A randomised controlled trial of pectoral nerve-2 (PECS 2) block vs. serratus plane block for chronic pain after mastectomy.

Thoracic interfascial plane blocks are effective for post-mastectomy acute analgesia. However, their effects on chronic pain are uncertain. We randomly allocated 80 women equally to pectoral nerve-2 (PECS 2) block or serratus plane block. The pectoral nerve-2 block reduced the rate of moderate or severe chronic pain from 13/40 (33%) with the serratus plane block to 4/40 (10%), p = 0.03, adjusted odds ratio (95%CI) 0.23 (0.07-0.80), p = 0.02. The rates of pain-free women at six postoperative months were indeterminate, 10/40 (25%) after serratus plane block vs. 19/40 (48%) after pectoral nerve-2 block, p = 0.06, adjusted odds ratio (95%CI) 2.9 (1.1-7.5), p = 0.03. Health-related quality of life at six postoperative months was similar after serratus plane and pectoral nerve-2 blocks, mean (SD) EQ-5D-3L scores 0.87 (0.15) vs. 0.91 (0.14), respectively, p = 0.21. The pectoral nerve-2 block reduced median (IQR [range]) morphine consumption in the first 24 postoperative hours from 6 (3-9 [1-25]) mg to 4 (2-7 [0-37]) mg, p = 0.04. However, acute pain scores after serratus plane and pectoral nerve-2 blocks were similar, median (IQR [range]) 23 (11-35 [0-70]) mm vs. 18 (11-27 [0-61]) mm, respectively, p = 0.44. Pectoral nerve-2 block reduced chronic pain 6 months after mastectomy compared with serratus plane block.

A novel assay for typing Rh antigens in blood-stains using a lectin specific to the bisecting N-acetyl-D-glucosamine side chain of glycoprotein.

A unique sandwich enzyme-linked immunosorbent assay for the determination of Rh antigens in blood stains has been developed using Rh antisera and phaseolus vulgaris E4/peroxidase conjugate (PHAE4/PO). The appropriate antiserum for detecting Rh C, c, D, E or e was coated on the inner surface of microplate wells, and the sample antigens from blood stains, solubilized with n-octyl-beta-D-glucopyranoside, were then placed in the wells. After washing the wells repeatedly, PHAE4/PO was added. Bound PHAE4/PO was detected by the development of colors using o-phenylenediamine/H2O2. All Rh antigens corresponding to the antisera were clearly detected using this technique. The detection limit expressed by sample dilution was more than 2 x 10(5) times (volume/dried blood weight) for the various antigens from the fresh 5 x 5 mm2 blood stain. Even when the blood stain samples were left beside a sunny window at room temperature for 2 months, Rh antigens were still detected. When the ABH, MN, P1, Kidd, Duffy and Lewis blood grouping systems were tested with similar ELISA procedures PHAE4 did not recognize any antigen. Since PHAE4 specifically recognizes and combines with the bisecting N-acetyl-D-glucosamine side chain, it was concluded that the glycoprotein was a component of all Rh antigens immune complexes.

Diagnosis of twin zygosity by hypervariable RFLP markers.

The application of variable number of tandem repeat (VNTR) markers to the determination of twin zygosity was investigated. In the first case, which was performed with the use of six VNTR markers, the probability of monozygosity, calculated from Essen-Möller's formula II, was 0.99972. In the other three cases in which four VNTR markers were analyzed, the probabilities were 0.98251-0.99557. These results suggest that VNTR markers are useful for determination of twin zygosity.

Monozygotic twins of different apparent sex.

We report on twins of unlike sex who shared a 45,X/46,X,+mar karyotype. The mar chromosome was found to be Yq- by DNA analysis. Marker studies, including 8 VNTR loci, yielded a probability of monozygosity of 0.99999996.

Potential bioactivated neurotoxicants, N-methylated beta-carbolinium ions, are present in human brain.

Potential bioactivated neurotoxicants, 2-N-methyl-beta-carbolinium and 2,9-N,N'-dimethyl-beta-carbolinium ions, as well as N-methylation activities which form these charged species, were analyzed for the first time in the parietal association cortex and the substantia nigra of human brain using GC/MS and HPLC. The brains were taken during forensic autopsies from corpses without obvious degeneration of substantia nigra. In the cortex, 2-methyl-norharmanium ion (2-MeNH) and 2,9-dimethyl-norharmanium ion (2,9-Me2NH) were detected in almost all samples. 2-Methyl-harmanium ions (2-MeHA) and 2,9-dimethyl-harmanium ions (2,9-Me2HA) were detectable in only two samples. In substantia nigra samples pooled from 3 or 4 brains for analysis, 2-MeNH and 2,9-Me2NH levels were higher than those in the cortex, whereas 2-MeHA and 2,9-Me2HA were below detection limits. Their precursors, norharman (NH) and harman (HA), were also measured using HPLC/fluorescence detection. In both regions, NH and HA were present in almost all samples; levels of NH and HA were also significantly higher in the nigra than in the cortex. Using 9-methyl-NH and 2-MeNH as substrates, in vitro N-methylation of the 2[beta] and 9[indole] nitrogens toward beta-carbolines was measured both in the cortex and in the nigra. 2[beta]-N-Methylation activity was significantly higher than 9[indole]-N-methylation activity in both regions. Recent studies show that beta-carbolinium ions resemble the synthetic parkinsonian toxicant, MPP+, with respect to structure and neurotoxic activity. Such 'bioactivated' carbolinium ions could be endogenous causative factors in Parkinson's disease.

1-Methyl-tetrahydro-beta-carboline-3-carboxylic acid is present in the rat brain and is not increased after acute ethanol injection with cyanamide treatment.

We conducted analyses of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (1Me3C-THBC) by gas chromatography-mass spectrometry (negative chemical ionization mode) to investigate its presence and the in vivo condensation between tryptophan and AcH. 1Me3C-THBC was found in the cerebellum and the cerebrum of normal rat [117.0 +/- 41.7 and 46.5 +/- 13.9 pmol/g tissue (mean +/- SEM), respectively]. The concentrations of 1Me3C-THBC and tryptophan were higher in the cerebellum than those in the cerebrum. The level of 1Me3C-THBC in both regions remained unchanged following a single oral ethanol administration alone or with cyanamide pretreatment. These data suggest that acetaldehyde is an unlike precursor of 1Me3C-THBC as a result of ethanol ingestion. 1Me3C-THBC also existed in the rat chow (282.0 +/- 24.2 pmol/g), so that most of brain 1Me3C-THBC detected in the rat brain might have originated from dietary sources. However, the possibility of a biosynthesis from tryptophan and alpha-keto acid still remained, especially after long-term ethanol treatment.

"First pass phenomenon" of inhaled gas in the fire victims.

We investigated the differences in the levels of carboxyhemoglobin (COHb), cyanide (HCN) and petroleum fuels (gasoline and kerosene) between left and right ventricular bloods from fire victims. COHb was slightly, and HCN and petroleum fuels were markedly higher levels in the left than those in the right. These effects were so called 'first pass phenomena' due to the circulation, diffusion and metabolization before the deaths of fire victims.

Derivative spectrophotometric studies on cytotoxic effects of carbon monoxide.

Characteristics of cytochrome oxidase prepared from hearts of Sprague-Dawley male rats were studied with the use of the fourth derivative spectrophotometry in respect to the cytotoxic effects of carbon monoxide (CO). CO-exposed rats tended to show lower specific activities of cytochrome oxidase than control and recovered rats. Moreover, there was a significant difference in the fourth derivative spectral features of the enzyme: CO-exposed groups indicated peaks at 412 nm in the spectra while controls at 408 nm. This spectral difference seemed to reflect specific effect of CO on cytochrome oxidase, though such trace remained for not more than a day after death.

Sex identification of forensic specimens by polymerase chain reaction (PCR): two alternative methods.

Sex identification of forensic samples (bloodstains and decomposed tissue) by polymerase chain reaction (PCR) was investigated. Amplification of a segment of the amelogenin gene using a pair of primers revealed both Y- and X-specific bands at the same time. The gene has counterparts in both the X and Y chromosomes and a small deletion in the former made it possible to distinguish them. Analysis of the X-specific band is the most reliable method for sex identification. THe locus includes a single copy gene so a sample of 250 ng/tube of deoxyribonucleic acid (DNA) is required for identification. Amplification of part of the DYZ1 locus was attempted as an alternative method for analysis of infinitesimal amounts of sample. Even DNA from putrefied tissue could be analyzed by PCR because the locus consists of thousands of copies of repeating units pHY10.

Detection of delta 9-THC in saliva by capillary GC/ECD after marihuana smoking.

A method is described for the determination of delta 9-tetrahydrocannabinol (delta 9-THC) in the saliva by the use of a combination of moving-precolumn injector and glass capillary gas chromatograph with electron capture detector (GC/ECD). There were no interfering peaks due to impurities around the peak of pentafluoropropyl derivative of delta 9-THC (delta 9-THC-PFP). This GC/ECD method was linear over the range of 5-200 ng/ml of delta 9-THC-PFP. The lower detection limit was approximately 1 ng/ml. delta 9-THC content in the saliva after experimental marihuana smoking was measured by this method. It was demonstrated that for at least 4 h after smoking the level of delta 9-THC was sufficient for detection.

Sex determination of forensic samples by dual PCR amplification of an X-Y homologous gene.

Sex determination by polymerase chain reaction (PCR) analysis of the X-Y homologous amelogenin gene is highly reliable since the detection of an X-specific amplified fragment validates the procedure. Previously, we reported that 250 ng of template DNA are required for sex determination by this method. We report here a refinement of the technique to include dual PCR. Dual PCR using two sets of primers results in the detection of X- and Y-specific amplified fragments from as little as 0.005 ng of template DNA. This is a powerful technique for the analysis of trace forensic samples and its application is discussed.

Determination of carboxyhemoglobin in heated blood--sources of error and utility of derivative spectrophotometry.

The cause for discrepancies in results from different methods of the carboxyhemoglobin (HbCO) analysis on the blood from bodies of burn victims was investigated. Blood samples with 0, 50, and 100% carbon monoxide (CO) saturation were heated at various temperatures for some time and then analyzed. Carboxyhemoglobin content was determined by the fourth-derivative spectrophotometric method and compared with results from the usual two-wavelength method. For total hemoglobin measurement, the fourth-derivative technique and cyanmethemoglobin method were used. Turbidity in blood samples, which occurred when samples were heated above 50 degrees C, affected the analysis. At about 70 degrees C, coagulation and hemoglobin degeneration occurred accelerating the errors of determined values. The fourth-derivative technique, however, proved to be independent of the turbidity and would be useful for the analysis on the blood without hemoglobin degeneration.

ABO genotyping by inverse PCR technique.

Inverse PCR technique was applied to type three major alleles (A(1), B and O(1)) of the ABO blood group by simultaneously detecting separated allele-determining nucleotides (the 261st base in exon 6 and the 796th and 803rd nucleotides in exon 7) of the ABO gene. A sequence of about 1.7 kb from exons 6 to 7 of each allele was amplified, both termini of the fragment ligated, and allele-typing performed by the inverse PCR-restriction fragment length polymorphism (IP-RFLP) and allele-specific inverse-PCR (ASIP) methods. For intramolecular ligation, primers for the first PCR were designed to have Acc I-restriction sites within the sequences, and both termini of the 1.7-kb fragment were digested with Acc I. Using the IP-RFLP method, the inverse PCR product was digested with Kpn I, Nla III and Dde I, A(1), B, O(1)-standard (O(A)) and O(1)-variant (O(G)) alleles were detected as 365-bp, 272-bp, 193-bp and 128-bp fragments, respectively. By the ASIP method using four allele-specific primers, 222-bp, 124-bp and 232-bp fragments were amplified from A(1), B and O(1) templates, respectively. These techniques would be applicable to detecting separated polymorphic regions of some other genes.

Effects of solvent displacement on sensitivity and specificity of monoclonal antibodies for ABO blood grouping of forensic specimens with an absorption-elution test.

Using commercially-available monoclonal antibodies (Bioclone, Neo Kokusai, Monoclonal Wako, Gamma Clone and Seraclone), ABO blood grouping of forensic specimens such as bloodstains, salivary stains, seminal stains, nails, hair and cerebral dura mater was performed with an absorption-elution test. Salivary stains, seminal stains and nails were not typed correctly using the antibodies other than Bioclone reagents, while precise grouping of bloodstains was performed using most antibodies. When hair and dura mater were tested, all of the antibodies induced weak or non-specific haemagglutination, hence correct grouping was not achieved. When the antibody solvents were displaced with 5-20% bovine serum albumin in saline, human serum of the group AB donor, or serum of chicken, sheep or bovine, titers of the reagents increased 2-8 times. Hair and dura mater were able to be typed using Bioclone reagents after solvent displacement with human AB or sheep serum, whereas displacement with the other solvents enhanced non-specific reactions.

A case of DNA analysis of ABO blood group variant allele A2.

The ABO phenotype of a bloodstain (B) on a knife that was used as a weapon in an attempted murder case was found to be different from that of the Peruvian victim's blood (AB). Serological analysis showed that the A-antigenicity was much weaker than B antigenicity, suggesting that the victim's phenotype was A(2)B or A(3)B. So, the ABO genotypes of the knife bloodstain and the victim's blood were determined by DNA analysis. The 261st G deletion, specific to the O(1) allele, was not detected in the specimens by restriction fragment length polymorphism analysis. Also, the 871st A, specific to the A(3) allele, was not found by the allele-specific amplification method. Amplified product length polymorphism and direct sequencing methods finally demonstrated that the typical B sequence was found in one allele and a single C deletion in the 1,059th-1,061st C stretch in the other allele, indicating that the ABO phenotype of the bloodstain and victim's blood were A(2)B.

Evaluation of prostate-specific antigen (PSA) membrane test for forensic examination of semen.

Prostate-specific antigen (PSA) is a glycoprotein produced by the prostatic gland and functions to liquefy the seminal fluid. Recently several PSA membrane test kits utilizing an immunochromatographic assay are commercially available for clinical screening of PSA concentration in serum. In this study, we applied the three PSA test kits to forensic examination of semen, and evaluated the sensitivity and specificity of the PSA test kits. The specificity were the same, but SMITEST PSA Card had the highest sensitivity among the three kits. Furthermore we certified that the SMITEST PSA Card was little affected by heat or contaminants such as other body fluids, reagent of preliminary test for semen stain and spermicides, and the PSA kit was applied to 30 casework samples.

Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.

The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochromogens digested by proteinase K. Alkaline and acid hematin were almost completely removed by phenol/chloroform treatment and ethanol precipitation, so as not to be copurified with DNA from the specimens. Spectrophotometric results indicated that the contaminant was likely to be the product of proteinase K digestion of some heme-blood protein complex, which was not completely extracted by organic solvents and remained in the ethanol precipitates of DNA. The results of polyacrylamide gradient gel electrophoresis and intensity of the inhibition of PCR suggested that the ligand of the contaminant was a somewhat large molecule, resistant to the proteolysis by proteinase K. The addition of bovine serum albumin to the reaction mixture prevented the inhibition of PCR by the heme compounds, probably by binding to the heme. This showed that the inhibition was not due to the irreversible inactivation of the enzyme.

Purification of forensic specimens for the polymerase chain reaction (PCR) analysis.

Purification methods of deoxyribonucleic acid (DNA) from degraded and contaminated forensic samples were investigated for polymerase chain reaction (PCR) analysis. DNA extracted from putrefied tissue or bloodstains sometimes contained the copurified contaminant, that was identified as the porphyrin compound (hematin). When contaminated but less degraded DNA was analyzed by PCR, it was necessary to eliminate the impurity by anion exchange column chromatography or chelating resin preparation, and ultrafiltration using Centricon microconcentrators. When highly degraded DNA was analyzed, trace amounts of high molecular weight DNA was recovered by electroelution method, and then further purified by both column chromatography and ultrafiltration. From thus purified samples, the amelogenin gene for sex determination could be amplified by dual PCR technique.

ABO genotyping following a single PCR amplification.

Using primers designed by Lee and Chang, 200 base-pair (bp) fragment of ABO locus was amplified by PCR, which spans the site of the single nucleotide deletion associated with O allele. O allele could be identified by Kpn I digestion of the PCR product as reported. A and B alleles were also distinguishable by Mae II digestion of the product. Thus restriction digestion by Kpn I and Mae II could genotype ABO blood group following the single amplification. The nucleotide substitution in the 200-bp product between A and B alleles was also found in O allele, resulting in 2 different suballeles OA and OG. The single-strand conformational polymorphism of the PCR product was also investigated for ABO genotyping following the single amplification.

Paternity testing: blood group systems and DNA analysis by variable number of tandem repeat markers.

Two recent paternity cases are reported. In the first case of paternity exclusion, deoxyribonucleic acid (DNA) restriction fragment length polymorphisms (RFLPs) on variable number of tandem repeat (VNTR) loci with multiple alleles were informative, as well as established systems of red blood antigens, red cell enzymes, serum proteins, and human leukocyte antigens. In the second case, in which both the alleged father and the first wife were deceased, the paternal genotype was determined by using genetic markers from the second wife and four children, which then were compared with the paternal alleles of the child in question, the plaintiff in this case. The high probability of paternity (0.999,998,7) made us conclude that the man probably was the actual father. The DNA analysis by VNTR probes appears to be quite valuable in the study of paternity cases.