Epidural infusion or combined femoral and sciatic nerve blocks as perioperative analgesia for knee arthroplasty.

BACKGROUND: Peripheral neural blockade appears to provide effective analgesia with potentially less morbidity than central neuraxial techniques. We compared the relative benefits of combined femoral (3-in-1) and sciatic nerve block with epidural blockade for postoperative knee arthroplasty analgesia. METHODS: Sixty patients, ASA I-III, undergoing unilateral knee replacement were prospectively randomized to receive either a lumbar epidural infusion or combined single-shot femoral (3-in-1) and sciatic blocks (combined blocks). All patients received standard general anaesthesia. Visual analogue pain scores and rescue opioid requirements were recorded at four time points postoperatively. Patient satisfaction, morbidity, block insertion time, perioperative blood loss and rehabilitation indices were also assessed. RESULTS: In both groups, pain on movement was well controlled at discharge from recovery and 6 h postoperatively but increased at 24 and 48 h. Median (95% CI) analogue scale scores were 0 (0-0), 15 (0-30), 55 (38-75) and 54 (30-67) mm for epidural block and 0.5 (0-22), 21.5 (10-28), 40 (20-50) and 34.5 (21-55) mm for combined block. VAS pain scores with the combined blocks were significantly lower at 24 h (P=0.004). Total morphine usage was low in both groups: median epidural group 17 mg (8-32) versus combined blocks 13 mg (7.8-27.5). Patient satisfaction was high in both groups with median (95% CI) scores of 100 (85-100), 83 (70-100) and 82 (57-90) mm for epidural and 90 (73-100), 100 (77-100) and 97 (80-100) mm for combined blocks (not significant). Perioperative blood loss and rehabilitation indices were also similar. CONCLUSIONS: Combined femoral (3-in-1) and sciatic blocks offer a practical alternative to epidural analgesia for unilateral knee replacements.

Efficacy of microfiltration in decreasing propofol-induced pain.

In a randomised, double-blinded, two-centre trial we evaluated the effect of a microbiological filter (Supor, Pall Life Sciences) on propofol injection pain. We studied 336 unpremedicated adult patients, who graded pain experienced during induction of anaesthesia with propofol on a 4-point verbal rating scale. Use of the microfilter reduced both the incidence and severity of propofol injection pain (p < 0.001). Incidence of severe pain in the filter group was 2.4% compared with 16.6% in the control group. Overall, 33.7% in the filter group experienced pain compared with 62.1% in the control group. A microbiological filter may provide a non-pharmacological alternative to a lidocaine/propofol mixture for reducing injection pain. It would also reduce the risk of any glass and bacterial contamination.

Evaluation of the HemoCue compared with the Coulter STKS for measurement of neonatal haemoglobin.

OBJECTIVE: To compare the measurement of haemoglobin concentration ([Hb]) using the HemoCue haemoglobinometer with that using the Coulter STKS haemoglobinometer. DESIGN: Thirty two EDTA samples were taken from neonates. [Hb] was measured in these samples using the HemoCue; the samples were then transferred to the haematology laboratory for [Hb] determination with the Coulter STKS. In addition, [Hb] was determined in 50 different random EDTA neonatal samples already held in the laboratory, using the HemoCue and Coulter STKS. PATIENTS: Neonates in the intensive care and low dependency Units of the Royal Devon and Exeter Hospital. INTERVENTIONS: Samples were collected from arterial lines or by venepuncture or heel prick into an EDTA bottle. MAIN OUTCOME MEASURES: [Hb] using the HemoCue and Coulter STKS methods. RESULTS: The mean [Hb] measured using the HemoCue was 150.3 g/l (range 78-215) compared with 152.8 g/l (range 78-217) measured using the Coulter STKS, with a mean of the differences of 2.5 g/l. The standard deviation of the differences of the 82 samples was 3.73 g/l. The limits of agreement of the two methods (mean difference +/- 2SD) were -4.8 to +9.8 g/l. CONCLUSION: With adequate training and monitoring, the HemoCue can be used directly on the neonatal unit for rapid determination of [Hb] to within 7.5 g/l compared with the laboratory Coulter STKS, using much smaller sample volumes.

A 46,XX fetus with external female and internal male genitalia, facial dysmorphic features and mildly dilated lateral ventricles of the brain: a new syndrome?

The clinical features of a 46,XX fetus with dysmorphic facial features, mild dilatation of the lateral ventricles of the brain, and female external and male internal genitalia are described. This combination of abnormalities does not appear to have been reported previously, and may represent a new syndrome.

Characterization of a chromosomally complex lung cancer cell line using multiwell fluorescence in situ hybridization.

The chromosomal characterization of a non-small cell lung cancer cell line (NCIH358) is described. This characterization was achieved using a simple, cheap and technically straightforward multiwell fluorescence in situ hybridization (FISH) method. The many and complex chromosome rearrangements identified by this method could not be defined using conventional G-banded chromosome analysis, and have not been previously described. For the detailed characterization of complex cell lines, multiwell FISH has many advantages over more technically demanding and expensive FISH techniques, and opens up the possibility of screening for consistent rearrangements, leading to the identification of unique fusion genes.

An interstitial deletion of 6p24-p25 proximal to the FKHL7 locus and including AP-2alpha that affects anterior eye chamber development.

The FKHL7 gene has been implicated in the pathogenesis of glaucoma/autosomal dominant iridogoniodysgenesis (IGDA) (IRID1). This has been supported by mutations in some glaucoma and IGDA patients and the development of anterior eye chamber anomalies in patients with 6p deletions affecting the 6p25 region. We report a case with anterior eye chamber anomalies and an interstitial deletion of 6p24-p25 that does not include the FKHL7 gene, suggesting the possible additional involvement of another locus, within 6p24-6p25, in anterior eye chamber development. A candidate gene is AP-2alpha, which is contained within the deleted segment and plays a role in anterior eye chamber development.

Delineation of two distinct 6p deletion syndromes.

Deletions of the short arm of chromosome 6 are relatively rare, the main features being developmental delay, craniofacial malformations, hypotonia, and defects of the heart and kidney, with hydrocephalus and eye abnormalities occurring in some instances. We present the molecular cytogenetic investigation of six cases with 6p deletions and two cases with unbalanced translocations resulting in monosomy of the distal part of 6p. The breakpoints of the deletions have been determined accurately by using 55 well-mapped probes and fluorescence in situ hybridization (FISH). The cases can be grouped into two distinct categories: interstitial deletions within the 6p22-p24 segment and terminal deletions within the 6p24-pter segment. Characteristics correlating with specific regions are: short neck, clinodactyly or syndactyly, brain, heart and kidney defects with deletions within 6p23-p24; and corneal opacities/iris coloboma/Rieger anomaly, hypertelorism and deafness with deletions of 6p25. The two cases with unbalanced translocations presented with a Larsen-like syndrome including some characteristics of the 6p deletion syndrome, which can be explained by the deletion of 6p25. Such investigation of cytogenetic abnormalities of 6p using FISH techniques and a defined set of probes will allow a direct comparison of reported cases and enable more accurate diagnosis as well as prognosis in patients with 6p deletions.

Isolation and chromosomal mapping of human glycogen synthase kinase-3 alpha and -3 beta encoding genes.

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that exists as two isoforms, alpha and beta, encoded by separate genes. Phosphorylation targets include a variety of cytoplasmic and nuclear proteins. Recent studies found that neurofilaments, amyloid precursor protein, and tau proteins are substrates of GSK-3 and that aberrant phosphorylation of these proteins is implicated in pathologies of the nervous system. To analyse the organisation of these two genes, a YAC library was screened by polymerase chain reaction, using primers specific for human GSK-3 alpha and GSK-3 beta cDNA. Two clones, 220 and 285 kb in size, containing the complete GSK-3 alpha coding sequence, and two clones, 365 and 285 kb in size, containing the 5' coding sequence of GSK-3 beta, were isolated. By somatic cell hybrid panel DNA amplification and radiation hybrid mapping, GSK-3 alpha was found to be located at 19q13.2. On the other hand, by somatic cell hybrid panel DNA amplification and fluorescence in situ hybridisation using the 285-kb YAC clone, GSK-3 beta was mapped to 3q13.3.

Further evidence for the involvement of human chromosome 6p24 in the aetiology of orofacial clefting.

Chromosomal translocations affecting the 6p24 region have been associated with orofacial clefting. Here we present a female patient with cleft palate, severe growth retardation, developmental delay, frontal bossing, hypertelorism, antimongoloid slant, bilateral ptosis, flat nasal bridge, hypoplastic nasal alae, protruding upper lip, microretrognathia, bilateral, low set, and posteriorly rotated ears, bilateral microtia, narrow ear canals, short neck, and a karyotype of 46,XX,t(6;9)(p24;p23). The translocation chromosomes were analysed in detail by FISH and the 6p24 breakpoint was mapped within 50-500 kb of other breakpoints associated with orofacial clefting, in agreement with the assignment of such a locus in 6p24. The chromosome 9 translocation breakpoint was identified to be between D9S156 and D9S157 in 9p23-p22, a region implicated in the 9p deletion syndrome.

Physical map of human 6p21.2-6p21.3: region flanking the centromeric end of the major histocompatibility complex.

We have physically mapped and cloned a 2.5-Mb chromosomal segment flanking the centromeric end of the major histocompatibility complex (MHC). We characterized in detail 27 YACs, 144 cosmids, 51 PACs, and 5 BACs, which will facilitate the complete genomic sequencing of this region of chromosome 6. The contig contains the genes encoding CSBP, p21, HSU09564 serine kinase, ZNF76, TCP-11, RPS10, HMGI(Y), BAK, and the human homolog of Tctex-7 (HSET). The GLO1 gene was mapped further centromeric in the 6p21.2-6p21.1 region toward TCTE-1. The gene order of the GLO1-HMGI(Y) segment in respect to the centromere is similar to the gene order in the mouse t-chromosome distal inversion, indicating that there is conservation in gene content but not gene order between humans and mice in this region. The close linkage of the BAK and CSBP genes to the MHC is of interest because of their possible involvement in autoimmune disease.

Generation of novel human MHC class II mutant B-cell lines by integrating YAC DNA into a cell line homozygously deleted for the MHC class II region.

The human B lymphoblastoid cell line (LCL) 721.174 sustains a large homozygous deletion in the major histocompatibility complex (MHC) class II region that results in an absence of DQ and DR molecules as well as a deficiency in the assembly and transport of class I molecules to the cell surface. The deleted genes include the transporters associated with antigen processing TAP1 and TAP2, DMA and DMB which are involved in editing class II bound peptides, and four genes whose roles in antigen processing are unclear; the low mass polypeptide genes LMP2 and LMP7, and DNA and DOB. To study this region we have integrated into 721.174 two overlapping yeast artificial chromosome (YAC) clones which cover the interval LMP2-DRA inclusive. Three clones (11.2A1.1, 4D1D10.1 and 4D1D10.2), containing complete copies of the transfected YAC, produced varying levels of mRNA from the LMP, TAP, DQ and DR genes and corresponding levels of LMP and TAP protein. Class I cell surface expression was restored in 11.2A1.1 and 4D1D10.1, as was DR expression in both 4D1D10 transfectants. These studies demonstrate the feasibility of introducing large groups of functional genes back into human lymphoblastoid cells sustaining deletions, with full restoration of biological function. The procedure could be exploited in order to restore all but one gene covered by the deletion, effectively producing a single gene defect. This could be used to introduce copies of genes engineered to contain mutations and to study cis regulatory elements at some distance from the chosen loci.

A detailed investigation of two cases exhibiting characteristics of the 6p deletion syndrome.

Deletions of the short arm of chromosome 6 are relatively rare, only 16 cases having been described in the literature so far. Here we present a detailed investigation by fluorescence in situ hybridisation of two further cases with different but overlapping interstitial deletions involving 6p22, 6p23 and 6p24. The main features involved are craniofacial malformations, heart and kidney defects, mental retardation/developmental delay, hypotonia and hydrocephalus. By using 36 yeast artificial chromosome and cosmid clones from a contig covering 6p22.3-6p25 and other probes with defined cytogenetic locations within 6p21-6p22 we have precisely localised the breakpoints involved in each of the cases, estimated the sizes of the deleted regions and defined the region that is hemizygously deleted in both cases.

An integrated map of human chromosome 6p23.

The human chromosomal band 6p23 is a Giemsa-negative (light) band that may be expected to be relatively gene rich. The genes for spinocerebellar ataxia type 1 (SCA1), guanosine monophosphate reductase (GMPR), DEK involved in a subtype of acute myeloid leukemia (AML), and the folate-sensitive fragile site FRA6A, have already been mapped to 6p23. Recent linkage data have suggested evidence for a susceptibility locus for schizophrenia in the region. We have constructed a single YAC contig of approximately 100 clones spanning the entire 6p23 band from 6p22.3 to 6p24.1 and covering 7.5-8.5 Mb of DNA. The YAC contig contains 55 markers including genetically mapped STSs, physically mapped STSs, anonymous STSs, anonymous ESTs, and ESTs from the genes mapped to the region. The order of the genetically mapped STSs is consistent with their order in the contig and some of the markers not resolved on the genetic map have been resolved by the YACs. Four of the YACs from 6p23 and covering approximately 3 Mb of DNA have been used to isolate approximately 300 cosmids from a flow-sorted human chromosome 6 cosmid library, which have been organized into pockets. The proposed susceptibility locus for schizophrenia is most closely linked to D6S260, which is located within the YAC contig along with genetic markers < or = 5 cM on either side. Therefore, the presented materials are valuable reagents for characterization of the genomic region implicated in schizophrenia.

A fetus with an X;1 balanced reciprocal translocation and eye disease.

A 19 week female fetus is described with a de novo X;1 reciprocal balanced translocation, with the breakpoint on the X chromosome at Xp11.4, and eye pathology consistent with the early stages of Norrie disease. The fetus seems to be an example of a female manifesting an X linked recessive disease, and it was shown that the normal X chromosome was completely inactivated in all cells examined. Norrie disease has been mapped to Xp11.3, and fluorescence in situ hybridisation studies showed that the Norrie disease gene had not obviously been disrupted. Mutation screening by SSCP analysis showed no aberrant fragments of the coding region of the gene. Several eye disease genes map to the same region of the X chromosome, but are excluded on grounds of pathology. One possibility is that this fetus has a Norrie-like eye disease caused by the mutation of another gene located at Xp11.4. If this is so, there are implications for prenatal diagnosis.

Evidence of a locus for orofacial clefting on human chromosome 6p24 and STS content map of the region.

Orofacial clefting is genetically complex, no single gene being responsible for all forms. It can, however, result from a single gene defect either as part of a syndrome (e.g. van der Woude syndrome, Treacher-Collins syndrome, velo-cardio-facial syndrome) or as an isolated phenotypic effect (e.g. X-linked cleft palate; non-syndromic, autosomal dominant orofacial clefting). Several studies have suggested that chromosome 6p is a candidate region for a locus involved in orofacial clefting. We have used YAC clones from contigs in 6p25-p23 to investigate three unrelated cases of cleft lip and palate coincident with chromosome 6p abnormalities. Case 1 has bilateral cleft lip and palate and a balanced translocation reported as 46,XY,t(6,7)(p23;q36.1). Case 2 has multiple abnormalities including cleft lip and palate and was reported as 46,XX,del(6)(p23;pter). Case 3 has bilateral cleft lip and palate and carries a balanced translocation reported as 46,XX,t(6;9)(p23;q22.3). We have identified two YAC clones, both of which cross the breakpoint in cases 1 and 3 and are deleted in case 2. These clones map to 6p24.3 and therefore suggest the presence of a locus for orofacial clefting in this region. The HGP22 and AP2 genes, potentially involved in face formation, have been found to flank this region, while F13A maps further telomeric in 6p24.3/25.

FISH detection of trisomy 21 in interphase by the simultaneous use of two differentially labelled cosmid contigs.

Techniques have been reported in which fluorescence in situ hybridisation (FISH) and cosmid probes are used to detect trisomy 21 (and other abnormalities involving chromosomes X, Y, 13, and 18) on uncultured amniocytes. However the detection rate of trisomy 21 is lower than for the other anomalies owing to a larger number of uninformative results and false negatives. We report the simultaneous use of two differentially labelled cosmid contigs to improve the detection rate of trisomy 21 on uncultured amniocyte samples thus allowing the prenatal diagnosis of Down's syndrome even if only few labelled nuclei are available.

Rapid molecular method for prenatal detection of Down's syndrome.

We have evaluated a rapid method that allows prenatal detection of Down's syndrome in less than 24 hours. DNA from uncultured amniotic fluid, fetal blood, and tissue samples was amplified with the small tandem repeat (STR) marker D21S11. Quantitative analysis of fluorescent STR products with evaluation of their sizes provided clear evidence for trisomy 21. Whilst most normal samples showed two amplification peaks of equal size, Down's syndrome samples were characterised by either three STR peaks or two peaks with a ratio of 2:1. Co-amplification with a non-polymorphic sequence allowed analysis of samples that were homozygous for the 21-derived STRs.