Ultrasound of the axilla: where to look for the sentinel lymph node.

AIMS: The aim of this paper is to guide the radiologist to the most likely location of the sentinel lymph node (SLN). MATERIALS AND METHODS: Patients with invasive breast cancer underwent axillary ultrasound examination. The position and morphological appearances of the lymph nodes were noted and core biopsy (CB) was performed of the largest or most suspicious node. Those patients whose biopsy revealed no evidence of malignancy proceeded to a surgical sentinel lymph node (SLN) biopsy (SLNB) looking for histopathological evidence of previous CB. RESULTS: Of 121 patients who underwent axillary ultrasound and CB no malignancy was identified in 73, all of whom subsequently underwent SLNB. Histological evidence of CB in the SLN was identified in 47 (64%) patients. The position of all the lymph nodes identified on ultrasound and the 47 patients whose SLNs were identified were drawn on composite diagrams of the axilla. Of the 36 nodes identified as sentinel whose position relative to other nodes could be determined, 29 (81%) represented the lowest node identified in the axilla, four (11%) were the second lowest, and three (8%) were the third lowest node. None of the four patients whose CB was from the fourth lowest node had the CB site identified at subsequent SLNB. CONCLUSION: Ultrasound of the axilla should be carried out in a systematic fashion focusing on level I nodes paying particular attention to the lowest one or two lymph nodes.

Use of ultrasound-guided axillary node core biopsy in staging of early breast cancer.

The aim of this study was to see how effective ultrasound-guided needle biopsy was at detecting lymph node involvement in patients with early breast cancer. Patients with newly diagnosed invasive breast cancer underwent axillary ultrasound (US) where lymph node size and morphology were noted. A core biopsy (CB) was undertaken of any node greater than 5 mm in longitudinal section. Patients with benign CBs proceeded to sentinel lymph node (SLN) biopsy, whereas those with malignancy underwent axillary lymph node dissection (ALND). US and CB findings were correlated with final surgical histology in all cases. One hundred and thirty-nine patients were examined, of whom 52.5% had lymph node metastases on final histology. One hundred and twenty-one patients (87%) underwent axillary node CB. The overall sensitivity of CB for detecting lymph node metastases was 53.4% (60.3% for macrometastases; 26.7% for micrometastases). The US morphological characteristics most strongly associated with malignancy were absence of a hilum and a cortical thickness greater than 4 mm. However, one third of patients with normal lymph node morphology had nodal metastases, and only 12% of these were diagnosed on CB. CB of axillary lymph nodes can diagnose a substantial number of patients with lymph node metastases, allowing these patients to proceed directly to ALND, avoiding unnecessary SLN biopsy.

[Diagnostic image (317). A man with cervical injury after an accident ]. / Diagnose in beeld (317). Een man met halsletsel na een ongeval.

A 20-year-old man suffered a larynx rupture and mucosal laceration, and had local subcutaneous emphysema, due to a motor vehicle accident.

Rat parathyroid hormone-(1-34) fragment: renal adenylate cyclase activity and receptor binding properties in vitro.

The rat PTH molecule contains five sequence differences from either the bovine or the human hormone within the biologically active 1-34 region. A synthetic rat 1-34 peptide was tested for activity by in vitro activation of canine and rat renal adenylate cyclase and binding to canine renal membrane receptors. The mean potency of 21,400 Medical Research Council units/mg in the canine adenylate cyclase system and 24,900 in the rat system was 8- to 10-fold higher than human 1-34 and 2- to 4-fold greater than bovine 1-34. These values represent the highest potency we have observed to date for a PTH preparation by these assay systems. In contrast, receptor binding of the rat fragment was comparable to that of bovine and human 1-34. Half-maximal inhibition of radioligand binding occurred at 1.7- 2.0 X 10(-9) M with all synthetic hormones. Hence, the amino acid substitutions in rat 1-34 appear to affect the cyclase-activating sequence domain without increasing avidity for the receptor. Analogs combining the rat sequence with modifications known to enhance receptor binding and/or retard enzymatic degradation offer a promising approach to the preparation of still more potent parathyroid agonists.

Establishment of a human squamous cell carcinoma cell line of the upper aero-digestive tract.

A human squamous cell carcinoma (SCC) cell line has been established from the surgical specimen of an untreated, upper aero-digestive tract tumour, diagnosed as a squamous carcinoma, grade III, of the pyriform fossa. The tumour tissue was grown as a xenograft in an athymic nude mouse and was designated as NT-8. Histological examination of the surgical specimen and the nude mouse tumour showed that the two were identical. NT-8 was subsequently passed by subcutaneous injections into nude mice. After the 6th passage in nude mouse, the tumour was cultured in vitro where it grew as an epithelial cell line, with a typical cobblestone appearance. This cell line was designated as NT-8e. Both the primary tumour as well as xenograft and the cells in culture have retained several common morphological and biochemical characteristics. Immunological markers for epithelial cells including epithelial membrane antigen and cytokeratins were seen in all three, confirming the epithelial lineage. Characterization of the NT-8e cell line including growth parameters, anchorage-independent growth and tumorigenicity in nude mice, chromosome counts and DNA content by flow cytometry have been carried out.

Cryopreservation of human germinal vesicle stage and in vitro matured M II oocytes: influence of cryopreservation media on the survival, fertilization, and early cleavage divisions.

OBJECTIVE: To study the influence of low-sodium cryopreservation media (CPM) on the survival and development of frozen-thawed germinal vesicle (GV) stage and in vitro matured human oocytes. DESIGN: Prospective experimental study. SETTING: Academic hospital-based fertility center. PATIENT(S): Experimental groups: Oocytes cryopreserved at the GV (group A, n = 63 and group B, n = 64) or M II stage (group C, n = 62) with use of conventional (group A) or low-sodium CPM (groups B and C). Control groups: Sibling GV stage oocytes subjected to in vitro maturation (IVM; control group A, n = 64; control group B, n = 64). INTERVENTION(S): IVM, intracytoplasmic sperm injection and subsequent culture. MAIN OUTCOME MEASURE(S): Rates of survival, maturation, fertilization, and cleavage. RESULT(S): The postthaw survival was significantly lower in groups A (57.1%) and B (48.4%) compared to C (84.4%). In group A, maturation and cleavage rates were significantly lower, and fertilization rate was similar to controls (GVBD: 72.2% vs. 90.6%; progression to M II: 33.3% vs. 76.6%; cleavage: 42.9% vs. 88.2%; and fertilization: 58.3% vs. 69.4% in group A vs. control group A, respectively). There was no such difference in group B. In group C, despite a slight but significant lowering of the rate of 2 PN and an increase in that of 3 PN (2 PN: 47.4% vs. 70.2% and 3 PN: 15.8% vs. 3.2% in group C vs. total controls, respectively), embryonic cleavage per GV oocyte was significantly higher (25.8%) compared to group A (4.8%) but not to group B (15.6%). The rate of maturation and cleavage per surviving GV oocyte was significantly higher in group B than group A. CONCLUSION(S): Low-sodium-based CPM is beneficial for in vitro matured M II stage oocytes and is significantly better than the conventional sodium-based media for the GV stage oocytes.

Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity.

OBJECTIVE: To study the effect of delayed intracytoplasmic sperm injection (ICSI) on the fertilization and cleavage of human in vitro matured (IVM) oocytes. DESIGN: Prospective experimental study. SETTING: Academic hospital-based fertility center. PATIENT(S): The experimental group consisted of 73 spare germinal vesicle-stage oocytes from 25 patients. The control group consisted of sibling in vivo matured oocytes from the same patients that were subjected to ICSI in the clinical program. INTERVENTION(S): Equal numbers of sibling IVM oocytes were subjected to ICSI either soon after maturation (30 hours, group 1) or after a 6-hour delay (36 hours, group 2). In a subsequent set of experiments, spermatozoa were labeled with a fluorescent mitochondria-specific vital dye and injected into 17 IVM oocytes that were intentionally aged by 6-8 hours. The resultant zygotes were fixed. MAIN OUTCOME MEASURE(S): Incidence of fertilization and cleavage, numbers and mean diameters of pronuclei, and incidence of zygotes with significant pronucleus size asynchrony. Identification of the male pronucleus by its proximity to the fluorescent sperm midpiece remnant. RESULT(S): Group 2 had significantly lower rates of normal fertilization (60%) than the control group (82.9%) and significantly lower cleavage rates (46.7%) than both group 1 (85%) and the control group (98.1%). The incidence of oocytes that developed one pronucleus and pronucleus size asynchrony was significantly higher in group 2 (32% and 40%, respectively) than in group 1 (4% and 5%, respectively) and in the control group (4.1% and 4.4%, respectively). All the zygotes with significant pronucleus size asynchrony that developed after delayed ICSI with labeled spermatozoa showed proximity of the fluorescent sperm midpiece remnant to the smaller pronucleus. CONCLUSION(S): For IVM oocytes, the incidence of one pronucleus, pronucleus size asynchrony (possibly related to a smaller male pronucleus), and cleavage failure increase when ICSI is delayed after maturation. Thus, the timing of ICSI is critical for optimum fertilization of IVM oocytes.

Umbilical cord blood as a source of CD34 positive haematopoietic stem cells.

In this paper we have used a monoclonal antibody to CD34 an antigen expressed solely on stem cells, and stem cell colony assays to show that umbilical cord blood has nearly the same number of functional stem cells as compared to normal bone-marrow. The number of CD34+ve cells in cord blood being 2 to 2.7 per cent, whereas bone-marrow had 3 to 3.5 per cent. The multi-potent colony forming cells (CFU-GEMM) were 60 +/- 18 in cord blood per 2 x 10(5) mononuclear cells (MNCs), whereas normal bone-marrow had 70 +/- 10 per 2 x 10(5) MNCs. Enrichment of these stem cells on Percoll gradients was successful for normal bone-marrow but not for cord blood.

Enhancement of haemopoietic stem cell adherence to bone-marrow derived stroma by phyto-haemagglutinin treated leucocyte conditioned medium (PHA-LCM).

Stem cell adhesion to bone-marrow derived stroma, plays a crucial role in haemopoiesis. However, there is very little information as to the nature of the adhesion molecule. In this paper we have shown that human bone-marrow derived stroma can be established in tissue culture. This stroma is able to adhere human bone-marrow mononuclear cells including the multipotent stem cell, viz. CFU-GEMM. Their adherence increases when the stroma is treated with lymphokines in the form of PHA-treated leucocyte conditioned medium (PHA-LCM). Triton X-100 extracts of the untreated and PHA-LCM treated stroma were analysed on single dimension PAGE. It was observed that PHA-LCM treated stromal extracts showed two extra bands and an increase in the density of a band of approximately 14 kDa. Whether these changes have anything to do with the increased adhesion of stem cell is not yet known.

Pain, physical and social functioning, and quality of life in individuals with multiple hereditary exostoses in The Netherlands: a national cohort study.

BACKGROUND: This study aimed to assess pain and quality of life in a large cohort of patients with multiple hereditary exostoses. METHODS: All 322 known patients with multiple hereditary exostoses in The Netherlands were asked to participate. An age-specific questionnaire was sent to children (less than eighteen years old) and adults. The questionnaire focused on pain, daily activities, and school and/or professional situation. Adults also filled out the RAND-36 questionnaire. Results were statistically analyzed with use of the SPSS 15.0 software and with the chi-square test and multiple logistic regression. A p value of <0.05 was regarded as significant. RESULTS: Two hundred and eighty-three patients (88%), including 184 adults (65%) and ninety-nine children (35%), completed the questionnaire. Multiple hereditary exostoses resulted in various physical and social consequences. The majority of adults (119) were employed; however, thirty-three (28%) had changed jobs because of the symptoms of multiple hereditary exostoses and twenty-five (21%) required adjustments in their working environment. Of the sixty-five adults who were not employed, thirteen were medically unfit to work. Of eighty-five children attending school, forty-five (53%) experienced problems at school. The symptoms of multiple hereditary exostoses caused twenty-seven children (27%) and eighty-five adults (46%) to stop participating in sporting activities. Pain was the greatest problem, with sixty-two children (63%) and 152 adults (83%) who reported recent pain. On multivariate analysis, pain in adults was correlated most significantly with age and problems at work, and pain in children was correlated with the perception of the disease and problems at school. Adult patients with multiple hereditary exostoses had a lower quality of life than the Dutch reference groups, with lower scores on six of eight RAND-36 subscales. CONCLUSIONS: Our study confirms that multiple hereditary exostoses is a chronic disease causing a profound impact on quality of life. The results suggest that pain is not the only problem associated with multiple hereditary exostoses, as it has an extensive influence on daily activities, as well as on social and psychological well-being, causing significant disability.

Breast surgical specimen radiographs: how reliable are they?

Radiography of the excised surgical specimen following wire guided localisation of impalpable breast lesions is standard surgical practice. The aims of the study were to establish the reliability of the breast specimen radiograph (SR) in determining lesion excision and to determine whether the radiographic margin correlated with the histological margin. The clinical, imaging, SR and pathological details of 106 patients with a pre-operative diagnosis of breast cancer were retrospectively reviewed. The reliability of orientation was estimated and the appearance and distance from the mammographic abnormality to each radial margin were measured and correlated with surgical histological findings. The overall accuracy of the specimen radiograph in determining whether the mammographic lesion was present was 99%. The SR could be orientated "very reliably" or "reliably" in 80% of patients however in only 48% of patients did the closest margin on the SR correspond with the same nearest margin at final histology. A maximum measurement of 11 mm or more from the lesion to the specimen edge was associated with a 77% likelihood of having a clear final histological margin (taken as 5mm or more) and if <11 mm a 58% chance of having involved final histological margins. There was however a wide overlap in the results with patients having an apparently wide SR margin but histologically involved margins and vice versa. The SR is reliable at determining whether the target lesion has been removed. The correlation of SR margin orientation and measurement with final histological measurement is however far less reliable.

Ultrasound guided percutaneous axillary lymph node core biopsy: how often is the sentinel lymph node being biopsied?

Patients with breast cancer now frequently undergo axillary ultrasound and core biopsy (CB) in an attempt to reduce the number of unnecessary sentinel lymph node (SLN) biopsies. This study aimed to establish the frequency of successful targeting of the SLN by ultrasound guided biopsy. A total of 137 patients had axillary ultrasound of which 121 underwent CB. 73 (60%) patients proceeded to SLN after negative CB. All SLNs were examined for evidence of metastases and previous CB. Of the 73 patients, 51 had no evidence of malignancy in the SLN (true negative=70%). However nodal deposits were found in the remaining 22 patients, representing a false negative rate for CB of 30%. Overall histopathological evidence of previous CB was identified in 47 (64%) of 73 patients undergoing SLN biopsy. The reason for false negative findings in the 22 (30%) patients was failure to sample the sentinel lymph node in 10 (45%) and failure to sample the metastatic disease in the sentinel node in 11 (55%). This study suggests that both better methods of identifying the sentinel lymph node and more adequate sampling are required.

Microtubule turnover in ooplasm biopsy reflects ageing phenomena in the parent oocyte.

Oviductal oocytes retrieved from superovulated B6D2F1 mice at 13.5, 16 and 19 h after human chorionic gonadotrophin (HCG) (groups A, B and C respectively, n = 382) were micromanipulated to obtain 12-20 mum sized ooplasm biopsy fragments. Experiments were divided into three sets. Ooplasmic microtubule dynamics were studied in ooplasm biopsy specimens and parent oocytes (set 1) and ooplasm biopsy specimens (set 2), whilst zona pellucida dissolution time, cortical granule loss and spindle/chromatin morphology using confocal microscopy were also studied in parent oocytes (set 2). Oocytes withstood oocyte biopsy with a high survival rate (98.2%) and the biopsied oocytes underwent successful fertilization and development (set 3). An absolute one-to-one correlation was seen between the oocyte biopsy specimens and the parent oocytes in terms of ooplasmic microtubule dynamics (set 1), and increased ooplasmic microtubule dynamics in oocyte biopsy specimens paralleled ageing phenomena in the parent oocytes (set 2). Zona pellucida dissolution time was significantly lower in parent oocytes from group A versus groups B (P = 0.032), and C (P < 0.001). (Groups A, B, C include minimal, moderate, increased ooplasmic microtubule dynamics in oocyte biopsy specimens respectively.) Oocyte cortical granule loss and spindle/chromatin abnormalities were mainly seen in group C (P < 0.001). Oocyte biopsy can thus be applied to judge age-related changes in the parent oocytes.

Synthesis and oral hypoglycemic properties of two 4-oxo-4,5,6,7-tetrahydroindole-3-acetic acids.

Condensation of beta-acetyl-2-hydroxy-4,4-dimethyl-6-oxo-1-cyclohexene-1-propionic acid (3) with n-butyl and isobutylamines affords the title acids 4 and 5, respectively, which show good oral hypoglycemic activity in normal rats. Acids 4 and 5, are also active in streptozocin-induced diabetic rats. Results of extensive pharmacological and acute toxicity tests are reported.

Presence and dynamic redistribution of type I inositol 1,4,5-trisphosphate receptors in human oocytes and embryos during in-vitro maturation, fertilization and early cleavage divisions.

We studied the presence and distribution of the intracellular calcium channel regulating type I inositol 1,4,5-trisphosphate receptors (IP3R) in human immature and mature oocytes, pronuclear zygotes and cleaved embryos using a specific antibody. Two approaches were used: (i) fluorescence immunocytochemistry using a confocal laser scanning microscope (CLSM) and (ii) Western blotting. With confocal microscopy, the receptors were found in the oocytes, fertilized zygotes as well as cleaved embryos at all stages studied. The pattern and distribution of the receptor staining in the oocytes changed gradually from a diffuse granular patchy one at the germinal vesicle (GV) stage to a reticular and predominantly peripheral one through the metaphase I and metaphase II (MII) stages. After fertilization, the distribution changed gradually to both, peripheral and central in the zygotes and early 2-4-cell embryos and predominantly perinuclear in the 6-8-cell embryos. Furthermore, an overall increase in the staining intensity was observed from GV to MII stage oocytes and from zygotes to 6-8-cell embryos. We also studied the spatial distribution of the receptor in detail by constructing three-dimensional images from the serial optical sections obtained on the CLSM. Peculiar peripheral aggregates of receptor clusters were noted in the MII stage oocytes, zygotes and some blastomeres from early cleaved embryos. Finally, Western blots performed on the extracts of 72 in-vitro matured oocytes and 50 spare cleavage stage embryos showed positive bands at approximately 260 kDa. These findings coincide with and thus possibly represent the dynamic changes occurring in the cellular Ca2+ release systems through oocyte maturation, fertilization and early embryogenesis. Thus, type I IP3R are likely to play a role during these stages of early development in the human.

Effect of post-ovulatory age and calcium in the injection medium on the male pronucleus formation and metaphase entry following injection of human spermatozoa into golden hamster oocytes.

The occurrence of parthenogenetic activation is a major hurdle in obtaining sperm chromosome metaphases after heterospecific intracytoplasmic sperm injection (ICSI) of golden hamster oocytes with human spermatozoa. We addressed two potential contributors to parthenogenetic activation namely, post-ovulatory age of the oocyte and Ca2+ content of the injection medium. In serial experiments, hamster oocytes were retrieved at 11.5, 13, 16 and 21 h after the ovulatory dose of human chorionic gonadotrophin (HCG) and microinjected with human spermatozoa suspended alternately in a regular (1.9 mM Ca2+) or a Ca2+-free medium. A progressive decrease in the rates of male pronucleus (MPN) formation and metaphase entry and increase in the rates of parthenogenetic activation without male pronucleus occurred with increasing post-ovulatory age. The favourable influence of Ca2+-free injection medium on the mean rates of MPN and metaphase entry was restricted to the relatively older oocytes (MPN 16 h: 49.5 versus 32.3%, P< 0.008; 21 h: 22.2 versus 11.1%, P< 0.001; metaphase entry 16 h: 36.8 versus 25.1%, P< 0.02; 21 h: 13.3 versus 5.2%, P< 0.01 in the Ca2+-free and regular groups respectively). Our data confirm the increased activation sensitivity with post-ovulatory ageing and its adverse influence on the MPN formation and metaphase entry after heterospecific ICSI of hamster oocytes.

Chromatin decondensation, pronucleus formation, metaphase entry and chromosome complements of human spermatozoa after intracytoplasmic sperm injection into hamster oocytes.

Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO2) and (ii) omission of Ca2+ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set 2, n=278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca2+-free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca2+ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.