Revisiting the Phadia/EliA cut-off values for anticardiolipin and anti-ß2-glycoprotein I antibodies: a systematic evaluation according to the guidelines.

Background Phadia/EliA fluorescence enzyme immunoassays are widely used automated assays for anticardiolipin (aCL) and anti-ß2-glycoprotein I (aß2GPI) antibodies. To date, cut-off values for these assays have not been evaluated systematically and the evidence behind manufacturer's recommended cut-off values is not clear. Objective To determine Phadia/EliA cut-off values for antiphospholipid antibodies (aPL) according to the procedures suggested by guidelines. Methods A total of 266 blood donors (135 females and 131 males) were included. The pre-handling and analysis of the samples were performed according to the International Society on Thrombosis and Hemostasis (ISTH) guideline for solid phase aPL assays. Cut-off values and corresponding 90% confidence intervals (CI) for each antibody were established and outliers were handled according to the Clinical and Laboratory Standards Institute (CLSI) guideline for reference intervals. Samples from 377 consecutive patients, referred to our thrombophilia center with evidence of thrombosis or pregnancy morbidity were included for aPL testing. Results The in-house 99th (97.5th) percentile cut-off values were 11 (8.7), 12 (6.9) 8.5 (5.0) AU/mL for aß2GPI IgG, IgM and IgA, and 21 (13) GPL-U/mL and 41 (25) MPL-U/mL for aCL IgG and IgM, respectively. The prevalence of positive results (%) defined by these cut-off values in patients with evidence of thrombosis or pregnancy morbidity was 9.5 (12.2), 1.6 (2.9), and 7.0 (9.9), and 0.8 (3.8) for aß2GPI IgG, IgM, and aCL IgG and IgM respectively. The use of in-house 99th percentile cut-off values compared to the manufacturer suggested cut-off values resulted in 1 and 39 fewer samples for aß2GPI and aCL to be classified as positive for aPL, respectively. Conclusions We present Phadia/EliA cut-off values with 90% CI for aPL determined systematically according to the ISTH and CLSI guidelines. These values are different from values previously determined, suggesting variation of aPLs in different populations. Our findings indicate the need for each laboratory to determine/validate assay specific cut-off values for aPL.

Quantitation of the mRNA expression of the epidermal growth factor system: selective induction of heparin-binding epidermal growth factor-like growth factor and amphiregulin expression by growth factor stimulation of prostate stromal cells.

The epidermal growth factor (EGF) system is a rapidly expanding system of growth factors involved in many aspects of normal and cancerous growth. We have developed a method for the quantitation of mRNA coding for all six growth factors activating the human EGF receptor (HER-1) and for the quantitation of mRNA for the receptors HER-1 and its preferred dimerization partner, HER-2. The method is based on the generation of specific RNA standards, which are amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with the sample RNA and a set of calibrators. The resulting calibration curve is used to quantitate the unknown samples, which require only a single RT-PCR reaction. Our method has the advantage that quantitation is based on coamplification of an internal RNA standard, thereby controlling both the PCR and RT reactions. In addition, the RNA standards for all growth factors and receptors are combined in a single RT reaction, which minimizes variation and allows the quantitation of all eight mRNA species with only 0.1 microg RNA. This makes the method suitable for studies in which the supply of material is limited. The developed method has enabled us to demonstrate that prostate stromal cells in primary culture express EGF, heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, and epiregulin as well as the HER-1 and HER-2 receptors, whereas no transforming growth factor-alpha mRNA is found. Furthermore, activation of the EGF system in these cells by stimulation with HB-EGF or EGF in mitogenic doses causes a selective increase in the expression of amphiregulin and HB-EGF mRNA (more than 15-fold and 25-fold, respectively), whereas there is no increase in the expression of mRNA for the other growth factors or receptors. In accord with the increase in amphiregulin mRNA, the amount of amphiregulin peptide released from the cells is also increased. The selective induction of amphiregulin and HB-EGF by growth factor stimulation may represent a mechanism to amplify the initial growth factor signal in prostate stromal cells.

Epidermal growth factor and insulin-like growth factor I upregulate the expression of the epidermal growth factor system in rat liver.

BACKGROUND/AIM: Both epidermal growth factor and insulin-like growth factor I play a role in connection with the liver. In the present study, the possible interaction of these two growth factor systems was studied by investigating the effect of epidermal growth factor or insulin-like growth factor I treatment on the expression of the epidermal growth factor receptor, and its activating ligands, transforming growth factor-alpha and epidermal growth factor. METHODS: Fifty-five male rats received no treatment, human recombinant epidermal growth factor or human recombinant insulin-like growth factor I for either 3 or 7 days. The amount of epidermal growth factor receptor, transforming growth factor-alpha, and epidermal growth factor mRNA was quantitated by a calibrated user-friendly RT-PCR assay (CURT-PCR), and the expression of transforming growth factor-alpha and epidermal growth factor peptides was quantitated by ELISA. RESULTS: Control liver (n=16) contained a mean (+/-SD) value of 12.7+/-7.4x10(-18) mol epidermal growth factor receptor mRNA, 3.8+/-2.0x10(-18) mol transforming growth factor-alpha mRNA and 0.8+/-0.4x10(-18) mol epidermal growth factor mRNA per microg total RNA and 9.8+/-1.6 fmol/mg protein epidermal growth factor and 144+/-22 fmol/mg protein transforming growth factor-alpha. Both epidermal growth factor and insulin-like growth factor I treatment increased the expression of mRNA for transforming growth factor-alpha and epidermal growth factor receptor, as well as the expression of transforming growth factor-alpha peptide. The level of epidermal growth factor receptor and transforming growth factor-alpha mRNA expression was found to correlate both in control and growth factor-treated animals, whereas the expression of epidermal growth factor receptor and epidermal growth factor showed no correlation. Marked differences were seen upon activation of the two growth factor systems, as epidermal growth factor, but not insulin-like growth factor I treatment, increased the plasma concentration of urea and decreased the concentration of insulin-like growth factor I and the liver enzymes, alanine aminotransferase and alkaline phosphatase. CONCLUSION: Our results show that epidermal growth factor and insulin-like growth factor I, which belong to two different growth factor systems, both induce a correlated upregulation of transforming growth factor-alpha and epidermal growth factor receptor mRNA in rat liver. Although marked differences were observed after treatment with either epidermal growth factor or insulin-like growth factor I on the liver as reflected in the plasma concentrations of e.g. liver enzymes, a common motif in their action involves an upregulation of the expression of the epidermal growth factor system.

An alternative parameter for monitoring the therapeutic benefits of allopurinol simultaneously in renal ischaemia-reperfusion injury: MDA/ATP Ratio.

The therapeutic benefits of allopurinol pretreatment in renal ischaemia-reperfusion injury were investigated by monitoring renal malondialdehyde (MDA) and ATP levels together with calculated MDA/ATP ratio in ischaemic (45 min) and reperfused (15 min) rat kidneys. MDA levels remained unchanged during ischaemia, but increased after the subsequent reperfusion. ATP content of the ischaemic kidney was decreased significantly and the recovery of ATP was incomplete after the reperfusion, whereas the MDA/ATP ratio increased at both periods. Allopurinol pretreatment (40 mg kg(-1) iv) maintained higher ATP levels during the ischaemia and inhibited the MDA formation during the reperfusion and decreased the MDA/ATP ratio at both periods. Our findings demonstrate that allopurinol exerts a biphasic protective action by preserving tissue ATP and by inhibiting lipid peroxidation during ischaemia and the reperfusion period, respectively. These findings suggest the selective involvement of two protective mechanisms in the different periods of renal ischaemia-reperfusion injury. The MDA/ATP ratio could be a useful parameter for monitoring these protective actions of allopurinol simultaneously.

Selenium levels and glutathione peroxidase activities in patients with acute myocardial infarction.

OBJECTIVE: Selenium (Se) is part of the enzyme glutathione peroxidase (GSH-Px) that plays an important role in the antioxidant defence of the body, including the myocardium, against the deleterious actions of free radicals and lipid peroxides. In order to evaluate the Se status and the GSH-Px activity in ischaemic heart disease, plasma, erythrocyte and urinary Se concentrations together with plasma and erythrocyte GSH-Px activities were determined in 27 patients diagnosed as acute myocardial infarction (AMI). The control group consisted of 24 age-matched healthy individuals. METHODS AND RESULTS: Fasting blood and urine samples were collected within 24 hours after the onset of chest pain. Mean plasma, erythrocyte and urine Se concentrations were significantly lower in the patient groups (63.7 +/- 12 micrograms/l, 0.48 +/- 0.04 microgram/g Hb and 49.6 +/- 27.7 micrograms/g creatinine, respectively), compared to controls (82.2 +/- 14.6 micrograms/l, 0.51 +/- 0.03 microgram/g Hb and 93.4 +/- 62.6 micrograms/g creatinine, p < 0.001, p < 0.02 and p < 0.003, respectively). No statistically significant difference was found between mean plasma GSH-Px activity in patients (0.36 +/- 0.1 U/ml) and controls (0.35 +/- 0.09 U/ml), whereas erythrocyte GSH-Px activity was higher in patients (48.1 +/- 10.2 U/g Hb) than in the controls (35.3 +/- 9.1 U/g Hb, p < 0.001). CONCLUSION: Our findings confirm the previous studies and demonstrate that patients suffering from AMI exhibit lower plasma, erythrocyte and urinary Se than the controls. Since the erythrocyte Se level represents a measure of the Se status over a period of several weeks due to its long biological half-life, low Se levels observed in the patient group might have been present before the acute event, thereby suggesting an aetiologic relevance. The presence of increased erythrocyte GSH-Px activity in these patients may be interpreted as an antioxidant defence against the chronic oxidant stress present before the AMI, presumably due to the process of coronary atherosclerosis.

The beneficial effect of 2'-deoxycoformycin in renal ischemia-reperfusion is mediated both by preservation of tissue ATP and inhibition of lipid peroxidation.

Renal ischemia injures the renal tubular cell by disrupting the vital cellular metabolic machinery. Further cell damage is caused when the blood flow is restored by oxygen free radicals that are generated from xanthine oxidase. Oxygen radicals cause lipid peroxidation of cell and organelle membranes, disrupting the structural integrity and capacity for cell transport and energy metabolism. In the present study, the possible therapeutic usefulness of the adenosine deaminase inhibitor, 2'-deoxycoformycin (DCF), during renal ischemia and reperfusion injury was investigated. The effects of DCF on renal malondialdehyde (MDA) and ATP levels were studied after 45 min ischemia and 15 min subsequent reperfusion in rat kidneys. MDA levels remained unchanged during ischemia, but increased after the subsequent reperfusion. DCF pretreatment (2.0 mg/kg i.m.) decreased MDA and increased ATP levels during the ischemia-reperfusion period. DCF exerts a dual protective action by facilitating purine salvage for ATP synthesis and inhibiting oxygen radical-induced lipid peroxidation. These results suggest that DCF therapy could be beneficial in the treatment of ischemia-reperfusion renal injuries.

Serum fructosamine and fructosamine-albumin ratio as screening tests for gestational diabetes mellitus.

To evaluate the role of fructosamine/albumin ratio as an alternative screening parameter for gestational diabetes mellitus (GDM), serum fructosamine, albumin, protein, fructosamine/albumin ratio, and oral glucose tolerance were measured in 56 non-pregnant control healthy subjects, and in 96 pregnant women who screened positive after a 50 g glucose challenge-test. Oral glucose tolerance test (OGTT) identified 12 of 96 pregnant women as having GDM. Fructosamine concentration of 1.98 +/- 0.32 mmol/L (mean +/- SD) and fructosamine/albumin ratio of 47 +/- 10 mumol/g (mean +/- SD) has been obtained in nonpregnant control subjects. During the second trimester a lower fructosamine level (1.84 +/- 0.29 mmol/L, p < 0.05) and a higher fructosamine/albumin ratio (62 +/- 15 mumol/g, p < 0.001) occurs in pregnant women, when compared to non-pregnant healthy control subjects, most likely due to the low serum albumin concentration (30 +/- 6 g/L). The serum fructosamine levels and fructosamine/albumin ratio were only slightly higher in the pregnant women with GDM than in normal pregnant women (2.05 +/- 0.47 mmol/L versus 1.84 +/- 0.29 mmol/L, 67 +/- 16 mumol/g versus 62 +/- 15 mumol/g, respectively) but the differences were not statistically significant. The fructosamine and fructosamine/albumin ratio values for normal and GDM groups overlapped considerably. Sensitivity, specificity, positive predictive and negative predictive values for fructosamine were 41.7%, 85.7%, 29.4% and 91%, and for fructosamine/albumin ratio 25%, 79.8%, 15% and 88% respectively. This suggests that both fructosamine and fructosamine/albumin ratio have low sensitivity as predictors of GDM and can therefore not be used as screening tests.

Calibrated user-friendly reverse transcriptase-PCR assay: quantitation of epidermal growth factor receptor mRNA.

We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CURT-PCR, a calibration curve was developed by plotting the ratio between the amount of PCR product originating from the calibrator and the RNA internal standard vs the amount of EGFR mRNA present in the calibrator. A fixed amount of RNA internal standard was coamplified with the EGFR mRNA present in the calibrator or in the sample, using the same primer set. The primers were chosen in regions of the EGFR mRNA that show 100% homology between human, rat, and mouse. The amount of EGFR in the unknown samples was calculated from the calibration curve based on the ratio between PCR product originating from the sample and the corresponding RNA internal standard. Competitive RT-PCR and CURT-PCR were used for rat liver samples from 21 different animals. Comparable results were obtained by the two methods. The imprecision of the CURT-PCR method was 8% (n = 20), and the imprecision of the traditional competitive RT-PCR was 16% (n = 17). We conclude that the CURT-PCR method developed is suitable for routine applications such as quantitation of EGFR expression in tumor biopsies. The imprecision is relatively low. Furthermore, the use of a calibration curve makes it possible to analyze a large number of samples in one analytical run and to accept or reject the results according to existing rules for quality assurance.

Influence of pulsed electromagnetic fields on healing of experimental colonic anastomosis.

PURPOSE: The study investigated the influence of pulsed electromagnetic fields (PEMFs) on the mechanical strength and collagen content of uncomplicated colonic anastomosis in rats. METHODS: A standardized left colonic resection was performed 3 cm above the peritoneal reflection, and end-to-end anastomosis was constructed with eight interrupted inverting sutures. Beginning immediately after surgery, randomly assigned groups were exposed to one of the following: 1) 100 Hz (frequency), 1 mT (intensity) PEMFs with 16-hour on/8-hour off cycles (n = 8); 2) 100 Hz, 2 mT PEMFs with 16-hour on/8-hour off cycles (n = 8); 3) 100 Hz, 1 mT PEMFs with 6-hour on/6-hour off cycles (n = 6), whereas the control group (n = 10) received no PEMFs. Relaparatomy was performed at 72 hours postoperatively, and the bursting pressure of the anastomotic segment was recorded in situ. The hydroxyproline contents of the anastomotic and adjacent perianastomotic segments of equal lengths were determined. RESULTS: Mean bursting pressure values of the groups that received 100 Hz, 1 or 2 mT PEMFs with 16-hour on/8-hour off cycles (90.88 +/- 19.13 and 83.88 +/- 7.08 mmHg, respectively) were significantly higher than those of the control group (61.66 +/- 10.6 mmHg) and the group with 6-hour on/6-hour off cycles (64.83 +/- 7.36 mmHg; P < 0.05 for all comparisons). Hydroxyproline contents of the anastomotic and perianastomotic segments were consistently higher in the 16-hour on/8-hour off PEMF groups, compared with those of the corresponding segments of the control group. CONCLUSIONS: PEMFs applied externally to unrestrained rats within a "window of PEMF parameters" provided a significant gain in the mechanical strength of the colonic anastomosis, at least 72 hours post-operatively. Associated relative increases in the hydroxyproline contents of the (peri)anastomotic colonic segments suggest that an altered collagen metabolism might contribute to this enhancement of the anastomotic repair. Further investigations based on these preliminary data and the definition of the exact measures regarding the effects of PEMFs on biologic systems, in general, may lead to an efficient and new adjunctive modality in colorectal surgery.