Deep venous thrombosis and pulmonary embolism after anterior cruciate ligament reconstruction: incidence, outcome, and risk factors.

AIMS: The aim of this study was to investigate the incidence, risk factors, and outcome of venous thromboembolism (VTE) following anterior cruciate ligament (ACL) reconstruction in a nationwide cohort. PATIENTS AND METHODS: All ACL reconstructions, primary and revision, that were recorded in the Swedish Knee Ligament Register (SKLR) between 2006 and 2013 were linked with data from the Swedish National Board of Health and Welfare. The incidence of VTE was determined by entries between the day of surgery until 90 days postoperatively based on diagnosis codes and the prescription of anticoagulants. Risk factors, outcome, and the use of thromboprophylaxis were analyzed. Descriptive statistics with multivariate analysis were used to describe the findings. RESULTS: The cohort consisted of 26 014 primary and revision ACL reconstructions. There were 89 deep venous thromboses (DVTs) and 12 pulmonary emboli (PEs) with a total of 95 VTEs (0.4 %). Six patients with a PE had a simultaneous DVT. The only independent risk factor for VTE was age greater than or equal to 40 years (odds ratio 2.31, 95% confidence interval 1.45 to 3.70; p < 0.001). Thromboprophylaxis was prescribed to 9461 patients (36%) and was equally distributed between those with and those without a VTE (37.9% vs 36.4%). All patient-reported outcome measures (PROMs) one and two years postoperatively were significantly lower in those with VTE. CONCLUSION: The incidence of VTE following ACL reconstruction is 0.4%, and the only significant risk factor is age. Patients with VTE had worse postoperative clinical outcome than patients without VTE. We recommend against the routine use of thromboprophylaxis, but it should be considered in older patients.

Description of a flow cytometry approach based on SYBR-14 staining for the assessment of DNA content, cell cycle analysis, and sorting of living normal and neoplastic cells.

RATIONALE: This study aimed to expand the utilization of a simplified flow cytometric approach that employing SYBR-14/PI staining into broader flow cytometry applications, including (i) measurement of the DNA content; (ii) performing cell cycle analysis on mammalian cells; and (iii) sorting of live SYBR-14-stained mammalian cells based on DNA content. MATERIAL AND METHODS: Cell lines of human origin were stained with SYBR-14 and propidium iodide (PI) and assessed by a dual-color flow cytometry. Finally, sorting of living SYBR-14-stained human cell lines was performed. RESULTS: Dual staining with SYBR-14 and PI of human cells followed by flow cytometry analysis demonstrates that in addition to quality assessment, this staining could be utilized to determinate the DNA content on mammal cells. In addition, it resolves the diploid, tetraploid, and aneuploid DNA content. Furthermore, the SYBR-14-stained mammal cells were efficiently sorted based on DNA content and live cells were obtained. All these features have not been previously described with the utilization of this staining approach. CONCLUSIONS: Results of this study demonstrate that this flow cytometric approach not only allows assessment of the viability of cells, but also the DNA content of mammal cells. In addition, this approach allows one to sort viable cells stained with SYBR-14. These findings open-up unexpected and unrestricted avenues for sorting of living mammal cells and provide significant advantages over the traditionally cumbersome sorting approaches for living cells, which demand very specialized and expensive UV light sources as well as sophisticated sorting procedures.

Metanephric adenosarcoma in a young adult: morphologic, immunophenotypic, ultrastructural, and fluorescence in situ hybridization analyses: a case report and review of the literature.

Metanephric neoplasms are uncommon renal tumors that arise in both children and adults. They may be composed of small epithelial cells or benign stroma, or both, and are termed metanephric adenoma, metanephric stromal tumor, or metanephric adenofibroma, respectively. Thus far, these tumors have been known for their benign behavior. We present the case of a 21-year-old woman who developed a neoplasm composed of a renal epithelial component identical to metanephric adenoma combined with a malignant spindle cell sarcoma. The epithelial component was positive for pankeratin AE1/3, whereas the sarcomatous component was negative for epithelial markers and positive for vimentin, CD34, and CD117. No smooth muscle differentiation was apparent in the sarcoma by immunohistochemistry or ultrastructural analysis. By fluorescent in situ hybridization analysis of the sarcomatous component there was monosomy of the X chromosome, but no apparent variation from the normal diploid pattern for chromosomes 3, 7, 12, and 17. We conclude that the spectrum of metanephric neoplasia should be expanded to include malignant stromal variants, and we propose the term "metanephric adenosarcoma" for the present case.

Do NF1 gene deletions result in a characteristic phenotype?

Neurofibromatosis-1 (NF1) is an autosomal dominant disorder with marked variability of expression. Analysis of the NF1 gene (NF1) has detected a variety of mutations without any clear correlation with phenotype. However, deletions which remove all of NF1 have been reported in a small number of patients who have minor facial abnormalities, mental retardation, learning disabilities, and early or excessive burden of cutaneous or plexiform neurofibromas. The purpose of this study was to determine whether these phenotypic traits are associated with whole gene deletions. Out of 406 of our NF1 patients, 70 patients had manifestations previously associated with gene deletions. Thirty-five of these patients from 26 families were available for study. By fluorescence in situ hybridization (FISH) analysis, 4 were found to have deletions of the entire gene, including 2 sporadic cases, 1 familial case, and 1 case where family history could not be verified. In addition, the mother of the familial case was found to be mosaic for the deletion. Our results suggest that although large NF1 deletions occur with relatively high frequency in patients with certain findings, the presence of a deletion cannot be predicted solely on the basis of clinical phenotype.

Monosomy 22 in two ovarian granulosa cell tumors.

Cytogenetic studies of ovarian sex cord stromal cell tumors, although limited in number, have found trisomy 12 to be a recurring abnormality, especially in fibromas and granulosa cell tumors (GCTs). However, recent fluorescence in situ hybridization (FISH) studies have failed to confirm a high prevalence of trisomy 12 in GCTs. We describe the karyotypic findings in one adult and one juvenile GCT. Only the juvenile GCT had an extra, abnormal chromosome 12, but both the adult and juvenile GCT had monosomy 22. In light of these findings and the data in the literature, we suggest that monosomy 22 may be important in the genesis of these relatively rare tumors.

Phenotypic, cytogenetic, and molecular studies of three patients with constitutional deletions of chromosome 5 in the region of the gene for familial adenomatous polyposis.

We have studied three patients, one with extensive polyposis of the colon, who have constitutional interstitial deletions of the long arm of chromosome 5. High-resolution banding studies indicated that the deletion in the patient with polyposis spans the region 5q21-q22, which includes APC, a gene involved in familial adenomatous polyposis and sporadic colon cancer. Molecular analysis with probes for sequences flanking APC confirmed this conclusion. The deletions in the other two patients, who are too young to have developed polyposis, had breakpoints within this region, precluding the use of cytogenetic analysis alone in making definitive predictions about their risks. Molecular studies resolved the uncertainty; in situ and quantitative Southern hybridizations of four probes for polymorphic segments revealed that one of the patients has a deletion of MCC, a gene which is approximately 150 kb proximal to APC, and two flanking markers. He is at increased risk for polyposis, while the other patient is not. The physical descriptions of these patients, in conjunction with cases in the literature, begin to allow delineation of two distinct 5q-syndromes. These studies also provide precise physical mapping data for D5S71, D5S81, D5S84, and MCC on 5q.

Somatic cell hybrid mapping of human chromosome band 5q31: a region important to hematopoiesis.

As a means of characterizing the distal long arm of chromosome 5, in particular, the region spanning 5q23-->q31, we analyzed somatic cell hybrids prepared from cells with overlapping chromosomal rearrangements. In one hybrid, the derivative chromosome 5 from a patient with acute myeloid leukemia (AML) de novo, whose bone marrow cells had a balanced translocation, t(5;7)(q31;q22), involving chromosome band 5q31, was isolated in a somatic cell hybrid (B294). In addition, we prepared somatic cell hybrids from a lymphoblastoid cell line (CC) derived from a patient who has a constitutional interstitial deletion of chromosome 5 spanning 5q23.1-->q31.1. By a combination of Southern hybridization analysis and fluorescent in situ hybridization, we constructed a map dividing 5q23-->q31 into four regions. We can assign genes to these regions and relate them to anonymous RFLP markers that have been genetically mapped.

der(3)t(3;5). Another recurring abnormality in myelodysplastic disorder.

Monosomy for chromosome 5 or a portion of the long arm is a common finding in acute nonlymphocytic leukemia (ANLL) and myelodysplastic syndrome (MDS), especially when the disorder is therapy related [1,2]. If only a portion of chromosome 5 is missing, the loss is usually accomplished by interstitial deletion of various bands, most frequently q12-14 to q31-33 [3]. Occasionally monosomy for 5q is the result of a translocation between chromosome 5 and another chromosome, with the loss of the derivative chromosome that contains 5q. A previously described unbalanced translocation involves chromosome 7: [der(5)t(5;7)(q11.2;p11.2)] and appears to be a recurring abnormality in these disorders [4]. We report here one case of therapy related MDS, one case of MDS which may be therapy related, and two cases of MDS with another "variant" 5q - abnormality, namely a derivative chromosome 3 composed of most of the short arm of chromosome 5 and the long arm of chromosome 3: [der(3)t(3;5)(?p11;?p11)].

Types of collagen in breast capsules.

Representative sections of the capsules that envelop surgically implanted silicone were analyzed for their collagen content. The specimens were biopsied (as incidental procedures) as the opportunities arose. All four classes of capsules as codified clinically and those surrounding tissue expanders are represented. The ages of the capsules ranged from 6 weeks to 13 years. Eighteen specimens were examined by slab gel electrophoresis. Type I collagen predominated in all of the electrophoretic patterns. Type III collagen was consistently present in lesser concentrations. Type V collagen was consistently present as a small fraction. There was no evident correlation between the concentrations of the component collagens and the clinically assessed class of the capsules or of those from around tissue expanders, but there was a correlation to the age of the capsules. The electrophoretic patterns of the collagens of this study are indistinguishable from the (previously reported) analyses of (cutaneous) scar.

Specific chromosomal abnormalities characterize fibrosarcomas of bovine papillomavirus type 1 transgenic mice.

In the BPV1.69 line of transgenic mice, the bovine papillomavirus type 1 genome elicits both benign dermal fibroblastic proliferation (fibromatoses) and malignant fibrosarcomas. Because these lesions arise only with time, nonviral factors appear to be involved. We have karyotyped several primary tumors as well as a series of low-passage cell lines derived both from fibromatoses and from fibrosarcomas. The fibrosarcomas, but not the preneoplastic fibromatoses, show consistent abnormalities of one or both of two chromosomes, chromosome 8 (trisomy or duplication) and chromosome 14 (monosomy or translocation). The chromosomal abnormalities are not a direct consequence of the viral integration, which we have mapped to chromosome 15 by in situ hybridization. These results suggest that transgenic mice can be used to study the role(s) of cytogenetic changes in tumorigenesis and may direct the search for genes involved in tumor progression.

Tumorigenic latency and separable stages during fibrosarcoma development in transgenic mice carrying papillomavirus genomes.

Transgenic mice have been established carrying the genomes of bovine papillomavirus type 1 (BPV-1), and human papillomaviruses types 5 and 18. Transcriptional dormancy is characteristic of all three viral genomes in transgenic mouse lines maintained for 2-4 years. Only BPV-1, which induces both dermal and epidermal pathology in its natural host, has been found to elicit abnormalities when carried in transgenic mice. The BPV-1 genome acts in these mice as a tissue specific oncogene, in that it elaborates the development of skin fibrosarcomas. Three abnormal stages are evident: two distinct and successive stages of a proliferative hyperplasia (a mild and an aggressive fibromatoses), and the solid tumors (fibrosarcomas). Analysis of tissue biopsies and of derivative cell cultures from each pathology confirms that this pathway is composed of separate stages. Notably, progression from hyperplasia to neoplasia is accompanied by specific cytogenetic changes, which appear necessary in addition to the actions of the BPV oncogenes. The reproducibility of the tumorigenic pathway induced by the BPV genome is providing inroads into the molecular genetic and biochemical mechanisms of tumor development.

Chromosomal localization of human Na+, K+-ATPase alpha- and beta-subunit genes.

Na+, K+-ATPase is a heterodimeric enzyme responsible for the active maintenance of sodium and potassium gradients across the plasma membrane. Recently, cDNAs for several tissue-specific isoforms of the larger catalytic alpha-subunit and the smaller beta-subunit have been cloned. We have hybridized rat brain and human kidney cDNA probes, as well as human genomic isoform-specific DNA fragments, to Southern filters containing panels of rodent X human somatic cell hybrid lines. The results obtained have allowed us to assign the loci for the ubiquitously expressed alpha-chain (ATP1A1) to human chromosome 1, region 1p21----cen, and for the alpha 2 isoform that predominates in neural and muscle tissues (ATP1A2) to chromosome 1, region cen----q32. A common PstI RFLP was detected with the ATP1A2 probe. The alpha 3 gene, which is expressed primarily in neural tissues (ATP1A3), was assigned to human chromosome 19. A fourth alpha gene of unknown function (alpha D) that was isolated by molecular cloning (ATP1AL1) was mapped to chromosome 13. Although evidence to date had suggested a single gene for the beta-subunit, we found hybridizing restriction fragments derived from two different human chromosomes. On the basis of knowledge of conserved linkage groups on human and murine chromosomes, we propose that the coding gene ATP 1B is located on the long arm of human chromosome 1 and that the sequence on human chromosome 4 (ATP 1BL1) is either a related gene or a pseudogene.

Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines.

We cloned and analyzed the integrated human papillomavirus type 16 (HPV-16) genomes that are present in the human cervical carcinoma cell lines SiHa and CaSki. The single HPV-16 genome in the SiHa line was cloned as a 10-kilobase (kb) HindIII fragment. Integration of the HPV-16 genome occurred at bases 3132 and 3384 with disruption of the E2 and E4 open reading frames (ORFs). An additional 52-base-pair deletion of HPV-16 sequences fused the E2 and E4 ORFs. the 5' portion of the disrupted E2 ORF terminated immediately in the contiguous human right-flanking sequences. Heteroduplex analysis of this cloned integrated viral genome with the prototype HPV-16 DNA revealed no other deletions, insertions, or rearrangements. DNA sequence analysis of the E1 ORF, however, revealed the presence of an additional guanine at nucleotide 1138, resulting in the fusion of the E1a and E1b ORFs into a single E1 ORF. Sequence analysis of the human flanking sequences revealed one-half of an Alu sequence at the left junction and a sequence highly homologous to the human O repeat in the right-flanking region. Analysis of the three most abundant BamHI clones from the CaSki line showed that these consisted of full-length, 7.9-kb HPV-16 DNA; a 6.5-kb genome resulting from a 1.4-kb deletion of the long control region; and a 10.5-kb clone generated by a 2.6-kb tandem repeat of the 3' early region. These HPV-16 genomes were arranged in the host chromosomes as head-to-tail, tandemly repeated arrays. Transcription analysis revealed expression of the HPV-16 genome in each of these two cervical carcinoma cell lines, albeit at significantly different levels. Preliminary mapping of the viral RNA with subgenomic strand-specific probes indicated that viral transcription appeared to be derived primarily from the E6 and E7 ORFs.

Human genes for U2 small nuclear RNA map to a major adenovirus 12 modification site on chromosome 17.

U2 RNA is one of the abundant, highly conserved species of small nuclear RNA (snRNA) molecules implicated in RNA processing. As is typical of mammalian snRNAs, human U1 and U2 are each encoded by a multigene family. In the human genome, defective copies of the genes (pseudogenes) far outnumber the authentic genes. The majority or all of the 35 to 100 bona fide U1 genes have at least 20 kilobases (kb) of nearly perfect 5' and 3' flanking homology in common with each other; these U1 genes are clustered loosely in chromosome band 1p36 (refs 5, 7) with intergenic distances exceeding 44 kb. In contrast, the 10 to 20 U2 genes are clustered tightly in a virtually perfect tandem array which has a strict 6-kb repeating unit. We report here the assignment, by in situ hybridization, of the U2 gene cluster to chromosome 17, bands q21-q22. Surprisingly, this region is one of three major adenovirus 12 modification sites which undergo chromosome decondensation ('uncoiling') in permissive human cells infected by highly oncogenic strains of adenovirus. The two other major modification sites, 1p36 and 1q21, coincide with the locations of U1 genes and class I U1 pseudogenes, respectively. We suggest that snRNA genes are the major targets of viral chromosome modification.

Minor Xp21 chromosome deletion in a male associated with expression of Duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and McLeod syndrome.

We are reporting a male patient who suffered from chronic granulomatous disease associated with cytochrome b-245 deficiency and McLeod red cell phenotype, Duchenne muscular dystrophy, and retinitis pigmentosa. On cytogenetic analysis, he seemed to have a very subtle interstitial deletion of part of band Xp21. Since it was impossible to know whether this material was truly deleted or inserted elsewhere in the genome, somatic cell and molecular studies were carried out. In somatic cell hybrids, the deleted X chromosome was isolated on a Chinese hamster background. Southern blot analysis with 20 single-copy probes, that had been mapped to the X short arm, led to the discovery of one (probe 754) that is missing from this patient's X chromosome and also from his total DNA. This proves that he, indeed, has a deletion rather than a balanced insertion. The results provide cytological mapping information for the X-linked phenotypes present in this patient. Furthermore, probe 754 recognizes a restriction fragment length polymorphism of high frequency that makes it the most powerful probe currently available for linkage studies with X-linked muscular dystrophy.

The pattern and clinical significance of karyotypic abnormalities in patients with idiopathic and postpolycythemic myelofibrosis.

Six of eight (75%) patients with postpolycythemic myelofibrosis (PPMF) and 11 of 20 (55%) patients with idiopathic myelofibrosis (MF), seen at the University of Chicago, had abnormal karyotypes in cells of bone marrow origin. The specific chromosomal findings and their clinical significance in these patients were analyzed. A review of the literature added the findings from abnormal karyotype studies in 10 patients with PPMF and 36 patients with MF to this series. The demonstration of an increased frequency of cytogenetic abnormalities after cytotoxic therapy in polycythemia vera (PV) implies that such therapy may have a role in the development of chromosomal changes seen in treated PV and PPMF. The cytogenetic abnormalities in MF appear to be unrelated to therapy except possibly for an association with partial or complete losses of chromosome 5 or 7. Trisomy 8 is the only finding that is more common in MF than in PPMF. Other abnormalities were more common in PPMF, particularly 20q-, loss of 7 or 7q-, and trisomy 9, and to a lesser extent trisomy 1q and 5q-. Cytogenetic abnormalities do not show a pattern that can be used to distinguish between PPMF and MF, nor are they useful in the prognosis of MF or in initial studies in PPMF. PPMF does appear to have a higher tendency toward leukemic transformation than does MF, and an evolution in karyotype appears to have serious prognostic implications in PPMF in regard to this transition.

Comparative analysis of mouse-human hybrids with rearranged chromosomes 1 by in situ hybridization and Southern blotting: high-resolution mapping of NRAS, NGFB, and AMY on human chromosome 1.

The human protooncogene NRAS and the genes for the beta-subunit of nerve growth factor (NGFB) and for amylase (AMY) have previously been assigned to the proximal short arm of chromosome 1, but their precise positions have not been unequivocally established. By in situ hybridization of DNA probes for the three genes, we have ascertained the location of complementary sequences in mouse-human somatic cell hybrids that contained translocations of chromosome 1. The results agreed with the presence or absence of the human sequences as determined by Southern blotting of hybrid cell DNA. The in situ data confirmed that the genes were present on the cytologically recognized rearranged chromosome. Compared to the autoradiographic silver grain distribution on normal human chromosome 1, our in situ results obtained with the translocation chromosomes allowed much greater precision of mapping. Both NRAS and NGFB map to band 1p22, and AMY was confirmed in band 1p21.

Chromosome replication in normal and transformed human lymphocytes.

We analyzed the late-replication patterns of human B-lymphocyte chromosomes before and after transformation by Epstein-Barr virus. There were no statistically significant differences between normal cells and transformed cells derived from the same male individual; therefore, the order of termination of chromosome replication was unchanged by transformation. We also examined the replication patterns of T lymphocytes from the same donor and found no differences between normal B and T cells.