Utility of plasma fluorometric emission scanning for diagnosis of the first 2 cases reports of variegate porphyria: a very rare type of porphyrias in Thai.

Two Thai women who are siblings presented with a history of recurrent pruritic vesicles on dorsum of both hands and extensor surface of forearms where the sun-exposed areas are. The excoriated vesicles were healed with depressed scars. They had no previous history of intense abdominal pain, seizure, or psychiatric disorder Urinary porphyrins were analyzed by High Performance Liquid Chromatography (HPLC). The level of coproporphyrin III was detected to be higher than the uroporphyrin level. Fluorescence emission scanning of both patients' plasma was performed and demonstrated typical emission peak at 626 nm, that confirmed the diagnosis of variegate porphyria.

Porphyrias diagnosed types by HPLC analysis: 10 years of Siriraj experience.

Objective: This study aims to report the HPLC patterns of urine porphyrin intermediates from Thai patients with various types of porphyrias in supporting the clinical diagnosis. Methods: A reverse phase HPLC method using kit reagents, was used to measure porphyrin intermediates in the urine of control subjects and patients with various types of porphyrias. Results: 22 control subjects showed very low levels of all urine porphyrin intermediates whereas 11 porphyriatic patients had increases of some specific isomers varying among each type of the disease. The results from 6 porphyria cutanea tarda (PCT) patients: marked increase of uroporphyrin and slight increase of the other porphyrin intermediates, 2 congenital erythropoietic porphyria (CEP), high elevation of uroporphyrin and coproporphyrin I – III ratio with slight increase of pentaporphyrin, 2 variegate porphyria (VP), marked increase of only coproporphyrin III and 1 acute intermittent porphyria (AIP) (non acute form), high increase of coproporphyrinIII with mild increase of ALA, PBG, uroporphyrin, and coproporphyrin I. Conclusion: The HPLC could provide data essential for differentiating common types of porphyrias in Thai patients, PCT, CEP, VP and AIP. Clinical findings of the patients and urine screening test for increased porphyrins were also helpful for the definite diagnosis.

Microalbuminuria analysis in Thai patients with diabetes and hypertension using albumin blue 580 fluorescence assay.

Objective: To evaluate the performance of the fluorescence assay using albumin blue 580 for microalbuminuria, which is one of the early signs of renal diseases and an important cardiovascular risk factor for patients with diabetes and hypertension. Methods: The fluorescence assay was tested for its precision and reliability by determining the intraassay and interassay coefficients of variation (CV). The correlation of the assay with the standard immunoturbidimetric assay (DCA 2000ฎ microalbumin/creatinine reagent kit), which is one of the methods routinely used for microalbuminuria, was evaluated by quantitating the urinary albumin levels in 13 urine samples by both methods and the results were compared. The fluorescence assay was also used to detect the presence of microalbuminuria in 11 healthy subject, 11 patients with hypertension, and 10 patients with diabetes and hypertension. Results: At the albumin concentrations of 5, 50, and 150 mg/L, the intraassay CVs of the fluorescence assay were 7.9, 4.4, and 3.5%, while the interassay CVs were 4.1, 8.0, and 0.4%, respectively. The fluorescence assay also showed a very good correlation with the standard immunoturbidimetric assay, with the intraclass correlation coefficient of 0.94 (0.81 to 0.98 at 95% confidence interval). When the assay was used to detect the presence of microalbuminuria (the excretion of 30-300 ตg albumin/mg creatinine), it identified two out of 11 patients with hypertension (18%) and three out of 10 patients with both diabetes and hypertension (30%) having microalbuminuria whereas none of the healthy subjects had the condition. In addition, the presence of clinical albuminuria (the excretion of more than 300 ตg albumin/mg creatinine) could also be identified in three patients with hypertension (27%) and one patient with both diabetes and hypertension (10%) respectively. Conclusion: The fluorescence assay using albumin blue 580 was found to be precise and reliable and also showed a very good correlation with the standard immunoturbidimetric assay. In addition, the fluorescence assay is simple and the assay cost is much cheaper compared with the immunoturbidimetric measurement. Therefore, it could be another alternative method for microalbuminuria, particularly for most hypertensive or diabetic patients in Thailand, who can benefit from the detection of microalbuminuria but cannot afford regular tests.

Comparative analysis of serum ceruloplasmin levels in wilson disease by conventional enzymatic assay and immunologic method.

Objective: Serum ceruloplasmin, which has markedly decreased in 95% of patients with Wilson disease, is one of the most useful markers in the diagnosis of this genetic disease. The disease is caused by an impairment of the excretion of hepatic copper, resulting in toxic accumulation of the metal in the brain, liver and other organs. Definite diagnosis leads to the need of continual, lifelong and effective treatment. Therefore, the accuracy of the measurement of this serum protein is clinically needed. Our study is aimed to compare the reliability of the two methods used in measuring serum ceruloplasmin: the conventional enzymatic assay and the recent immunologic method by using kit reagents. Methods: Serum ceruloplasmin levels were performed by the conventional enzymatic assay as reported by Ravin in 1961, and compared to the immunologic method using kit reagents, Dade Behring Inc., Newark, USA. Seven patients with clinically proven Wilson disease and twenty-two controls were recruited for the study. Results: The mean  SD levels of serum ceruloplasmin from all patients and controls as measured by the enzymatic assay were 1.58  2.28 mg/dl and 28.94  9.60 mg/dl, respectively. The serum levels from those patients measured by the kit assay were less than 8 mg/dl while the mean  SD of controls were 25.91  7.71 mg/dl. All serum ceruloplasmin levels after measurement by both assays showed a strong correlation coefficient (r = 0.8713; p-value < 0.01), with a significant decrease in all patients with Wilson disease when compared to controls. Conclusion: Our study supported the high correlation between the conventional enzymatic assay and the recent immunologic method in measuring serum ceruloplasmin. Although the analysis kit is expensive, it is more advantageous for routine laboratory service because of its simpler, automated test with a well-accepted quality control.

Comparative study of two screening tests for urinary porphyrins in the diagnosis of porphyrias.

Objective: To evaluate the efficiency of two urinary porphyrins screening tests: routine fluorescent detection and semi quantitative spectrophotometric scanning. Methods: Minimal-level detection was performed by adding standard coproporphyrin of 0, 25, 50, 100, 250 and 500 ตg/L in urine and then screened by the two methods. Urine samples from 39 controls, 7 patients with porphyrias and 20 patients with liver impairment were quantitated for total porphyrins, followed by a comparison of the two qualitative tests. Results: The fluorescent test detected the minimal porphyrin level at 250 ตg/L whereas spectrophotometric scanning could detect a lower level, at 100 ตg/L. Total control subjects showed negative results from both tests while all 7 patients with porphyrias and 6 out of 20 cases of liver impairment showed definite positive results. Conclusion: Urinary screening for porphyrins from both tests revealed the same accuracy from this study. Still, the spectrophotometric method which is simpler, more sensitive and easily interpretable seemed more practical as a screening test in general laboratories. Keywords: Screening test; Porphyrias; Urinary porphyrins

Cytotoxicity and apoptosis of ovarian and breast cancer cell lines induced by OVS1 monoclonal antibody and paclitaxel.

OVS1 monoclonal antibody (MAb) produced against ovarian cancer is currently used to identify mucinous cystadenocarcinoma antigen as a tumor marker secreted in serum. The potential of OVS1 MAb in ovarian cancer treatment was studied by evaluating the induction of cytotoxicity and apoptosis of SKOV3 ovarian cancer and BT549 breast cancer cell lines induced by OVS1. Paclitaxel, an antitumor drug, was used as positive control and applied as a combined drug together with OVS1 MAb. OVS1 MAb and paclitaxel were found by MTT assay to induce cytotoxicity against both cell lines. The ED50 of OVS1 MAb were 26.25 and 25.00 microg/ml and of paclitaxel were 21.88 and 9.20 nM against SKOV3 and BT549 cell lines, respectively. The quantitative amount of cells determined by fluorimetric assay was correlated to the results of the MTT assay. The combined application of OVS1 MAb and paclitaxel on these two cell lines resulted in a greater cytotoxicity than observed by either agent alone. OVS1 MAb and paclitaxel applied against both cell lines induced the morphological changes of apoptotic cell death at 24 hours visualized by two color fluorescence dyes, Ho33342 and propidium iodide. Combination of the two substances enhanced the rate of apoptosis compared to either OVS1 MAb or paclitaxel given alone. DNA fragmentation was detected in an agarose gel electrophoresis after treating cells with OVS1 MAb and paclitaxel at 24 hours. These findings on the induction of cytotoxicity and apoptosis by OVS1 MAb on cancer cell lines have implications on the potential application of OVS1 MAb for clinical therapy.

Comparative analysis of venous blood lactate levels by automatic lactate strip kit (accusport) and by standard marbach’s enzymatic method.

Lactic acidosis is an emerging life-threatening condition that needs to be diagnosed and treated as early as possible. The complete analysis of blood lactate levels by standard method takes at least a few hours and is not available at all times. The automatic lactate strip kit (Accusport) will be more practical for diagnosis and treatment of lactic acidosis patients. This study showed the results of blood lactate determined by standard enzymatic method of Marbach compared to the lactate strip. The results showed a strong correlation between the two methods (r2=0.966). The correlation increased in the case of high blood lactate levels (r2=0.978) and decreased in normal blood lactate levels (r2=0.943). Overall lactate values measured from the strip method were lower than those from the enzymatic method. From this study we can calculate a constant factor of 0.981 which when multiplied with the value of blood lactate analysed by lactate strip then added with 0.532, the result will be equal to that from Marbach’s enzymatic method.