Francisco V. Álvarez

Cell-Free DNA as a Noninvasive Acute Rejection Marker in Renal Transplantation

BACKGROUND Acute rejection (AR) is a key conditioning factor for long-term graft function and survival in renal transplantation patients. The standard care with creatinine measurements and biopsy upon allograft dysfunction implies that AR is usually detected at advanced stages. Rapid noninvasive biomarkers of rejection are needed to improve the management of these patients. We assessed whether total cell-free DNA (tCF-DNA) and donor-derived cell-free DNA (ddCF-DNA) were useful markers for this purpose, both in plasma and in urine. METHODS Plasma and urine samples from 100 renal transplant recipients were obtained during the first 3 months after transplantation. tCF-DNA and ddCF-DNA were analyzed by quantitative PCR for the HBB (hemoglobin, beta) and the TSPY1 (testis specific protein, Y-linked 1) genes, respectively. We observed 19 episodes of AR, as well as other complications, such as acute tubular necrosis, nephrotoxicity, and infections. RESULTS Plasma tCF-DNA concentrations increased markedly during AR episodes, often before clinical diagnosis, and returned to reference values after antirejection treatment. A cutoff plasma tCF-DNA concentration of 12 000 genome equivalents/mL correctly classified AR and non-AR episodes in 86% of posttransplantation complications (diagnostic sensitivity, 89%; specificity, 85%). Although similar increases were observed during severe posttransplantation infections, use of the combination of plasma tCF-DNA and procalcitonin (PCT), a specific marker of sepsis, significantly improved the diagnostic specificity (to 98%; 95% CI, 92%-100%), with 97% of the episodes being correctly classified. Use of transrenal DNA and ddCF-DNA concentrations did not add relevant information. CONCLUSIONS Given that renal biopsy is the gold standard for detecting AR, analysis of both plasma tCF-DNA and PCT could permit a more selective use of this invasive procedure.

Usefulness of cell-free plasma DNA, procalcitonin and C-reactive protein as markers of infection in febrile patients

Background Circulating nucleic acids were discovered more than 60 y ago. With the recent developments in the study of circulating nucleic acids, its application in the diagnostic field has increased. The objective of this study was to assess the usefulness of the quantification of cell-free plasma DNA (CF-DNA) concentration in the diagnosis of infections in febrile patients and as a prognostic marker in septic patients. Methods Concentrations of CF-DNA, procalcitonin (PCT) and C-reactive protein (CRP) were measured in 110 febrile patients who were clinically diagnosed with fever of unknown origin, localized infection, sepsis or septic shock. Results Concentrations of CF-DNA increase according to the severity of the infection. The best cut-off point for predicting infection was 2800 GE (genome equivalents)/mL (sensitivity: 95.0%; specificity: 96.7%) and 14,000 GE/mL for sepsis prediction (sensitivity: 77.8%; specificity: 94.6%). Higher concentrations of CF-DNA were found in exitus septic patients than in survivors. The diagnostic efficiency of CF-DNA was similar to PCT and higher than CRP in infectious processes. Conclusions Normal concentrations of CF-DNA can exclude the presence of an infection in febrile patients, and very high concentrations (>10-fold over the normal reference range) stratify the severity of infections, showing a high prognostic value to predict mortality in the absence of other causes for elevated CF-DNA.

New biomarkers in diagnosis of early onset preeclampsia and imminent delivery prognosis.

Abstract Background: Several studies have revealed a high soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth factor (PlGF) ratio in preeclamptic women. However, its role in patients with suspected preeclampsia (PE) at triage in the emergency department remains an issue and a controversial unique cutpoint of 85 has been proposed regardless of gestational age. A new cutpoint for sFlt-1/PlGF ratio was investigated to rule out PE at obstetric triage, and to assess its prognostic value for risk of imminent delivery. Methods: Blood samples from 257 pregnant women with suspected PE were obtained at obstetric triage admission. Serum PlGF and sFlt-1 were measured by an electrochemoluminiscence immunoassay (ECLIA) on the immunoanalyzer Cobas e601 (Roche Diagnostics) and the corresponding ratio was calculated. Final outcomes (mainly development of PE) were reviewed and time between clinical presentation and delivery was calculated. Results: The best ratio cutpoint to diagnose PE changed according to gestational age: 23 (92.0% sensitivity, 81.1% specificity) and 45 (83.7% sensitivity, 72.6% specificity) for women <34 and ≥34 weeks’ gestation, respectively. Furthermore, sFlt-1/PlGF ratio inversely correlated with time elapsed between clinical presentation and delivery, and a cutpoint of 178 could predict complications such as imminent delivery or fetal/neonatal death with a sensitivity of 70.6% and a specificity of 97.8%. Conclusions: The new cut-off values for the sFlt-1/PlGF ratio adjusted by the gestational age at clinical presentation can be used to rule out PE at obstetric triage and to predict imminent delivery with better accuracy than the cutpoint currently accepted.

Non-invasive fetal RHD genotyping in the first trimester of pregnancy.

Abstract Background: Hemolytic disease of the fetus and newborn (HDN) is caused primarily by feto-maternal RhD incompatibility. Although all RhD negative pregnant women undergo routine antenatal RhD prophylaxis at 28 weeks of gestation, and following delivery if the newborn is RhD positive, HDN has not been eradicated. Here, we investigated fetal Rhesus D (RHD) genotype in maternal plasma during the first trimester of pregnancy in our area. Methods: Plasma samples were obtained from 111 RhD negative pregnant women, between 9 and 13 weeks of gestation. DNA from maternal plasma containing cell-free fetal DNA (cffDNA) was analyzed by quantitative PCR (qPCR) to detect RHD exons 5 and 7. A β-globin (HBB) sequence was quantified to estimate total DNA concentration. qPCR results were compared with newborn RhD determined in cord blood serum. The influence of several gestational parameters on DNA concentration was also analyzed. Results: The specificity and sensitivity of the assay was 93% and 100%, respectively, with 97% diagnostic accuracy. Cell-free DNA concentrations during the first trimester of pregnancy were not affected by the gestational parameters studied (free-β fraction of human chorionic gonadotropin and pregnancy-associated plasma protein A concentrations, fetal sex, materno-fetal ABO blood group incompatibility, maternal weight and gestational age). Conclusions: Non-invasive fetal RHD genotyping during the first trimester of pregnancy can be determined with a high specificity, thus representing a valuable tool for improving the management of RhD negative pregnant women. As a high percentage of pregnant women participate in the routine first trimester combined screening program for aneuploidies, the fetal RHD study could be of immediate implementation, since the same blood collection could be used. Clin Chem Lab Med 2010;48:1121–6.

Optimization of nucleated red blood cell (NRBC) recovery from maternal blood collected using both layers of a double density gradient

The isolation of fetal nucleated red blood cells (NRBC) from maternal blood represents a promising approach to non‐invasive prenatal diagnosis. However, the number of fetal NRBC in maternal circulation is quite low and therefore difficult to isolate. An enrichment procedure in which both layers from a double density 1.077/1.107 g/ml gradient are collected was optimized, followed by MACS selection using non‐commercial monoclonal antibodies. The influence of the delay in processing maternal blood on the NRBC distribution in both interfaces of the gradient was also studied in cord blood and peripheral maternal blood samples. A significant increase in the number of NRBC isolated from maternal blood was achieved by collecting both layers of the double density gradient compared with the previous protocol in which only the lower layer was recovered. Cord blood samples showed significant differences in the number of NRBC recovered when processed at 24 instead of within 3 h. This effect was also observed in the number of NRBC collected only from the upper layer of peripheral maternal blood samples. Therefore, in order to minimize the target cell losses, it is advisable to process the maternal blood samples as soon as possible. Copyright

Plasma procalcitonin measured by time-resolved amplified cryptate emission (TRACE) in liver transplant patients. A prognosis marker of early infectious and non-infectious postoperative complications

Abstract Background: Elevated procalcitonin (PCT) levels are observed after major surgery, such as orthotopic liver transplantation (OLTx). The aim of this observational study was to evaluate PCT kinetics during the first 5 following days after surgery to establish the prognostic value of PCT changes in the outcome of OLTx, and to predict medical, technical and infectious complications. PCT was also evaluated in the differential diagnosis of infection vs. rejection. Methods: A total of 64 OLTx were performed in 58 patients; they were split into two groups: with and without complications. Out of these patients, 18 developed infection, and nine rejection. PCT was measured before and during surgery, 12 h after transplantation and daily for the 5 following days. PCT was also measured the day when infection or rejection was diagnosed, and on the previous day. PCT was determined by time-resolved amplified cryptate emission (TRACE) technology. Results: PCT elevation began at 12 h after surgery, reaching a peak on the 1st day in both groups. Significantly higher PCT concentrations were found in the group of patients developing complications, on the 5 postoperative days. It was found that a 24 h PCT value higher than 1.92 μg/L increased by 9.1-time-fold the risk of complications. When infection was diagnosed, a second peak of PCT was observed, but no PCT elevation was shown in rejection. Conclusions: Daily monitored PCT provides valuable information about the early outcome of OLTx. Clin Chem Lab Med 2008;46:660–6.

Cord blood plasma reference intervals for potential sepsis markers: Pro-adrenomedullin, pro-endothelin, and pro-atrial natriuretic peptide

OBJECTIVES To establish reference values in cord blood of the following new sepsis markers: pro-adrenomedullin (MR-proADM), pro-endothelin (CT-proET-1), and pro-atrial natriuretic peptide (MR-proANP). METHODS MR-proADM, CT-proET-1, MR-proANP, and procalcitonin (PCT) were measured in cord blood of newborn infants by Time Resolved Amplified Cryptate Emission (TRACE) technology. The inclusion criteria in the control group (n=194) was the absence of any clinical sign or risk factor of sepsis. A group of 73 newborn infants presenting with risk factors of sepsis at delivery was also studied. RESULTS The median values (reference interval) of CT-proET-1, MR-pro-ADM, and MR-proANP measured in cord blood plasma were 72 pmol/L (39-115), 0.84 nmol/L (0.5-1.38), and 163 pmol/L (76-389), respectively. The PCT reference interval was not significantly different from that previously described in cord blood serum. CONCLUSIONS The reference intervals established will serve as a starting point for further clinical investigations aimed to elucidate the potential prognostic/diagnostic value of these markers in neonatal sepsis management.

Umbilical cord blood serum procalcitonin by Time-Resolved Amplified Cryptate Emission (TRACE) technology : reference values of a potential marker of vertically transmitted neonatal sepsis

BACKGROUND Neonatal infection remains a major diagnostic problem because of non-specific clinical signs and symptoms, as well as low sensitivity and specificity of routine laboratory tests. C-reactive protein (CRP), white blood cell count, absolute neutrophil count and immature/total neutrophil ratio are the most widely used tests in the diagnosis of sepsis and provide useful information, but none of these has demonstrated to be reliable in detecting all septic infants. Procalcitonin (PCT) has been suggested as a potentially useful laboratory test performed in umbilical cord blood when perinatal bacterial sepsis is under investigation. METHODS In this study, the reference interval for umbilical cord blood serum PCT was established for the first time by Time-Resolved Amplified Cryptate Emission (TRACE) technology. RESULTS The reference interval for PCT in umbilical cord blood serum ranged from 0.04 to 0.43 microg/L in 168 non-infected newborn infants (95% CI 0.02-0.06 and 0.35-0.60 microg/L, respectively). Cord blood serum PCT correctly classified one infected patient out of 90 newborn infants at risk of vertically transmitted sepsis and identified another neonate as a potentially infected patient despite having negative blood cultures. However, cord blood CRP misclassified 21 out of the 90 patients as infected neonates. CONCLUSIONS Cord blood PCT measured by TRACE is a potentially more useful early marker of neonatal sepsis than cord blood CRP.

N-Terminal pro-Brain natriuretic peptide as a potential non-invasive marker of cardiac transplantation rejection

Background: Transplantation is the main palliative treatment for patients with heart failure. Clinical signs of cardiac rejection can be very non-specific or even absent. Thus, successfull management relies on early diagnosis, ideally before the onset of clinical features of cardiac dysfunction. Although endomyocardial biopsy (EMB) is the reference diagnostic method, several non-invasive methods have been proposed to reduce the number of EMB performed during the follow-up of the transplanted patient. The aim of the present work was to study the potential relationship between rejection and serum concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) as well as cardiac troponin T (cTnT) in post-transplantation patients. Methods: Twenty-three consecutive orthotopic heart transplantation recipients with a mean age of 51 years (range 22-66) were prospectively recruited from the cardiac transplantation programme at the Hospital Universitario Central de Asturias. Serum NT-proBNP and cTnT were measured during the follow-up of these patients (ranging from 9-13 months post-transplantation) and compared with the results of EMB. Results: Serum NT-proBNP concentrations progressively decrease during the first year post-transplantation, reaching concentrations slightly higher than the reference values. NT-proBNP concentrations increase significantly in those patients with a rejection episode graded ≥3A on the basis of the EMB (P < 0.001, Mann-Whitney U-test). No relation between cTnT and rejection was observed. Conclusions: The potential of NT-proBNP as a non-invasive marker of transplantation rejection shows promising results, since NT-proBNP concentrations increase whenever a significant rejection event takes place in the first year of follow-up.

Role of vitamin D and sFlt-1/PlGF ratio in the development of early- and late-onset preeclampsia

Abstract Background: The imbalanced production of placental biomarkers and vitamin D deficiency have been proposed as risk factors for the development of preeclampsia (PE). However, little is known about the relationship between them and their role in early- versus late-onset PE. The objectives were to assess the role of 25-hydroxyvitamin D [25(OH)D] concentrations and the soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth factor (PlGF) ratio in the development of early- and late-onset PE; and to evaluate the relationship between 25(OH)D and the biomarkers. Methods: A retrospective, full-blinded cohort study was conducted at the Obstetric Emergency Service of a tertiary care hospital. Pregnant women (n=257) attending obstetric triage with suspicion of PE were included. sFlt-1, PlGF and 25(OH)D concentrations were measured by electrochemoluminescence (ECLIA) immunoassay and pregnancy outcome (development of PE) was registered from patients records. Results: PE women showed lower 25(OH)D concentrations at clinical presentation than non-PE women (median: 35.0 nmol/L and 39.6 nmol/L, respectively; p=0.027). Women with 25(OH)D levels <50 nmol/L experienced an increased risk of developing late-onset PE [odds ratio (OR) 4.6, 95% confidence interval (CI) 1.4–15], but no association was found for early-onset PE. However, a sFlt-1/PlGF ratio above the corresponding cutpoints increased the risk of developing both early- and late-onset PE [ORs 58 (95% CI 11–312) and 12 (95% CI 5.0–27), respectively]. No association was found between 25(OH)D levels and sFlt-1/PlGF ratio. Conclusions: Low vitamin D status in women with suspected late-onset PE increases the risk of imminent development of the disease.