Neonatal Blood Samples Research Articles

Closure Times Measured by the Platelet Function Analyzer PFA-100® Are Longer in Neonatal Blood Compared to Cord Blood Samples

Background: Although neonatal platelets have been shown to be hyporesponsive to most agonists in vitro, several groups have reported shorter closure times (CT) in term cord blood samples than in children and adults. It is unknown whether this is also true for preterm neonates, or for neonates of any gestational age (GA) during the 1st week of life, since limited studies have evaluated neonatal blood samples. Objectives: We designed this study to determine the effects of GA and postconceptional age on platelet function using the platelet function analyzer PFA-100®. Methods: We measured CTs in cord blood samples and in neonatal blood samples of varying GAs on days of life 1–2, and ≧7. Results: CTs were determined in 51 cord blood samples, 34 neonatal blood samples obtained on day of life 1–2, 16 neonatal blood samples from preterm neonates ≧7 days old, and 10 adults. We found a significant inverse relationship between ADP CTs and GA in both cord blood and neonatal blood day of life 1–2 samples (p = 0.02 and p = 0.01, respectively). When cord blood samples were compared with neonatal and adult blood, epinephrine and ADP CTs were significantly longer in adult blood as well as in neonatal samples obtained at either of the two time points (p ≤ 0.01 for all). Conclusions: Platelet function in response to ADP appears to improve with advancing GA. The differences between cord blood and neonatal blood CTs indicate that substantial changes in primary hemostasis occur shortly after birth. The reasons underlying these changes are unknown.

Efficacy of remifentanil-propofol-atracurium combined anesthesia for caesarean section

Objective To investigate the efficacy of remifentanil-propofol-atracurium combined anesthesia for caesarean section. Methods Forty ASA Ⅰ or Ⅱ patients, aged 24-30 yr, weighing 60-89 kg, at 38-41 week gestation, scheduled for elective cesarean section under general anesthesia, were studied. Remifentanil 1 μg/kg was slowly injected iv and then infused iv at a rate of 0.15 μg·kg-1·min-1 until the end of operation. Propofol 1.5 mg/kg and atracurium 0.6 mg/kg were injected in turn 30 s after remifentauil injection. Mechanical ventilation was performed after tracheal intubation. Atracurium 0.3 mg/kg was intermittently injected iv. Blood samples were taken from umbilical artery (UA) within 1 min after the delivery of the baby for blood gas analysis. Blood samples were simultaneously drawn from UA, umbilical vein (UV), and maternal artery (MA) for determination of plasma remitentanil concentrations (CUA, CUV and CMA) using gas chromatography-mass spectrometry. Systolic blood pressure (SP), diastolic blood pressure (DP), MAP and HR were monitored and recorded before intubation, at the time of inserting the catheter, at skin incision, and at the time of extubation. The analepsia time and satisfactory rate of patients were also recorded. The patient's awareness during operation was noted after operation. Apgar score was performed at 1, 5 and 10 min after the delivery of the newborn. The neonatal behavioral neurological assessment (NBNA) was conducted 1 and 24 h after the delivery of the baby. Results The hemodynamics was stable and varied within the normal range at each time point in all the patients. The analepsia time was (5±4) min and no awareness during operation occurred after postoperative follow-up. The pH value, PaCO2, PaO2 and SaO2 were in the normal range in the neonatal umbilical arterial blood samples. The neonatal Apgar scores at 1, 5 and 10 min after the delivery of the baby were more than 7. NBNA scores were normal. CUV, CUA and CMA were (0.65±0.14), (0.22±0.09) and (0.86±0.25) ng/ml respectively. CUV/CMA and CUA/CUV were 0.7±0.4 and 0.35±0.11 respectively. Conclusion The remifentanil-propofol-atracurium combined anesthesia can produce good anesthetic effect in the patients undergoing cesarean section, with high safety. Key words: Piperidinos; Propofol; Atracurium; Cesarean section

Acinetobacter Septicemia in Neonates Admitted to Intensive Care Units

Background:Acinetobacter species are gaining importance as potential pathogens in neonatal septicemia because of their frequent isolation and multidrug resistance.Aims and Objectives:The aim of the present study was to evaluate the role of Acinetobacter spp. as important pathogens in neonatal blood stream infection, to identify the associated risk factors, and to evaluate the drug sensitivity pattern.Materials and Methods:Blood samples of infected neonates were studied bacteriologically. Cases of Acinetobacter septicemia were identified. Speciation of Acinetobacter species was done. Various risk factors were identified. The drug-sensitivity test was done.Results:A total of 26 Acinetobacter septicemia cases were identified by blood culture. Acb complex strains predominated. Institutional birth and preterm birth were identified as the most frequent significant risk factors. 11.3% mortality rate was recorded. Acb complex strains exhibited a multi-drug resistant pattern. No carbapenem resistance was observed.Conclusion:Acinetobacter should be added to the list of organisms causing severe nosocomial infection in neonatal intensive care units. Continuous bacteriological surveillance, implementation of infection control policies, careful disinfection of intensive care equipment, and rational antibiotic use are required for control of such infections.

Spontaneously arising red cells with a McLeod-like phenotype in normal donors

Very few human genes can be used to identify spontaneous inactivating somatic mutations. We hypothesized that because the XK gene is X-linked, it would be easy to identify spontaneously arising red cells with a phenotype resembling the McLeod syndrome, which results from inherited XK mutations. Here, by flow cytometry, we detect such phenotypic variants at a median frequency of 9 × 10 −6 in neonatal cord blood samples and 39 × 10 −6 in healthy adults ( p = 0.004). It may be possible to further investigate the relationship between aging, mutations, and cancer using this approach.

Cardiac troponin I at birth is of fetal-neonatal origin

Objective:Neonates produce predominantly skeletal muscle troponin I (TnI) in the myocardium; however, in asphyxiated neonates, high levels of cardiac troponin I (cTnI) have been found. We hypothesised that in these...

Hb Barts's Quantitation in Neonatal Cord Blood Screening Samples

Background: a-thalassaemia is known to be prevalent in the Sultanate of Oman. However, there are no studies on the effects of a-thalassaemia on red cell indices in the new born children from the Sultanate of Oman.Materials & Methods: In a prospective study over a period of 2 years, cord blood samples from 7837 neonates, from the two prominent cities i.e Muscat and Sohar in the Sultanate of Oman, were analyzed for complete blood counts [CBC] and high performance chromatography[HPLC]; as part of ongoing studies related to the national neonatal cord blood screening programme. HPLC was performed on the Biorad Variant II™ using the beta thalassaemia short programme for qualitative assessment and alpha thalassaemia short programme for quantitation of Haemoblobin Barts, which was considered as diagnostic of alpha thalassaemia on the cord blood samples.(Biorad Laboratories, Hercules, CA, USA) The positive samples were then further studied with molecular methods for confirmation by GAP PCR with direct sequencing and Genescan studies on ABI 3100 Genetic Analyzer®. (Applied Biosystems, Foster City, CA, USA)Results: There were 4042 samples(51.58%) with normal HPLC (HbA + HbF). [Table] The remaining 3795 cases(48.42%) were associated elevated Hb Barts, indicative of the presence of a-thalassaemia. Amongst these subjects, 229 cases additionally also had HbS, 28 cases had HbD, 30 cases had HbE, and 3 cases had HbC. Overall, a total of 773 cases (9.86%) showed an abnormal hemoglobin like HbS(429), HbC(6), HbD(73), HbE (60)or beta thalassaemia(206).Reference range for Cord Blood in Ethnic Omani newborn children(Mean ± SD)HbRBCHctMCVMCHMCHCRDWHbAHbF(g/dl)(×1012/L)(%)(fl)(pg)(g/dl)(CV%)%%Normal [A+F]15.38 ±2.044.69 ±0.6850.5 ±7.18107.66 ±7.7533.61±4.0730.98 ±3.4417.01 ±2.1722.88±8.0377.02±8.04a-thal [A+F+Barts]14.79† ±2.95.09† ±0.7749.7† ±7.497.29† ±13.829.74† ±11.830.39† ±3.618.09† ±2.5625.74† ±9.0573.96† ±8.91#Barts <1% N=62515.81 ±1.584.54 ±0.5247.92 ±5.03105.72 ±5.6434 ±2.2733.03 ±1.2316.27 ±1.2423.78 ±8.681.55 ±10.2#Barts 1–3% N=184715.78 ±2.034.74 ±0.6648.25 ±6.99101.57 ±5.4433.38 ±2.6932.71 ±1.1716.25 ±6.4621.14 ±8.184.65 ±9.75#Barts 3–10% N=132314.96 ±2.015.29 ±0.6947.46 ±6.889.87 ±5.3928.47 ±2.131.65 ±1.3917.3 ±1.8224.11 ±8.581.42 ±9.5†Significant (P<0.05) compared with Normal (A+F)#Barts Subgroups classified as per alpha thalassaemia short programme [Manufacturer's instructions]Hb Barts levels between 1–3% and 3–10% were seen amongst 48.68% and 34.87% cases respectively. Genescan studies revealed that 61.23% cases had nondeletional type and 38.77% subjects showed deletional.(-a3.7 ) types of a-thalassaemia.[figure] which were confirmed by GAP PCR for deletional cases whereas in non-deletional cases both a2 and a1 genes were selectively amplified and directly sequenced.Conclusions : The incidence a-thalassaemia in this cohort of neonates was 48.42%. a-thalassaemia gene significantly reduced the MCV, MCH, MCHC, Hct, RBC count and the Hb and increased the RDW in the cord blood samples of neonates screened in this study. There was an excellent correlation between qualitative and quantitative estimation of Hb Barts by the two techniques. Importantly, we did not encounter any case of Hb H disease or hydrops faetalis in this study. [Display omitted]

Review: venepuncture is less painful than heel lance for blood sampling in neonates

V Shah Dr V Shah, Mount Sinai Hospital, Toronto, Ontario, Canada; vshah@mtsinai.on.ca Is venepuncture less painful than heel lance for blood sampling in healthy neonates? Studies selected compared venepuncture and...

Possible Risk Factors for Congenital Malaria at a Tertiary Care Hospital in Sagamu, Ogun State, South-West Nigeria

Congenital malaria, defined as the presence of malaria parasites in the erythrocytes of newborns aged <7 days, was considered rare in endemic areas until recent studies started reporting high prevalence rates. Various theories have been postulated to explain this phenomenon, but they are not proven conclusively from research. Against this background, a prospective study was designed with the following objectives. To determine the prevalence of congenital malaria parasitaemia and identify possible risk factors amongst newborns delivered in O.O.U.T.H Sagamu, Ogun State. Over a 6-month period, 192 live newborns and their mothers were consecutively recruited into the study. Within 3 days of life, neonatal peripheral blood samples were collected for malaria screening by blood film microscopy and detection of plasmodium lactate dehydrogenase (pLDH) with the OptiMAL Rapid Malaria Test kit. Maternal peripheral blood samples were taken simultaneously, to check for malaria infestation by blood film microscopy, and questionnaires were administered on the mothers to identify possible factors associated with the development of neonatal parasitaemia. Neonatal clinical and laboratory data were recorded in a pro forma designed for the study. Data analysis was done with Epi-info version 6 software and level of significance set at <5%. Twenty-one of 192 newborns delivered in O.O.U.T.H within the study period were diagnosed as having congenital malaria by blood film microscopy, giving a prevalence rate of 10.9%. The main identified innate neonatal risk factor for congenital malaria parasitaemia was prematurity. First-order pregnancy, history of fever within 3 months of delivery and peripheral parasitaemia at delivery (p < 0.001) were the variables that were significantly higher in the mothers of the parasitemic newborns. We conclude that congenital malaria parasitaemia in tropical endemic areas is not rare. Pre-term neonates, infants of primigravidae, women with history of fever within 3 months of delivery and women with post-partum peripheral parasitaemia may benefit from routine screening for malaria.

Peripheral Blood Leukocyte Production of BDNF following Mitogen Stimulation in Early Onset and Regressive Autism.

Brain-derived neurotrophic factor (BDNF) is critical for neuronal differentiation and synaptic development. BDNF is also implicated in the development of psychological disorders including depression, bipolar disorder and schizophrenia. Previously, elevated BDNF levels were observed in neonatal blood samples from infants who were later diagnosed with autism when compared with children who developed normally, suggesting that BDNF may be involved in the development of autism. BDNF is produced by activated brain microglial cells, a cellular phenotype that shares several features with peripheral macrophages, suggesting an important role for the immune system in BDNF production. We hypothesized that under mitogenic stimulation, peripheral blood mononuclear cells obtained from children with autism may have altered BDNF production compared with age-matched typically developing control subjects. In addition, we examined the differences between the production of BDNF in classic/early-onset autism and children who had a regressive form of autism. We show here that plasma levels of BDNF levels are increased in children with autism, especially in early onset autism subjects. Furthermore, under mitogenic stimulation with PHA and LPS, BDNF production is significantly increased in children with autism compared with typically developing subjects. However, stimulation with tetanus toxoid results in a decreased response in children with autism. This data suggest that immune cell-derived production of BDNF could be an important source for the increased BDNF that is detected in some subjects with autism. As a neurotrophic factor produced by immune cells, BDNF could help elucidate the role of the immune system in neurodevelopment and neuronal maintenance, which may be dysregulated in autism.

Incomplete Platelet Dense Granule Formation in Normal Neonates.

Platelets are important in maintaining hemostasis in newborns, where bleeding can arise from abnormal platelet function and/or thrombocytopenia. It is well established that plasma coagulation factor concentrations are lower in neonates compared to children and adults, but less is known about the development and function of neonatal platelets. It has been postulated that platelets from neonates, and to a greater extend from premature neonates, are dysfunctional due to low dense granule counts (Blood 2006; 108:331a), however, other studies have shown normal neonate platelet function. Our previous studies indicated a slightly decreased number of dense granules per platelet in neonates (Blood 2005; 106:4159–4156). We have now extended these studies to a larger cohort of 19 normal neonatal cord blood samples (gestational age 37.5–40 weeks) from planned Caesarean sections, which were analyzed under optimal sample handling conditions and compared to platelets from 10 children (age 8–10 years). 50 platelets from each subject were evaluated for dense granule content utilizing whole mount and thin section electron microscopy (EM) for the quantification of dense granules (detected via their electron-dense calcium content) and ultrastructural assessment. A subset of samples was tested via flow cytometry for P-selectin expression as a measure of platelet activation, and platelet structural integrity was also assessed using thin section EM. Our data revealed that platelets in neonatal cord blood had a mean dense granule count of 2.3 (SD=2.2) per platelet, compared to 4.4 dense granules per platelet (SD=2.7) in blood from older children; t-test comparisons showed the difference between these groups to be highly significant (P<0.001). Interestingly, 22% of cord blood platelets contained no measurable dense granules, whereas only 3% of platelets from older children where devoid of dense granules. We suspected that the mean dense granule counts of <1 per platelet in neonatal cord blood reported by others may have arisen due to high levels of platelet activation during sample acquisition or handling. In our samples platelet activation as measured by P-selectin expression was similar in both populations and did not exceed 7.5%, and platelet morphology as assessed by thin section EM was also comparable. Our studies confirm that neonatal cord blood platelets contain fewer recognizable dense granules than those found in older children. Two possible explanations for this observation are:1.normal numbers of dense granules are present in neonatal platelets, but a subset cannot be detected via EM owing to insufficient calcium uptake;2.there are fewer dense granules in neonatal platelets owing to peculiarities in the development of megakaryocytes, where recent studies have suggested that dense granules originate by an active transport mechanism and move into proplatelets.These possibilities point to the usefulness of studying fetal and neonatal megakaryopoiesis.

Haemolysis in neonatal blood samples: a survey of practice

To survey how laboratories report, and neonatologists perceive them to report, the presence of haemolysis in neonatal blood samples. A questionnaire was sent to clinical biochemists and neonatologists in 88 hospitals believed to be using Roche methodology. A 49% response rate was obtained. Seventeen hospitals were excluded from analysis owing to incomplete questionnaires or altered methodology. Of the remaining 71 hospitals, 30 (42%) laboratories and 18 (25%) neonatologists responded - where both responded, none gave identical replies. Eighteen laboratories admitted the use of serum indices to make decisions on haemolysed samples. Although recommended interference limits are available from Roche, there was little consensus between laboratories. Laboratories should have clear guidelines on the processing of haemolysed samples from neonates and these should be adequately communicated to those responsible for their direct care.

Azathioprine and breastfeeding—is it safe?

Traditionally, women receiving azathioprine have been discouraged from breastfeeding because of theoretical potential risks of neonatal bone marrow suppression, susceptibility to infection, and pancreatitis. The aims of this study were to measure the concentration of 6-mercaptopurine (6-MP) in breast milk of mothers receiving azathioprine and in the blood of their babies and to investigate any immunosuppressive effects on the babies. Women receiving azathioprine, who after appropriate counselling wished to breastfeed their babies, were approached for inclusion in the study. Breast milk samples were obtained from recruited women, and 6-MP levels were measured in each breast milk sample. Haemoglobin level, white cell and platelet counts, and 6-MP and 6-thioguanine nucleotides (6-TGN) levels were measured in the respective neonatal blood samples. Clinical signs of immunosuppression in the neonates were noted. Thirty-one breast milk samples were collected from ten women. Low concentrations of 6-MP (1.2 and 7.6 nanograms/ml, compared with therapeutic immunosuppressant level of 50 nanograms/ml in serum) were detected in two breast milk samples obtained from one woman. 6-MP was not detected in any of the other 29 samples. 6-MP and 6-TGN were undetectable in the neonatal blood. There were no clinical or haematological signs of immunosuppression in any of the ten neonates. We conclude that breastfeeding should not be withheld in infants of mothers receiving azathioprine.

Quantitative assessment of human whole blood RNA as a potential biomarker for infectious disease

Infection remains a significant cause of morbidity and mortality especially in newborn infants. Analytical methods for diagnosing infection are severely limited in terms of sensitivity and specificity and require relatively large samples. It is proposed that stringent regulation of the human transcriptome affords a new molecular diagnostic approach based on measuring a highly specific systemic inflammatory response to infection, detectable at the RNA level. This proposition raises a number of as yet poorly characterised technical and biological variation issues that urgently need to be addressed. Here we report a quantitative assessment of methodological approaches for processing and extraction of RNA from small samples of infant whole blood and applying analysis of variation from biochip measurements. On the basis of testing and selection from a battery of assays we show that sufficient high quality RNA for analysis using multiplex array technology can be obtained from small neonatal samples. These findings formed the basis of implementing a set of robust clinical and experimental standard operating procedures for whole blood RNA samples from 58 infants. Modelling and analysis of variation between samples revealed significant sources of variation from the point of sample collection to processing and signal generation. These experiments further permitted power calculations to be run indicating the tractability and requirements of using changes in RNA expression profiles to detect different states between patient groups. Overall the results of our investigation provide an essential first step toward facilitating an alternative way for diagnosing infection from very small neonatal blood samples, providing methods and requirements for future chip-based studies.

Neonatal Cytomegalovirus Blood Load and Risk of Sequelae in Symptomatic and Asymptomatic Congenitally Infected Newborns

Human cytomegalovirus (CMV) is a ubiquitous human-specific DNA virus and is the main cause of congenital virus infection in developed countries leading to psychomotor impairment and deafness. Diagnostic techniques for CMV detection have greatly improved during recent years with the advent of sophisticated serological and virological methods. The aim of the present study was to assess the diagnostic and prognostic value of detection and quantification of virus in neonatal blood samples of symptomatic and asymptomatic newborns with CMV congenital infection. Between January 1997 and December 2003, we studied 99 newborns who were born to women with primary, recurrent, and undefined CMV infection during pregnancy. CMV congenital infection was identified by isolation of the virus in urine within the second week of life. Fifty-eight of 99 infants were infected and were assessed clinically for disease in the newborn period and classified as having symptomatic or asymptomatic infection on the basis of physical, instrumental, and laboratory findings. The infants were followed up from birth according to a protocol of the tertiary NICU at the University of Bologna in a prospective study of long-term sequelae of congenital infection. Forty-seven blood samples were obtained from 47 infants in the neonatal period: 34 were examined for pp65 antigenemia test and 44 for qualitative and quantitative polymerase chain reaction (PCR and qPCR). Sequelae at 12 months were evaluated in a group of 50 infants. Antigenemia was positive in only 10 of 34 samples of infected newborns (29.4% sensitivity). PCR was performed in 44 samples of infected newborns and was positive in all (100% sensitivity). qPCR showed a finding of > or =100 copies per 10(5) of polymorphonuclear leukocytes (PMNLs) in 39 of 44 samples; in the other 5 cases, the number of copies per 10(5) PMNLs was <100. Between symptomatic and asymptomatic newborns, the mean values of viral blood load determined by qPCR turned out to be significantly higher in symptomatic newborns. Mean values of neonatal blood viral load were statistically higher in newborns who developed sequelae than in those who did not. Of 20 children with a neonatal viral blood load of <1000 copies per 10(5) PMNLs, 19 did not develop sequelae (negative predictive value: 95%), whereas 2 of 3 with a viral blood load of >10,000 copies did develop sequelae. Different viremia value ranges are correlated to a different risk of sequelae: approximately 70% sequelae were found in newborns with a qPCR higher than 10,000 copies per 10(5) PMNLs. Low neonatal viral blood load detected by pp65 antigenemia test and qPCR was highly predictive of absence of sequelae: DNAemia <1000 copies per 10(5) PMNLs has a negative predictive value of 95%. As an independent predictive factor of outcome, neonatal viremia is another useful element for neonatal counseling and therapeutic choices in symptomatic and asymptomatic newborns.

Determination of total bilirubin in whole blood from neonates: results from a French multicenter study

Jaundice is frequent in neonates and can cause severe complications, especially in premature neonates, particularly the risk of developing acute bilirubin encephalopathy. Our purpose was to verify if determination of total bilirubin (TBIL) in whole blood on an ABL 735 blood gas analyzer with a spectrophotometer module could provide an analytical alternative to chemical methods of TBIL measurement. Our multicenter comparative study involved four hospital laboratories. We studied the repeatability and reproducibility of ABL 735 TBIL measurements in two control sera of medium (N1, 58.1 micromol/L) and high (N2, 275.3 micromol/L) TBIL levels. The same study was simultaneously conducted on four chemistry instruments (two LX 20, one Integra 800 and one Hitachi 917) using four Jendrassik-Grof derived methods. At one site, repeatability was performed with two adult whole-blood samples containing low and high TBIL levels (55.1 and 312.6 micromol/L). Repeatability tests provided coefficients of variation (CVs) between 0.67% and 1.86% on the ABL 735 system, vs. 0.35% and 1.96% for the chemistry instruments. Reproducibility tests for the same control sera resulted in CVs between 1.01% and 3.55% for the ABL 735 and between 0.52% and 3.65% for the chemistry instruments. Recovery for the N1 and N2 control sera was 102-120%. A correlation study of TBIL determination in whole blood vs. plasma was conducted on 473 neonatal blood samples. Correlation coefficients between whole blood and plasma TBIL ranged from 0.969 to 0.994. Passing-Bablok equations were y=1.17x+9.7 [site 1 (IP)], y=1.01x+5.6 [site 2 (JPB, MR)], and y=1.00x-20 [site 3 (AS)]. Only 10% of the results fell outside the 10% range in the bias-corrected Bland-Altman difference plot for the ABL 735 method compared to traditional laboratory methods. The ABL 735 instrument is reliable for measuring TBIL in 70-microL whole blood samples from neonates. Thus, this method might allow significant blood savings in preterm neonates. Correlation with the reference method for plasma or sera must be established to ensure good follow-up of patients.

Quantification of trimethylsilyl derivatives of amino acid disease biomarkers in neonatal blood samples by gas chromatography-mass spectrometry

A novel analytical procedure was developed for the rapid determination of disease biomarkers of maple syrup urine disease (MSUD), L-valine, L-leucine, L-isoleucine, and L-phenylalanine in dried blood spots. Amino acids extracted from neonatal dried blood spots were rapidly derivatized with bis-(trimethylsilyl)trifluoroacetamide (BSTFA) and then analyzed by gas chromatography-mass spectrometry (GC-MS). Derivatization conditions and the method validation were studied: optimal derivatization conditions were acetonitrile as reaction solvent, a temperature of 100 degrees C, and a reaction time of 30 min. The proposed method provided a detection limit lower than 2.0 microM, recovery between 92% and 106%, and relative standard deviation less than 8.0%. The method was further tested in screening for neonatal MSUD by determination of L-valine, L-leucine, L-isoleucine, and L-phenylalanine in blood samples. The experimental results show that GC-MS following BSTFA derivatization is a rapid, simple, and sensitive method for the determination of amino acid disease biomarkers in blood samples, and is a potential tool for fast screening of MSUD.

Fast Diagnosis of Neonatal Phenylketonuria by Gas Chromatography-Mass Spectrometry Following Microwave-Assisted Silylation

Phenylketonuria (PKU) is a fairly common autosomal recessive disease. Phenylalanine (Phe) and tyrosine (Tyr) are the biomarkers of PKU, and it can be diagnosed by the measurement of Phe and Tyr in neonatal blood samples. A fast diagnostic procedure for neonatal PKU has been developed using microwave-assisted silylation followed by gas chromatography-mass spectrometry. Amino acids extracted from neonatal blood samples are rapidly derivatized with N, O-bis(trimethylsilyl)-trifluoroacetamide under microwave irradiation; the derivatives are then analyzed by GC-MS. The silylation conditions have been optimized, and the method validated for linear range, detection limit, precision and recovery. The proposed method is linear from 20 to 800 µM, detection limit is less than 0.35 µM, relative standard deviation (RSD) is less than 9.0%, and recoveries of 87% and 83% were obtained for Phe and Tyr. The proposed method was tested by the determination of Phe and Tyr in blood spots from eight PKU-positive neonates and twelve control neonates. Microwave irradiation considerably accelerates the derivatization reaction of amino acid with BSFTA, and shortens the whole analysis time. Microwave-assisted silylation coupled to GC-MS is a powerful tool for fast screening of neonatal PKU.

Neonatal screening of glucose-6-phosphate dehydrogenase deficiency in Yanbu, Saudi Arabia

To determine the prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in the population tested, and to evaluate the prevalence of neonatal jaundice in newborns with G6PD deficiency. Cord blood of all babies born between October 1996 and October 1998 at the Royal Commission Medical Center in Yanbu, Saudi Arabia, was screened for G6PD deficiency by fluorescent spot test. The results of screening of cord blood samples were reported to the physician in charge, and also placed on the files of the babies and their mothers. These babies were observed for 72 h and discharged if no jaundice developed. During this two-year period, 2,505 neonatal cord blood samples from 1,278 boys and 1,227 girls were screened for G6PD. There were 50 positive results for G6PD deficiency (39 boys and 11 girls), and the prevalence was estimated to be around 2%. The sex-specific prevalence for boys was 3.05%, and for girls 0.9%. Male to female ratio was 3:1. Neonatal jaundice developed in six (12%) babies, five male and one female. All were treated with phototherapy and discharged within one week of birth. The prevalence of G6PD is relatively high in Yanbu. Routine neonatal screening in areas with a high prevalence of G6PD in Saudi Arabia is justifiable.

Inborn Errors of Metabolism Masquerading as Hypoxic-Ischemic Encephalopathy

After completing this article, readers should be able to: 1. Compare the clinical features of hypoxic-ischemic encephalopathy and inborn errors of metabolism. 2. Review the metabolic disorders that may present with isolated neonatal seizures. 3. Review the metabolic disorders that may present with lactic acidosis, hypotonia, and systemic involvement. 4. Compare patterns of brain injury in hypoxic-ischemic encephalopathy and inborn errors of metabolism. Because inborn errors of metabolism can present in the neonatal period with neurologic distress, metabolic acidosis, and multiorgan system involvement, similar to features encountered in hypoxic-ischemic encephalopathy (HIE), an underlying metabolic disorder may go undiagnosed unless the clinician maintains a high index of suspicion and performs appropriate diagnostic testing. Although expanded newborn screening using tandem mass spectrometry (MS/MS) undoubtedly will detect many children who have metabolic disorders, this testing will not necessarily find neonates who have metabolic disorders that present with HIE-like features. In such cases, specialized testing of blood, urine, or cerebrospinal fluid (CSF) frequently is necessary to make the diagnosis and allow accurate genetic counseling. HIE typically occurs in term infants (gestation >37 wk) who display signs of distress before delivery, require resuscitation at birth, and develop neurologic abnormalities in the first 24 hours after delivery. (1) Perinatal asphyxia affects 3 to 5 infants per 1,000 live births, with encephalopathy occurring in 25% and death in an additional 30%. (2)(3) Although inborn errors of metabolism are rare individually, as a group they affect approximately 1 in 1,000 neonates, (4) mandating inclusion in the differential diagnosis of patients who present with nonspecific features suggestive of sepsis or asphyxia. Three criteria are essential to diagnose the presence of an acute intrapartum hypoxic event: 1) evidence of a metabolic acidosis in intrapartum fetal, umbilical arterial cord, or very early neonatal blood sample (pH <7.00 and base deficit ≥12 mmol/L); 2) …

Development of microwave‐assisted derivatization followed by gas chromatography/mass spectrometry for fast determination of amino acids in neonatal blood samples

Analysis of amino acids in blood samples is an important tool for the diagnosis of neonatal amino acid metabolism disorders. In the work, a novel, rapid and sensitive method was developed for the determination of amino acids in neonatal blood samples, which was based on microwave-assisted silylation followed by gas chromatography/mass spectrometry (GC/MS). The amino acids were derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) under microwave irradiation. The controlled reaction was carried out employing BSTFA under conventional heating at 120 degrees C for 30 min. Experimental results show that microwave irradiation can accelerate the derivatization reaction of amino acids with BSFTA, and much shorten analysis time. The method validations (linear range, detection limit, precision and recovery) were studied. Finally, the method was tested by determination of amino acids in neonatal blood by the measurement of their trimethylsilyl derivatives by GC/MS in electron impact (EI) mode. Two biomarkers of L-phenylalanine and L-tyrosine in phenylketonuria (PKU)-positive blood and control blood were quantitatively analyzed by the proposed method. The results demonstrated that microwave-assisted silylation followed by GC/MS is a rapid, simple and sensitive method for amino acid analysis and is also a potential tool for fast screening of neonatal aminoacidurias.