Paediatrics and Pregnancy Flashcards
Newborn screening encompasses more than just screening.
Screening
Follow-up
Diagnostic testing
Management
Program evaluation and quality assurance
How to select tests for newborn screening?
Modified Wilson and Jungner criteria
- suitable test available
- recognisable “latent” phase
- evidence for benefit to infant from early detection and management
- availability of efficacious diagnostic and clinical management program
- cost effective - cost of screening and management of outcome more cost effective than not screening
What is a suitable/ideal test method for newborn screening?
- validated methods available for screening and diagnosis
- amenable to dried blood spot testing
- high throughput with low cost
- multiple analytes/secondary targets detected by test method
- multiplex platform - LCMS/MS, HPLC, bead-based immunocapture, microarray
Most common amino acid/urea cycle disorder?
PKU 1:10 000
What is the most common enzyme deficiency in PKU?
Phenylalanine hydroxylase
How is PKU detected by newborn screening?
Tandem MS detection of increased phenylalanine and increased phenylalanine/tyrosine ratio
In general, how is newborn screening of amino acid/urea cycle disorders performed?
After dried blood spot sample collection, tandem MS quantification of amino acid/metabolite profiles with interpretation of disease specific patterns
Describe amino acid/urea cycle disorders
Autosomal recessive disorders of amino acid or protein metabolism, in which a specific enzyme defect causes accumulation of neurotoxic amino acids, metabolites or ammonia
Describe organic acid disorders
Autosomal recessive disorders due to a specific enzyme defect in the organic acid metabolic pathway, resulting in accumulation of organic acids in blood/urine
How are organic acid disorders detected by newborn screening?
Tandem MS quantification of acylcarnitines with interpretation of disease-specific profiles
What factors affect newborn screening of inborn errors of metabolism?
Feeding status, diet type, transfusion (PKU)
Describe the fatty acid oxidation disorders.
Autosomal recessive disorders causing specific enzyme defects in the fatty acid metabolic pathways which affect utilisation of dietary and stored fats
What is the risk associated with fatty acid oxidation disorders?
During illness or fasting, the inability to utilise fatty acids for energy results in hypoglycaemia. Death from first crisis in pts with MCAD is 1 in 4!
How is CAH detected in newborn screening protocols?
Detection of increased 17-OHP by DELFIA
List factors causing false positives and false negatives for CAH newborn screening
False positives:
- low birth weight
- prematurity
- physiologic stress
- day 1 17-OHP surge
False negatives:
- treatment with glucocorticoids prior to sample collection
How is newborn screening for congenital hypothyroidism performed?
Measurement of TSH on dried blood spot
Causes of congenital hypothyroidism
Athyreosis
Small, maldescended gland
Iodination defect
Clinical implications of hypothyroidism
Long term: Intellectual and physical disability
Short term: prolonged neonatal jaundice, feeding problems, FTT, constipation, lethargy, hypotonia, coarse facial features, thick tongue, hoarse cry, distended abdomen, umbilical hernia
Causes of false positives and negatives in newborn screening for hypothyroidism
False +ves: Very high TSH in first 24 hours of life, prematurity
False -ves: critically ill, premature, post-transfusion
What is galactosaemia?
An austosomal recessive disorder in which there is a deficiency of a specific enzyme required for conversion of galactose (from dietary lactose) to glucose. Accumulation of galactose in the blood causes organ toxicity
List the enzymes that may be deficient in galactosaemia. Which is the most common deficiency? Which of these enzyme deficiencies is detected by your state’s neonatal screening program?
GALT: Galactose-1-phosphate uridyltransferase (GUT) - the most common
GALK: Galactokinase
GALE: Uridine diphosphate-galactose-4-epimerase
In WA, GALT deficiency is screened for, however, GALK and GALE deficiency may be detected.
How is newborn screening for galactosaemia performed?
Detection of increased galactose and galactose-1-phosphate, and decreased GUT (galactose-1-phosphate uridyltransferase) activity on neonatal blood spot. Enzyme assay.
False negatives on galactosaemia testing may result from?
Extreme heat or delayed submission due to enzyme degradation
Baby not on milk feeds (do enzyme activity testing only)
Exchange transfusion (perform testing before transfusion)
Diagnostic testing for galactosaemia?
Genotyping. Could do plasma galactose and blood galactose-1-phosphate. RBC GUT activity.
Treatment of galactosaemia
Lactose-free diet
Clinical features of galactosaemia
Infants with classical galactosaemia are usually normal at birth; however, they soon develop jaundice and vomiting, and they fail to gain weight. Other symptoms of the condition include:
Liver failure and renal dysfunction
E.coli sepsis
Intellectual disability
Cataracts
Infants who are diagnosed early have good long-term outlooks although some may develop problems with their speech and women may suffer from infertility. Galactokinase deficiency only causes cataracts.
Rare causes of PKU?
Biopterin deficiencies
Clinical features of PKU
Normal for the first few months then development slows.
Microcephaly
Intellectual disability
Seizures
Autistic-like behaviour
Fair-light complexion, hair and eye colour
Musty odour
Diagnostic tests for PKU
Plasma amino acids
Urine pterins
Bloodspot DHPR enzyme
Treatment of PKU
Phenylalanine-restricted diet
Tyrosine supplementation
Tetrahydrobiopterin cofactor in some
How is newborn screening for cystic fibrosis performed in your state?
Increased immunoreactive trypsin (IRT)
Common CFTR variant analysis in those with IRT>99% (tested for p.Phe508del, p.Gly551Asp, p.Gly542*, and c.489+1G>T)
Diagnostic testing for cystic fibrosis
Sweat test
Extended CFTR gene analysis
Clinical assessment and genetic counselling
Limitations of newborn screening for cystic fibrosis in WA
May miss uncommon variants (95% affected individuals will have one of the four variants tested for)
Newborn screening program should be informed if there is any history of meconium ileus or a family history of CF, so variant analysis can be performed regardless of IRT result
Some healthy carriers may have a positive screening test
False positives occur more commonly if collected within first 24 hours of life
Most common fatty acid oxidation defect?
Medium-chain acyl-CoA dehydrogenase deficiency
Clinical presentation of MCAD deficiency?
Hypoketotic hypoglycaemia with risk of permanent neurological deficit/death
How is medium-chain acyl-CoA dehydrogenase screened for in your state?
By tandem MS.
Increased medium-chain acylcarnitines (C6, C8, C10, C10:1)
Increased C8/C2 ratio
Diagnostic tests for MCAD
Plasma acylcarnitine profile
Urine organic acids
ACADM gene analysis
Treatment of MCAD
Low-fat diet
Carnitine supplementation
Avoidance of fasting, particularly when sick
IV glucose during illness
What is the urea cycle
A metabolic pathway in the liver. Used to convert toxic nitrogenous waste (ammonia) to harmless urea which can be excreted in the urine.
Which amino acids are the main nitrogen containing acids?
Glutamine and alanine
What amino acids are produced by the urea cycle?
Arginine
Non-protein forming citrulline and ornithine
Which urea cycle disorder is X linked?
OTC deficiency (also most common of them)
Key biochemical feature of urea cycle disorders?
Hyperammonaemia
Causes of hyperammonaemia
Urea cycle disorders
Citrullinaemia type 2
Arginase deficiency
Biochemical pattern of UCDs
Ammonia > 100-150 umol/L with normal glucose and normal anion gap
Useful diagnostic tests for UCDs
Blood amino acids and urine organic acids (orotic acid) help to differentiate between UCDs
Why should you check lactic acid if you suspect a UCD?
To help rule out disorders of pyruvate metabolism
Factors affecting ammonia result interpretation
Heparinised plasma from stasis-free veins is the preferred collection
Timing - sample collection during an episode, otherwise it will be missed
Delayed processing can cause false increases due to RBC degradation of AAs - send on ice/freeze
Rejection criteria for ammonia sample
Haemolysed, lipaemic or grossly icteric specimens
Delayed or room temp specis
Most common UCD?
OTC deficiency
Acute management of urea cycle disorders
- Remove ammonia
- nitrogen scavenger
- haemodialysis - Reverse catabolic state
- Reduce risk of neurologic damage (judicious fluid management to prevent worsening cerebral oedema)
Acute management of urea cycle disorders
- Remove ammonia
- nitrogen scavenger
- haemodialysis - Reverse catabolic state
- fluids
- L-arginine in arginase deficiency
- stop protein intake - Reduce risk of neurologic damage (judicious fluid management to prevent worsening cerebral oedema)
Chronic management of urea cycle disorders
- Dietary restrictions
- Prophylaxis for viral infections esp in infants/children
- Disease specific treatments, including liver transplant
Screening test for mucopolysaccharidosis
Urine glycosaminoglycans
Normal placental transport
No transport
- Most proteins
- Maternal IgM, IgA
- Maternal and fetal erythrocytes
Limited passive transport
- Unconjugated steroids
- Steroid sulfates
- Free fatty acids
Passive transport
- Molecules up to 5000 Da having lipid solubility
- Oxygen
- Carbon dioxide
- Sodium and chloride
- Urea
- Ethanol
Active transport across cell membranes
- Glucose
- Many amino acids
- Calcium
Receptor-mediated endocytosis
- Maternal IgG
- Low-density lipoprotein
True or false: Placental hormone production increases in proportion to placental mass
True EXCEPT for HCG which peaks at the end of 1st trimester
Steroid hormones produced by the placenta
Progesterone
Estradiol and estrone
Estriol
Protein hormones produced by the placenta
HCG
Placental lactogen
ACTH
Chronionic thyrotropin
Thyrotropin releasing hormone
Gonadotropin releasing hormon
Corticotropin releasing hormone
Growth hormone
Somatostatin
Inhibin A
Pregnancy-associated plasma protein A
Schwangerschaft protein (~20 different pregnancy specific proteins)
Historical use of human placental lactogen (hPL)?
Monitor fetal wellbeing
Biological roles of hPL
- Prepare mammary glands for lactation
- Mimic GH metabolic effects (switch mother to lipolysis and spare glucose for fetus)
T or F: Placenta has no 17alpha hydroxylase
True
How does the composition of amniotic fluid change during pregnancy?
Early in gestation, amniotic fluid is mostly a complex dialysate of maternal serum. Towards the end of first trimester, fetal kidneys begin to produce urine which becomes the main component of amniotic fluid. Concentrations of sodium and osmolality decrease and concentrations of uric acid, urea and creatinine increase.
Amniotic fluid also becomes more turbid, filling with fetal cells, lanugo and lamellar bodies (surfactant particles from the fetal lung) and towards term, vernix caseosa (fetal sebum and skin epithelium).
In what domains of physiology/biochemistry do maternal adaptations occur in pregnancy?
Haematologic
Renal
Endocrine
General biochemical changes
Describe haematologic adaptations during pregnancy
Physiologic anaemia of pregnancy - blood volume increases disproportionate to red cell mass
Fibrinogen increased by 65%
Describe the general biochemical changes during pregnancy
Electrolytes show little change (slightly lower Na)
Bicarb decreases
40% increase in cholesterol, phospholipids and FFAs
Tg increase 3fold - possibly resulting in chylomicronaemia if familial disposition
Alb decreases
Globulins increase
Bilirubin decreases
ALP activity triples (heat stable placental ALP variant)
CK increases (3rd trimester/delivery)
Ferritin and Tf sats decrease
Alpha-1 antitrypsin increases
Describe the changes in renal function during pregnancy
GFR increases to 170mL/min/1.73m^2 by 20 weeks
Urea, creatinine and uric acid clearnce increases - decreased for much of pregnancy
In the last 4 weeks, as GFR returns to normal, urea and creatinne rise slightly but uric acid increases dramatically
Glucosuria and proteinuria due to increased GFR may occur
Describe the endocrine changes during pregnancy
Early preg - progesterone produced by corpus luteum in response to hcg. Then produced by placenta in quantities sufficient to maintain pregnancy.
PTH increase by 40% with almost no change in ionised calcium (new set point)
Calcitriol increases - increases intestinal calcium absorption to support fetal skeletal development
Increased plasma aldosterone and deoxycorticosterone concentrations
Estrogens increase CBG, reduce hepatic clearance of cortisol and increase cortisol levels
Estrogens increase SHBG and prolactin (10x)
FSH and LH suppressed.
Estrogens induce thyroxine binding globulin with decreases in total thyroid hormones but also slight decrease in free thyroxine
Thyroglobulin significantly increased in 3rd trimester
At what level of HCG does an intrauterine pregnancy become visible by ultrsonography?
1500-2000U/L
Sensitivity 42%, specificity of 81% for detection of ectopic pregnancy
When is serial testing of HCG helpful in diagnosis of ectopic pregnancy?
When HCG <1500U/L and sonography is non-diagnostic. Follow HCG every 48-72 hrs if rising normally until 1500U/L at which point, repeat ultrasonography.
What is the HCG doubling rate in 2-5th week of preg and 5th-10th week of preg?
48 hours before 5 weeks and up to 72 hours after 5 weeks
Diagnostic criteria pre-eclampsia
htn (>/= 140/90) and proteinuria (+1 dipstick, 300mg/24hr)
or htn with new onset of any of the following: plt <100, creat >97 umol/L, doubling of serum creat (in absence of other renal disease), liver transaminases >/=2xURL, pulm oedema, cerebral/visual sx
Diagnostic criteria severe pre-eclampsia
SBP >/= 160 and/or diastolic >/= 110 on 2 occasions 6 hr apart
Oliguria <500mL in 24 hrs
Cerebral/visual disturbances
Pulm oedema/cynosis
Epigastric/RUQ pain
Impaired liver function
Thrombocytopaenia
What is HELLP syndrome?
Haemolysis
Elevated liver enzymes
Low platelets
in association with pre-eclampsia
Liver diseases unique to pregnancy
- Hyperemesis gravidarum
- Cholestasis of pregnancy
- Fatty liver of pregnancy
Which LFTs remain normal in pregnancy
Total bilirubin
GGT
ALT
AST
Cholestasis of pregnancy - clinical presentation
Pruritis, 10% have jaundice, dark urine, pale stools.
Risks of cholestasis of pregnancy
Preterm delivery, fetal death, recurrence in subsequent pregs
Biochemical changes in cholestasis of pregnancy
Bili up to 85.5 umol/L
ALP 2-4xURL
AST/ALT mildly increased
Increased serum bile acids (however, often not available)
Increased PT due to vit K malabsorption
DDx cholestasis of pregnancy
Other causes of cholestasis outside of preg ie
1. Drugs
2. PBC
3. Cholelithiasis
4. Dubin-Johnson
Clinical presentation of fatty liver of pregnancy
Usually at 37/40
Rapid onset malaise, nausea, vomiting, abdo pain
Small/normal sized liver (c.f. PET/HELLP where liver is enlarged)
If untreated (with immediate delivery), fulminant hepatic failure and encephalopthy ensues
Biochemical changes in fatty liver of pregnancy
AST>ALT increase usually <6x URL
Bili > 102.6 umol/L
Hypoalbuminemia
Hypoglycaemia
Hyperuricemia from tissue destruction and renal failure
DIC
Cause/association of fatty liver of pregnancy
Inherited mitochondrial fatty acid oxidation disorder in fetus - long-chain 3-hydroxyacyl CoA dehydrogenase deficiency.
Historical indication for amniotic fluid bilirubin? What has it been replaced by?
Determining disease severity in Haemolytic disease of the newborn. Now we use noninvasive ultrasonographic determination of middle fetal cerebral artery velocity
What conditions can NIPT (NGS cfDNA) not be used for?
Neural tube defects
Multiple pregnancies
What is the multiple of the median and why is it used?
Wide variability in AFP assay results in the original UK study of maternal-fetal screening. Multiple of the median developed to normalise results. The median values for each week or day of gestation are determined using the lab’s own assay measurements in the population being screened. Individual test result is divided by the median for gestational age to produce the MoM.
How is risk calculated in prenatal screening?
Individual patient risk is calculated by:
1. Measure all analytes in that patient’s serum
2. Convert measurements into multiples of the median for that gestataional age
3. Determine pt’s a priori risk by maternal age, weight and/or other relevant factors
4. Determine the likelihood ratio of the condition in question based on the MoMs
5. Calculate individual patient risk my multiplying a priori risk and likelihood ratio.
The patient specific risk is used for clinical decision making, rather than the analyte concentrations themselves.
How is the likelihood ratio determined in prenatal screening?
Ratio of heights of the distributions of MoMs in affected and unaffected populations. When more than one analyte is used, a single likelihood ratio is calculated taking into account the correleation between analytes.
Factors which affect prenatal screening test performance (detection and false-positive rates)
- Analyte combination chosen
- Risk cutoff chosen
- Method of dating used to determine gestational age
- Maternal ages of the women being tested
Information that should be included in the maternal serum screening report
- Concentrations and MoM values
- Interpretation as screen positive or negative
- Down syndrome risk estimate (along with risks for othe abnormalities eg trisomy 18)
- Recommendations for possible further action
What information needs to be provided by the physician prior to prenatal screening?
- Speci collection date
- Date of LMP or gestational age confirmed by ultrasound
- Maternal birth date/age
- Relevant pregnancy history
- Number of fetuses if knwon
- Maternal race
- Presence/absence of pre-existing maternal diabetes
Causes of a high AFP on prenatal screening
- Neural tube defect
- Recent fetal demise
- Inaccurate gestational dating
- Twins
Which two analytes combine to make amniocentesis diagnostic for neural tube defect?
Amniotic fluid AFP and acetylcholinesterase
What analytes make up the second trimester triple test?
AFP, hCG, unconjugated estriol
What analytes make up the second trimester quadruple test?
AFP, hCG, unconjugated estriol and inhibin A
What analytes make up first trimester screening?
free betahCG and PAPP-A (and nuchal translucency)
Pattern of results associated with neural tube defect on second trimester screening
High AFP +/- low unconjugated estriol
Pattern of results associated with Down syndrome on second trimester screening
Low AFP, high hCG, low uE3, high inhibin A
Pattern of results associated with trisomy 18 on second trimester screening
Low AFP, hCG and uE3
Pattern of results associated with underestimated gestational age on second trimester screen
High AFP, low hCG, high uE3
Pattern of results associated with overestimated gestational age on second trimester screening
Low AFP, high hCG, low uE3
Pattern of results associated with molar pregnancy on second trimester screening
Low AFP, very high hCG, low uE3
Pattern of results associated with Down syndrome in first trimester screening
Low PAPP-A, high free betaHCG, increased NT
Main benefit of combining first and second trimester screen into integrated screening test?
Improved detection rate (from 85% to 90%) and false positive rate (from 5% to 1%)
Explain how each of the following factors adjusts risks for neural tube defects and Down syndrome
a) Weight
b) Race
c) Diabetes
d) Multiple pregnancy
e) IVF/ART
a) Weight
As weight increases, blood volume decreases and therefore analyte concentration decreases. This affects all analytes apart from uE3. Screening without adjusting for weight may miss cases.
b) Race
African American women have higher AFP and hCG and lower inhibin A concentrations, which should be accounted for when calculating MoMs. The risk of NTD is approximately 50% that of Caucasian women.
c) Diabetes
AFP and uE3 values systematically lower in women who require insulin therapy for diabetes prior to pregnancy. Whether this is explained entirely by a difference in weight is still being debated. Risk of NTD is approx 5x that of the general population
d) Multiple pregnancy
Twin pregnancy does not necessarily double the expected MoM. The most common scenario is that one twin will have disease while the other does not, however, in some cases, both twins will have disease. The analyte distributions between affected and unaffected pregnancies is not as clearly separated in twin pregnancy. Hence screening is not as effective and calculation of an approximate or pseudorisk is all that can be achieved.
e) IVF/ART
Women who achieve pregnancy by IVF are twice as likely to ahve a positive result after second trimester Down syndrome screening than women who conceive spontaneously. This can be adjusted for as it is known that uE3 tends to be reduced and hCG and inhibin A increased in IVF pregnancies. Effects on first trimester screening is less pronounced.
Most common cause of poor lab performance in prenatal screening?
Incorrect median.
How can a lab ensure an accurate median?
Epidemiological monitoring of the initial positive rate. Gather data from screened population to determine screen-positive proportion and compare to expectations based on maternal age distribution of pouplation studied. This rate will be shifted up or down if medians are incorrect for population being screened.
Obtaining an actual detection rate is difficult because complete ascertainment of all pregnancy outcomes is required and it can take years to acquire enough outcome data to allow reliable estimates.
Role of external proficiency testing in prenatal screening
- Evaluate ability to convert analyte values to MoM
- Abiliy to make screening recommendations
- Adjust for variables that influence analyte values
- Calculate patient specific risks.
Physiological adaptations of newborn
Cardiorespiratory - removal of fetoplacental unit, maturation of lung
GI - initiation of enteral feeding, immaturity of hepatic function
Body fluids - maternal biochemistry, insensible water losses, immaturity of renal function
Causes of neonatal hypoglycaemia
- Inadequate gluconeogenesis/glycogenolysis
a) Transient - sepsis, hypothermia, LBW/SGA
b) Hormonal deficiency - cortisol, GH, hypopit
c) Severe liver disease
d) IEM - glycogen storage, gluconeogenic defects, fatty acid oxidation, ketogenesis defects, organic acidaemias, galactosaemia, tyrosinaemia type 1 - Hyperinsulinism
a) Transient - premature, maternal diabetes, maternal PET, birth asphyxia
b) Familial - Beckwith-Wiedemann syndrome
Biochemical investigation of neonatal hypoglycaemia
Insulin, GH, cortisol
Ketones, lactate
Amino acids, acyl carnitines, ammonium
Acid-base, LFTs
Urine organic and amino acids
Identification of neonatal hypoglycaemia?
Similar clinical manifestations occur with other neonatal problems, so use an operational threshold for intervention of 2 mmol/L, <2.5 mmol/L if symptomatic. Target for treatment is >2.5mmol/L
Neonatal hypocalcaemia definition
total < 1.8 mmol/L (if alb < 20g/L, rely on ionised)
ionised < 0.8 mmol/L
Causes of hypocalcaemia in a newborn within the first 72 hours
Prem
Maternal diabetes
Birth asphyxia
Resp distress
Sepsis
Low milk intake
Causes of hypcalcaemia in a newborn after 72 hours
Iatrogenic
Maternal vit D deficiency
Mat hyperparathyroidism
Hypoparathyroidism
Vit D resistance
Hypomg
Renal/liver disease
Organic acidaemias
Biochemical investigation of hypocalcaemia in a neonate
Albumin
Phosphate, Mg
ALP
Creatinine
Acid-base
PTH
25OHD, 1,25OHD
Inherited hypocalcaemic disorders
Familial hypoparathyroidism
- developmental defects in parathyroid gland eg DiGeorge syndrome
- activating mutations in CASR gene (autosomal dominant hypocalcaemia)
- mutations in PTH gene
- defects in stimulatory guanine-nucleotide binding protein (pseudohypoparathyroidism)
