Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa/Va and automation of assay.

INTRODUCTION: Antithrombin resistance (ATR) is a novel thrombotic risk in abnormal prothrombins. A manual ATR assay using Oxyuranus scutellatus (Ox) venom as a prothrombin activator was established for detecting antithrombin-resistant prothrombin. However, this assay was limited because of Ox snake venom availability and its throughput capacity. Here, we have improved the ATR assay using bovine factors Xa and Va (FXa/Va) as prothrombin activators and have optimised assay conditions for an automated instrument (ACL TOP 500). METHODS: Diluted plasma was incubated with a prothrombin activator mix (phospholipids, CaCl2 , and bovine FXa/Va), followed by inactivation with antithrombin for 10, 20 and 30 minutes. We added a chromogenic substrate S-2238, and assessed changes in absorbance/min at 405 nm. We also adapted assay conditions for ACL TOP 500. RESULTS: Optimum conditions for FXa/Va treatment were 6.25% phospholipids, 5 mM CaCL2 , 0.01 µg/mL FXa and 0.1 µg/mL FVa. ATR assay kinetics with the FXa/Va activator was comparable with that with the Ox activator in heterozygous reconstituted plasma with the recombinant wild-type or antithrombin-resistant prothrombin. Using ACL TOP 500, optimum conditions for the FXa/Va treatment were 10.0% phospholipids, 5 mM CaCl2 , 0.02 µg/mL FXa and 0.2 µg/mL FVa. The automated ATR assay with the FXa/Va activator demonstrated good detectability for antithrombin-resistant prothrombin in plasma from a heterozygous carrier with prothrombin Yukuhashi or Belgrade. CONCLUSION: We optimised the ATR assay with the FXa/Va activator and adapted the assay for ACL TOP 500; the assay showed the ability to clearly detect antithrombin-resistant prothrombin in manual and automated procedures.

Ophthalmic artery velocimetry in normotensive and preeclamptic women with or without photophobia.

OBJECTIVE: To compare ophthalmic arterial velocimetry in normotensive and preeclamptic gravidas with and without photophobia. METHODS: Ophthalmic arteries were studied by color-flow Doppler ultrasonography in 118 normotensive pregnant women, 20 gravidas with preeclampsia and no visual symptoms, and 11 with preeclampsia, photophobia, and retinal edema. RESULTS: The ophthalmic arterial pulsatility index (PI) correlated negatively with gestational age (y = -0.01x + 1.84, r = -0.41, P<.01). Pulsatility index in preeclamptics with photophobia (0.71+/-0.17) was lowest among the three groups (P<.01) and was highest in normotensive pregnant women (1.41+/-0.21, P<.01). Mean velocity in normotensive pregnant women (0.19+/-0.05 m/second) was highest among the groups (P<.01) and was not significantly different in preeclamptic women with no visual symptoms (0.27+/-0.03 m/second) and with photophobia (0.30+/-0.02 m/second). CONCLUSION: Preeclamptic women, especially those with photophobia, have orbital vascular vasodilation or hyperperfusion, or both.

A case of Guillain-Barré syndrome developed minimal change nephrotic syndrome simultaneously.

A case who developed Guillain-Barré syndrome (GBS) and nephrotic syndrome (NS) simultaneously is reported. In this case, the onset of nephrotic change of proteinuria coincided with the development of the neurological disturbance. Analysis of renal biopsy by light, immunofluorescent and electron microscopy showed minor glomerular abnormalities. The occurrence of GBS in association with NS is rare, and moreover most cases in the previous reports had membranous nephropathy. Only 1 case had minimal change nephrotic syndrome (MCNS). To our knowledge, this is the first report of a case beneficially treated with glucocorticoids, suggesting that immune disorder may play an important role in the association between MCNS and GBS.

Transcranial assessment of maternal cerebral blood flow velocity in patients with pre-eclampsia.

BACKGROUND: To clarify the cerebral hemodynamics in pre-eclamptic pregnant women, we investigated the blood flow velocity of the cerebral arteries. METHODS: The mean blood flow velocity and pulsatility index (PI) of the middle cerebral artery (MCA) and internal carotid artery (ICA) in normal pregnant women (n = 35) and pre-eclamptic patients (n = 18) were examined transcranially using pulsed-wave Doppler technique with a 2 MHz probe. In two pre-eclamptic women with post-partum visual disturbance, we examined the mean blood flow velocity and PI of the MCA and ICA every day. RESULTS: The mean blood flow velocity of the MCA in the pre-eclamptic patients (89.7 +/- 20.5 cm/s) was significantly higher than that in the normal pregnant women (53.6 +/- 16.9 cm/s) (p < 0.05). PI of the MCA in the former group (0.67 +/- 0.13) was significantly lower than that in the latter (0.78 +/- 0.02) (p < 0.05). There was no significant difference between these two groups in these variables of the ICA. In the two patients with visual disturbance, the mean blood flow velocity of the MCA was increased before the onset of visual disturbance and decreased gradually following the disappearance of this symptom. In these patients, spasm of the MCA was confirmed by magnetic resonance angiography. CONCLUSIONS: In pre-eclamptic patients, we found increased MCA mean velocity before the onset of visual disturbance. Transcranial Doppler may be useful for the evaluation of cerebral hemodynamics and the prediction of eclampsia.

Systemic phaeohyphomycosis caused by Exophiala dermatitidis.

We report a case of systemic phaeohyphomycosis due to Exophiala dermatitidis in a 24-year-old man. At the age of 17, the patient had noticed cervical swellings. On palpation at his initial examination, more than a dozen firm lymph nodes between 1 and 5 cm in diameter were found on both sides of the neck and in both axillae. Examination of biopsy specimens of lymph nodes revealed pale brown hyphae in granulomatous lesions and Exophiala dermatitidis was isolated from mycological cultures of the local tissues. The clinical course was marked by an episode of jaundice six months later, and surgery disclosed a fist-sized inflammatory mass in the region of the head of the pancreas and the duodenum. The patient was treated by providing external biliary drainage and by the administration of antifungal agents, but two years later complained of diplopia and a slight heaviness in the head. Computed tomographic scans were made of the head, revealing multiple plum-sized masses in the brain. The patient died a year later. In Japan, 10 cases of systemic infection by this organism (including the present one) have been reported, all in patients of up to 30 years of age, with lesions appearing in the brain, lung, liver, digestive organs and lymph nodes. The prognosis is grave.

A case of polycystic kidney disease with nephrotic syndrome.

Nephrotic syndrome developed in a patient with autosomal-dominant polycystic kidney disease. Renal biopsy revealed minor glomerular abnormalities. This type of nephrotic syndrome complication in an autosomal-dominant polycystic kidney disease has only rarely been reported.

Parathyroid hormone enhances gentamicin uptake by opossum kidney cells but not by LLC-PK1 cells.

Parathyroidectomy is proposed to be protective against gentamicin nephrotoxicity in rats. In the present study, we evaluated the effects of bovine PTH(1-34) on gentamicin accumulation in cultured kidney epithelial cell lines, opossum kidney (OK) cells which possess PTH receptors and LLC-PK1 cells which are devoid of PTH receptors. Ten days after seeding, the culture medium was exchanged for medium containing 1 mM gentamicin and bovine PTH. The cell gentamicin concentration was measured by a substrate-labeled fluorescence immunoassay (TDA gentamicin kit). Gentamicin uptake was accelerated by bovine PTH in OK cells but not in LLC-PK1 cells. The enhancing effect of bovine PTH seems to be mediated by a cAMP-dependent process. The results suggest that PTH accelerates gentamicin accumulation in renal tissues and potentiates gentamicin nephrotoxicity.

Comparative effects of cyclosporine A and FK-506 on endothelin secretion by a cultured renal cell line, LLC-PK1.

Cyclosporine A (CSA) stimulated endothelin secretion by a cultured renal epithelial cell line, LLC-PK1. A less nephrotoxic immunosuppressant (FK-506) did not affect endothelin secretion. Putative endothelin converting enzyme inhibitors or specific receptor antagonists may therefore prevent CSA nephrotoxicity.

[Acute phase protein (alpha 1 AT) as a tumor marker in patients with ovarian cancer (OC)].

Serum alpha 1 AT was measured in 30 healthy women, 30 pregnant women, in 97 patients with various benign gynecologic diseases, and 38 patients with OC. The positive ratio (cut off level: 317 mg/dl) in OC patients was 78.9% (30/38), 11.3% (11/97) in patients with a benign gynecologic disease, 90% (27/30) in pregnant women, and 0% (0/30) in healthy women. Histologically 92.3% of the patients (12/13) with a serous cystadenocarcinoma, 50% (4/8) with an endometrioid carcinoma, and 77.8% (7/9) with a mucinous cystadenocarcinoma were positive, but only 5.9% of the patients with a chocolate cyst were positive. Thus it was found that alpha 1 AT is a useful tumor marker in diagnosing OC.

[Changes in CA125 concentration in ovarian cancer patients during the remission state--mean attenuating curve and the effectiveness of surgery and chemotherapy].

We have periodically monitored CA125 in 21 primary ovarian cancer patients who were clinically diagnosed as in the remission state after cytoreductive surgery and a combination chemotherapy 200 days after surgery. In this monitoring, the mean attenuating curve (exponential curve) of CA125 within 75 days after surgery was obtained, Y = 2.4831 exp (-0.11281 x 10-1X) [Y:log10 (CA125), X:days from the surgery]. According to this formula, the half life of CA125 is 11.5 days and CA125 is supposed to decrease to under 35U/ml in 42.1 days. In the patients whose preoperative CA125 was over 1,000U/ml (n = 8), the half life was 6.8 days and CA125 fell below 35U/ml within 45.8 days. In the rest of the patients, (35U/ml less than preoperative CA125 less than 1,000U/ml), these figures were 16.3 days and 38.2 days, respectively. Then in the patients whose tumors were completely resected (n = 13), the mean attenuating curve was Y = 2.3655 exp (-0.99935 x 10(-2)X), the half life was 13.6 days, and CA125 decreased to under 35U/ml in 42.7 days. After primary surgery, CA125 decreased to 50.0 +/- 54.2% of the preoperative concentration. Among 16 patients (without 5 P-MFC chemotherapy) 13 (13/16, 81.3%) showed CA125 under 35U/ml after 2 courses of chemotherapy. Thus, it became clear that CA125 decreases in the remission state of ovarian cancer.

[Clinical effect of combined chemotherapy with UFT, MMC and OK-432 in recurring or advanced cancer of the digestive organs].

The usefulness of UFTMO therapy (combined chemotherapy with UFT, MMC and OK-432) performed in 40 cases of recurring or advanced cancer of the digestive organs was investigated. According the response criteria by Koyama et al., of 40 eligible cases, the treatment was judged effective in 13, 2 CR and 11 PR cases with a response rate of 32.5%, while of the 35 complete cases, 2 CR and 9 PR cases made for 11 effective cases and a response rate of 31.4%. Side effects were observed in 58.3% of the 36 evaluated cases; of the subjective and objective side effects, however, none were serious enough to require cessation of administration, while stopping administration in the cases of abnormal laboratory findings resulted in rapid recovery. UFTMO therapy, therefore, is considered to be one of the beneficial treatments for recurring or advanced cancer of the digestive organs.