Concordance between creatinine- and cystatin C-based eGFR in clinical practice.

The mean of GFR-estimates based on serum creatinine (eGFRcrea) and cystatin C (eGFRcys) has superior accuracy than each estimate alone. Recent studies have shown that agreement between eGFRcrea and eGFRcys is an indicator for the accuracy of the mean of the two estimates. As long as the difference between the two (|ΔeGFR|) is below 40%, a high P30 accuracy rate of more than 90% was documented in research settings using gold-standard GFR measurements. This was the case in approximately 80% of the measurements. The study was set out to explore |ΔeGFR| in a broader pediatric nephrological population and identify factors influencing the discrepancy between eGFRcrea and eGFRcys. We retrospectively analyzed 1596 simultaneous cystatin C and creatinine measurements in 649 unique patients at the pediatric nephrology outpatient clinic of VU university medical center. The FASage equation was used to calculate eGFRcrea, FAScys for eGFRcys. |ΔeGFR| was calculated as 100x(|eGFRcrea-eGFRcys|)/(0.5x(eGFRcrea+eGFRcys). ΔeGFR below 40% was considered high agreement. Patient characteristics like age, diagnosis, glucocorticosteroid use, eGFR, BMI and sex were analyzed for their effect on ΔeGFR below or above 40% using non-parametric tests and a potential explanation for measurements with low agreement was sought. Eighty-seven percent of the population had a |ΔeGFR| lower than 40%. Measurements with |ΔeGFR| above 40% were significantly more frequent from patients with neural tube defects. In 102 out of 208 measurements with low agreement, a potential explanation was found. In a broad pediatric nephrological population, |ΔeGFR| is below 40% in the vast majority of measurements. In this group, the mean of eGFRcrea and eGFRcys can be used as an accurate estimate of GFR.

Expression of the nucleoside transporters hENT1 (SLC29) and hCNT1 (SLC28) in pediatric acute myeloid leukemia.

Cellular uptake of clinically important deoxynucleoside analogs is mediated by nucleoside transporters including the human equilibrative nucleoside transporter 1 (hENT1) and the concentrative nucleoside transporter-1 (hCNT1). These transporters are responsible for influx of cytarabine and reduced hENT1 expression is a major resistance mechanism in acute myeloid leukemia. We determined hENT1 and hCNT1 protein expression by immunocytochemistry in 50 diagnostic pediatric acute myeloid leukemia patient samples. All samples expressed hENT1 [9/43 (21%) low; 26/43 (60%) medium and 8/43 (19%) high] and hCNT1 [2/42 (5%) low; 35/42 (83%) medium and 5/42 (12%) high] at the cell membrane and cytoplasm. Statistical analysis showed a non-significant relationship between survival and transporter expression and in vitro drug sensitivity. In conclusion, the nucleoside transporters hENT1 and hCNT1 are broadly expressed in pediatric acute myeloid leukemia at diagnosis.

Evidence for shrunken pore syndrome in children.

The link between cystatin C and mortality independent of glomerular filtration rate (GFR) in adults has prompted the "Shrunken Pore Syndrome" (SPS) hypothesis, where high serum cystatin C with normal creatinine is explained by smaller glomerular pores, through which creatinine can pass freely, while the larger cystatin C, beta-trace protein (BTP) and pro-inflammatory molecules are retained. This study set out to apply the definition of SPS to children. In 294 children who underwent inulin clearance (Cin) test, serum creatinine, cystatin C and BTP were measured. For all three markers eGFRx was calculated using the full age spectrum equations. The ratio eGFRcys/eGFRcrea was plotted against the error of eGFRBTP(%) (i.e. eGFRBTP-Cin)/Cin*100%). Patients with and without SPS according to different cut-off points of eGFRcys/eGFRcrea and eGFRcys/Cin (i.e. ≤0.6,0.7,0.8) were compared in terms of eGFRx, Cin, error of eGFRx(%) and eGFRBTP/eGFRcrea-ratio. The ratio eGFRcys/eGFRcrea and error of eGFRBTP(%) were positively correlated. The prevalence of SPS by eGFRcys/eGFRcrea with a cut-off of 0.6 was 4.8%. Patients with SPS had a more negative error of eGFRcys(%) and eGFRBTP(%) and higher Cin regardless of the definition. Overestimation of eGFRcrea in patients with SPS was only present when using the eGFRcys/eGFRcrea rather than the eGFRcys/Cin definition. Cystatin C and BTP are related independent of creatinine, suggesting glomerular pore size as a common denominator. The prevalence of SPS in children is comparable to adults. For research in SPS, a definition based on eGFRcys/exogenous clearance study may be useful to study the effect of SPS on creatinine metabolism.

Diagnostic performance of Elecsys immunoassays for cerebrospinal fluid Alzheimer's disease biomarkers in a nonacademic, multicenter memory clinic cohort: The ABIDE project.

INTRODUCTION: We compared the automated Elecsys and manual Innotest immunoassays for cerebrospinal fluid (CSF) Alzheimer's disease biomarkers in a multicenter diagnostic setting. METHODS: We collected CSF samples from 137 participants in eight local memory clinics. Amyloid ß(1-42) (Aß42), total tau (t-tau), and phosphorylated tau (p-tau) were centrally analyzed with Innotest and Elecsys assays. Concordances between methods were assessed. RESULTS: Biomarker results strongly correlated between assays with Spearman's ρ 0.94 for Aß42, 0.98 for t-tau, and 0.98 for p-tau. Using Gaussian mixture modeling, cohort-specific cut-points were estimated at 1092 pg/mL for Aß42, 235 pg/mL for t-tau, and 24 pg/mL for p-tau. We found an excellent concordance of biomarker abnormality between assays of 97% for Aß42 and 96% for both t-tau and p-tau. DISCUSSION: The high concordances between Elecsys and Innotest in this nonacademic, multicenter cohort support the use of Elecsys for CSF Alzheimer's disease diagnostics and allow conversion of results between methods.

Estimation of GFR in children using rescaled beta-trace protein.

INTRODUCTION: Beta-trace protein (BTP) is a low molecular weight protein, produced mainly in the cerebrospinal fluid. It has been proposed as a marker for kidney function. Recently, a new method for GFR estimation using mean normal values to rescale GFR marker concentrations has been described for creatinine and cystatin C, two commonly used endogenous markers for kidney function. The aim of this study is to apply this approach to BTP in children. METHOD: We retrospectively analyzed serum concentrations of creatinine, cystatin C and BTP measured during inulin clearance tests in children. BTP was measured using a particle-enhanced immunonephelometric assay (Siemens Healthcare). A novel BTP-based eGFR equation was developed using published normal values for children: eGFRBTP[ml/min/1.73m2] = 107.3/BTP/QBTP with QBTP = 0.69. Performance of this equation was compared to the established creatinine-based full age spectrum equation FASage and the cystatin C-based FAScys equations as well as the BTP-based Benlamri equation in terms of bias, % prediction error and P30 and P10 accuracy rates. RESULTS: 322 inulin clearance tests were studied. Overall, our novel equation performed comparably to the creatinine-based FASage and the BTP-based Benlamri equations but was less accurate than FAScys (P30: 79.2 vs 86.3%, p = .008). Combining markers significantly enhanced performance compared to the single marker equations, with the exception of FAScys. CONCLUSION: Rescaled BTP concentrations are a simple method for estimating GFR in children. However, the additional value of BTP for the estimation of GFR compared to rescaled creatinine and cystatin C still remains to be demonstrated.

GFR-estimation by serum creatinine during glucocorticosteroid therapy.

BACKGROUND: While glucocorticosteroids (GCS) are widely used in patients with kidney disease, little is known about their effect on serum creatinine, the most commonly used endogenous marker of kidney function. METHODS: We assessed the effect of GCS on the relationship between estimated GFR using the Schwartz equation (eGFR) and measured GFR using a single-injection inulin clearance (Cin) in children both in a paired analysis and a cross-sectional study. Primary outcome variable was the difference between eGFR and Cin (ΔGFR) in a paired analysis involving 22 patients during and off GCS treatment (mean GFR 103.8 ml/min/1.73 m2, mean prednisone dose 34.8 mg/m2/day). In a cross-sectional analysis in 42 patients receiving GCS (mean dose of 25.7 mg/m2/day), a dose-dependent effect was explored using univariate and multivariate linear regression of various variables including GCS dosage with serum creatinine as dependent variable. RESULTS: The paired analysis showed no significant difference in ΔGFR with or without GCS [- 23 (SD 53) vs. - 9 (SD 41) ml/min/1.73 m2, p = 0.203]. Stepwise multivariate linear regression analysis showed a significant correlation between age and Cin, while GCS dose was not related to serum creatinine. CONCLUSION: GCS use had no significant effect on serum creatinine as a marker for kidney function in a mixed population of renal outpatient clinic children.

Combining GFR estimates from cystatin C and creatinine-what is the optimal mix?

BACKGROUND: Combining estimated glomerular filtration rate (eGFR) equations based on creatinine and cystatin C has been shown to improve the accuracy of GFR estimation. This study aims to optimize this strategy for height-independent GFR estimation in children. METHODS: Retrospective study of 408 inulin clearance tests with simultaneous International Federation of Clinical Chemistry-calibrated measurements of creatinine, cystatin C, and urea in children (mean age 12.5 years, GFR 91.2 ml/min/1.73m2) comparing the arithmetic (meanarith) and geometric means (meangeom) of a height-independent creatinine-based (full age spectrum, based on age (FASage)) and a cystatin C-based equation (FAScys), with the complex height-dependent CKiD3 equation incorporating gender, height, cystatin C, creatinine, and urea. RESULTS: Meangeom had a P30 accuracy of 89.2% compared to meanarith 87.7% (p = 0.030) as well as lower bias and %precision error and performed almost as well as CKiD3 (P30 accuracy 90.9%). Modifying the weight of FASage and FAScys when calculating the means showed that an equal contribution was most accurate in most patients. In spina bifida patients, FAScys alone outperformed any combination. Malignancy or nephritis patients had slightly higher accuracy with weighted means favoring cystatin C or creatinine, respectively. Disagreement between FAScys and FASage was inversely correlated with the accuracy of meangeom. When disagreement exceeded 40%, application of weighted means based on diagnosis improved the performance of eGFR. CONCLUSIONS: In the absence of height data, the optimal strategy for estimating GFR in children is by using the geometric mean of FASage and FAScys. When there is large disagreement between the two, weighted means based on diagnosis improve accuracy.

Accurate eGFR reporting for children without anthropometric data.

INTRODUCTION: Reporting estimated glomerular filtration rate (eGFR) instead of serum concentrations is advised in current guidelines. Most creatinine-based eGFR equations for children require height, a parameter not readily available to laboratories. Combining height-dependent creatinine- and cystatin C-based eGFR improves performance. Recently, a height-independent creatinine-based eGFR equation has been developed. AIM: To compare the combination of height-independent creatinine- and cystatin C-based equations with a combination of equations using anthropometric data. METHODS: Retrospective analysis of 408 pediatric inulin clearance studies with simultaneous height, creatinine, cystatin C and urea measurements. eGFR calculation using the recalibrated Schwartzcrea (height-dependent), FASage (height-independent) and the Schwartzcys equation. The means (Schwartzcrea+Schwartzcys)/2 and (FASage+Schwartzcys)/2 were compared with the CKiD3 equation incorporating cystatin C, creatinine, urea, height and gender in terms of %prediction error and accuracy. RESULTS: All three single parameter equations performed similarly (P30 accuracy around 80%). (FASage+Schwartzcys)/2 (P30 89.2%) and (Schwartzcrea+Schwartzcys)/2 (P30 89.0%), performed comparably to CKiD3 (P30 90.0%). If the difference between the creatinine- and the cystatine C based eGFR was <40%, P30 accuracy of the mean exceeded 90%. CONCLUSION: Combining the height-independent FASage and SchwartzCys equations substantially improves accuracy and performs comparably to height-dependent equations. This allows laboratories to directly report eGFR in children.

Presence of human papillomavirus in semen in relation to semen quality.

STUDY QUESTION: Is the presence of human papillomavirus (HPV) in semen associated with impairment of semen quality? SUMMARY ANSWER: In a large cohort of males seeking fertility evaluation, no associations were observed between seminal HPV presence and semen parameters. WHAT IS KNOWN ALREADY: HPV is commonly detected in semen samples. Whether the presence of HPV is related to impairment of semen quality, remains unclear. STUDY DESIGN, SIZE, DURATION: This cross-sectional study included a cohort of 430 males. PARTICIPANTS/MATERIALS, SETTING, METHODS: Male partners in couples seeking fertility evaluation provided one semen sample per person. Semen samples were tested for HPV-DNA using GP5+/6+-PCR. Sperm concentration was counted and motility was assessed in a Makler counting chamber at a magnification of ×200. The presence of antisperm antibodies was assessed by a mixed agglutination reaction (MAR)-test. MAIN RESULTS AND THE ROLE OF CHANCE: Overall HPV was detected in 14.9% (64/430) of semen samples, including 2.1% (9/430) that contained both high-risk (hr) HPV and low-risk (lr) HPV types, 8.8% (38/430) with exclusively hrHPV types and 4.0% (17/430) with exclusively lrHPV types. The presence of HPV in semen was not associated with the age of the participants, seminal pH, semen volume, total sperm count, sperm concentration, progressive motility or the presence of antisperm antibodies. LIMITATIONS, REASONS FOR CAUTION: This study did not observe an association between HPV presence in semen and impairment of semen quality. However, we cannot exclude an effect of seminal HPV on early embryo development and clinical reproductive outcomes. WIDER IMPLICATIONS OF THE FINDINGS: As HPV is frequently present in semen, screening of donor semen for HPV should be considered to prevent iatrogenic cervical HPV infections in the recipient. However our findings do not support standardized HPV testing of semen in the diagnostic work-up of subfertile couples. STUDY FUNDING/COMPETING INTERESTS: This study was sponsored by an unrestricted grant of Stichting Researchfonds Pathology Amsterdam, the Netherlands. P.J.F.S. has been on the speakers bureau of Roche, Gen-Probe, Abbott, Qiagen and Seegene and has been a consultant for Crucell B.V. J.B. has been on the speakers bureau of Qiagen and has been a consultant for Roche, DDL Diagnostic Laboratory, GlaxoSmithKline and Merck. D.A.M.H. has been member of the scientific advisory boards of Amgen and Pfizer, and has been on the speakers bureau of Hologic/Gen-Probe. C.J.L.M.M. has been on the speakers bureau of GlaxoSmithKline, Qiagen, Merck, Roche, Menarini and Seegene, has served occasionally on the scientific advisory board of GlaxoSmithKline, Qiagen, Merck, Roche and Genticel, and has occasionally been a consultant for Qiagen. Formerly, C.J.L.M.M. was a minority shareholder of Delphi Biosciences, which bankrupted in 2014. C.J.L.M.M. is a minority shareholder of Diassay B.V. P.J.F.S., D.A.M.H. and C.J.L.M.M. have minority stake in Self-Screen B.V., a spin-off company of VU University Medical Center. R.L., M.G.D., P.G.A.H., D.T.M.P., and I.H. do not have any conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Technical performance of a novel, fully automated electrochemiluminescence immunoassay for the quantitation of ß-amyloid (1-42) in human cerebrospinal fluid.

INTRODUCTION: Available assays for quantitation of the Alzheimer's disease (AD) biomarker amyloid-beta 1-42 (Aß [1-42]) in cerebrospinal fluid demonstrate significant variability and lack of standardization to reference measurement procedures (RMPs). We report analytical performance data for the novel Elecsys ß-amyloid (1-42) assay (Roche Diagnostics). METHODS: Lot-to-lot comparability was tested using method comparison. Performance parameters were measured according to Clinical & Laboratory Standards Institute (CLSI) guidelines. The assay was standardized to a Joint Committee for Traceability in Laboratory Medicine (JCTLM) approved RMP. RESULTS: Limit of quantitation was <11.28 pg/mL, and the assay was linear throughout the measuring range (200-1700 pg/mL). Excellent lot-to-lot comparability was observed (correlation coefficients [Pearson's r] >0.995; bias in medical decision area <2%). Repeatability coefficients of variation (CVs) were 1.0%-1.6%, intermediate CVs were 1.9%-4.0%, and intermodule CVs were 1.1%-3.9%. Estimated total reproducibility was 2.0%-5.1%. Correlation with the RMP was good (Pearson's r, 0.93). DISCUSSION: The Elecsys ß-amyloid (1-42) assay has high analytical performance that may improve biomarker-based AD diagnosis.

Engagement of SIRPα inhibits growth and induces programmed cell death in acute myeloid leukemia cells.

BACKGROUND: Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia. DESIGN AND METHODS: We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs. RESULTS: By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0-M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs. CONCLUSIONS: Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.

In vitro sensitivity and cross-resistance to deoxynucleoside analogs in childhood acute leukemia.

BACKGROUND AND OBJECTIVES: Cytarabine (ara-C) is a key drug in the treatment of acute leukemia. Resistance to ara-C might be circumvented by the use of other deoxynucleoside analogs. DESIGN AND METHODS: Using the MTT assay, we determined in vitro sensitivity and cross-resistance to deoxynucleoside analogs in 362 acute leukemia samples from untreated children and 32 normal bone marrow mononuclear cell samples. RESULTS: Normal bone marrow samples were significantly more resistant to ara-C, cladribine and fludarabine than were acute myeloid leukemia (AML) samples and significantly more resistant to ara-C and fludarabine than were acute lymphoblastic leukemia (ALL) samples. The only drug to which AML samples were more sensitive in vitro than ALL was cladribine. AML FAB M5 was significantly more sensitive in vitro to ara-C and cladribine than FAB M1/2 or FAB M4. T-ALL was significantly more resistant to cladribine than B-cell precursor ALL. A paired analysis of 60 AML and 99 ALL samples demonstrated significant cross-resistance between all four deoxynucleoside analogs. Cross-resistance was also observed between ara-C and etoposide (Rp=0.54, p<0.0001), and ara-C and daunorubicin (Rp=0.48, p<0.0001) in AML. In ALL blasts, cross-resistance was observed between ara-C and vincristine (Rp=0.50; p<0.0001), and between ara-C and daunorubicin and L-asparaginase (Rp=0.25; p=0.01; Rp=0.28; p=0.005). INTERPRETATION AND CONCLUSIONS: Cladribine appears to be a useful drug in AML, particularly in FAB M5. We observed cross-resistance between ara-C and other deoxynucleoside analogs, as well as between ara-C and drugs with different modes of action in childhood acute leukemia.

In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant.

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.

The effect of G-CSF on the in vitro cytotoxicity of cytarabine and fludarabine in the FLAG combination in pediatric acute myeloid leukemia.

The combination of fludarabine, cytarabine (ara-C) and G-CSF (FLAG) is routinely used in the treatment of acute myeloid leukemia (AML). In this study we characterized the interactions between fludarabine, ara-C and G-CSF in vitro using AML blasts. Exposure to G-CSF alone resulted in a higher leukemic cell survival (LCS), which might be indicative of increased proliferation. The LCS decreased significantly from 69.7 to 54.0% when blasts were exposed to G-CSF 21 h prior to incubation with ara-C (p=0.01). In contrast, LCS increased significantly (from 55.6 to 69.0%; p=0.04) after sequential exposure to G-CSF and fludarabine. Exposure to 4 combinations of fludarabine (4 h; 0.14 microM and 0.55 microM) and ara-C (96 h; 0.21 and 0.82 microM) (FLA) resulted in additive cytotoxicity. The triple combination (FLAG), 21 h 5 microM G-CSF followed by 4 h fludarabine (0.14 and 0.55 microM) and finally ara-C (0.21 and 0.82 microM) for 96 h also resulted in an additive cell kill. In conclusion, these data support the clinical use of G-CSF in combination with ara-C, and the combination of ara-C and FLA. Pre-exposure to G-CSF before FLA (FLAG) did not result in increased cytotoxicity in our experiments, indicative of similar anti-leukemic activity.

Aphidicolin decreases ex vivo resistance to cytosine arabinoside in childhood acute leukaemia.

In order to assess the effect of the DNA polymerase inhibitor aphidicolin on resistance to cytosine arabinoside, blast cells from 15 children with ALL and 9 with AML were exposed to a range of concentrations of ara-C +/- aphidicolin. Cell survival was measured using the MTT assay. Aphidicolin significantly increased sensitivity to ara-C in blast cells from both ALL (p=0.001) and AML (p<0.01). The median fold increase (sensitisation ratio) for ALL was 3.4 (range 1.2-13.6) compared to 12.4-fold (range 6.0-148) for AML blasts (p=0.005). There was a striking relationship between increasing ara-C resistance and increasing effect of aphidicolin in AML (p<0.001) but not ALL (p>0.05). These remarkable results suggest that aphidicolin should be considered for future clinical trials as a modulator of ara-C resistance, particularly in AML.