Robert de Jonge

Polymorphisms in folate-related genes and risk of pediatric acute lymphoblastic leukemia

Polymorphisms in folate pathway genes may influence the susceptibility to acute lymphoblastic leukemia (ALL). DNA was isolated from 245 pediatric ALL patients (cases) and from 500 blood bank donors (controls). Polymorphisms in methylene-tetrahydrofolate reductase (MTHFR 677C>T, 1298A>C), methionine synthase (MTR 2756A>G), methionine synthase reductase (MTRR 66A>G), methylenetetrahydrofolate dehydrogenase (MTHFD1 1958G>A), nicotinamide N-methyltransferase (NNMT IVS -151C>T), serine hydroxymethyl transferase (SHMT1 1420C>T), thymidylate synthase (TS 2R3R), and the reduced folate carrier (RFC1 80G>A) were detected. In ALL patients, an increased occurrence was observed of the RFC1 80AA variant (odds ratio [OR] = 2.1; 95% confidence interval [CI] = 1.3-3.2; P = .002) and the RFC1 80A allele (OR = 1.5; 95% CI, 1.1-2.1; P = .02). Likewise, the NNMT IVS -151TT genotype showed a 2.2-fold increased ALL risk (OR = 2.2; 95% CI, 1.1-4.6; P = .04). A 1.4-fold reduction in ALL risk was observed for (heterozygous or homozygous) carriers of the TS 2R allele and the MTHFR 677T allele (OR = 0.7; 95% CI, 0.5-1.0; P < .05). Furthermore, interactions between NNMT and MTHFR 677C>T and RFC1 were observed. NNMT IVS -151CC/MTHFR 677CT + TT patients exhibited a 2-fold reduction in ALL risk whereas RFC1 80AA/NNMT IVS -151CT + TT subjects had a 4.2-fold increase in ALL risk (P = .001). For the first time, we associate the RFC1 80G>A and NNMT IVS -151C>T variants to an increased ALL susceptibility.

Plasma N-terminal pro-B-type natriuretic peptide as long-term prognostic marker after major vascular surgery

Objective: To assess the long-term prognostic value of plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) after major vascular surgery. Design: A single-centre prospective cohort study. Patients: 335 patients who underwent abdominal aortic aneurysm repair or lower extremity bypass surgery. Interventions: Prior to surgery, baseline NT-proBNP level was measured. Patients were also evaluated for cardiac risk factors according to the Revised Cardiac Risk Index. Dobutamine stress echocardiography (DSE) was performed to detect stress-induced myocardial ischaemia. Main outcome measures: The prognostic value of NT-proBNP was evaluated for the endpoints all-cause mortality and major adverse cardiac events (MACE) during long-term follow-up. Results: In this patient cohort (mean age: 62 years, 76% male), median NT-proBNP level was 186 ng/l (interquartile range: 65–444 ng/l). During a mean follow-up of 14 (SD 6) months, 49 patients (15%) died and 50 (15%) experienced a MACE. Using receiver operating characteristic curve analysis for 6-month mortality and MACE, NT-proBNP had the greatest area under the curve compared with cardiac risk score and DSE. In addition, an NT-proBNP level of 319 ng/l was identified as the optimal cut-off value to predict 6-month mortality and MACE. After adjustment for age, cardiac risk score, DSE results and cardioprotective medication, NT-proBNP ⩾319 ng/l was associated with a hazard ratio of 4.0 for all-cause mortality (95% CI: 1.8 to 8.9) and with a hazard ratio of 10.9 for MACE (95% CI: 4.1 to 27.9). Conclusion: Preoperative NT-proBNP level is a strong predictor of long-term mortality and major adverse cardiac events after major non-cardiac vascular surgery.

ABCB1 and ABCC3 Gene Polymorphisms Are Associated with First-year Response to Methotrexate in Juvenile Idiopathic Arthritis

Objective. Although methotrexate (MTX) is the most widely prescribed drug in juvenile idiopathic arthritis (JIA), 30% of patients fail to respond to it. To individualize treatment strategies, the genetic determinants of response to MTX should be identified. Methods. A cohort of 287 patients with JIA treated with MTX was studied longitudinally over the first year of treatment. MTX response was defined as the American College of Rheumatology pediatric 70 criteria (ACRped70). We genotyped 21 single-nucleotide polymorphisms in 13 genes related to MTX polyglutamylation and to cellular MTX uptake and efflux. Potential associations between ACRped70 and genotypes were analyzed in a multivariate model and corrected for these 3 covariates: disease duration prior to MTX treatment, physician’s global assessment of disease activity at baseline, and MTX dose at all study visits. Results. MTX response was more often achieved by patients variant for the adenosine triphosphate-binding cassette transporter B1 (ABCB1) gene polymorphism rs1045642 (OR 3.80, 95% CI 1.70−8.47, p = 0.001) and patients variant for the ABCC3 gene polymorphism rs4793665 (OR 3.10, 95% CI 1.49−6.41, p = 0.002) than by patients with other genotypes. Patients variant for the solute carrier 19A1 (SLC19A1) gene polymorphism rs1051266 were less likely to respond to MTX (OR 0.25, 95% CI 0.09−0.72, p = 0.011). Conclusion. ABCB1 rs1045642, ABCC3 rs4793665, and SLC19A1 rs1051266 polymorphisms were associated with response to MTX in 287 patients with JIA studied longitudinally. Upon validation of our results in other JIA cohorts, these genetic determinants may help to individualize treatment strategies by predicting clinical response to MTX.

Evaluation of the new body fluid mode on the Sysmex XE-5000 for counting leukocytes and erythrocytes in cerebrospinal fluid and other body fluids

Abstract Background: We evaluated the body fluid (BF) mode on the new Sysmex XE-5000 analyzer. Methods: Red (RBC) and white blood cell (WBC) (differential) counts of BFs (139 patient samples and 87 normal samples) were measured and compared to the Fuchs-Rosenthal chamber and stained cytospin slides. Results: Extended cell counting using the BF mode was noted to have an improved WBC detection limit (CV20%) of 10×106/L. Excellent agreement with the manual method was observed for most BFs [mean bias +2 to 6×106/L for cerebrospinal fluid (CSF) and –1 to 12×106/L for other fluids]. In CSF, the BF-mode counted more WBC (polymorphic nuclear cells) compared with the manual method (mean bias +5 to 6×106/L), especially in samples with low cell counts (<20×106/L). Carry over was negligible (mostly <0.17%) and linearity was excellent (mean bias <5%). The reference ranges for CSF (n=87) were RBC 0×106/L, WBC and mononuclear <7×106/L, and polymorph nucleated cells <3×106/L. Conclusions: The BF mode on the Sysmex XE-5000 offers rapid and accurate RBC and WBC (differential) counts in clinically relevant concentration ranges in CSF and other fluids. In addition, the exclusion of high fluorescent cells, such as mesothelial cells and macrophages from WBC counting may reduce the number of manual analyses in pleural fluids and ascites. Clin Chem Lab Med 2010;48:665–75.

β-d-Glucan and S-adenosylmethionine serum levels for the diagnosis of Pneumocystis pneumonia in HIV-negative Patients: A prospective study

OBJECTIVE To prospectively assess the diagnostic utility of S-adenosylmethionine (AdoMet) and (1→3)-β-D-glucan (β-D-glucan) serum markers for Pneumocystis pneumonia (PCP) in HIV-negative patients. METHODS HIV-negative, immunocompromised patients suspected of PCP based on clinical presentation and chest imaging were included. PCP was confirmed or rejected by results of direct microscopy and/or real-time PCR on broncho-alveolar lavage (BAL) fluid. Measurement of serum β-D-glucan and AdoMet was performed on serum samples collected at enrollment and during follow-up. Both serum β-D-glucan and AdoMet were assessed for diagnostic accuracy and correlation with clinical and laboratory parameters. RESULTS In 31 patients enrolled (21 PCP-positive, 10 PCP-negative), AdoMet levels did not discriminate between patients with and without PCP. Elevated serum β-D-glucan was a reliable indicator for PCP with a sensitivity of 0.90 and specificity of 0.89 at the 60 pg/ml cut-off. In PCP-positive patients β-D-glucan serum levels decreased during treatment and inversely correlated with Pneumocystis PCR cycle threshold values in BAL fluid. CONCLUSIONS The level of β-D-glucan--but not AdoMet--was diagnostic for PCP within the clinical context and may serve as marker for pulmonary fungal load and treatment monitoring.

Body Mass Index Is an Important Determinant of Methylation Biomarkers in Women of Reproductive Ages

B vitamin deficiencies lead to moderate hyperhomocysteinemia, which has been associated with health and disease. However, concomitant derangements in cellular methylation, reflected by altered plasma S-adenosylmethionine (SAM) or S-adenosylhomocysteine (SAH) concentrations, may be the primary cause. Therefore, we identified determinants of homocysteine, SAM, and SAH concentrations in 336 women, aged 20-48 y, as part of a large study focusing on risk factors for reproductive disorders. Blood was obtained to determine plasma SAM, SAH, and total homocysteine (tHcy), serum vitamin B-12 and folate, RBC folate concentrations, and the related single nucleotide polymorphisms 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C > T and 1298A > C, methionine synthase reductase (MTRR) 66A > G, and nicotinamide N-methyltransferase IVS1-151G > A. Questionnaires provided information on demographics, lifestyles, and nutrient intakes. Correlation coefficients were calculated and multivariable associations were assessed with a general linear model. Serum folate was positively correlated with SAM concentrations (r = 0.159; P = 0.004). Folate and vitamin B-12 were not correlated with SAH concentrations or the SAM:SAH ratio but were inversely correlated with tHcy concentrations (serum folate r = -0.324; RBC folate r = -0.294; vitamin B-12 r = -0.307; P < 0.01). From the multivariable analysis, BMI was the strongest determinant of SAM (standardized beta = 19.145; P < 0.001) and SAH concentrations (standardized beta = 3.241; P = 0.010). MTHFR 677TT (standardized beta = 0.195; P = 0.001), B vitamin supplement use (standardized beta = -0.156; P < 0.001) and dietary protein intake (standardized beta = -0.011; P < 0.001) were the strongest determinants of tHcy concentrations. Thus, the determinants of SAM and SAH differ from those of tHcy concentrations. Given that BMI was a strong determinant of SAM concentrations, it should be included in future studies on cellular methylation.

Screening for metabolic vitamin B12 deficiency by holotranscobalamin in patients suspected of vitamin B12 deficiency: a multicentre study

Background Vitamin B12 deficiency occurs frequently, especially among the elderly. However, screening for vitamin B12 deficiency is hampered by poor sensitivity of the existing total vitamin B12 assay. Methylmalonic acid (MMA) is considered as the most representative indicator of metabolic vitamin B12 deficiency and is used as such in this study. The aim of this study was to validate the clinical usefulness of holotranscobalamin (holoTC) as an initial screening assay for metabolic vitamin B12 deficiency in a mixed patient population. Methods Three hundred and sixty blood samples were collected by five Dutch hospitals. Vitamin B12 and holoTC in serum were measured (AxSYM; Abbott). MMA in serum was measured by tandem mass spectrometry (LC-MS/MS). Results Receiver operating curve (ROC) analysis demonstrated a greater area under the curve (AUC) for holoTC than for vitamin B12 in detecting vitamin B12 deficiency characterized by three predefined cut-off levels of MMA. A cut-off value of 32 pmol/L of holoTC resulted in the highest sensitivity (83%) with acceptable specificity (60%) in detecting MMA concentrations above 0.45 μmol/L. The combination of vitamin B12 and holoTC did not improve diagnostic accuracy at this cut-off level. Conclusions HoloTC has a better diagnostic accuracy than vitamin B12 and can replace the existing vitamin B12 assay as a primary screening test in patients suspected of vitamin B12 deficiency. Critical evaluation of cut-off values of holoTC indicated that a cut-off value of 32 pmol/L can be considered in screening for metabolic vitamin B12 deficiency (defined by MMA > 0.45μmol/L) in a mixed patient population.

Congenital heart defects and biomarkers of methylation in children: a case–control study

Eur J Clin Invest 2011; 41 (2): 143–150

Validation of the body fluid module on the new Sysmex XN-1000 for counting blood cells in cerebrospinal fluid and other body fluids

Abstract Background: We evaluated the body fluid (BF) module on the new Sysmex XN-1000 for counting blood cells. Methods: One hundred and eighty-seven BF samples [73 cerebrospinal fluid (CSF), 48 continuous ambulatory peritoneal dialysis (CAPD), 46 ascites, and 20 pleural fluid] were used for method comparison between the XN-1000 and manual microscopy (Fuchs-Rosenthal chamber and stained cytospin slides) for counting red blood cells (RBCs) and white blood cells (WBCs) (differential). Results: Good agreement was found for counting WBCs (y=1.06x+0.09, n=67, R2=0.96) and mononuclear cells (MNs) (y=1.04x–0.01, n=40, R2=0.93) in CSF. However, the XN-1000 systematically counted more polymorphonuclear cells (PMNs) (y=1.48x+0.18, n=40, R2=0.99) compared to manual microscopy. Excellent correlation for RBCs >1×109/L (y=0.99x+116.56, n=26, R2=0.99) in CSF was found. For other fluids (CAPD, ascites and pleural fluid) excellent agreement was found for counting WBCs (y=1.06x+0.26, n=109, R2=0.98), MNs (y=1.06x–0.41, n=93, R2=0.96), PMNs (y=1.06x+0.81, n=93, R2=0.98) and RBCs (y=1.04x+110.04, n=43, R2=0.98). By using BF XN-check, the lower limit of quantitation (LLoQ) for WBC was defined at 5×106/L. Linearity was excellent for both the WBCs (R2=0.99) and RBCs (R2=0.99) and carry-over never exceeded 0.05%. Conclusions: The BF module on the XN-1000 is a suitable tool for fast and accurate quantification of WBC (differential) and RBC counts in CSF and other BFs in a diagnostic setting.

Automated analysis of pleural fluid total and differential leukocyte counts with the Sysmex XE-2100.

Abstract Background: Determination of leukocyte (WBC) counts in pleural fluid is routinely performed by microscopic examination. In this study, we evaluated the performance of automated (differential) WBC counting in comparison with manual counting. Methods: Pleural fluid samples (n=45) were obtained from patients undergoing diagnostic thoracocentesis. The manual total WBC count was determined after Samson staining in a Fuchs-Rosenthal hemocytometer; microscopic differential counts were performed on May-Grünwald Giemsa-stained cytospin slides. The Sysmex XE-2100 hematology analyzer was used for automated (differential) WBC counting. The functional detection limit was determined by serial dilution of continuous ambulatory peritoneal dialysis (CAPD) fluid and replicate measurements of each dilution. Results: The automated WBC count (×106/L) was highly correlated with that of the microscopic reference method (r2=0.95; WBC-analyzer=0.97×WBC-reference method+16; n=45). Good agreement was also observed for the absolute lymphocyte count (r2=0.92; WBC-analyzer=0.99×WBC-reference method+32; n=36), neutrophil count (r2=0.94; WBC-analyzer=0.91×WBC-reference method+6; n=35), and monocyte count (r2=0.73; WBC-analyzer=0.83×WBC-reference method+6; n=38). The functional detection limit for WBCs was calculated at 50×106/L (coefficient of variation 20%). Conclusions: With some limitations, total and differential WBC counts in pleural fluid can be reliably determined using the Sysmex XE-2100 instrument. Clin Chem Lab Med 2006;44:1367–71.