Genotyping of hemochromatosis-associated mutations in the HFE gene by PCR-RFLP and a novel reverse hybridization method.

Comparative analysis of the hemochromatosis-associated mutations C282Y, H63D and S65C in the HFE gene in 51 patients using three different methods is reported. One PCR-RFLP method was based on general primers, whereas another employed mutation-specific mismatched primers. The third method was a new PCR-based reverse hybridisation line probe assay (LiPA), comprising DNA amplification by general primers followed by a single step reverse hybridization to specific probes, immobilized on a nitrocellulose strip. Forty-eight (94%) of the 51 samples yielded identical results by all three methods. Three discrepant results were obtained, caused by polymorphisms in the primer binding region, resulting in no amplification at all or selective amplification, leading to misinterpretation of the HFE genotype by PCR-RFLP. The design of the assay and the stringency of the reaction conditions used are crucial to obtain a correct HFE genotype. PCR-LiPA offers an easy and reliable alternative to currently used conventional methods.     

Ultrastructure of preimplantation rabbit embryos exposed to visible light and room temperature

Summary Early cleavage stage embryos (day 1 p.c.) and morulae (day 3 p.c.) of rabbits were exposed to visible (standard) lighting (1600 lx) and room (standard) temperature (23°C) during a 24 h in-vitro culture. Control embryos were cultured in darkness at 37°C. Development was assessed by light and electron microscopy as well as by the cytochemical demonstration of glycogen.In day 1 and day 3 embryos standard temperature induced swelling of the SER and Golgi complex vesicles. Major changes in day 1 embryos consisted of smallish microtubules-like crystalloids, and in day 3 embryos of unusually large SER vesicles. In both embryonic ages cleavage rate and development was more retarded by standard temperature than by standard lighting. Standard lighting, however, led to distinct signs of degeneration and cell death. The mode of cell damage seemed to be different in light exposed early cleavage stages and morulae: In day 1 embryos cytoplasmic degeneration was predominant while the majority of cells in day 3 embryos died by apoptosis. Despite clear indications of cell damage, cleavage rate was not notably impaired compared with non-exposed controls. Glycogen increased during development from cleavage stages to early blastocysts. The distribution was not changed either by exposure to standard temperature nor by standard lighting.The results demonstrate that day 1 embryos were clearly more susceptible to lighting whereas day 3 embryos were more affected by temperature. The mode of damage exerted by both the physical environmental factors was different. Reduction to standard temperature interfered mainly with the organization of the cytoskeleton and intracellular transport of organelles, while exposure to standard lighting led to cell degeneration and death.     

Influence of suction cup oculopression on corneal astigmatism

In 11 (right) eyes of 11 ocularly healthy subjects an artificial stepwise intraocular pressure (IOP) elevation was applied by suction cup oculopression. The initial negative pressure in the suction cup was 80 mmHg; it was raised in steps of 40 mmHg. The median of the corneal astigmatism, measured with a Zeiss ophthalmometer, was 0.50 D before oculopression (the values of the 10th and 90th percentiles were 0.30 and 1.10 D, respectively). Corneal astigmatism increased to 2.00 (0.00–5.10) D, 2.25 (1.00–5.55) D, 2.63 (0.63–7.00) D, 3.38 (1.88–6.88) D, 3.38 (2.83–7.25) D, and 4.38 (2.85–5.63) D with 80, 120, 160, 200, 240, and 280 mmHg oculopression, respectively. The astigmatism dropped to 0.75 (0.00–2.30) D immediately after removal of the suction cup. Thus, suction cup oculopression not only influenced IOP but also ocular refraction. This is of particular importance since visual evoked potential (VEP) amplitude is to a high degree dependent on refractive changes. This fact has to be considered if changes in VEP amplitude during suction cup oculopression are used as a tolerance test in glaucoma diagnosis.     

Bewertung von Wirkstoffen für Externa unter Berücksichtigung ihrer Anwendungsgebiete Eine Auswahl

Zusammenfassung Zwischen 1988 und 1994 wurden im Bundesanzeiger (BAnz) über 40 Monographien von Arzneistoffen, die in der dermatologischen Verschreibungspraxis einen relevanten Stellenwert einnehmen, ver?ffentlicht. Diese sog. Aufbereitungsmonographien stellen neutrale Bewertungen dar, die basierend auf dem damaligen aktuellen wissenschaftlichen Erkenntnisstand von einer unabh?ngigen Expertenkommission erarbeitet wurden. In den vergangenen Jahren haben sich nur in wenigen F?llen gravierende ?nderungen in dem publizierten Erkenntnismaterial ergeben, weshalb diese „offiziellen” Bewertungen auch heute noch für den Arzt ein wichtiges Instrument bei der Entscheidung für oder gegen den Einsatz von bestimmten Therapeutika darstellen. Angesichts der Tatsache, da? die im BAnz publizierten Monographien in der ?rzteschaft nur wenig bekannt sind, wird im Rahmen dieses Beitrages eine kurze Zusammenfassung der wichtigsten Fakten für ausgew?hlte Substanzen vorgestellt. Eingegangen am 12. Juni 1997 Angenommen am 28. November 1997     

Morphological and cytochemical findings in 150 cases of T-lineage acute lymphoblastic leukaemia in adults

We evaluated the morphological findings in 150 consecutive cases of T-lineage acute lymphocytic leukaemia (T-ALL). Cytochemistry including PAS staining and acid phosphatase reaction proved of limited value for the diagnosis of ALL.
The diagnosis of acute leukaemia was easy to establish in most instances. However, in a few cases the leukaemic cells were difficult to recognize as blasts. The nuclei of such cells showed condensed chromatin and nucleoli were lacking, and was encountered particularly in thymic ALL.
Basophilic cytoplasm combined with prominent vacuolization suggestive of mature B-ALL (ALL-L3 type), was observed in 16 cases. Other features, however, such as cell size, polymorphism, chromatin structure, sparse cytoplasm or focal positivity for acid phosphatase, excluded a diagnosis of ALL-L3 in those cases.
Distinction from hybrid leukaemia was difficult in 20 cases, because of a low percentage of peroxidase-positive blasts or other features which suggested a separate myeloid leukaemia component. In nine of these the hybrid nature of the leukaemia was considered as certain on the basis of morphology. Seven cases had been diagnosed as biphenotypic with coexpression of myeloid and lymphoid markers by immunological techniques.
In conclusion, our analysis showed some serious pitfalls of the morphology in T-ALL, clearly indicating the need for immunological analysis of the leukaemic cells. However, morphology remains an essential component of the diagnostic repertoire, especially when the marrow is difficult to aspirate and in cases with equivocal immunological findings. Furthermore, recognition of a separate myeloid leukaemic component in addition to the lymphatic one requires a morphological analysis.     

Lymphatic Mapping and Sentinel Lymph Node Biopsy

The status of the regional nodal basin remains the most important prognostic indicator of survival. The current standard of care for the management of invasive breast cancer is the complete removal of the tumor, with documentation of negative margins by either mastectomy or lumpectomy, followed by complete axillary lymph node dissection. Data suggest that complete lymph node dissection (CLND) provides better local control of the disease and may actually offer a survival advantage. Lymphatic mapping and sentinel lymph node (SLN) biopsy are clearly changing this long-held paradigm and have the potential to change the standard of surgical care of the breast cancer patient. The purpose of this report is to describe the lymphatic mapping experience at the H. Lee Moffitt Cancer Center and Research Institute. From April 1994 to January 1999, 1,147 consecutive breast cancer patients were enrolled in an institutional review board-approved lymphatic mapping protocol. Lymphatic mapping was performed using Tc99m-labeled sulfur colloid and isosulfan blue dye. An SLN was defined as any blue node and/or any hot node with ex vivo radioactivity counts >/=10 times an excised non-SLN or in situ radioactivity counts >/=3 times the background counts. Lymphatic mapping was successful in identifying the SLN in 1,098 of 1,147 (95.7%) cases. In the first 186 patients, all of whom underwent CLND following SLN biopsy, one false-negative biopsy was encountered for a false-negative rate of 0.83%. The method of diagnosis (excisional versus minimally invasive) does not appear to impact on lymphatic mapping. Tumor size, however, is directly related to the probability of axillary lymph node involvement. Advances in technology and the development of minimally invasive surgical techniques have heralded a new era in surgery. Lymphatic mapping and SLN biopsy may actually prove to be a more accurate method of identifying metastases to the axilla by allowing a more focused pathologic examination of the axillary node(s) at highest risk for metastasis. With adequate training, this technique can be readily implemented as a valuable tool in the surgical treatment of breast cancer.     

Increased hsCRP is associated with higher risk of aortic valve replacement in patients with aortic stenosis

Objective To investigate relations between inflammation and aortic valve stenosis (AS) by measuring high-sensitivity C-reactive protein, at baseline (hsCRP₀) and after 1 year (hsCRP₁) and exploring associations with aortic valve replacement (AVR). Design We examined 1423 patients from the Simvastatin and Ezetimibe in Aortic Stenosis study. Results During first year of treatment, hsCRP was reduced both in patients later receiving AVR (2.3 [0.9–4.9] to 1.8 [0.8–5.4] mg/l, p?<?0.001) and not receiving AVR (1.90 [0.90–4.10] to 1.3 [0.6–2.9] mg/l, p?<?0.001). In Cox-regression analyses, hsCRP₁ predicted later AVR (HR?=?1.17, p?<?0.001) independently of hsCRP₀ (HR?=?0.96, p?=?0.33), aortic valve area (AVA) and other risk factors. A higher rate of AVR was observed in the group with high hsCRP₀ and an increase during the first year (AVR_{highCRP0CRP1inc?}=_?47.3% versus AVR_{highCRP0CRP1dec?}=_?27.5%, p?<?0.01). The prognostic benefit of a 1-year reduction in hsCRP was larger in patients with high versus low hsCRP₀ eliminating the difference in incidence of AVR between high versus low hsCRP₀ (AVR_{highCRP0CRP1dec?}=_?27.5% versus AVR_{lowCRP0CRP1dec?}=_?25.8%, p?=?0.66) in patients with reduced hsCRP during the first year. Conclusions High hsCRP₁ or an increase in hsCRP during the first year of follow-up predicted later AVR independently of AVA, age, gender and other risk factors, although no significant improvement in C-statistics was observed.