The composition of human preimplantation embryo culture media and their stability during storage and culture.

STUDY QUESTION: What is the composition and stability during storage and culture of fifteen commercially available human preimplantation embryo culture media? SUMMARY ANSWER: No two culture media had the same composition, and both storage and culture had an effect on the concentrations of multiple components. WHAT IS KNOWN ALREADY: The choice of embryo culture medium not only affects the success rate of an IVF treatment, but also affects the health of the future child. Exact formulations of embryo culture media are often not disclosed by manufacturers. It is unknown whether the composition of these media changes during storage or culture in the IVF laboratory. Without details on the exact concentrations, it is not possible to determine which components might be responsible for the differences in IVF success rates and health of the resulting children. STUDY DESIGN, SIZE, DURATION: Between October 2014 and October 2015, all complete human preimplantation embryo culture media, i.e. ready to use for IVF, that were commercially available at that time, were included (n = 15). Osmolality and the concentration of thirty seven components including basic elements, metabolites, immunoglobulins, albumin, proteins and 21 amino acids were tested immediately upon arrival into the IVF laboratory, after three days of culture without embryos (sham culture) starting from the day of arrival, just before the expiry date, and after three days of sham culture just before the expiry date. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ions, glucose, immunoglobulins, albumin and the total amount of proteins were quantified using a combination of ion selective electrodes and photometric analysis modules, and lactate, pyruvate and 21 amino acids were analysed by ultra performance liquid chromatography mass spectrometry. Osmolality was analysed by an advanced micro-osmometer. Statistical analysis was done using multivariate general linear models. MAIN RESULTS AND THE ROLE OF CHANCE: The composition varied between media, no two media had the same concentration of components. Storage led to significant changes in 17 of the 37 analyzed components (magnesium, chloride, phosphate, albumin, total amount of proteins, tyrosine, tryptophan, alanine, methionine, glycine, leucine, glutamine, asparagine, arginine, serine, proline, and threonine). Storage affected the osmolality in 3 of the 15 media, but for all media combined this effect was not significant (p = 0.08). Sham culture of the analyzed media had a significant effect on the concentrations of 13 of the 37 analyzed components (calcium, phosphate, albumin, total amount of proteins, tyrosine, alanine, methionine, glycine, leucine, asparagine, arginine, proline, and histidine). Sham culture significantly affected the osmolality of the analysed culture media. Two media contained 50% D-lactate, which a toxic dead-end metabolite. In a secondary analysis we detected human liver enzymes in more than half of the complete culture media. LIMITATIONS, REASONS FOR CAUTION: The analyzed culture media could contain components that are not among the 37 components that were analyzed in this study. The clinical relevance of the varying concentrations is yet to be determined. WIDER IMPLICATIONS OF THE FINDINGS: The presence of D-lactate could be avoided and the finding of human liver enzymes was surprising. The wide variation between culture media shows that the optimal composition is still unknown. This warrants further research as the importance of embryo culture media on the efficacy and safety in IVF is evident. Companies are urged to fully disclose the composition of their culture media, and provide clinical evidence supporting the composition or future changes thereof. STUDY FUNDING/COMPETING INTEREST(S): None.

Improved assay protocol for measurement of ammonia on the Roche Cobas 8000 automated platform.

INTRODUCTION: Ammonia is a metabolite of protein catabolism that, when elevated, may be toxic for tissues, especially for the central nervous system. Elevated ammonia in blood is an indicator and a prognostic factor for hepatic and kidney disease or inherited metabolic disorders in nitrogen metabolism. The accuracy of ammonia determination is influenced by sampling condition, handling, storage and assay itself. Our and other laboratories have been experiencing high frequencies sample error flags while measuring ammonia with glutamate dehydrogenase method on Roche Cobas 8000 platform. To reduce the number of error flags we adapted Roche NH3L protocol by incorporation of an additional onboard routine step for sample pre-dilution. MATERIAL AND METHODS: The AMC NH3L is an adaptation of Roche protocol that uses four fold pre-dilution of the sample in the rerun prior to the analysis. It was assessed for 1.occurrence of absorbance error flags, 2.precision, 3.correlation with Roche method and 4.interference by hemolysis, icterus and lipemia. RESULTS: The AMC NH3L adaptation demonstrates acceptable within-run and total precision. Comparison studies show no differences between the Roche rerun application and AMC NH3L adaptation. The AMC NH3L adaptation solves 78% of absorbance errors and for samples with high ammonia concentration is less affected by interferences from icterus and hemolysis than the Roche rerun application. CONCLUSION: The AMC NH3L adaptation is less prone to instrument error flags and for samples with high ammonia concentration, is more robust to endogenous interferences. The AMC NH3L adaptation is viable alternative to the Roche protocol for the ammonia measurement.

High levels of soluble MICA are significantly related to increased disease-free and disease-specific survival in patients with cervical adenocarcinoma.

Downregulation of major histocompatibility complex class I chain-related molecule A (MICA) and upregulation of human leukocyte antigen G (HLA-G) on the tumor cells are important immune escape mechanisms for different epithelial tumors. In addition, upregulation of the soluble forms of the latter molecules in serum leads to peripheral T-cell and natural killer (NK)-cell tolerance. As for cervical cancer, it remains unknown whether soluble MICA (sMICA) and soluble HLA-G (sHLA-G) concentrations are related to tumor characteristics or patient survival rates. We measured sMICA and sHLA-G in pre-treatment sera of a large cohort of cervical cancer patients (n = 366) by enzyme-linked immunosorbent assay (ELISA). We detected a median sMICA of 174.73 pg/ml and a median sHLA-G of 5.35 U/ml. We did not find an association between sHLA-G levels and clinicopathological characteristics. In adenocarcinoma, low sMICA concentration was positively related to recurrent disease, a higher International Federation of Gynecology and Obstetrics (FIGO) stage and vaginal involvement (Mann-Whitney U-test; P = 0.018, P = 0.042 and P = 0.013, respectively). In the latter patient group, high sMICA levels were associated with better disease-free survival (DFS) and disease-specific survival (DSS) (P = 0.011 and P = 0.047). After adjusting for confounding factors, high sMICA proved to be an independent predictor for a better DFS and DSS [HR 0.16; 95% confidence interval (CI) 0.04-0.64; P = 0.009 and HR 0.12; 95% CI 0.03-0.50; P = 0.004]. sHLA-G did not influence survival in cervical cancer patients, regardless of histology. We conclude that cervical adenocarcinoma patients with high sMICA levels have an increased DFS and DSS. This data warrants a prospective trial to study the functional role of sMICA in cervical adenocarcinoma.

A simple non-invasive diagnostic algorithm for ruling out chronic thromboembolic pulmonary hypertension in patients after acute pulmonary embolism.

BACKGROUND: Our aim was to construct a diagnostic model for ruling out chronic thromboembolic pulmonary hypertension (CTEPH) in symptomatic patients after acute pulmonary embolism (PE) that is based on simple, non-invasive tests. METHODS: Plasma levels of various CTEPH associated biomarkers and conventional ECG criteria for right ventricular pressure overload were assessed in 82 consecutive patients with confirmed CTEPH and 160 consecutive patients with a history of PE who were suspected to have CTEPH, but in whom this disease was ruled out. RESULTS: ECG criteria of right ventricular hypertrophy were detected more frequently in patients with CTEPH (77%) than in the patients without CTEPH (11%, Odds ratio 26, 95% confidence interval [CI] 13-53). Also, clotting factor FVIII activity and the levels of N-terminal-pro-brain-type natriuretic peptide (NT-pro-BNP), Growth Differentiation Factor-15, C-reactive protein and urate, but not D-dimer level, were higher in patients with CTEPH. A diagnostic model including ECG criteria and NT-pro-BNP levels had a sensitivity of 94% (95% CI 86-98%) and a specificity of 65% (95% CI 56-72%). The area under the receiver-operator-characteristic curve was 0.80 (95% CI 0.74-0.85) for the diagnosis of CTEPH. Even with high disease prevalences of up to 10%, the negative predictive value of this model proved to be very high (99%, 95% CI 97-100%). CONCLUSIONS: Ruling out CTEPH in patients after acute PE seems to be safe without additional diagnostic testing in absence of ECG criteria indicative of right ventricular hypertrophy and a normal NT-pro-BNP level.

Increases in myeloperoxidase levels after exercise in myocardial perfusion scintigraphy are not induced by myocardial ischemia.

BACKGROUND: Increased systemic levels of myeloperoxidase (MPO) have been reported in patients with acute myocardial ischemia. We studied the association between exercise-induced myocardial ischemia measured by myocardial perfusion scintigraphy (MPS) and the magnitude and time course of changes in MPO levels in humans. METHODS: One hundred and twenty six patients underwent symptom limited exercise MPS. Myocardial ischemia was assessed semi-quantitatively. Plasma samples were taken before the start of exercise (baseline), at maximum exercise and every hour up to 6 h after maximum exercise. RESULTS: Myocardial ischemia was present in 42 (33%) patients. MPO levels rapidly increased during exercise in patients with and without ischemia (to 131% (106-172%) and 145% (121-199%) of baseline, respectively). MPO levels and absolute changes in MPO did not differ between patients with and without ischemia at any time point. None of the patient characteristics, including presence of ischemia, was independently predictive of the absolute increase in MPO levels during exercise. CONCLUSIONS: Exercise related immediate increases in MPO levels do not reflect myocardial ischemia.

The prognostic value of pre-procedural plasma C-reactive protein in patients undergoing elective coronary angioplasty.

AIMS: The acute phase reactant C-reactive protein is an important prognostic risk factor in patients with both stable and unstable coronary artery disease. The potential prognostic implications of an abnormal pre-procedural C-reactive protein concentration in patients undergoing elective coronary angioplasty may be relevant for subsequent treatment. METHODS AND RESULTS: Pre-procedural plasma levels of C-reactive protein were measured in 501 patients with stable coronary artery disease undergoing elective coronary angioplasty. The incidence of death or myocardial infarction during a 2-year follow-up was 10.6% (24/227) in patients with an increased C-reactive protein level (>3 mg. l(-1)) and 2.9% (8/274) in patients with a normal C-reactive protein level (RR 3.9, 95% CI 1.7-8.9). Survival without death, myocardial infarction, urgent revascularization or hospital admission for unstable angina was significantly lower in patients with an increased C-reactive protein vs patients with a normal C-reactive protein (log-rank 14.62, P<0.0001). Logistic regression analysis identified an increased C-reactive protein level as a strong independent predictor of event-free survival (RR 2.54, 95% CI: 1.44-4.47, P=0.001). CONCLUSION: Pre-procedural C-reactive protein levels are increased in 45% of patients undergoing elective coronary angioplasty. An increased C-reactive protein level is a powerful independent prognostic indicator for subsequent cardiac events, suggesting that late clinical outcome is markedly influenced by pre-procedural systemic activation of inflammation.

Troponin T in the first 24 hours after the administration of chemotherapy and the detection of myocardial damage in children.

Early detection of damage to cardiac myocytes after cardiotoxic chemotherapy in paediatric patients may allow timely preventive measures to be taken. We investigated the diagnostic value of cardiac troponin T (cTnT) after the administration of cardiotoxic chemotherapy. In 38 children, cTnT levels were measured at three time points during the first 24 h after 58 cardiotoxic chemotherapy cycles (163 samples). An abnormal cTnT level, defined as a cTnT>0.010 ng/ml, was measured in only six samples from 3 patients. After completion of chemotherapy, 7 out of the 38 patients had left ventricular dysfunction (LV dysfunction). Only 1 of these 7 patients had an elevated cTnT level. 2 other patients with elevated cTnT levels did not develop LV dysfunction until 2 and 7 months after the cTnT measurement. Our data show that the measurement of cTnT within 24 h after administration of chemotherapy does not have a high sensitivity for the identification of patients with subsequent subclinical cardiotoxicity.

C-Reactive protein and cardiac troponin T in risk stratification: differences in optimal timing of tests early after the onset of chest pain.

BACKGROUND: Increased C-reactive protein (CRP) is an important prognostic indicator for early risk stratification in patients with an acute coronary syndrome (ACS), independent of, and in combination with, increased cardiac troponin T (cTnT). However, increases in both cTnT and CRP also occur secondary to myocardial damage. METHODS AND RESULTS: In 156 consecutive patients, early release kinetics of CRP and cTnT were analyzed. The cutoff values were 3.0 mg/L for CRP and 0.1 microgram/L for cTnT. In the 75 patients with a CRP below the cutoff on admission, there was little change in CRP until 8 h after the onset of symptoms. At 12 h after the onset of symptoms, the cumulative proportions of abnormal CRP and cTnT in non-ST elevation ACS patients were 27% and 89%, respectively (P <0.01). During the first 24 h after the onset of symptoms, the median time above the cutoff was 20 h for CRP and 5 h for cTnT (P <0.0001). CRP was below the cutoff on admission significantly more often among patients receiving thrombolytic therapy than in patients without an indication for reperfusion therapy (51% vs 28%; P = 0.004). CONCLUSIONS: Increased CRP as an early independent risk indicator should be measured as soon as possible after the onset of symptoms, whereas increased cTnT is most reliable at 12 or more hours after the onset of symptoms.

Different time frames for the occurrence of elevated levels of cardiac troponin T and C-reactive protein in patients with acute myocardial infarction.

The baseline plasma level of C-reactive protein (CRP) is considered to be a parameter for risk stratification in patients with an acute coronary syndrome, independent of the level of cardiac troponin T (cTnT) or cardiac troponin I. However, myocardial tissue necrosis following prolonged arterial occlusion also induces release of CRP. Both phenomena may have their own kinetic behaviour with respect to changes in concentration of CRP. Therefore, in this study the time frame after onset of symptoms for measurement of CRP as an independent parameter is established. For this purpose, we evaluated patients with proven myocardial damage due to acute myocardial infarction (AMI) with respect to changes of creatine kinase (CK)-MB mass, cTnT and CRP during 24 hours after onset of symptoms. Our results show that two subgroups can be discerned in patients with AMI: those with initially normal and those with already elevated concentration CRP on admission. Furthermore, based on the results of this study we conclude that for use of CRP as an independent prognostic parameter in patients with acute coronary syndrome, CRP should be measured in blood samples drawn as early as possible after the onset of symptoms to avoid contribution of a process of myocardial tissue necrosis, whereas estimation of cTnT should be performed at 6-12 hours.

[The relationship between the serum level of prostate specific antigen and bone and CT scans in the staging of the primary carcinoma of the prostate]. / De samenhang tussen de serumconcentratie van prostaatspecifiek antigeen en de skelet- en CT-scan bij de stagering van primair prostaatcarcinoom.

OBJECTIVE: To determine the relationship between the serum level of prostate-specific antigen (PSA) and the presence of abnormalities in a skeletal or CT scan in patients with primary carcinoma of the prostate. DESIGN: Retrospective. SETTING: Academic Medical Centre, Amsterdam. PATIENTS AND METHODS: The serum PSA levels were compared with the findings in the skeletal and CT scans of 440 patients with carcinoma of the prostate without clinical signs of metastases, seen in the period from January 1990 to December 1994 in the outpatient clinics for Urology of the Academic Medical Centre (AMC) in Amsterdam, Hospital Gooi-Noord in Blaricum and Hospital De Heel in Zaandam. CT scan data were analysed only from the AMC and Hospital Gooi-Noord. RESULTS: There were 76 patients with a positive bone scan (17.3%) and 31 (out of 337; 9.2%) with a positive CT scan. Higher PSA serum levels went together with increasing risk of abnormalities in bone or CT scan. Of 85 patients with PSA values < 10 micrograms/l, none had a positive bone scan and one (out of 73; 1%) a positive CT scan; of the 180 patients with PSA levels < 20 micrograms/l, 4 (2.2%) had a positive bone scan and 2 (out of 154; 1.3%) a positive CT scan. The T stage, the histological grading and the serum alkaline phosphatase activity appeared not to have any supplementary value. CONCLUSION: In view of the low frequency of abnormalities in a bone or CT scan in patients with low PSA levels, it appears justified no longer to recommend bone or CT scanning for staging of patients for a clinically non-metastasized carcinoma of the prostate and serum PSA levels < 20 micrograms/l.

Critical difference between serial measurements of CK-MB mass to detect myocardial damage.

To assess the critical difference in serial measurements of CK-MBmass and the ability of this critical difference to detect myocardial damage, we studied 110 patients in whom an acute myocardial infarction (AMI) had been ruled out. Blood samples were drawn at 3, 4, 5, 6, 7, 8, 12, 16, 20, and 24 h after onset of symptoms. With a critical difference of 72.6%, an increase of >2.0 microg/L between two CK-MBmass measurements was determined to be significant. Twenty-three of the non-AMI patients had an increase in CK-MBmass >2.0 microg/L, but five of these did not have an abnormal concentration of troponin T (i.e., not >0.1 microg/L). Also among the 110 non-AMI patients, 22 did have an abnormal troponin T value, 18 of whom (82%) also had CK-MBmass increased by >2.0 microg/L. In 20 of the 23 patients with an increase in CK-MBmass >2.0 microg/L, this increase was detected from the values for two samples collected at 5 and 12 h after onset of symptoms. In conclusion, using the critical difference for CK-MBmass defined as an increase >2.0 microg/L detected myocardial damage in patients without AMI.

Prognostic value of troponin T, myoglobin, and CK-MB mass in patients presenting with chest pain without acute myocardial infarction.

OBJECTIVE: To assess the prognostic value of minor myocardial damage in patients presenting with chest pain without myocardial infarction. DESIGN: The relative risk of suffering a cardiac event in the next six months was assessed in patients with minor myocardial damage assessed by the cardiac markers CK-MB, myoglobin, and troponin T. SETTING: Emergency department of a large university hospital. PATIENTS: In 128 consecutive patients with chest pain, acute myocardial infarction (by WHO criteria) was ruled out; of these, 39 had a rise and fall of one or more markers, indicating minor myocardial damage. The presence of a documented history of coronary artery disease was assessed on admission. RESULTS: 24 patients had a subsequent event (cardiac death, acute myocardial infarction, percutaneous transluminal coronary angioplasty, coronary artery bypass grafting) in the next six months. An abnormal troponin T predicted a subsequent event while abnormal CK-MB or myoglobin did not. The relative risk for troponin T was 2.8 (95% confidence interval: 1.0 to 7.9), for myoglobin 1.0 (0.3 to 3.2), and for CK-MB 0.9 (0.2 to 3.4). A documented history of coronary artery disease predicted subsequent events with a relative risk of 3.9 (1.3 to 11.3). CONCLUSIONS: Troponin T was the only marker that predicted future events, but a documented history of coronary artery disease was the best predictor in patients in whom an acute myocardial infarction had been ruled out.

Evaluation of the Hitachi 911 for routine urine analysis and for measurement of various special serum analytes.

The Boehringer Mannheim Hitachi 911 is a selective analyzer for 35 different methods including 3 ion-selective electrode (ISE) methods. We have evaluated this analyzer primarily to obtain objective information on its applicability for routine urine analyses in our laboratory. We also implemented appropriate assays for various special serum- and whole blood-tests, some for the first time on the Hitachi 911 and some with modified settings. Analytical evaluation involved NCCLS EP5-T2 (imprecision), NCCLS EP6-P (linearity), Krouwer 27 (multifactor) and Passing & Bablok (method comparison) evaluation protocols. With the exception of evidence of systematic erroneous sample predilution, overall results were favourable. Practicability of the Hitachi 911 was judged by simulating daily routine. During a period of two weeks, daily urine samples were rerun on the Hitachi 911, leading to a gain of about 50% in total processing time. It was concluded that the Hitachi 911 meets the requirements in terms of analytical performance, reliability, versatility and speed for an analyzer to be used in a routine (urine) setting, while having a distinct role in special (serum/whole blood) measurements.

Kinetics of prostate-specific antigen after manipulation of the prostate.

Kinetics of prostate-specific antigen (PSA) were investigated after manipulation of the prostate in two groups of patients: those treated with digital rectal examination (DRE), and those with needle biopsy. 8 patients had serial PSA measurements to study the effect of DRE (group 1). 7 of 8 patients had PSA baseline values < 10 ng/ml. Blood samples were taken at 1 min, 30 min, 1, 3, 6, 12 and 24 h after DRE. Some patients were further monitored for 5 days with one blood sample taken at the same time each day. Statistically significant increased PSA levels were found after DRE (P < 0.001). Maximal increase was 70%. In most patients, peak levels were found between 30 and 60 min after DRE. Based on the results, it is concluded that after DRE it is prudent to wait 3 days before PSA is determined. 7 patients had serial PSA measurements after transrectal prostate needle biopsy (group 2). PSA sampling was similar as in the previous group. All patients had increased PSA levels after biopsy (range 1.3-9.5-fold). After 5 days, only 2 of 7 patients had returned to baseline levels. We conclude that biopsies of the prostate induce an important and long-lasting PSA elevation.

Biological half-life of prostate-specific antigen after radical prostatectomy.

The disappearance pattern of prostate-specific antigen from serum after a standard radical prostatectomy was studied in eight patients with cancer confined to the prostate. The results were used to plot an elimination curve and calculate the best fit. A biphasic pattern was found with an average biological half-life of 1.63 hours in the alpha-phase, and 4.63 days in the beta-phase. Based on these results it is concluded that determination of prostate-specific antigen concentrations less than one month after a standard radical prostato-vesiculectomy has no value for the detection or exclusion of residual malignant processes.

Bone-alkaline phosphatase as indicator of bone formation.

Bone-alkaline phosphatase was determined in patients at risk of osteoporosis due to treatment with oral corticosteroids, and in patients at risk of increased bone synthesis because of treatment with cyclosporin. Both a significant decrease of bone-alkaline phosphatase during corticosteroid treatment, and a significant increase of bone-alkaline phosphatase during cyclosporin treatment could be demonstrated. It is concluded that bone-alkaline phosphatase is a useful parameter for monitoring changes in bone formation.

Inhibition of lactate dehydrogenase isoenzymes by sodium perchlorate evaluated.

We evaluated a method of measuring lactate dehydrogenase isoenzyme 1 (LD-1) selectively (Clin Chem 1987;33:991-2), in which all other LD isoenzymes were inhibited by adding sodium perchlorate to the reaction medium to a final concentration of 0.825 mol/L. In this study we used the different isoenzymes purified from human autopsy tissue and found that the originally published amount of inhibitor (a) increased the original LD-1 activity and (b) did not eliminate all enzyme activity of LD-2 and LD-3. Interference by LD-2 was further demonstrated. Thus we conclude that this method cannot be used for the selective determination of LD-1 because its results do not accurately reflect the original LD-1 activity.

An unusual case of black teeth.

Loss of enamel and a deep black stain of the teeth in a 40-year-old diabetic patient are strongly suggested to be caused by the daily consumption of a cheap white wine and, possibly, by the chewing of cayenne. The wine proved to be rather acid, thereby promoting abrasion as a result of gnashing, and to contain a high concentration of tannin. The exact role of the tannins is described.