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1 1 Laboratory Testing for Laboratory Testing for Cystic Fibrosis in Newborns Cystic Fibrosis in Newborns Candace Williams, B.S. Candace Williams, B.S. Medical Laboratory Specialist, CF Medical Laboratory Specialist, CF NC State Laboratory of Public Health NC State Laboratory of Public Health Newborn Screening/Clinical Chemistry Newborn Screening/Clinical Chemistry 2 Objectives Objectives Overview of the disorder Overview of the disorder Affected organs Affected organs Life Expectancy Life Expectancy Diagnosis of CF Diagnosis of CF The importance of early detection The importance of early detection Symptom Management Symptom Management Newborn Screening Newborn Screening Methodology Methodology 3 What is Cystic Fibrosis? What is Cystic Fibrosis? Cystic Fibrosis (CF) is a life threatening Cystic Fibrosis (CF) is a life threatening genetic disorder that causes: genetic disorder that causes: Severe lung damage Severe lung damage Serious breathing problems Serious breathing problems Nutritional deficiencies Nutritional deficiencies

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Page 1: Laboratory Testing for Cystic Fibrosis in Newborns · Cystic Fibrosis in Newborns Candace Williams, B.S. ... Life Expectancy for individuals with Cystic Fibrosis On an average people

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Laboratory Testing for Laboratory Testing for Cystic Fibrosis in NewbornsCystic Fibrosis in Newborns

Candace Williams, B.S.Candace Williams, B.S.Medical Laboratory Specialist, CFMedical Laboratory Specialist, CF

NC State Laboratory of Public HealthNC State Laboratory of Public HealthNewborn Screening/Clinical ChemistryNewborn Screening/Clinical Chemistry

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ObjectivesObjectives

�� Overview of the disorderOverview of the disorder�� Affected organsAffected organs�� Life ExpectancyLife Expectancy�� Diagnosis of CFDiagnosis of CF�� The importance of early detectionThe importance of early detection�� Symptom ManagementSymptom Management�� Newborn Screening Newborn Screening �� MethodologyMethodology

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What is Cystic Fibrosis?What is Cystic Fibrosis?

�� Cystic Fibrosis (CF) is a life threatening Cystic Fibrosis (CF) is a life threatening genetic disorder that causes:genetic disorder that causes:�� Severe lung damageSevere lung damage�� Serious breathing problemsSerious breathing problems�� Nutritional deficienciesNutritional deficiencies

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Organs most frequently affected by Organs most frequently affected by Cystic FibrosisCystic Fibrosis

�� A thick accumulation A thick accumulation of mucus causes:of mucus causes:�� Frequent lung Frequent lung

infectionsinfections�� Airway blockagesAirway blockages�� Obstruction to the Obstruction to the

pancreaspancreas

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What is the severity of CF?What is the severity of CF?

�� Limits the organLimits the organ’’s ability to breakdown s ability to breakdown food and absorb nutrients.food and absorb nutrients.

�� Opens a perfect breeding ground for Opens a perfect breeding ground for bacteria and viruses.bacteria and viruses.

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Life Expectancy for individuals with Life Expectancy for individuals with Cystic FibrosisCystic Fibrosis

�� On an average On an average people live into their people live into their mid to late 30s.mid to late 30s.

�� Steadily increasing Steadily increasing past the age of 40 past the age of 40 due to new due to new treatments.treatments.

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How is CF diagnosed?How is CF diagnosed?

�� Through routinely Through routinely screening newborns screening newborns at birth.at birth.

�� Newborn Screening is Newborn Screening is designed to detect designed to detect babies with CF early babies with CF early to improve:to improve:�� GrowthGrowth�� Lung functionLung function�� Increase lifespanIncrease lifespan

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April 13, 2009April 13, 2009

�� NCSLPH implemented NCSLPH implemented Cystic Fibrosis screening Cystic Fibrosis screening for NC newborns.for NC newborns.

�� Each year we see about Each year we see about 35 confirmed cases.35 confirmed cases.

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Benefits of CF screening in early Benefits of CF screening in early detectiondetection

�� Since there is no cure for CF, early Since there is no cure for CF, early symptom management results in:symptom management results in:�� Better nutritional outcomesBetter nutritional outcomes�� Slower progression of lung damageSlower progression of lung damage�� Improved quality of lifeImproved quality of life

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CF Newborn Screening and CF Newborn Screening and MethodologyMethodology

Two tier strategy on a single sample: Two tier strategy on a single sample: IRT/DNAIRT/DNA

IRTIRT�� ImmunoImmuno--reactive reactive trypsinogen(IRTtrypsinogen(IRT))-- a pancreatic enzyme a pancreatic enzyme

which exhibits increased circulating blood levels in CF which exhibits increased circulating blood levels in CF affected patients.affected patients.

�� Automation (Automation (AutodelfiaAutodelfia®®) using a solid phase, two) using a solid phase, two--site site fluorofluoro--immunoassay is used to immunoassay is used to quantitatequantitate IRT levels. IRT levels. The The analyteanalyte is measured in is measured in ngng/ml./ml.

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DNA Mutation TestingDNA Mutation Testing

�� DNA testing will be DNA testing will be conducted on the initial conducted on the initial NBS sample only if IRT NBS sample only if IRT value is abnormalvalue is abnormal..

�� Highest 5% of IRT values Highest 5% of IRT values are identified each day of are identified each day of testing. These testing. These specimens are then specimens are then tested with a DNA tested with a DNA mutation panel.mutation panel.

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Basis of CF Molecular TestBasis of CF Molecular Test

�� A commercial genotyping test A commercial genotyping test kit is used.kit is used.

�� It is an It is an in vitro in vitro diagnostic diagnostic device used to simultaneously device used to simultaneously detect and identify a panel of detect and identify a panel of mutations and variants in the mutations and variants in the cystic fibrosis cystic fibrosis transmembranetransmembraneconductance regulator (CFTR) conductance regulator (CFTR) gene in genomic DNA gene in genomic DNA samples.samples.

�� The CF mutation panel The CF mutation panel consists of 46 different consists of 46 different mutations.mutations.

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Mutation ListMutation List�� E60XE60X Q493XQ493X G551DG551D R1162XR1162X 3120+1G>A3120+1G>A�� G85EG85E deltaI507deltaI507 R553XR553X D1270ND1270N 3659delC3659delC�� R117HR117H deltaF508deltaF508 R560TR560T W1282XW1282X 3849+10kbC>T3849+10kbC>T

�� Y122XY122X F508CF508C 621+1G>T621+1G>T N1303KN1303K 3849+4A>G3849+4A>G�� I148TI148T V520FV520F 711+1G>T711+1G>T 17171717--1G>A1G>A 3876delA3876delA�� R334WR334W G542XG542X 1078delT1078delT 1898+1G>A1898+1G>A 3905insT3905insT�� R347PR347P S549NS549N Y1092X C>AY1092X C>A 2183AA>G2183AA>G IVS8IVS8--5T5T�� R347HR347H S549R A>CS549R A>C Y1092X C>GY1092X C>G 2184delA2184delA IVS8IVS8--7T7T�� 394delTT394delTT S549R T>GS549R T>G D1152HD1152H 2789+5G>A2789+5G>A IVS8IVS8--9T9T

*The variant IVS8*The variant IVS8--5T/7T/9T is a reflex test for individuals positive for the R117H5T/7T/9T is a reflex test for individuals positive for the R117Hmutation. Variant F508C is a reflex test to confirm results witmutation. Variant F508C is a reflex test to confirm results with DI507 or DF508 h DI507 or DF508 homozygosityhomozygosity..

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NCSLPH mutationsNCSLPH mutationsCommon MutationsCommon Mutations

Jan 2011Jan 2011-- May 2011May 2011

0

10

20

30

40

50

60

70

80

90

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DelF508R117HD1270N3120+1G>AG551DD1152H

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CF Molecular test has three CF Molecular test has three processes:processes:

�� DNA extractionDNA extraction�� PCR amplification of PCR amplification of

specific regions of the specific regions of the CFTR gene in CFTR gene in genomic DNAgenomic DNA

�� InvaderInvader®® chemistry to chemistry to detect mutationsdetect mutations

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DNA technology DNA technology

�� A A microfluidicmicrofluidic card card contains a separate contains a separate reaction chamber for reaction chamber for each mutation.each mutation.

�� 8 specimens per card8 specimens per card�� 48 reaction chambers 48 reaction chambers

per specimenper specimen�� 384 different reactions 384 different reactions

per card.per card.

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Two isothermal reactions take Two isothermal reactions take place simultaneouslyplace simultaneously

�� A primary reaction A primary reaction detects mutations on detects mutations on PCR amplified DNA PCR amplified DNA fragments.fragments.

�� A secondary reaction A secondary reaction produces fluorescent produces fluorescent signal from cleaved signal from cleaved DNA structures used DNA structures used for signal amplification for signal amplification and result readout.and result readout.

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Automated ID of mutationsAutomated ID of mutations

�� The card is read on a The card is read on a fluorescent plate fluorescent plate reader. Results are reader. Results are summarized summarized electronically by the electronically by the reader to identify any reader to identify any mutations detected.mutations detected.

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CFTR Executive SummaryCFTR Executive Summary

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DNA analysisDNA analysis

�� What does an abnormal test mean?What does an abnormal test mean?--An abnormal CF screening test means An abnormal CF screening test means that the IRT was elevated and DNA that the IRT was elevated and DNA analysis resulted in 1 of 3 possible analysis resulted in 1 of 3 possible outcomes.outcomes.

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CF ResultsCF Results

�� MutantMutant�� HeterozygoteHeterozygote�� UltraUltra--high IRT >175ng/mL with no mutations. high IRT >175ng/mL with no mutations.

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Report Format Report Format (Laboratory Results)(Laboratory Results)

�� RESULTS RESULTS for IRT and DNA with for IRT and DNA with mutation(smutation(s) ) listed listed

�� INTERPRETATIONINTERPRETATION-- carrier, diseasecarrier, disease�� RECOMMENDATIONRECOMMENDATION-- sweat test at sweat test at

accredited CF center.accredited CF center.

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What should happen when CF test What should happen when CF test is abnormal?is abnormal?

�� The 3 steps:The 3 steps:�� NBS CF FollowNBS CF Follow--up Coordinator is notified.up Coordinator is notified.�� Health care provider is contacted.Health care provider is contacted.�� Sweat test is performed on child.Sweat test is performed on child.

�� Sweat testingSweat testing-- measures how much salt is measures how much salt is in a babyin a baby’’s sweat and should be done at a s sweat and should be done at a CF Foundation accredited care center.CF Foundation accredited care center.

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Goal of CF Newborn ScreeningGoal of CF Newborn Screening

�� Identify newborns with Identify newborns with cystic fibrosis, which cystic fibrosis, which will will notnot include every include every carrier.carrier.

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Some important facts about CF to Some important facts about CF to remember!remember!

�� A hereditary chronic diseaseA hereditary chronic disease�� There is NO cure!There is NO cure!�� Two faulty genes, one from each parentTwo faulty genes, one from each parent�� Most common in Caucasian and Most common in Caucasian and

Ashkenazi Jewish originAshkenazi Jewish origin

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For laboratory questions regarding For laboratory questions regarding Cystic Fibrosis contact:Cystic Fibrosis contact:

NCSLPHNCSLPHNewborn Screening/Clinical ChemistryNewborn Screening/Clinical Chemistry

919919--733733--39373937http://http://slph.ncpublichealth.comslph.ncpublichealth.com//

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ReferencesReferences�� http://www.nhlbi.nih.govhttp://www.nhlbi.nih.gov�� http://www.mayoclinic.com/health/genehttp://www.mayoclinic.com/health/gene--therapy/MY00105therapy/MY00105�� http://http://en.wikipedia.org/wiki/CFTR_(geneen.wikipedia.org/wiki/CFTR_(gene))�� KlevenKleven D, D, McCuddenMcCudden C, Willis M. Cystic fibrosis: newborn screening in C, Willis M. Cystic fibrosis: newborn screening in

America. MLO, July 2008, pp 16America. MLO, July 2008, pp 16--27.27.�� Third Wave Technologies, Third Wave Technologies, ““InplexInplex CF Protocol SummaryCF Protocol Summary””�� North Carolina State Laboratory of Public Health, SOPs for CF IRNorth Carolina State Laboratory of Public Health, SOPs for CF IRT and T and

DNA Testing and Reporting, Mar 2009.DNA Testing and Reporting, Mar 2009.