Solving a cold case of haemolysis: back to the basics.

Membrane disorders comprise an important group of inherited haemolytic anaemias. Diagnostic work-up starts with examination of the blood smear, followed by osmotic gradient ektacytometry. In special cases DNA analysis is performed to confirm the diagnosis. For this purpose a next-generation sequencing-based method has been developed. The combination of these techniques established the correct diagnosis in a case of haemolytic anaemia of unknown cause.

Genetic polymorphisms in UDP-glucuronosyltransferases and glutathione S-transferases and colorectal cancer risk.

Colorectal cancer (CRC) is one of the most common malignancies in the Western world showing an increasing incidence, and has been associated with genetic and lifestyle factors. Individual susceptibility to CRC may be due partly to variations in detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes may result in variations in detoxification activities, which subsequently might influence the levels of toxic/carcinogenic compounds, and this may influence the risk for CRC. To determine whether genetic polymorphisms in detoxification enzymes predispose to the development of CRC, 371 patients with sporadic CRC and 415 healthy controls were genotyped for polymorphisms in the important detoxification enzymes UDP-glucuronosyltransferase UGT1A1, UGT1A6, UGT1A7 and UGT1A8, and glutathione S-transferase GSTA1, GSTM1, GSTP1 and GSTT1. Patients and controls were all of Caucasian origin. DNA was isolated from either blood or tissue and tested by polymerase chain reaction followed by restriction fragment length polymorphism analyses. Logistic regression analyses showed significant age- and gender-adjusted risks for CRC associated with variant genotypes of UGT1A6 [OR 1.5, 95% (confidence interval) CI 1.03-2.3] and UGT1A7 (OR 2.4, 95% CI 1.3-4.6), whereas no associations were found between CRC and the other polymorphic genes as mentioned above. In conclusion, the data suggest that the presence of variant UGT1A6 and UGT1A7 genotypes with expected reduced enzyme activities, might enhance susceptibility to CRC.

Survivin mRNA expression is elevated in malignant urothelial cell carcinomas and predicts time to recurrence.

BACKGROUND: Expression of the inhibitor of apoptosis protein survivin is up-regulated in many tumors of epithelial origin and frequently shows a relationship with disease prognosis. MATERIALS AND METHODS: We investigated survivin mRNA expression in 32 urothelial cell carcinomas by use of real-time quantitative PCR. Expression values were normalized to transcript levels of the housekeeping gene cyclophilin. RESULTS: All bladder tumor tissues expressed survivin mRNA. The median normalized survivin mRNA expression values were 0.26 for superficial tumors (n = 17) and 0.78 for invasive tumors (n = 15). A significant relationship with increasing pathological stage (p < 0.001) and grade (p < 0.001) was observed. Although survivin mRNA expression did not relate to disease progression or the patient survival period, patients with superficial bladder tumors and normalized survivin values over 0.26 had an increased risk of recurrence (log-rank test: p = 0.018). CONCLUSION: Our results suggest that quantitative measurement of survivin mRNA 1) can identify invasive and high-grade urothelial cell carcinomas and 2) may be used as an indicator for early recurrence of superficial tumors.

Increased total cell-free DNA in the serum of pregnant women carrying a fetus affected by trisomy 21.

OBJECTIVE: Analysis of the levels of cell-free fetal and total DNA in serum of women carrying a male fetus affected by trisomy 21, and comparison of these levels with those in women carrying a normal male fetus. METHODS: DNA was extracted from archived second-trimester maternal serum samples collected as part of a prenatal screening program. A total of 10 cases with trisomy 21 male fetuses were compared with 10 controls (male fetuses) with samples matched for duration of storage and gestational age. Real-time quantitative PCR of the SRY and albumin genes was used to quantify fetal and total DNA respectively. RESULTS: The median fetal DNA level in the group of 10 pregnancies with trisomy 21 was 31.98 cell-equivalents per mL compared to 34.06 in the control group. The difference was not significant. The median total DNA level in women with a trisomy 21 fetus was significantly higher (P = 0.029) than that in controls (36 152.6 vs 5832.81 cell-equivalents per mL). CONCLUSIONS: Although we could not confirm previous studies of an increased amount of fetal DNA in pregnancies affected by trisomy 21, we did find increased levels of total DNA. The possible reasons for these observations are discussed with respect to previous findings. Larger studies are needed to elucidate the true value, if any, of measurement of fetal and total DNA in maternal serum in the context of prenatal screening for chromosomal abnormalities.

Clinical value of bile for the detection of mutant K-ras from colorectal liver metastases.

In 25% of patients diagnosed with colorectal cancer, hepatic metastases are not detected at presentation of the colorectal primary but develop during follow-up. Early detection of these metastases may improve the chance of cure by surgical resection. We hypothesised that in patients with occult hepatic metastases, tumour DNA might be detected in bile which could be collected during resection of the colorectal primary. To test this hypothesis, bile from the gall bladder was collected from 17 patients scheduled for resection of evident hepatic metastases (> 2 cm3) from a previously resected colorectal primary. Mutation analysis of the metastases identified five patients (34%) with a K-ras gene mutation in the tumour tissue. These cases were selected for bile analysis for mutant K-ras. Non-mutated DNA could be amplified from all the bile samples, but mutant K-ras could only be detected in bile from one patient. False negative results due to technical deficits could be ruled out by control experiments showing a high DNA isolation efficiency and high sensitivity of the mutation detection method. It is concluded that hepatic metastases, in contrast to pancreatic cancers, do not (regularly) shed mutated DNA into the bile. Hence, molecular screening of bile seems of only limited clinical value for the detection of occult liver metastases.

Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas.

Telomerase reverse transcriptase (hTERT) messenger RNA has been detected in 95% of bladder tumors using RT-PCR. In this study, we quantified the expression of hTERT in 35 bladder urothelial cell carcinomas and in 6 normal bladder epithelia using a real-time quantitative PCR assay. hTERT expression was detected in all 35 urothelial cell carcinomas of varying grade and stage, but not in normal tissue samples. An increase in both pathological grade and clinical stage as prognostic parameters correlated with increased hTERT expression. Using different cutoff values for grades and stages, normalized hTERT expression values could discriminate among low, medium, and high grade tumors and between superficial and muscle-invasive tumors. We conclude that standardized real-time measurement of hTERT expression can be used for early tumor detection and may be used for determination of prognosis in urothelial cell carcinomas of the bladder.

Real-time quantification of human telomerase reverse transcriptase mRNA in tumors and healthy tissues.

BACKGROUND: Expression of the hTERT gene, which codes for the catalytic subunit of telomerase, is associated with malignancy. We recently developed a real-time reverse transcription-PCR assay, based on TaqMan technology, for accurate and reproducible determination of hTERT mRNA expression (Lab Investig 1999;79:911-2). This method may be of interest for molecular tumor diagnostics in tissues and corresponding body fluids, washings, or brushes. METHODS: In this study, we measured hTERT expression in a subset of healthy tissues and tumors to select those tumor types with the best potential for quantification of hTERT in corresponding body fluids. To demonstrate the use of the method in body fluids, we quantified hTERT expression in voided urine of patients with bladder cancer and controls. RESULTS: Real-time measurement of hTERT expression could discriminate between all healthy and malignant tissue samples from pancreas, lung, esophagus, and bladder, but not for colon tissues. Moreover, in five of nine (55%) urine samples, hTERT could be quantified. CONCLUSIONS: The present study demonstrates that accurate quantitative measurement of hTERT expression has high potential for discrimination between healthy and tumor cells in tissues and urine and supports future measurements in pancreatic fluid, bronchoalveolar lavage fluid, esophageal brushings, and urine or bladder washings.

Trends in trace element determinations in blood, serum, and urine of the Dutch population, and the role of neutron activation analysis.

A critical review on the quality of literature data on trace elements in human blood, serum, and urine of inhabitants in the Netherlands has shown that many of the currently available data have been established 15-20 years ago. Only in a few publications are quality indicators mentioned, which should be considered typical--and minimal--for studies resulting at reference values. The use of neutron activation analysis for determination of trace elements in human body fluids was restricted to a few studies in the 1970s. However, although it is frequently assumed that the sensitivity of NAA might be insufficient, it is demonstrated that modern, large, well-type Ge detectors may serve well for the determination of trace elements in human body fluids via radiochemical NAA, for example.

Detection of tumour DNA in serum of colorectal cancer patients.

Circulating tumour DNA has previously been detected in serum and plasma of patients with lung cancer and head and neck cancer. These observations could potentially lead to new, specific and non-invasive tools for diagnosis, prognosis and follow-up in neoplastic disease, if found to be a more general phenomenon. To test if tumour DNA is also present in serum of patients with colorectal cancer, we selected 14 colorectal cancer patients with advanced disease. In seven patients, K-ras mutations were detected in the primary tumour, using mutant-specific primers for point mutations in codon 12 or 13 of the K-ras gene. All patients were analysed for mutant DNA in serum. Tumour-specific point mutations, corresponding to the K-ras mutations found in the primary tumour were detected in the serum of all patients but one. No mutant K-ras could be detected in the serum of seven patients without K-ras mutations in the primary tumour. These results may be useful in assessing tumour burden in patients with neoplastic disease. Moreover, consecutive testing of serum tumour DNA after surgery or chemotherapy may be used as a tumour marker for recurrent disease.

On the uptake and release of zinc (65Zn) in the growing alga Selenastrum capricornutum Printz.

A Zn- and pH-buffered medium was used to study the accumulation of Zn in batch-cultured algae (Selenastrum capricornutum Printz.). All experiments were carried out using (65)Zn radiotracers, which were measured by gamma-ray spectrometry. Zinc was applied in the presence of nitrilotriacetic acid (NTA), leading to free Zn(2+) concentrations ranging from 4.2 x 10(-11) to 1.6 x 10(-7) mol litre(-1). During the 75-h experiments, the rates of Zn influx and efflux were determined, the latter with full consideration for algal growth rates. Algal growth stopped completely at a free Zn(2+) concentration of 1.6 x 10(-8) mol litre(-1). Initial Zn binding was determined and, based on free Zn(2+) concentrations, presented by the apparent dissociation constant K(diss) and capacity C as 4.6 x 10(-9) mol litre(-1) and 19.8 x 10(6) mol gDW(-1), respectively. The rate constant of Zn efflux could be calculated as a constant 0.021 +/- 0.003 h(-1), irrespective of the Zn concentrations applied. The rates of Zn influx were expressed by the V(max) and K(M) values as 3.3 x 10(-10) mol gDW(-1) s(-1) and 6.3 x 10(-9) mol litre(-1), respectively. These values, which are based on applied free Zn(2+) concentrations, are compatible with the operation of a high-affinity low-concentration carrier mechanism. The results suggest that, under the conditions applied, free Zn(2+) may be the most relevant Zn species for Zn uptake; furthermore, relative growth rate may be regarded as a relatively sensitive signal for Zn stress circumstances.

Detection of the protozoan parasite Theileria annulata in Hyalomma ticks by the polymerase chain reaction.

Adult Hyalomma ticks were examined for the presence of Theileria annulata infection using the Polymerase Chain Reaction (PCR). A 372 bp DNA fragment derived from the small ribosomal RNA gene of T. annulata was amplified from 45 out of 50 (90%) H. dromedarii ticks and from 36 out of 50 (72%) H. marginatum marginatum ticks. No product was amplified from non-infected control ticks. Restriction enzyme digestion with Sac II confirmed that the product was derived from the targeted T. annulata gene. As a further confirmation it was shown that both species of Hyalomma ticks were able to transmit T. annulata to experimental calves. PCR detection of Theileria parasites in ticks was compared with conventional staining of dissected salivary glands using methyl green pyronin and its comparative advantages are discussed.

The uptake of zinc by erythrocytes under near-physiological conditions.

The in vitro uptake of zinc by erythrocytes was measured under near-physiological conditions, using 65Zn as a radioactive tracer. Because of the presence of serum albumin--a strong zinc ligand--a low concentration of medium free zinc was maintained. Under these conditions a high-affinity carrier for zinc transport over the cell membrane was identified. With human erythrocytes, a Michaelis constant (Km) of 0.2 nM with respect to free medium zinc was measured and a Vmax of 4.5 nmoles Zn transported per h/g dry wt. The Km for medium Zn increases when the size of the internal erythrocytic Zn pool is augmented, whereas Vmax remains virtually unchanged. A model to explain this phenomenon is proposed. It is suggested that this phenomenon could underlie observations, confirmed here, that the in vitro uptake of Zn by animal erythrocytes depends on the Zn status of the animal.