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SCREENING WHAT TESTS WHEN & WHAT NEXT?

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SCREENING WHAT TESTS WHEN & WHAT NEXT? by Dr. Narendra Malhotra

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SCREENING WHAT TESTS WHEN & WHAT NEXT?

  1. 1. SCREENING WHAT TESTS WHEN & WHAT NEXT? NARENDRA MALHOTRA JAIDEEP MALHOTRA RISHABH BORA NEHARIKA BORA KANIKA BANSAL MANJEET MEHTA Global rainbow health care India
  2. 2. Reference Material • New WHO guidelines on antenatal care – Systematic review BJOG 2016;123:519-28 • Guidelines by Government of western Australia • SOGC guideline on Prenatal Screening • RCOG / NICE Guidelines • FOGSI expert group(TOG)
  3. 3. “I get by with a little help from my friends” Dr S Suresh Dr Ashok Khurana Dr Pratima Radhakrishnan Dr Deepika Deka Dr Anita Kaul Dr Feroza Begum THANK YOU FRIENDS FOR HELPING AND CONTRIBUTING TO THIS
  4. 4. SCREENING PRINCIPLES Screening is done for: • Disorders with common prevalence • Have significant impact on perinatal mortality & morbidity Conditions to be fulfilled: • Accessibility, Availability , Affordability • Must understand Cost of burden of the disease vs cost of screening
  5. 5. What Do Screening Tests Do? 1. Identify patients “at risk” for a disorder in an “unsuspected” population 2. “Filters” patients for further diagnostic testing 3. Results are given as “high risk” or “low risk” based on a “cut off” value
  6. 6. PRENATAL SCREENING • I TRIMESTER • History, MAP, Ut A doppler / Biochem • II TRIMESTER(?) • UT A doppler • CERVICAL LENGTH – II trimester • I & II TRIMESTERS • ULTRASOUND • BIOCHEMISTRY • CELL FREE DNA • I & II TRIMESTERS ANEUPLOIDY ANOMALY PRE- ECLAMPSIA PRETERM BIRTH
  7. 7. PRENATAL TESTING SCREENING TEST •Provides individual risk assessment •Advantages • Lowers the number of procedures done for diagnosis • Lowers the procedure related complications •Disadvantages • Non diagnostic • May miss target DIAGNOSTIC TEST •A test that WILL identify a disease or defect •Advantages • Definitive •Disadvantages • Risk associated with diagnostic procedure/s
  8. 8. Magnitude of Problem Congenital & Genetic Disorders in India Disorder Frequency at birth in stillbirths in NN Deaths Congenital 2-5% (1:50 , 6 lacs/yr) 13.6 % 7 – 10 % Malformations Chromosomal 0.5% ( DS – 1:916 , 22,000 / yr ) Single Gene 0.6% ( B- thal – 1:2700 , 9000/ yr ; SCD - 5,200 /yr ; DMD + SMA - 4,500 / yr ) ( IC Verma , Preventive Genetics ,2006 )
  9. 9. Chromosomal defects Source: Kagan KO et al. Screening for Chromosomal Abnormalities by First Trimester Combined Screening and Noninvasive Prenatal Testing. Ultraschall in Med 2015; 36:40-46)
  10. 10. FOGSI OLD CHECK LIST OF 2009,MODIFIED IN 2017 AT FOGSI T.O.G.
  11. 11. SCREENING FOR ANEUPLOIDY • Trisomy 21 - Down syndrome - 1 in 800 • Trisomy 18 – Edwards synd. - 1 in 10,000 • Trisomy 13 – Patau’s syndrome > 1 in 10,000 Apriori Risk MATERNAL AGE, Previous screening) Identify markers (USG, Biochem) Modify Risk Identify Patients for invasive testing
  12. 12. 12 TEST FIRST TRIMESTER SECOND TRIMESTER CRL 45-84 BPD 30mm -51mm 10 11 12 13 14 15 16 17 18 19 20 Sequential Screen NT, PAPP-A, hCG +PLGF AFP, hCG, uE3, Inhibin A CONTINGENT SCREEN NB/ DV/TR Serum Integrated Screen hCG , PAPP-A AFP, hCG, uE3, Inhibin A Quad Screen E-FTS PAPP-A, hCG +AFP +PLGF AFP, hCG, uE3, Inhibin A 1 4 w G r a y Z o n e THE TIMELINES FOR SCREENING NIPS – after 10 weeks – ideal before 17 weeks to help decision making < 20 weeks
  13. 13. First trimester screening FOR CHROMOSOMAL ANOMALIES FETAL CROWN FETAL RUMP NT/NB DV TR
  14. 14. First Trimester Screening – The yield Test Name Time of Test(in Weeks) Markers DR(%) FPRs ( % ) Early Biochemistry 9-10^+6 MA+hCGB, PAPP-A ~60-65 5 % COMBINED FIRST TRIMESTER SCREENING 11-13^+6 MA+hCGB, PAPP-A+NT ~90-91 3 % 11-13^+6 MA+hCGB, PAPPA+NT +NB+tri cuspid flow+facial angle+Ductus Venoses ~95% 3 % Penta Screening 10- 13^+6 MA+ hCGB, PAPPA, PIGF, DIA+NT+NB 98 % 1.2 % BE HAPPY TEST
  15. 15. JOGI – Feb 2019
  16. 16. Objective • To derive a risk calculation algorithm suitable for use in India when screening for Down’s • Syndrome using four first-trimester maternal serum markers either alone or with ultrasound nuchal translucency (NT).
  17. 17. Enhanced Combined test 1: 250 cutoff Enhanced Combined EFTS COMBINED TEST DETECTION RATE 90% 85% FALSE +ve rate 1.4% 1.8%
  18. 18. Screening for Trisomy 21 at 11- 14 weeks 2-stage (contingency) screening- UK system COMNINED TEST (NT+BIOCHEM) FOR ALL Fetal NT and free BhCG and PAPP-A at 12 wks High risk >1:250 low risk <1:1000 CVS Reassure Borderline risk 1 in 251-1:1000 Further screening Nasal bone DV, TR NIPS
  19. 19. 99
  20. 20. First Trimester Second Trimester Third Trimester
  21. 21. Second Trimester Screening Screening Strategy Time of Test(in Weeks) Markers Used DR(%) FPRs ( %) Triple Test 15-21^+6 MA+AFP,hCGB, uE3 ~65-70 5-7% Quadruple Test 15-21^+6 MA+AFP,hCGB, uE3 ,Inhibin-A ~70-75 5-7% Integrated Test 15-21^+6 1st Trimester – PAPP-A 11-13+6/7weeks (11 Wks is Ideal) 2nd Trimester –Quad AFP, uE3, FBhCG, Inhibin-A (15-17 Wks is ideal) 93% 3 %
  22. 22. Contingent II trimester “Genetic sonogram” To modify risks after FTS combined, Quadruple Or Integrated screening Needs high USG expertise Software for calculation ARSA Absent NB
  23. 23. When there are major structural defects NO screening is offered – Direct testing is preferred
  24. 24. SCREENING FOR FETAL ANOMALIES- I TRIMESTER 70% detection rate if a systematic scan is done
  25. 25. Pre-Eclampsia • Adaptive disease when there is relative placental imbalance (Utero Placental mismatch) – Late onset may be beneficial to the baby – improved quality of survival in late mild PE • Early onset may have implications for mother / baby
  26. 26. SCREENING FOR PE • Maternal history • Maternal blood pressure • Uterine artery doppler • Biochemical markers • Combination .. PAPP-A / PLGF
  27. 27. First trimester screening for Preeclampsia Maternal serum PIGF, Mean arterial Blood pressure - Implantation - Maternal artery remodelling Detection rate – 90% 0 10 20 30 Falsepositive(%) Doppler 12% 30% PP13 9% Doppler & PP13
  28. 28. History + MAP + UT A doppler + PLGF & PAPP-A • 93% detection rate (5% FPR) for early preeclampsia (<34 weeks) Poon LC et al Hypertension 2009 A combination of markers at 11- 13 weeks is ideal
  29. 29. • Low-dose aspirin initiated in early pregnancy is an efficient method of reducing the incidence of preeclampsia and IUGR. (Obstet Gynecol 2010;116:402–14) Is there intervention??
  30. 30. Is there intervention if screened in first trimester? • Low-dose ASA may prevent as many as 50% of cases of PE & IUGR when treatment is initiated before 16 weeks of gestation Bujold E, et al Obstet Gynecol 2010;116(2 Pt 1):402–14. Bujold E et al J Obstet Gynaecol Can 2009;31:818–26.
  31. 31. DIAGNOSTIC TESTS AFTER SCREENING WHAT TO ORDER AND WHAT TESTS TELLS WHAT
  32. 32. DIAGNOSTIC CONFIRMATORY TESTING AFTER SCREEN POSITIVE RESULT • What tests: –Karyotyping • Aneuplodies , structural rearrangements –Microdeletions • 22q most common –Microarray • Increased NT – Normal Karyotype • Deletions & duplications
  33. 33. 1. Karyotyping 2. Fluorescence In Situ Hybridization (FISH) 3. Polymerase Chain Reaction (PCR) 4. Array‐based comparative genomic hybridization (array CGH) i.e. chromosomal microarray analysis (CMA) ,next-generation sequencing (NGS) , whole-exome sequencing Shaffer LG, Bui T‐H. 2007. Molecular cytogenetic and rapid aneuploidy detection methods in prenatal diagnosis. Am J Med Genet Part C Semin Med Genet 145C:87–98. Testing with foetal cells
  34. 34. Karyotype can detect • Some specific abnormalities e.g. - Trisomies (21,18,13), Klinefelter's syndrome (XXY and other variations), Triple X syndrome (XXX) - Monosomy : Turner syndrome (X0) • Some Chromosomal deletions - Cri-du-Chat syndrome (missingCh5) - Williams syndrome (missing Ch7) • Translocations : Robertsonian translocations Limitation : Cannot detect specific gene mutations e.g. cystic fibrosis , Thalassemia. Resolution limited to around 5 Mb.
  35. 35. Molecular cytogenetics :FISH (Florescence In Situ Hybridization) • Is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes • Allows visualization of small region on chromosome too small to be identified by karyotyping(submicroscopic deletion) • Unlike Karyotype(21days) results available with in 24 to 72 hrs as done on interphase nuclei
  36. 36. QF-PCR  has recently entered the field of prenatal diagnosis to overcome the need to culture fetal cells, allow rapid diagnosis of some selected chromosomal anomalies.  has the advantage of being much less expensive and allowing the simultaneous processing of much larger number of samples than FISH.  Can detect mosaicism of about 20–30% and is superior to both traditional karyotyping and interphase FISH for the identification of maternal cell contamination [ Donaghue et al., 2005]
  37. 37. QF-PCR: Offerings QF-PCR BASIC QF-PCR EXTENDED Coverage • Trisomies :13,18,21 and Gonsomal Aneuploidies  Trisomies :13, 18,21, 15,16,22, Gonosomal Aneuploidies  20 common microdeletions and microduplications:  DiGeorge Syndrome,Langer- Giedons Syndrome,Smith- Magnenis Syndrome,Miller- Dieker Syndrome,Williams- Beuren Syndrome,Potoscki- Lupuski Syndrome
  38. 38. Microarray Analysis Can identify chromosomal aneuploidy , large structure changes as well as submicroscopic abnormalities that are too small to be detected by traditional modalities A DNA microarray is a thin-sized chip that has been spotted at fixed locations with thousands of single- stranded DNA fragments corresponding to various genes of interest A single microarray may contain 10,000 or more spots, each containing pieces of DNA from a different gene
  39. 39. Introduction of chromosomal microarray analysis into prenatal diagnosis • CMA is now the first-tier genetic diagnostic test for children and adults with multiple congenital anomalies, genetic syndromes, and intellectual and developmental disabilities, diagnostic yield - 15 to 20% • CMA is recommended as a primary test, by ACOG ,after USG finding of structural abnormality unless the abnormality is “strongly suggestive” of a particular aneuploidy • CMA suitable for analysis of stillbirth samples & early miscarriages , it does not require actively dividing cells Miller DT, Adam MP, Aradhya S, et al. Am J Hum Genet. 2010;86(5):749–64. Hillman SC, McMullan DJ, Hall G, et al. : Ultrasound Obstet Gynecol. 2013;41(6) ACOG Practice Bulletin,163:2016
  40. 40. Next Generation Sequencing(NGS) • Sequence each of the 3 billion bases in the human genome multiple times providing an insight into unexpected DNA variation missed by other methods. • Can sequence entire genomes or specific areas of interest, including all 22 000 coding genes (a whole exome) or small numbers of individual genes • By providing a base-by-base view of the genome, NGS can identify single nucleotide variants (SNV), small structural changes, and balanced translocations • increasing information while decreasing costs with a genome-wide view of variation. Arch Dis Child Educ Pract Ed. 2013 Dec; 98(6): 236–238
  41. 41. SEQUENCING KARYOTYPE NEXT GENERATION SEQUENCING MICROARRAY
  42. 42. Whole-exome sequencing(WES) • Congenital abnormalities identified USG , karyotype and CMA reveal a diagnosis in up to 20–30%, for the remainder, single-gene tests or gene panels, may be useful • CMA and NGS based methods, such as targeted gene- panel sequencing and, recently, whole-exome sequencing (WES), has enabled to diagnose more fetal genetic conditions • In WES, majority of coding exons, which represent only 2% of the genome but contain 85% of disease-causing mutations, are sequenced Lee H, Deignan JL, Dorrani N, et al. : . JAMA. 2014;312(18). Gilissen C, Hehir-Kwa JY, Thung DT, et al. : Nature. 2014;511(7509) Beaulieu CL, Majewski J, Schwartzentruber J, et al. : . Am J Hum Genet. 2014;94(6)
  43. 43. Cont-- • The American College of Medical Genetics and Genomics recommends considering WES when specific genetic tests available for a phenotype, including targeted sequencing , have failed to determine a diagnosis in a fetus with multiple congenital anomalies suggestive of a genetic disorder • Routine use of whole-genome or whole-exome sequencing is not recommended outside of the context of clinical trials ACMG Board of Directors. Genet Med 2012;14:759–61 , ACOG Practice bulletin ,Number 682, December 2016
  44. 44. Take home message SCREEING SHOULD BE OFFERED TO ALL POPULATION IRRECPECTIVE OF RISK While • Testing should be focused on the individual patient's risks, reproductive goals, and preferences Patients should understand the benefits and limitations , including the conditions that will not be detected by tests PRE AND POST TEST COUNCELLING IS VERY IMPORTANT
  45. 45. Various Integrated screening in strategies (1st and 2nd trim) Main strategies: • Fully Integrated • Step-wise sequential • Contingent screening 1st trimester: NT, PAPP-A No risk estimate 2nd trimester: Fb-hCG, AFP, uE3, (± Inhibin) Final risk estimate: All markers 1st trimester: NT, PAPP-A, Fb-hCG Risk estimate Final risk estimate: All markers CVS NIPT High risk Low risk 2nd trimester: Fb-hCG, AFP, uE3, (± Inhibin) 1st trimester: NT, PAPP-A, Fb-hCG Risk estimate 2nd trimester: Fb-hCG, AFP, uE3, (± Inhibin) Final risk estimate: All markers CVS NIPT HR Borderline risk No further screening LR
  46. 46. Thank you Screen with the right test at the right time

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