Malignant histiocytosis and related tumors. A clinicopathologic study of 42 cases using cytological, histochemical and ultrastructural parameters

Light and electron microscopical, immunohistochemical and clinical characteristics in 42 cases of malignant neoplasms, arising from true histiocytes, are described. These were separated in a lymphoma-like subtype, called true histiocytic lymphoma (29 patients) and a disseminated variant, called malignant histiocytosis (9 patients). In addition 4 related histiocytic tumors are discussed, including 2 tumors arising from interdigitating cells. Sinus pattern and cytologic features, especially 'window' nuclei, are emphasized as diagnostic criteria. Erythrophagocytosis was not a constant finding. Electron microscopic features, presence of acid phosphatase, acid alpha-naphthylacetate esterase, lysozyme, alpha 1-antitrypsin, alpha 1-antichymotrypsin, Ia-antigen and absence of B- and T-cell markers, were important in establishing the histiocytic nature or excluding a non-histiocytic tumor. A distinct male predominance existed (male:female = 2.5:1) with a higher relapse free period in females (p = 0.032). A high number of mitotic figures appeared to be a favourable sign, p = 0.020 and 0.019, for remission rate and relapse free period respectively. The degree of cell differentiation and the immunohistochemical pattern did not show a correlation with remission and relapse free period. Extranodal involvement and the presence of short profiles of endoplasmic reticulum were prognostically unfavourable signs. True histiocytic lymphomas showed a higher remission rate (p = 0.041) and relapse-free period (p = 0.017) than malignant histiocytosis.     

非致病性分枝杆菌多糖促红系祖细胞生长的研究

使用无血清培养体系观察非致病性分枝杆菌多糖(MPS)对正常人红系祖细胞CFU—E形成的作用。MPS及MPS与rhIL—3联合呈浓度依赖性促CFU—E形成,而MPS与rhGM—CSF联合,则降低后者的CFU—E产率,结论:单—MPS以及MPS与rhIL—3联合能有效促进正常人CFU—E形成。     

Fingerprint profiles in lymphocytic vacuoles of mucopolysaccharidoses I-H,II, III-A,and III-B

Summary Fingerprint (FP) profiles in vacuolated lymphocytes of mucopolysaccharidoses I-H, II, III-A, and III-B are a numerically rare, but possibly consistent finding as they have not been seen in vacuolated lymphocytes of other non-neuronal lipofuscinosis (NCL) lysosomal diseases. Their nosologic significance is not clear, but they may be as non-specific as tubular inclusions in lymphocytes and they are identical to those FP profiles seen in juvenile NCL.     

Bone marrow transplant in a case of mucopolysaccharidosis I Scheie phenotype: skin ultrastructure before and after transplantation

Summary An 11-year-old girl with mucopolysaccharidosis I Scheie phenotype (MPS I-S) received a bone marrow transplant (BMT) from her heterozygous HLA-identical LMC-non-reactive mother. Multidisciplinary studies were carried out and results evaluated 21 months after transplantation. Herein we report the ultrastructural findings pre-and post-BMT in skin. Multidisciplinary studies are commonly used to evaluate the benefits of metabolic correction following BMT in some MPS and other inherited metabolic disorders, and changes in morphology have been described in liver and few other tissues. In this case, we elected skin, since connective tissue is universally involved in MPS and is safely and easily obtainable. Comparison of skin biopsy specimens taken before and after BMT showed a considerable change in dermal fibroblast morphology, with marked reduction in cell size and the number and size of abnormal lysosomes, thus indicating the clearance of storage. Our results demonstrate that dermal cells respond to enzyme replacement therapy in MPS I-S, with the clearance of glycosaminoglycan lysosomal accumulation in connective tissue fibroblasts, which had near-normal morphology 21 months after BMT. Therefore, the practice of skin biopsy after BMT in MPS and other metabolic disorders in which dermal cells are involved should be encouraged.Supported in part by Fondo de Investigaciones Sanitarias de la Seguridad Social Grant 89/0389. Clinical aspects of this report were presented in Munich, September 1989, at the 27th Annual Meeting of the Society for the Study of Inborn Errors of Metabolism, and also at Réunion de la Société Française de Neuropathologie held in Toulouse, July 1990     

Genotyping polymorphic microhaplotype markers through the Illumina® MiSeq platform for forensics

Microhaplotype markers are emerging forensic genetic markers that have received broad attention in forensics and may supplement existing genetic marker panels. Short tandem repeat polymorphisms (STRPs) and single nucleotide polymorphisms (SNPs) are the general genetic markers at present. Stutter and the high mutation rate of STR markers and the low polymorphism of SNP markers obstruct the solving of certain cases. Kidd proposed microhaplotype markers that encompass 2–4 SNPs. In this study, we screened microhaplotype loci through three criteria, and chose the Illumina^® MiSeq platform to sequence the new markers. A new nomenclature was proposed and Perl-based tool FLfinder was designed to genotype the microhaplotype marker. After counting the number of haplotypes in samples that were sequenced and calculating common forensic parameters, 13 loci with high polymorphism were reported. Twelve of the 13 loci had an average allele coverage ratio (ACR) of 0.72 to 0.92. Structure analysis showed that 2504 samples (1000 genome project) could be divided into 5 groupings of populations, and each one representing a continental origin. The finding indicates that microhaplotype markers could be used for individual identification and ancestry inference, and a new choice is provided for forensic practice in the future.     

Forensic Y-SNP analysis beyond SNaPshot: High-resolution Y-chromosomal haplogrouping from low quality and quantity DNA using Ion AmpliSeq and targeted massively parallel sequencing

Y-chromosomal haplogroups assigned from male-specific Y-chromosomal single nucleotide polymorphisms (Y-SNPs) allow paternal lineage identification and paternal bio-geographic ancestry inference, both being relevant in forensic genetics. However, most previously developed forensic Y-SNP tools did not provide Y haplogroup resolution on the high level needed in forensic applications, because the limited multiplex capacity of the DNA technologies used only allowed the inclusion of a relatively small number of Y-SNPs. In a proof-of-principle study, we recently demonstrated that high-resolution Y haplogrouping is feasible via two AmpliSeq PCR analyses and simultaneous massively parallel sequencing (MPS) of 530 Y-SNPs allowing the inference of 432 Y-haplogroups. With the current study, we present a largely improved Y-SNP MPS lab tool that we specifically designed for the analysis of low quality and quantity DNA often confronted with in forensic DNA analysis. Improvements include i) Y-SNP marker selection based on the “minimal reference phylogeny for the human Y chromosome” (PhyloTree Y), ii) strong increase of the number of targeted Y-SNPs allowing many more Y haplogroups to be inferred, iii) focus on short amplicon length enabling successful analysis of degraded DNA, and iv) combination of all amplicons in a single AmpliSeq PCR and simultaneous sequencing allowing single DNA aliquot use. This new MPS tool simultaneously analyses 859 Y-SNPs and allows inferring 640 Y haplogroups. Preliminary forensic developmental validation testing revealed that this tool performs highly accurate, is sensitive and robust. We also provide a revised software tool for analysing the sequencing data produced by the new MPS lab tool including final Y haplogroup assignment. We envision the tools introduced here for high-resolution Y-chromosomal haplogrouping to determine a man’s paternal lineage and/or paternal bio-geographic ancestry to become widely used in forensic Y-chromosome DNA analysis and other applications were Y haplogroup information from low quality / quantity DNA samples is required.     

Mitigating the effects of reference sequence bias in single-multiplex massively parallel sequencing of the mitochondrial DNA control region

Sequence analysis of the mitochondrial DNA (mtDNA) control region can provide forensically useful information, particularly in challenging samples where autosomal DNA profiling fails. Sub-division of the 1122-bp region into shorter PCR fragments improves data recovery, and such fragments can be analysed together via massively parallel sequencing (MPS). Here, we generate mtDNA data using the prototype PowerSeq™ Auto/Mito/Y System (Promega) MPS assay, in which a single PCR reaction amplifies ten overlapping amplicons of the control region, in a set of 101 highly diverse samples representing most major clades of the mtDNA phylogeny. The overlapping multiplex design leads to non-uniform coverage in the regions of overlap, where it is further increased by short amplicons generated alongside the intended products. Primer sequences in targeted amplification libraries are a potential source of reference sequence bias and thus should be removed, but the proprietary nature of the primers in commercial kits necessitates an alternative approach that minimises data loss: here, we introduce the bioinformatic selection of sequencing reads spanning putative primer sites (Overarching Read Enrichment Option, OREO). While OREO performs well in mitigating the effects of primer sequences at the ends of sequence reads, we still find evidence of the internalisation of primer-derived sequences by overlap extension, which may compromise the ability to call variants or to measure heteroplasmy in primer-binding regions. The commercially available PowerSeq™ CRM Nested System design prevents primer internalisation, as shown in a reanalysis of a subset of 57 samples that contain possible heteroplasmies. In combination with OREO, the CRM Nested kit mitigates reference sequence bias, allowing heteroplasmic variants to be estimated down to a 5% threshold. Provided appropriate steps are taken in data processing, single-reaction multiplex assays represent robust tools to analyse mtDNA control region variation. The OREO approach will allow users to bypass the effects of unknown primer sequences in any single-reaction tiled multiplex and eliminate primer-derived bias in overlapping amplicon sequencing studies, in both forensic and non-forensic settings.     

Synthesis of 13C‐labelled sulfated N‐acetyl‐d‐lactosamines to aid in the diagnosis of mucopolysaccharidosis diseases

Morquio A syndrome is an autosomal mucopolysaccharide storage disorder that leads to accumulation of keratan sulfate. Diagnosis of this disease can be aided by measuring the levels of keratan sulfate in the urine. This requires the liquid chromatography tandem mass spectrometry (LCMS/MS) measurement of sulfated N‐acetyl‐d ‐lactosamines in the urine after cleavage of the keratan sulfate with keratanase II. Quantification requires isotopically‐labelled internal standards. The synthesis of these ¹³C₆‐labelled standards from ¹³C₆‐galactose and N‐acetylglucosamine is described. The required protected disaccharide is prepared utilising a regioselective, high yielding β‐galactosylation of a partially protected glucosamine acceptor and an inverse addition protocol. Subsequent synthesis of the ¹³C₆‐labelled mono and disulfated N‐acetyllactosamines was achieved in five and eight steps, respectively, from this intermediate to provide internal standards for the LCMS/MS quantification of keratan sulfate in urine.     

Added sampling improves reproducibility of multipoint motor unit estimates

Motor unit number estimation (MUNE) has been used to track motor unit attrition. Studies have used the modified multiple‐point stimulation (MPS) technique, collecting three surface motor unit action potentials (sMUAPs) from 3 sites to calculate MUNE. Factoring additional sMUAPs should theoretically improve reproducibility, but the optimal number has not been defined. We evaluated the effect of increased sMUAP sampling on test–retest reproducibility of the modified MPS MUNE technique and found that MUNE reproducibility increased with additional sampling. Muscle Nerve, 2010