphoid tissues.

Identification and classification of B cell subpopulations has been shown to be challenging and inconsistent among different species. Our study tested aspects of ontogeny, phenotype, tissue distribution, and function of equine CD5hi B cells, which represented a greater proportion of B cells early in development and in the peritoneal cavity. CD5hi and CD5lo B cells differentially expressed B cell markers (CD2, CD21, IgM) measured using flow cytometry, but similar mRNA expression of signature genes (DGKA, FGL2, PAX5, IGHM, IL10) measured using quantitative RT-PCR. Sequencing lambda light chain segments revealed that CD5hi B cells generated diverse immunoglobulin repertoires, and more frequently bound to fluorescence-labeled phosphorylcholine. This study shows developmental characteristics and tissue distribution of a newly described subpopulation of B cells in the horse.

An ordered BAC contig map of the equine major histocompatibility complex.

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.

Expression of essential B cell development genes in horses with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p<0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p<0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients.

Development and characterization of an equine infectious anemia virus Env-pseudotyped reporter virus.

We developed a replication-defective reporter virus pseudotyped with the envelope glycoprotein of equine infectious anemia virus (EIAV). The in vitro host range and neutralization phenotype of EIAV Env-pseudotyped virus were similar to those of replication-competent virus. An EIAV Env pseudovirus will improve antigenic characterization of viral variants and evaluation of lentivirus vaccines.