P‐11THE IMPACT OF INTRODUCING LIQUID BASED CYTOLOGY INTO A ROUTINE SCREENING LABORATORY

Introduction: A glacial acetic acid wash performed retrospectively or prospectively on visibly bloodstained cervical ThinPrep® specimens can reduce the unsatisfactory rate and increase the number of diagnostic epithelial cells. This study was undertaken to determine which specimens are most likely to benefit from a prospective glacial acetic acid wash. Methods: Bloodstained ThinPrep® specimens selected for routine lysing prior to processing were macroscopically assessed and scored based on the level of blood present (+ to +++). Both unlysed and lysed slides were prepared from each specimen and microscopically examined. Results: Fifty‐eight specimens (32 scored +, 12 ++ and 14+++) were assessed. Three unlysed slides prepared from the ++ specimens and 12 from +++ specimens were evaluated as unsatisfactory due to excessive blood and inadequate numbers of squamous cells. In contrast, only one of the unlysed slides from the 32 + specimens was considered to be unsatisfactory. Almost all the lysed slides were satisfactory and generally more cellular than the unlysed slides. Abnormal cells were found in four cases (both unlysed and lysed paired slides). Discussion: Although the acetic acid wash increases cellularity of bloodstained ThinPrep® specimens, prospectively lysing all bloodstained specimens is an unnecessary procedure. Lysing only very heavily bloodstained specimens prior to processing will reduce laboratory workload, costs and the possibility of specimen mix up. Occasionally a retrospective wash may be required but screening staff should be aware that although blood may be present on an unlysed ThinPrep® slide, a lysed slide should not be requested unless there are insufficient numbers of squamous cells present.     

Evolutionary relationships among hares from North Africa (Lepus sp. or Lepus spp.), cape hares (L. capensis) from South Africa, and brown hares (L. europaeus), as inferred from mtDNA PCR-RFLP and allozyme data

Systematics and taxonomy of hares of the genus Lepus (Lagomorpha) are under contentious debate, and phylogenetic relationships among many taxa are not well understood. Here we study genetic differentiation and evolutionary relationships among North African hares, currently considered subspecies of Lepus capensis , cape hares ( L. capensis ) from the Cape province in South Africa, and brown hares ( L. europeaus ) from Europe and Anatolia, using maternally (mtDNA) and biparentally (allozymes) inherited markers. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of a c. 1.8 kb long segment of the mitochondrial control region using eight hexanucleotide-recognizing restriction endonucleases yielded 28 haplotypes, and horizontal starch gel electrophoresis of proteins encoded by 25 structural gene loci revealed 52 alleles at 18 polymorphic loci. Diverse phylogenetic analyses (neighbor joining dendrogram, median joining network, multidimensional scaling of pairwise distances, AMOVA, F -statistics, hierarchical F -statistics) of genetic variants revealed marked substructuring of mtDNA into three phylogeographic groups, namely an African, a central European, and an Anatolian, but a somewhat less pronounced overall differentiation of the nuclear genome, despite a relatively high number of population-specific (private) alleles. However, all our results are not incongruent with Petter's (1959: Mammalia 23 , 41; 1961: Z. f. Säugetierkunde 26 , 30; 1972 : Société Des Sciences Naturelles et Physiques du Maroc 52 , 122) hypothesis that North African hares generally belong to L. capensis and that brown hares should be included in this species as well.     

Conditional Maximum Likelihood Estimation Following a Group Sequential Test

We consider estimation after a group sequential test. An estimator that is unbiased or has small bias may have substantial conditional bias (Troendle and Yu, 1999, Coburger and Wassmer, 2001). In this paper we derive the conditional maximum likelihood estimators of both the primary parameter and a secondary parameter, and investigate their properties within a conditional inference framework. The method applies to both the usual and adaptive group sequential test designs. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Double trouble for grasshopper molecular systematics: intra-individual heterogeneity of both mitochondrial 12S-valine-16S and nuclear internal transcribed spacer ribosomal DNA sequences in Hesperotettix viridis (Orthoptera: Acrididae)

Abstract.  Hesperotettix viridis grasshoppers (Orthoptera: Acrididae: Melanoplinae) exhibit intra-individual variation in both mitochondrial 12S-valine-16S and nuclear internal transcribed spacer (ITS) ribosomal DNA sequences. These findings violate core assumptions underlying DNA sequence data obtained via polymerase chain reaction (PCR) amplification for use in molecular systematics investigations. Undetected intra-individual variation of this sort can confound phylogenetic analyses at a range of taxonomic levels. The use of a DNA extraction protocol designed to enrich mitochondrial DNA as well as an initial long PCR of approximately 40% of the grasshopper mitochondrial genome failed to control for the presence of paralogous mitochondrial DNA-like sequences within individuals. These findings constitute the first demonstration of intra-individual heterogeneity in mitochondrial DNA-like sequences in the grasshopper subfamily, Melanoplinae, and only the second report of intra-individual variation in nuclear ITS ribosomal DNA sequences in grasshoppers. The fact that intra-individual variation was detected in two independent DNA marker sets in the same organism strengthens the notion that the orthology of PCR-derived DNA sequences should be examined thoroughly prior to their use in molecular phylogenetic analyses or as DNA barcodes.     

Genetic and morphometric differentiation among island populations of two Norops lizards (Reptilia: Sauria: Polychrotidae) on independently colonized islands of the Islas de Bahia (Honduras)

Aim Anole lizards (Reptilia: Sauria: Polychrotidae) display remarkable morphological and genetic differentiation between island populations. Morphological differences between islands are probably due to both adaptive (e.g. differential resource exploitation and intra‐ or interspecific competition) and non‐adaptive differentiation in allopatry. Anoles are well known for their extreme diversity and rapid adaptive speciation on islands. The main aim of this study was to use tests of morphological and genetic differentiation to investigate the population structure and colonization history of islands of the Islas de Bahia, off the coast of Honduras. Location Five populations of Norops bicaorum and Norops lemurinus were sampled, four from islands of the Islas de Bahia and one from the mainland of Honduras. Methods Body size and weight differentiation were measured in order to test for significant differences between sexes and populations. In addition, individuals were genotyped using the amplified fragment length polymorphism technique. Bayesian model‐based and assignment/exclusion methods were used to study genetic differentiation between island and mainland populations and to test colonization hypotheses. Results Assignment tests suggested migration from the mainland to the Cayos Cochinos, and from there independently to both Utila and Roatán, whereas migration between Utila and Roatán was lacking. Migration from the mainland to Utila was inferred, but was much less frequent. Morphologically, individuals from Utila appeared to be significantly different in comparison with all other localities. Significant differentiation between males of Roatán and the mainland was found in body size, whereas no significant difference was detected between the mainland and the Cayos Cochinos. Main conclusions Significant genetic and morphological differentiation was found among populations. A stepping‐stone model for colonization, in combination with an independent migration to Utila and Roatán, was suggested by assignment tests and was compatible with the observed morphological differentiation.