Testing Options Flashcards
What is the timing of a first trimester screen and what is it used for?
10-13w6d; For T13, T18, and T21
What is included in the first trimester screen?
NT measurement, PAPP-A, hCG measurements
What is PAPP-A
protein produced by the growing placenta and increases until delivery
What is hCG
hormone produced by the placenta in large quantities. Levels rapidly rise for the first 8-10 weeks then decrease and stabilize for the remainder of the pregnancy
What is the hCG trend for T13 and T18
Decreased
What is the hCG trend for T21
Increased
What is the hCG trend for triploidy/molar pregnancy
Extremely increased
What is the NT measurement
Increased fluid in the back of the baby’s neck; above the 95th percentile (3.0mm)= increases the possibility of an aneuploidy; greater than or equal to 3.5mm is above the 99th percentile and could also mean a cardiac defect
What are the cutoff values of a first trimester screen based off of
Selected based off of a less than or equal to 5% false positive rate (for T21 screen positive it is greater than or equal to 1 in 230; for T18 screen positive it is greater than or equal to 1 in 100)
What are the advantages and disadvantages of the first trimester screen
Advantages: 1st tri results, 1 visit, CVS available if +, incorporates maternal factors: age, prior aneuploidy, weight, race, # of fetuses
Disadvantages: lower detection rate compared to integrated screen (~85% for T21), false + could be due to incorrect dating
What population is first trimester screen usually offered to
If a physician with an early to care pop has access to and NT provider and CVS and prefers to complete screening in one visit
What are the trends in a first trimester screen that would indicate T21
Increased NT, decreased PAPP-A, increased hCG
What are the trends in a first trimester screen that would indicate T18
Increased NT, decreased PAPP-A, decreased hCG
Everything DownWARD (Edward, Everything Low)
What are the trends in a first trimester screen that would indicate T13
Increased NT, decreased PAPP-A, decreased hCG
When the NT is high, what biochemical marker is always low
PAPP-A
What is the timing of a second trimester screen and what is it used for?
Timing is 15-21w6d; For ONTDs, T18, T21, T13 (sometimes)
What is included in the second trimester screen?
AFP, hCG, uE3, Inhibin A
Three types: QUAD screen, Integrated MSS, Sequential MSS
What is AFP
Protein produced by the fetal tissue and rises until the 12th wk then gradually falls until birth. If there are problems with the baby, AFP crosses the placenta into mom’s blood (which originated in the amniotic fluid)
What is the detection rate for ONTDs using AFP
85%
What can cause elevations in AFP
GA younger than calculated, spina bifida/anencephaly, pilonidal cysts, AWDs, liver necrosis, GI obstruction, cystic hygroma, urinary obstruction, polycystic kidney, absent kidney, congenital nephrosis, OI, low birth weight, oligohydramnios, multiple gestation, low maternal weight
What can cause low levels of AFP
GA older than expected, trisomies, hydatiform mole, fetal demise, high maternal weight
What can effect hCG levels?
maternal weight, parity
>3.5MoM= poor fetal outcome
What is uE3
form of estrogen produced by fetal metabolism, which involves the liver, adrenals, and placenta. Some crosses the placenta. Levels rise until ~8th week and continue to increase shortly before delivery
What is very low uE3 (<0.15MoM) and indicator of?
SLO or X-linked ichthyosis (Steroid sulfatase deficiency)
If you cannot produce enough cholesterol (like the conditions above), you cannot produce estrogen or testosterone (both will be decreased on NBS)
What is the QUAD screen?
Does not include the NT measurement and does NOT assess risk for T13
Low analyte levels can also be associated with placental insufficency
Incorporates factors like age, weight, race, DM
Inhibin A increases the detection rate for DS
What is the ideal timing for ONTD screening
16-18wks
What is a PENTA screen
Incorporates invasive trophoblast antigen (ITA); Detection rates are not substantially increase over QUAD
What is the integrated MSS?
Two step process to adjust the maternal age- related risk for DS and T18. Pt is given a single RA after 1st tri screening and QUAD screening
Detection rate for T21 is 94-96% and T18 is 91-96%
Eliminates the possibility of CVS
What is the stepwise sequential MSS?
Combines 1st tri screen and maternal age, then are grouped into high or low risk categories. High risk is offered dx testing, low risk group moves onto QUAD screen
Detection rate for T21 is 94-96% and T18 is 91-96%
What are the trends in a second trimester screen that would indicate T21
Decreased AFP, Decreased uE3, increased hCG, increased Inhibin A (only detects T21)
What are the trends in a second trimester screen that would indicate T18
Decreased AFP, decreased uE3, decreased hCG
Everything DownWARD (Edward, Everything Low)
What are the trends in a second trimester screen that would indicate T13
Decreased hCG
What are the trends in a second trimester screen that would indicate ONTDs
Very high AFP
What is inhibin A
hormone produced by the placenta that decreases slightly from 14-17wks GA then rise again
ONLY DETECTS T21
What is the timing of ultrasound and what is it used for?
Timing: ~11-13wks for NT, ~18-20wks for anatomy scan
Goal: congenital anomalies occur in ~3% of live births. Identify anomalies that may not be compatible with life, are associated with a high level of morbidity, or may benefit from specific interventions before/after birth
What are soft markers
Minor anomalies on u/s that are often normal variants with no clinical significance but are associated Wwith increased risk of fetal abnormality
What is the timing of CVS and what is it used to test
10-13wks, aneuploidies, microdel/dup syndromes, single-gene disorders
What is the procedure of a CVS, what is the miscarriage rate, and what kinds of indications are recommended for this test
insert a small catheter transabdominally or transvaginally (dependent on placement of placenta) to take up small pieces of the chorion (placenta)
0.5-1% risk of pregnancy loss (can damage the placental barrier= mixing of fetal + maternal blood)
indications may include: abnormal NIPS, prior child with structural defect, prior child with trisomy, AMA/APA, parental carrier of a chromosomal rearrangement, parental aneuploidy/aneuploidy mosaicism, parental carrier of a genetic dz
What are the advantages and disadvantages of a CVS
advantages: know accurate results early on
disadvantages: risks of false positive and negatives
What are some ways that a false positive result can occur on CVS
not sampling the actual embryo= mosaicism
What are some ways that a false negative result can occur on CVS
in sampling the chorion close to the maternal tissue, could take up mom’s tissue in sample and cause contamination
Rh factor (protein factor on RBCs) either have the protein (Rh+) or don’t (Rh-); ITS AND AD TRAIT
What are the implications of the Rh factor on pregnancy outcomes
If MOM has Rh+ and FETUS has Rh- there are no issues
If MOM has Rh- and FETUS has Rh+, mom’s body recognizes the fetus as a foreign invader in which the maternal immune system damages the fetus (Tx mom with Rh immunoglobin to repress response to the Rh immune system)
What is the timing of amniocentesis and what is it used to test
Timing: 16-20wks, for aneuploidies, microdel/dup syndromes, single gene dz’s, ONTDs, biochemical conditions, TORCH
What is the procedure of an amniocentesis and what are the complications that can occur for mom and fetus
insert hollow needle to sample fetal skin cells and urine inside amniotic cavity
In mom: estimated 2.6% of fetomaternal hemorrhage, minimal chance of introducing skin bacteria into the amniotic cavity (<0.1%), preterm labor in 3rd tri amnio, 2-3% risk of vaginal bleeding
in fetus: miscarriage risk is 0.5-0.1%, amniotic fluid leak in ~1-2% of cases, fetal injuries like bleeding from the cord, ocular injuries, clubfoot can occur but is VERY rare
What is the timing of cord blood sampling and what is it used to test
timing: after 18wks, for: fetal anemia, Rh discrepancies, biochemical analysis (Arginase deficiency)
What is the procedure of a cord blood sampling and what are the advantages/disadvantages
under u/s guidance, is performed through maternal abdomen; blood sample sent to eh lab for hematological, immunological, and biochemical analysis
miscarriage rate bwn 1-3%
advantage: fetal transfusion, which can be life saving
What is the timing of NIPS and what is used to detect
beginning at 10wks, used to screen for fetal aneuploidies, sex chromosome abnormalities, microdel/dup syndromes, fetal sex
How does NIPS work on a SNP array platform?
involves analyzing thousands of SNPs along the genome; arrays have probes that hybridize to specific regions of the genome, allowing for detection of variations at those positions
in cases of aneuploidies, there is an imbalance in the number of copies of specific chromosomes. This imbalance is detected through allele ratio analysis. By comparing the relative abundance of alleles on the chromosomes of interest to those on other chroms, deviations from the expected ratios can indicate the presence of an aneuploidy
genotyping data is compared to reference genomes to identify variations/abnormalities
vanishing twin/bone marrow transplant: SNPs will fail
ex: Panorama by Natera
What are the advantages/disadvantages of NIPS on a SNP array platform?
advantages: ability to detect large chromosomal abnormalities and SNVs
disadvantages: not as sensitive/specific as newer technology like NGS
How does NIPS work using the counting/ relative dosage analysis method?
uses qPCR to measure the copy # of target sequences corresponding to to chroms of interest
relative ratios of target sequences to reference sequence is calculated, deviations=aneuploidies
CANNOT DETECT TRIPLOIDY
How does NIPS work on an NGS platform? What are its advantages
isolated cffDNA undergoes library prep where DNA fragments are enzymatically fragmented and adapters are added
libraries are sequenced simultaneously in a massively parallel manner
detects aneuploidies by analyzing the relative abundance of sequencing reads mapping to each chrom. Deviations from expected ratios indicate presence of aneuploidy
advantages: higher resolution, sensitivity, ability to detect a wider range of genetic variations
What is the approximate sensitivity and PPV for the trisomies using SNP based NGS
T21: 99%/95%
T18: 95%/91%
T13: >99%/68%
Triploidy: >99%/7.5%
22qdel: 83%/53%
PWS:93.8%/5%
What are some confounding factors that can affect NIPS results
low ff, maternal autoimmune dz, benign uterine tumors, maternal obesity, twins
false +’s: placental mosaicism, vanishing twin, malignancies
What is analytical validity
ability of a test to accurately measure a given analyte (enzyme, mutation, protein). Based on analytical accuracy, analytical precision, analytical sensitivity, analytical specificity, and analytical PPV
What is analytical precision
reproducibility, agreement between measurements
What is analytical accuracy
degree of agreement of the test result with the “gold standard” of the analyte
What is analytical sensitivity
ability to detect an analyte and gives a lower limit of detection of the analyte
What is analytical specificity
ability of the test to detect ONLY the analyte of interest (the proportion of negative results given that the analyte is not there)
presence of VUS lowers the analytic specificity of the test bc the lab cannot determine whether the VUS qualifies as an analyte
What is analytical PPV
% of all positive results that are true positives
When PPV decreases what else decreases
prevalence
What can a karyotype identify
large del/dups and balanced t’s
What can FISH identify
uses probes that bind to target DNA sequence and light up to show how many copies there are
consider a prelim result; does not have 100% detection, should not make pregnancy decisions on these results
aneuploidies and del dups at specific point using a probe
can detect del/dups >0.5-1Mb
What can a microarray identify
detect large and small del/dups and ROH
oligos: detect del/dups; SNPs detect different SNPs and number of alleles at a point
but cannot detect balanced translocations or sequence variants
What are the exceptions when you do NOT want to start with gene sequencing following del/dup as a first line test
DMD: caused by a del in DMD gene
SMA: del in SMN1 (start with CMV analysis)
What are ROH? What should you consider if they are present
Review genes in affected region that could cause other conditions
if a whole chrom is ROH, consider UPD
What is clinical validity
ability of a test to predict the presence of the associated disorder or phenotype and includes clinical sensitivity, clinical specificity, and clinical utility
What is clinical sensitivity
ability to detect dz given that the dz is present, or the frequency of + tests when the dz is present
estimations/measurements generally assume a high degree of penetrance
if test is 100% analytically valid and penetrance is low, clinical sensitivity may be very low
What is clinical specificity
ability of a test to discriminate between those with the suspected phenotype or dz and those without it
frequency of a negative test when the dz is absent
What is clinical utility
ability of the test to detect and assist with management of dz , improve tx effectiveness, and/or improve quality of life of affected individuals
based on the presumption of analytical validity
include and balance clinical, economic, and psychological measures of possible benefit and harm
What is the predictive value
ability of a test to identify the presence of the dz
What is clinical positive predictive value
% of + results given that the dz is present
population based measurement, accuracy is based on the population prevalence of a dz
Describe how you would calculate the clinical sensitivity, clinical specificity, PPV, NPV, false positive, and false negative rate for a given test
clinical sensitivity: probability that a person with a dz will have a positive test results (True positives/ TPs+ False Negatives)
clinical specificity: probability that a person without a dz will have negative test results (True Negatives/ TN’s +False positives)
PPV: probability that the person has the dz, given the test is + (TPs /TPs +FPs)
NPV: probability that the person does not have the dz, given the test result is - (TN’s/ TN’s +FNs)
False positive rate: proportion of all + test results occurring in individuals who are NOT affected (FPs /FPs +TNs)
False negative rate: proportion of all negative results occurring in individuals who ARE affected (FNs/ TPs+ FNs)
What are some causes for a false + result on a test
lack of perfect analytical specificity
test is cross reacting (Testing for polymorphic marker near real dz causing variant)
genotype result is correct but phenotype is not visible due to reduced penetrance
dz has been clinically misclassified
What are some causes for a false - on a test
insufficient analytical sensitivity- test does not detect the dz-associated phenotype
untested genes are responsible (genetic heterogeneity)
untested variants (allelic heterogeneity) are responsible for the dz
dz has been misclassified (penetrance or expressivity)
genotype is not present in the cell population tested (mosaicism)
ethnicity-dependent variations in frequency of the genotype
What are level 1 standards/guidelines for ensuring quality control in clinical genetic testing
CLIA (clinical laboratory improvement amendments)
government regulated and designed to be a set of minimal lab requirements to ensure safety of the public
assures tests are accurate, timely, reliable, confidential, and do not harm pts
NY and Washington have state regulations that supersede CLIA (have additional specific requirements)
What are level 2 standards/guidelines for ensuring quality control in clinical genetic testing
ACMG, College of American Pathologists (CAP), Association of Molecular Pathologists (AMP)
The centers for Medicare and Medicaid services currently do NOT recognize what laboratories
Lab accreditation programs outside the US
Although all genetic testing labs are overlooked by CLIA, what is not required?
most lab developed genetic tests are not subject to direct government oversight (including DTC and research)
CLIA requires internal testing of the tests’ analytical performance but does NOT require outside verification (only analytical validity; does not take into account clinical validity or clinical utility)
Under CLIA regulations, the dispersing the results of research is non-compliant with federal law. However, how can this CLIA regulation be superseded
Some IRBs allow the release of results to pts before confirmation by a certified CLIA lab
research results CAN be released in aggregate but individuals should NOT be told of their genetic status
If a prenatal pt presents with research results and wants to test her fetus for the finding, can they do it?
No, clinical labs will NOT accept research results as the basis for prenatal testing if results are not confirmed in a timely manner
What can Sanger sequencing test? What are its advantages/disadvantages?
can only sequence one fragment of DNA at a time
each based is only read one time by the camera
cost effective for small sequences of DNA
What can Next Gen sequencing test? What are its advantages/disadvantages
can sequence as many fragments as needed, even a whole genome
multiple sequence reads per base pair and higher sensitivity (each base is detected in multiple different fragments)
computer aligns all of the different fragments to compile DNA sequence
WES/WGS both use NGS (sanger sequencing used to rescue low coverage areas)
Describe the tests and their corresponding genetic condition that do NOT test DNA
IHC tumor testing (mismatch repair proteins)
hexA enzyme testing (Tay-Sachs)
urine/serum screening for aas and oas when a metabolic condition is suspected
immunoreactive trypsinogen analysis (CF for NBS, followed by sweat chloride when +)
sister chromatid exchange assay for Bloom
Chromosome breakage analysis for FA
What are the criteria that need to be met to have a condition on NBS
suitable screening test
early dx is beneficial
the natural hx is understood
an accepted tx is available
there are facilities for dx and tx
dx and tx is cost effective
Give an example testing strategy for imprinting disorders
start w methylation studies which are able to detect all three mechanisms of methylation differences; if it is negative can do gene del/dup sequencing to rule in or out dx; if it is positive, need to to karotype and FISH/microarray for dels larger than 5Mb and translocations; if positive, do microarray to detect deletion size; if negative, need to do UPD testing w microarray; if that is negative, then do gene del/dup of the imprinting center and gene
