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[Hodgkin's disease--an entity?].

Since 26 years Hodgkins disease is classified according to the Rye classification into 4 types. This classification is based on morphology and has turned out to be clinically relevant. However, sometimes the classification on morphological and immunohistochemical ground can be difficult to put a special case in a defined category of the 4 types. In addition, there seems to be no sharp, or well defined borders between Hodgkin's disease and Non-Hodgkin's lymphomas, especially T-cell lymphomas. Immunophenotyping of small lymphocytes and detection of follicular dendritic cells can demonstrate typical patterns in different types of Hodgkin's disease. In all types of Hodgkin's disease there is the same amount of proliferating small T-cells present. Hodgkin cells are lymphoid cells with B- or T-cell markers. Hodgkin cells of nodular para-granuloma (lymphocyte predominant type of Hodgkin's disease) show m-RNA for one light chain in the cytoplasm which can be visualized by in situ-hybridization. A new technique called "molecular histology" is applied to Hodgkin's disease. This is a single cell PCR of immunostained cells extracted from tissue sections by a micromanipulator. This technique enables us for the first time to demonstrate light chain and heavy chain gene rearrangements in Hodgkin cells of nodular sclerosis and mixed cellularity type. Hodgkin's disease seems to be no single entity but a heterogenous group of B- and possibly T-cell lymphomas. In the B-cell types Hodgkin cells are probably pre-B and B-cells.

Chromosomes, Human, Pair 14↗

[Current application development of IR microscopy and mapping].

Basing on the development of FTIR microscope technique and infrared imaging technique, the newest FTIR microscope--AIM-8800 was made by Shimadzu Co. High level of automatic technique and accessories (diamond cell and MMS-77 micromanipulator) of AIM-8800 is briefly introduced. Infrared imaging technique (Mapping) of AIM-8800, which is very important for obtaining multidimensional infrared map, is discussed with regard to the different formats of plots. Two typical examples are introduced.

Electronic Data Processing↗

In vitro fertilisation in immunological infertility.

Fertilisation occurs through a complex and orderly sequence of changes in both the oocyte and the spermatozoon. Although mechanisms have evolved to protect spermatozoa from recognition by the immune system, there is evidence that a breakdown in protection can occur resulting in homologous or heterologous antisperm antibodies which can immobilise or agglutinate spermatozoa leading to infertility. Antisperm antibodies may affect the reproductive process by interfering with sperm survival and motility and sperm transport through cervical mucus. Fertilisation is also affected, and those oocytes that do fertilise go on to develop poorly. Antisperm antibodies appear to inhibit sperm attachment to and penetration of the zona pellucida. Immune recognition of sperm antigens, which are normally present on the surface of the embryo once fertilisation has occurred, may lead to abortion after implantation. Despite the adverse effects of antisperm antibodies on fertilisation and embryo development, acceptable pregnancy rates can be achieved with assisted reproductive techniques using sperm-washing to remove antibody and using a protein source other than female serum. Further study is necessary to understand the mechanism of immunological infertility and to evaluate newer techniques using micromanipulation of gametes to achieve fertilisation.

Antibodies↗

Cryopreservation of semen, oocytes, and embryos.

Human embryo cryopreservation is widely applied by programs of assisted reproductive technology. However, recent surveys have shown that relatively few in vitro fertilization programs in the United States substantially increase their per retrieval delivery rates through cryopreservation. While embryos may be successfully frozen and thawed at a range of developmental stages using several different cryoprotectants, the majority of in vitro fertilization programs use 1,2 propanediol to freeze early (pronuclear four-cell) embryos. Ultrarapid freezing techniques are under continued study; they have not yet been shown to offer any clinical advantage over slow freezing protocols. Although interest in oocyte cryopreservation remains high, experimental evidence shows that cryoinjury to human oocytes results in chromosome sorting defects, reduced fertilization, and compromised embryonic development. Micromanipulation of mammalian oocytes appears to have no deleterious effect on subsequent cryopreservation.

Cryopreservation↗

[Experimental branch vein occlusion: the effect of carbogen breathing on preretinal PO2].

PURPOSE: To evaluate the variations in preretinal PO2 in normal and in ischemic postexperimental branch vein occlusion (BRVO) retinal areas during normoxia, hyperoxia (100% O2) and carbogen (95% O2, 5% CO2) breathing. METHODS: Preretinal PO2 measurements were obtained in intervascular retinal areas far from the retinal vessels of 13 anesthetized miniature pigs with oxygen-sensitive microelectrodes (10 microm tip diameter) introduced through the vitreous cavity by a micromanipulator. The microelectrode tip was placed at 50 microm from the vitreoretinal interface in the preretinal vitreous. PO2 was measured continuously for 10 minutes in systemic normoxia, hyperoxia (100% O2 breathing) and carbogen (95% O2, 5% CO2) breathing. A BRVO was induced with an argon green laser, and oxygen measurements were repeated in normoxia, hyperoxia and carbogen breathing. RESULTS: In hyperoxia, preretinal PO2 remained almost constant in both normal retinas (DeltaPO2=1.33 mmHg +/- 3.39; n=13) and ischemic retinas (DeltaPO2=3.73 mmHg +/- 2.84; n=8), although systemic PaO2 significantly increased. Carbogen breathing induced a significant increase in systemic PaO2 and PaCO2. Furthermore, it significantly increased preretinal PO2: DeltaPO2=23.05 mmHg +/- 17.06 (n=12) in normal retinas, and DeltaPO2=22.54 mmHg +/- 5.96 (n=6) in ischemic retinal areas. CONCLUSIONS: Systemic hyperoxia does not increase preretinal PO2 significantly in normal and ischemic post-BRVO retinal areas of miniature pigs, as hyperoxia induces a decrease in the retinal blood flow. Carbogen breathing significantly increases preretinal PO2 in normal and in ischemic post-BRVO retinal areas. This effect is probably due to the vasodilatation of the retinal arterioles induced by the intravascular PaCO2 increase.

Animals↗

[Experiment study of infusing tPA in retinal vein for treatment of retinal vein occlusion].

OBJECTIVE: To Investigate the effectiveness and complications of injection of tissue plasminogen activator into a retinal vein for treatment of retinal vein occlusion via micropuncture of the retinal vein. METHODS: Thirty miniature pigs (one eye of each pig) were produced photodynamic thrombi of retinal vein by injecting Rose bengal (20 mg/kg) into the ear vein, and randomly divided into three groups: 1. control (n = 8), 2. infusion of sterile water (n = 8) or 3. infusion of tissue-type plasminogen activator (n = 14) in obstructed retinal vein. In the first group no micropuncture of retinal vein was performed. In the others micropuncture of retinal vein were performed by a micromanipulator that we designed and sterile water was infused in retinal vein in the second group or tissue-type plasminogen activator was infused in the third one. Fluorescent angiography and histological study were performed to document the morphological alterations with clinical observations. RESULTS: 18 eyes were successfully performed micropuncture of retinal vein in the 22 eyes (81.4%). Except stopping operation due to surgical accident, there were 7 eyes that the thrombi were lysed in 9 eyes by infusion of tissue-type plasminogen activator in obstructed retinal vein (77.8%). If the micropipette was not accurate for puncturing the vessels, the vessels' wall could be ruptured and hemorrhage appeared. If thrombi of retinal veins were lysed, no bleeding and no leakage of the infused drug were observed from the site of micropuncture after micropipette was withdrawn from the vessel. Sterile water failed to flush the clot downstream. When the thrombus failed to be dislodged, the infusion fluid would back flow into the distal retinal vein and, an exudative retinal detachment would occur. In the first group, the retina exhibited hemorrhages and edema due to the vein occluded. In the second group, sterile water resulted in exaggerated retinal edema and exudative retinal detachment. In the third group, after thrombi of retinal veins were lysed, the hemorrhages and edema of retinal tissues were reduced significantly in few days. CONCLUSIONS: Infusing thrombolytic agents into the thrombosed retinal vein is a new method for treatment of retinal vein occlusion. This surgery must performed accurately by a special design. This study demonstrated that the rate and dose of thrombolytic agents infused in retinal vein should be limited in case of injuring retina tissues.

Animals↗

[Mitochondria transfer from self-granular cells to improve embryos' quality].

OBJECTIVE: To improve embryos' quality and pregnancy rate with the method of self-mitochondria transfer. METHODS: All the 18 cases of women with repeated in vitro fertilization-embryo transfer (IVF-ET)-treatment failure or older than 37 years were treated with the long gonadotropin releasing hormone-agonist (GnRH-a) regimens. The oocytes were divided into two groups: intracytoplasmic sperm injection (ICSI) group and mitochondria transfer group if the number of the individual patients' oocytes was more than four. In mitochondria transfer group, mitochondria were prepared by different centrifugation after the granulosa cells were trimmed from the oocytes or collected from follicular fluid and homogenated. Mitochondria and immobilized sperm were injected into oocytes by micromanipulation. The fertilization rate and embryos' quality were compared. RESULTS: The fertilization rate was 74.4% in mitochondria transfer group, and 76.8% in ICSI group, with no statistical difference (P > 0.05). The good quality embryo rate in mitochondria transfer group was 59.4%, and it was 34.9% in ICSI group. The difference was significant (P < 0.05). There were 7 clinical pregnancies in the 18 cases. CONCLUSION: Self-mitochondria transfer can improve embryos' quality and pregnancy rate without influence on fertilization rate.

Adult↗

[Usefulness of ND:YAG laser in head and neck surgery].

Nd:YAG laser is widely used in surgery as well as in otolaryngology and head and neck surgery for 25 years. This type of laser is characterized by low absorption in water and haemoglobin, deep penetration to the tissue and high ability for vessels coagulation. The laser light can be guided with glassfiber and can be focussed with handpices and micromanipulators. These characteristics make Nd:YAG laser very useful surgical instrument, especially in ORL and head and neck surgery. One institution's experiences, based on 300 operations with Nd:YAG laser are presented and discussed. Main indications to Nd:YAG laser operations in our material were: malignant neoplasms of oral cavity, pharynx and larynx, papillomas and haemangiomas of mucosa of upper respiratory tract, tracheal stenoses, scars of the larynx after partial laryngectomies and snoring and sleep apnoea syndrome. In our opinion, Nd:YAG laser has high usefulness in treatment of malignant and benign head and neck neoplasms, as well as in laryngeal scars and treatment of snoring. In cases of post intubation tracheal stenoses and in cases of post strumectomy bilateral paralysis of larynx the treatment results were not satisfactory. High usefulness of Nd:YAG laser results from very good coagulation ability and wide possibility of transmission of laser light with glassfiber.

Head and Neck Neoplasms↗

[Antizonal antibodies in ovulatory cervical mucus and in serum of patients with fertility disorders].

OBJECTIVE: Detection of antizonal antibodies (aZPs) in cervical ovulatory mucus and in serum in patients with fertility failure. DESIGN: Retrospective study. SETTING: Department of Gynaecology and Obstetrics, Medical Faculty of Charles University, and Faculty Hospital, Plzen. METHODS: Detection of aZPs (passive hemagglutination test) in cervical ovulatory mucus (OM) and in serum from 105 patients with reproductive failure and in 25 fertile healthy women (control group). RESULTS: Positive levels of aZP in OM were found in 79 (75.2%) patients, in 56 of them aZP were observed in OM and in serum together, only ovulatory aZPs in 23 (21.9%), and only serum aZPs in 18 (17.1%) were found. aZPs predominate in 47 patients (of 48 women) with more than two in vitro fertilization processes, in thirty of them aZPs in serum and in OM together were observed. On the other hand, repeated spontaneous miscarriage and obesity have no connection with detection of aZPs in our patients. CONCLUSION. The increased levels of serum and OM aZPs are very often seen in patients after repeated unsuccessful IVF. This very important result in reproductive immunology means to go on the micromanipulation technics combined with immunosuppression in the next planned IVF procedure.

Adult↗

[Use of specific antibody to detect fetal erythroblasts in maternal circulation].

OBJECTIVE: To investigate the feasibility of using immunohistochemical technique to detect the presence of fetal erythroblasts in the maternal circulation for prenatal diagnosis. METHODS: Maternal blood was obtained from 30 pregnant women at 8 to 26 weeks of gestation. Nucleated red blood cells (NRBCs) were separated with Percoll using a discontinuous density gradient method, and then smeared on microscope slides using cytocentrifugation. Slides were stained with antibody against the gamma-chain of fetal hemoglobin (HbF). All positive NRBCs were collected by micromanipulator under microscopic observation, and then amplified by improved primer extension preamplification(PEP). Sex and Duchenne's musclar dystrophy (DMD) genetic diagnosis were determined from a small aliquot of the PEP reaction. RESULTS: NRBCs stained with HbF were found in all of the blood from the 30 pregnant women at 8 to 26 weeks of gestation. 17 male fetuses and 13 female fetuses were detected in the 30 cases. These results coincided with those of induced labor or amniotic fluid control, and 8 fetuses at the risk of DMD were diagnosed. CONCLUSION: This diagnostic method using immunohistochemical technique to mark fetal NRBC shows good application prospects.

Antibodies, Monoclonal↗

[DMDUse of fetal specific antibody-HbF to detect fetal erythroblasts for non-invasive prenatal diagnosis of DMD].

Maternal blood was obtained at 8-26 weeks of gestation. After discontinuous density gradient centrifugation with Percoll, HbF antibody was used to identify fetal NRBC. The precise differentiation between fetal and maternal NRBC is based on the constitutional difference between fetal and adult hemoglobin (Hb). Fetal cells appear yellow cytoplasmic staining,while adult cells colorless. NRBCs were collected by micromanipulation and whole genome amplification was performed. DMD was prenatally diagnosed by using the combination of sex determination,multiplex PCR and linkage analysis of several STR sites of dystrophin. NRBCs stained with HbF were found in all of 20 maternal blood samples with gestations, and 7 fetuses with risk of DMD were diagnosed. We concluded that HbF antibody could identify fetal NRBC efficaciously, and this is a kind of more prospective application method.

Antibodies↗

[The Erbium-Yag laser in stapes surgery].

Laser has been used for 21 years in otology. This technique was introduced to decrease the number of complications after micromanipulations within the middle ear. Continuous-wave laser and pulsed laser have clinical application. In the ENT Dept. in Poznań pulsed Er-Yag laser (Zeiss Corp.) is used in the treatment of otosclerosis. First experiences and results of treatment of 35 patients using this method were presented. In all patients recovery of hearing was observed. No damage of the inner ear (deafness or hypoacusis) was found.

Adult↗

[Characteristics of dynamic membrane based on oxygen microelectrode].

Self-forming dynamic membrane coupled bioreactor is a technology using coarse pore-sized material modules to separate solid and liquid in the activated sludge. The cake layer formed on the coarse pore-sized material, i.e. dynamic membrane, can remove turbidity efficiently and some organic pollutants. Its biological activity can be studied by microelectrode. For measurement in-situ, a small bioreactor is designed to simulate the real filtration process. A microelectrode with micromanipulator is used to measure the dissolved oxygen profile in the dynamic membrane. The results indicate that bubble emerged in the dynamic membrane during operation of bioreactor. The dissolved oxygen concentration decreased with the increasing depth, and fell to 0 at 2.0 - 2.5mm depth. The specific oxygen uptake rates of the surface part of dynamic membrane were calculated to be 34.3, 10.6 and 12.4 mg/(g x h) respectively at the 1st, 5th and 8th day of the filtration.

Bioreactors↗

Current status of in vitro fertilization and the new reproductive technologies.

The rapid pace of development and refinement of in vitro fertilization and ancillary assisted reproductive techniques warrants periodic review to keep the reader abreast of the growth in this clinical and scientific arena. Although not exhaustive, this review targets key areas of special interest and significance, including oocyte and embryo micromanipulation for assisted fertilization and genetic analysis, advances in the approach to ovarian stimulation, and examination of the scientific model afforded by ovum donation.

Female↗

Computer-aided formation of the whole-cell patch-clamp recording configuration.

The conventional patch-clamp technique requires well-trained experimenter. Few commercial automated patch-clamp systems, designed for drug development, are better suited for large-scale research then for standard electrophysiological experiments. Here we describe a state machine for automated recognition of recording states of the patch-clamp experiment. The principle of the state machine is based on evaluation of the charge carried by membrane current during specific time segments in responses to square wave voltage stimulation. The state machine may serve for generating various sound alerts, signals for automated control of other devices, assistance in micromanipulation, internal pipette pressure control, and holding potential adjustments. Algorithm of the state machine, designed to cover wide variety of cell types, was successfully tested on rat ventricular myocytes.

Algorithms↗

Treatment of snoring--with and without carbon dioxide laser.

Thirty patients with bedpartners complaining of their heavy snoring which had resulted in separate bedrooms were treated surgically. Three different methods were used, each group consisted of 10 patients. In group A conventional scalpel excision was used for uvulopalatopharyngoplasty including tonsillectomy. In groups B and C, patients with small tonsils were selected and therefore the tonsils were saved. Group B was operated with carbon dioxide laser under a microscope and using micromanipulator. The same kind of excision was used as in group A but the tonsils were saved. Group C was operated as group B but using conventional scalpel excision technique. With a starting-point of snoring score 3 preoperatively in all groups, the mean postoperative snoring score in Group A was 1.0, in Group B 0.2 and in Group C 1.1. Patients with postoperative snoring scores of 0 and 1 were regarded as successful responders to surgery. No serious side effects were noted due to treatment.

Airway Obstruction↗

[Biological and clinical aspects of in vitro fertilization].

Roughly 15% of the French population is concerned by a problem of unfertility. Assisted reproductive technique may be proposed as a way to answer to this problem. In an IVF laboratory, several procedures may be carried out to obtain embryos in vitro, including micromanipulation as intracytoplasmic sperm injection. Among a high number of embryos obtained, only few will be selected for embryo transfer. The aim of this activity is to define which embryo present the best characteristics to undergone a positive implantation. Several criteria have been identified and positively correlated with pregnancy rates, however these criteria are not absolute. Finally, the medical team is often pressed to propose a double or a triple embryos transfer, in order to maintain an acceptable pregnancy rate but exhibiting the couple to a risk of multiple pregnancy.

Embryo Culture Techniques↗

[From single disseminated tumor cells to metastasis insights from molecular genetic analyses of single cells].

The prevailing models of cancer metastasis postulate that, after a series of accumulating genetic and epigenetic changes during transformation and invasive growth, the most advanced clone within a primary tumour acquires the critical cellular phenotype enabling dissemination and metastasis. This postulate is particularly based on observations that metastases usually display more genetic changes than the primary tumour. The development of several novel techniques is now enabling experimental testing of the model: The detection of single disseminated cancer cells by epithelial specific antibodies directed against cytokeratin in mesenchymal tissues has become possible even before manifestation of metastasis. After their isolation by micromanipulation comprehensive amplification of the single cell genomes allows the application of several molecular genetic methods for further characterisation. Our first data from single cytokeratin-positive cells that were isolated from the bone marrow of breast cancer patients indicate that the model of cancer progression should be revised. Cancer cells already disseminate in an early stage of genomic development and still have to acquire the critical chromosomal aberrations needed for metastatic outgrowth and full malignant potential. These observations apparently imply consequences for the development of novel adjuvant therapies. The early diversification of primary tumours and disseminated cancer cells precludes a simple extrapolation from local to systemic disease eventually necessitating increasing efforts for the direct analysis of single disseminated cancer cells as the cellular targets of adjuvant therapies.

Bone Marrow↗