Development of an algorithm for the identification of leukemic hematolymphoid neoplasms in Primary Care patients.

BACKGROUND: Diagnosis of hematolymphoid neoplasm (HLN) requires different technologies which are performed on a patient basis instead of per protocol. We hypothesize that integration of hematimetric and cytological analysis along with multiparametric flow cytometry (MFC) provides a framework to evaluate peripheral blood (PB) samples from Primary Care. METHODS: Samples from patients with persistent (>3 months) lymphocytosis (>5 × 109/L) and/or monocytosis (>109/L) or the presence of atypical and/or blast cells upon the smear review were analyzed by MFC concurrent to cytological analysis. MFC studies were carried out following standardized procedures. RESULTS: In a 3-year period, smear review and MFC were performed simultaneously in 350 samples, demonstrating HLN in 194 cases (55.4%). In 156 cases, reactive cell populations were found. The combination of age, absolute lymphocyte count (ALC), hemoglobin and platelets provided the best correlation with MFC for the presence of a chronic lymphoproliferative disorder (CLPD) in lymphocytosis [area under the curve (AUC) 0.891, p < 0.05]. A model evaluating the probability of CLPD has been proposed and validated in an independent cohort. CONCLUSIONS: A strategy to perform MFC studies following standardized procedures has proven to be useful to evaluate samples from patients in Primary Care centers for HLN diagnosis or reactive conditions, providing a sensitive and rapid clinical orientation and avoiding unnecessary consultations in routine clinical practice. The probability for the presence of CLPD in PB can be calculated and help guide decision-making regarding further testing.

Strontium and its role in preeclampsia.

BACKGROUND: Preeclampsia (PE) is considered a specific vascular disease in which endothelial dysfunction may be the crucial factor of its pathogenesis. It has been suggested that strontium (Sr) may play a role in the pathophysiology of PE. Our group established in a previous study the serum levels of Sr in healthy pregnancies, and the main aim of the present study was to evaluate Sr concentrations and oxidative status in preeclamptic women. METHODS: The study population included women with early-onset PE (E-PE, n = 39), late-onset PE (L-PE, n = 67) and serial samples from a subset of preeclamptic women (PE-ss, n = 20). The control group included women with gestational hypertension (GH, n = 56) and healthy pregnancies (samples collected in the 1st (n = 50), 2nd (n = 51) and 3rd trimesters (n = 53)). Strontium, calcium (Ca), uric acid (UA), placental growth factor (PlGF), soluble fms-like tyrosine kinase 1 (sFlt-1), N-terminal pro-brain natriuretic peptide (NT-proBNP), lipid peroxidation and total antioxidant activity (TAA) were measured in these samples. RESULTS: Mean Sr levels were significantly higher in PE than in control groups (p ≤ 0.0001). Calcium values were found to be significantly lower in E-PE compared to control groups (p = 0.03). Higher levels of NT-proBNP were found in PE vs. control groups (p < 0.001). sFlt-1/PlGF ratio was higher in E-PE compared to L-PE and GH (p < 0.001). Uric acid levels in PE were significantly higher than in control groups (p < 0.0001). There was a strong positive correlation between UA and Sr in the E-PE serial samples (r = 0.80, p < 0.0001). Lipid peroxidation and lipid peroxidation/TAA ratios were found to be higher in PE, with lower values of TAA. CONCLUSION: The higher levels of Sr and the alterations of redox status found in preeclamptic women, along with the strong correlation between UA and Sr suggest that this element may be involved in the pathogenesis of PE.

Strontium and oxidative stress in normal pregnancy.

BACKGROUND: Pregnancy brings about metabolic and oxidative changes that involve various trace elements and oxidative stress. Strontium (Sr) is a trace element scarcely studied in this context, although it has been suggested that it may play a role in the pathophysiology of preeclampsia. The main aim of this study was to evaluate Sr concentrations and oxidative status in normal pregnancy. METHODS: The study population included non-pregnant women (n=31), healthy pregnant women in the first (n=50), second (n=51) and third (n=53) trimesters of gestation, and women in postpartum period (n=31). Additionally, samples from another twenty pregnant women were obtained in the three trimesters. Strontium, copper, selenium and zinc were measured by inductively coupled plasma-mass spectrometry. Calcium (Ca), uric acid (UA), lipid peroxidation and total antioxidant activity (TAA) were measured by spectrophotometric assays. RESULTS: Strontium remained unchanged until the third trimester of pregnancy, in which significantly higher levels were found (p=0.001). The other elements showed diverse trends during pregnancy. Uric acid levels were significantly different in all groups (p<0.001), increasing gradually as the pregnancy progresses. In serial samples, there was a statistically significant positive correlation between Sr and gestational week of sampling (r=0.31, p=0.01), UA (r=0.40, p=0.001) and lipid peroxidation/TAA ratio (r=0.38, p=0.0002). Additionally, Sr correlated negatively with TAA (r=-0.40, p=0.0001). CONCLUSION: Strontium seems to play a physiological role in the oxidative status of the human organism. Further studies involving Sr and pathologies of pregnancy are warranted.

Variation of trace element concentrations in patients undergoing hemodialysis in the north of Spain.

BACKGROUND: Trace elements are essential substances for the proper physiological and biochemical functioning of the organism. Hemodialysis patients are potentially at risk of deficiency or excess of these elements. The application of inductively coupled plasma mass spectrometry (ICP-MS) allows the simultaneous quantification of very small amounts of multiple trace elements. The aim was to measure the serum concentration of copper (Cu), zinc (Zn), selenium (Se), and nickel (Ni), and the whole blood concentration of arsenic (As), lead (Pb), and manganese (Mn), in patients undergoing hemodialysis as well as in controls. METHODS: The study was carried out in 57 hemodialysis patients compared with 57 controls with normal renal function. Serum and whole blood samples from the dialysis group were collected before and after hemodialysis sessions and Cu, Zn, Se, Ni, As, Pb and Mn levels were determined using ICP-MS. RESULTS: Hemodialysis patients showed significantly lower blood levels of Cu, Zn and Se than controls (p < 0.001) and higher concentrations of Ni, As and Pb (p < 0.0001). The levels of Mn were similar in both groups. After performing hemodialysis, Cu, Zn, Se and Ni concentrations were significantly higher than the pre-hemodialysis levels (p < 0.0001). However, the concentration of As decreased (p < 0.0001) and Pb and Mn levels were not significantly altered after the dialysis session. CONCLUSION: Hemodialysis patients are at increased risk of trace elements deficiency (especially for Zn and Se) or excess (Ni) in respect to healthy subjects. Monitoring of blood levels and supplementation of some trace elements may be indicated in patients undergoing hemodialysis.

Challenges for Worldwide Harmonization of Newborn Screening Programs.

BACKGROUND: Inherited metabolic disorders (IMDs) are caused by a defect in a metabolic pathway, leading to malfunctioning metabolism and/or the accumulation of toxic intermediate metabolites. To date, hundreds of IMDs have been identified. Many of these diseases are potentially fatal conditions that are not apparent at birth. Newborn screening (NBS) programs involve the clinical and laboratory examination of neonates who exhibit no health problems, with the aim of discovering those infants who are, in fact, suffering from a treatable condition. CONTENT: In recent years, the introduction of tandem mass spectrometry has allowed the expansion of screening programs. However, this expansion has brought a high degree of heterogeneity in the IMDs tested among different NBS programs. An attempt to harmonize the metabolic conditions recommended to be screened has been carried out. Two uniform screening panels have been proposed in the US and European Union, by knowledgeable organizations. Here, we review current evidence-based processes to assess and expand NBS programs. We also discuss the IMDs that have recently been introduced in some screening programs, such as severe combined immunodeficiencies, lysosomal storage disorders, and adrenoleukodystrophy. SUMMARY: NBS programs have been an established public health function for more than 50 years to efficiently and cost-effectively identify neonates with severe conditions. However, NBS is not yet optimal. This review is intended to elucidate the current degree of harmonization of NBS programs worldwide as well as to describe the major controversial points and discuss the multiple challenges that must be confronted in expanded NBS strategies.

Neurofilament medium polypeptide (NFM) protein concentration is increased in CSF and serum samples from patients with brain injury.

BACKGROUND: Brain injury is a medical emergency that needs to be diagnosed and treated promptly. Several proteins have been studied as biomarkers of this medical condition. The aims of this study were to: 1) evaluate the selectivity and precision of a commercial ELISA kit for neurofilament medium polypeptide (NFM) protein; and 2) evaluate the concentration in cerebrospinal fluid (CSF) and serum of healthy individuals and patients with brain damage. METHODS: An ELISA from Elabscience was used. The selectivity was evaluated using size-exclusion chromatography and mass spectrometry. Intra- and inter-batch coefficients of variation (CV) were also studied. Fifty-one CSF samples from 36 age-matched patients with hemorrhagic stroke (HS) (n=30), ischemic stroke (IS) (n=11) and healthy individuals (n=10) were assayed. In addition, serum samples from healthy volunteers (n=47), 68 serum samples from seven patients with HS, 106 serum samples from 12 patients with traumatic brain injury (TBI) and 68 serum samples from 68 patients with mild traumatic brain injury (mTBI) were also analyzed. RESULTS: NFM was identified in the chromatographic fraction with highest immunoreactivity. The intra- and inter-batch CVs were ≤10% and ≤13%, respectively. The CSF-NFM concentration in HS was significantly higher (p<0.0001) than in IS and controls. Serum NFM concentration ranged from 0.26 to 8.57 ng/mL in healthy individuals (median=2.29), from 0.97 to 42.4 ng/mL in HS (median=10.8) and from 3.48 to 45.4 ng/mL in TBI (median=14.7). Finally, 44% of patients with mTBI had increased NFM concentration, with significantly higher levels (p=0.01) in patients with polytrauma. CONCLUSIONS: To our knowledge this is the first study describing increased NFM levels in CSF and serum from patients with brain damage.

Diagnosis of infection in critical care.

Sepsis is the primary cause of death in the intensive care unit. The prevention of sepsis complications requires an early and accurate diagnosis as well as the appropriate mon itoring. A deep knowledge of the immunologic basis of sepsis is essential to better understand the scope of incorporating a new marker into clinical practice. Besides revising this theoretical aspect, the current available tools for bacterial iden tification have been briefly reviewed as well as a variety of new markers showing either well-recognized or potential usefulness for diagnosis and prognosis of infections in crit ically ill patients. Particular conditions such as community acquired pneumonia, pedi atric sepsis, or liver transplantation, among others, have been separately treated, since the optimal approaches and markers might be different in these special cases.

Identification of novel biomarkers of brain damage in patients with hemorrhagic stroke by integrating bioinformatics and mass spectrometry-based proteomics.

Hemorrhagic stroke (HS) is a significant cause of mortality that requires rapid diagnosis and prompt medical attention. A time-efficient diagnostic test to assist in the early classification of patients with stroke would be of great value. The aims here were to (a) select "brain-specific" proteins using a bioinformatics approach, (b) develop selected reaction monitoring (SRM) assays for candidate proteins, and (c) quantify these proteins in cerebrospinal fluid (CSF). "The Human Protein Atlas" and the "Peptide Atlas" were used to select proteins specifically and abundantly expressed in brain tissue, excluding high-abundance plasma proteins. Protein extracts from brain tissue were used for SRM assay development of proteins of interest. The levels of 68 "brain-specific" proteins were measured by SRM in 36 age-matched patients, including individuals with HS (n = 15), ischemic stroke (n = 11), and controls (n = 10). Additionally, S100B was measured using an electrochemoluminometric immunoassay. CSF levels of S100B and eight of the "brain-specific" proteins (NSE, GFAP, α-Inx, MBP, MT3, NFM, ß-Syn, and γ-Syn) were increased in a subset of samples from HS patients, especially in those individuals with intraventricular hemorrhage and poor outcome. Seven of these proteins (S100B, NSE, GFAP, α-Inx, MBP, NFM, and ß-Syn) showed significant differences between patients with and without brain hemorrhage. Novel biomarkers of brain injury (α-Inx, NFM, and ß-Syn) were identified in the CSF of patients with HS. Investigating the role of these proteins in blood with more sensitive methods is warranted.

Falso incremento de la hormona estimulante del tiroides asociado a la presencia de macro-TSH / Falsely elevated levels of thyrotropin caused by macro-TSH

Mujer que presentó un importante incremento de la hormona estimulante del tiroides (TSH) (62,2 mU/L) con hormonas tiroideas dentro de los intervalos de referencia. La paciente se encontraba eutiroidea y no presentaba bocio. Se realizó un estudio inicial para determinar la posible causa del incremento en la concentración de TSH. La recuperación de TSH tras precipitación con polietilenglicol fue del 1%, sugiriendo la presencia de alguna molécula de elevado peso molecular que podría interferir en la determinación. Mediante cromatografía de exclusión, se confirmó la presencia de macro-TSH, un complejo autoinmune formado por TSH unido a una Inmunoglobulina G que es inmunorreactivo pero biológicamente inactivo, por lo que, si no se detecta, induce a una interpretación errónea de la concentración de TSH (AU)

Woman showing an important increase of serum TSH (62.2 mU/L) with thyroid hormones within the reference interval. The patient was clinically euthyroid and without goitre. Investigations were carried out to determine the origin of the unexpected high TSH. Polyethylene glycol precipitation test showed low TSH recovery (1%), indicating the presence of large molecules that could interfere with the measurement. The serum sample was fractionated by gel filtration chromatography and the presence of a macro-TSH form was confirmed, an immunoreactive but biologically inactive TSH-Immunoglobulin G autoantibody complex. Its detection is important to avoid a misleading interpretation of the TSH concentration (AU)

Valor pronóstico postoperatorio del extremo aminoterminal del propéptido natriurético tipo B en el trasplante cardíaco / Prognostics value of NT-proBNP after heart transplantation

Introducción. El trasplante cardíaco es una opción terapéutica disponible para algunos pacientes con insuficiencia cardíaca avanzada. Es importante establecer el pronóstico que presentan estos pacientes para poder diferenciar aquéllos con peor expectativa de vida y adoptar medidas adicionales en estos casos. Los péptidos natriuréticos cardíacos han demostrado su utilidad diagnóstica y pronóstica en diferentes enfermedades. El objetivo de este estudio es valorar la utilidad del extremo aminoterminal del propéptido natriurético tipo B (NT-proBNP) postoperatorio en el pronóstico de los pacientes trasplantados cardíacos a corto plazo. Materiales y métodos. determinó la concentración de NT-proBNP a los 15 días postoperatorios en 50 pacientes que recibieron trasplante cardíaco para valorar la utilidad pronóstica de mortalidad a 6 meses de seguimiento. Resultados. Los pacientes que fallecieron mostraron concentraciones de NT-proBNP significativamente superiores que los que sobrevivieron, con una mediana de la concentración de NT-proBNP a los 15 días postrasplante de 20.632ng/l (intervalo interculartílico: 6.183 a 37.925ng/l) frente a 3.923ng/l (intervalo interculartílico: 1.752 a 6.890ng/l) respectivamente. Se observó que el hazard-ratio de mortalidad se multiplica por 8,5 veces (IC del 95%: 1,7–44,2) en el grupo de pacientes con concentraciones de NT-proBNP, cuantificadas a los 15 días postrasplante, superiores al valor discriminatorio de 7.500ng/l (AU)

Introduction. Cardiac transplantation is a widely accepted option for the treatment of end-stage congestive heart failure. In order to identify those patients at risk of short life expectancy, it could be worthwhile to establish an individual prognosis factor. The prognostic and diagnostic values of natriuretic peptides have been studied in different areas of clinical practice. The objective of this study was to evaluate the short-term prognostic ability of NT-proBNP concentration in heart transplantation patients. Materials and methods. The group studied consisted of 50 adult heart transplant patients. NT-proBNP concentration was measured in each patient 15 days after surgery to evaluate the prognostic value at 6 months follow up. Results. The non-survivor patients showed higher NT-proBNP concentrations than survivors, with a median of NT-proBNP concentration on the 15th day post-transplantation of 20632ng/L (IIC: 6183 to 37925ng/L) and 3923ng/L (IIC: 1752 to 6890ng/L) in the non-survivors and the survivors groups, respectively. The hazard ratio of mortality was 8.5 times higher (95% CI: 1.7 to 44.2) in those patients with NT-proBNP concentrations over 7500ng/L on the 15th day post-transplantation (AU)

Determinación de procalcitonina en un Cobas e411 / Determination of procalcitonin on a Cobas e411

En los últimos años, la procalcitonina (PCT) ha demostrado ser la prueba más sensible para el manejo del proceso infeccioso, el diagnóstico y la estratificación de la sepsis. Ésta aporta valor pronóstico de complicaciones en pacientes postoperados y críticos, e incluso es útil como guía para la retirada del tratamiento antibiótico.Recientemente se está comercializando un nuevo inmunoensayo de electroquimioluminiscencia para la determinación de PCT (Elecsys BRAHMS PCT) con características similares a la prueba más eficaz descrita hasta ahora (PCT KRYPTOR). En el presente trabajo se procesaron 140 especímenes de pacientes con concentraciones de PCT de entre 0,02 y 47,13ng/ml, en un Cobas e411 y en un KRYPTOR; se observó que los resultados no son transferibles entre ambos métodos. Si bien la clasificación diagnóstica con los puntos de corte actuales no varía sensiblemente, es necesario un estudio más amplio para redefinir estos puntos de corte con el nuevo método. Por otra parte, se ha verificado que, aunque no hay efecto de arrastre significativo para una concentración de PCT cercana al límite superior del intervalo dinámico de la prueba, sí se observa arrastre para concentraciones más altas. Sin embargo, la modificación producida para una concentración cercana al límite de decisión clínica de 0,5ng/ml es clínicamente irrelevante, por lo que no parece necesario incorporar ninguna acción correctiva para evitar este arrastre(AU)

Over the last few years, Procalcitonin (PCT) has been shown to be the most sensitive test for the management of the infectious process, diagnosis and stratification of sepsis, prognosis of postoperative complications, and even useful for monitoring antibiotic therapy.A new electrochemical luminescence immunoassay (ECLIA) has recently been developed for measuring Procalcitonin (Elecsys BRAHMS PCT), with similar characteristics to the most efficient method described up to now (PCT KRYPTOR). In the present work, 140 patient specimens were analysed with PCT concentrations ranging 0.02–47.13ng/mL, in a Cobas e411 and a KRYPTOR analysers, and the results were found not to be transferable between methods. Although diagnostic classification using the current cut-off points did not vary significantly, a larger study will be required to redefine these cut-off points for the new method. On the other hand, there was no significant carryover effect at PCT concentrations close to the upper limit of the dynamic range, but it was observed at higher concentrations of PCT. However, at a concentration of PCT close to the clinical decision limit of 0.5ng/mL this effect seems to be clinically insignificant, thus no corrective action would be necessary to prevent carryover(AU)

Elevated transrenal DNA (cell-free urine DNA) in patients with urinary tract infection compared to healthy controls.

OBJECTIVE: To study whether levels of transrenal DNA (Tr-DNA) are influenced by the presence of urinary tract infections (UTI). METHODS: Tr-DNA concentrations were measured in 124 donors and 55 patients with UTI. Two methods for DNA extraction were compared. RESULTS: UTI patients showed higher concentrations than the donors (normal range: 0-147 GE/mL). QIAamp MiniElute Virus Spin Kit was more efficient than QIAamp DNA Blood Mini Kit. CONCLUSION: Increased concentrations of Tr-DNA urine DNA are shown in presence of UTI.

Situación actual del diagnóstico prenatal no invasivo / Current state of the non-invasive prenatal diagnosis methods

El diagnóstico prenatal se basa en la obtención de tejido fetal, mediante métodos invasivos, para su posterior análisis genético. La amniocentesis y la biopsia corial son las técnicas más utilizadas, cuyo principal inconveniente es que conllevan un riesgo de aborto, entre el 0,5¿1% y el 3,9%, respectivamente. El aislamiento de células fetales en sangre materna permite obtener material genético del feto de forma no invasiva, pero su escasez en sangre materna dificulta su utilización como método diagnóstico. La relativa abundancia del ADN fetal en la circulación materna, junto con la sensibilidad de la técnica utilizada para su cuantificación, convierten la detección de ADN fetal en plasma materno en un método alternativo para el diagnóstico de las enfermedades ligadas al cromosoma X o la determinación del estado RhD. En este artículo se hace una revisión del estado actual de los métodos de diagnóstico prenatal no invasivo(AU)

Prenatal diagnosis is based on genetic analysis of invasively obtained fetal tissue. Amniocentesis and chorial biopsy are the most used techniques, but carry a potential risk of miscarriage, from 0.5¿1% up to 3.9% respectively. Isolation of fetal cells from maternal blood allows a non-invasive obtention of fetal genetic material, but their scarcity difficults an efficient diagnosis method. The relative abundance of fetal DNA in maternal blood, as well as the sensitivity of quantitative PCR based methods, constitute an attractive alternative method for X-linked diseases and RhD status diagnosis. In this paper, a review of the present state of non-invasive prenatal diagnosis methods is described(AU)

Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR.

BACKGROUND: Recently cell-free plasma DNA has been described as a marker of apoptosis during hemodialysis (HD), but little is known about how different dialysis membranes may contribute to this process or whether pre-HD levels are restored afterwards. Here we evaluate the influence of the dialysis membrane on cell-free plasma DNA levels and investigate the clearance of plasma circulating DNA after HD. METHODS: Cell-free plasma DNA was measured using a real-time quantitative PCR for the beta-globin gene. Reference values for plasma DNA were established in a group of 100 healthy voluntary blood donors. Pre- and post-HD levels were also measured in 30 patients with end-stage renal disease on regular HD (52 sessions; 104 samples). The sessions lasted for 2.5-5 h. Different dialysis membranes were compared: high-flux (n=37) vs. low-flux (n=15) and polysulfone (n=42) vs. modified cellulose (n=10). To determine the time at which pre-HD levels are restored, DNA was quantified in serial plasma samples obtained from 10 of these 30 patients, just before and immediately after HD, as well as at 30, 60 and 120 min after HD. RESULTS: Reference plasma DNA values for healthy blood donors ranged from 112 to 2452 gEq/mL (median 740 gEq/mL). Cell-free plasma DNA levels significantly increased during HD (Wilcoxon test for paired samples, p<0.0001), with increases of more than four-fold observed in 75% of the patients after HD. There was no significant linear association between the length of the HD session (between 2.5 and 5 h) and the increase in cell-free plasma DNA concentration (Pearson correlation). No significant differences were observed between different types of membranes (Mann-Whitney U-test). Plasma DNA returned to pre-HD levels by 30 min after HD, regardless of the starting concentration. CONCLUSIONS: Plasma DNA levels significantly increase after a conventional 2.5-5-h HD session. Therefore, HD patients require special consideration for correct interpretation of plasma DNA concentrations. This parameter can be considered a reliable diagnostic tool for certain pathologies when measured at least 30 min after a HD session without further complications. The different dialysis membranes used in this study had no influence on cell-free plasma DNA concentrations, so the level of circulating DNA is not an appropriate marker of dialysis membrane biocompatibility.