An Introduction to An Introduction to FISHFISH

Genzyme GeneticsGenzyme Genetics

OverviewOverview Introduction to FISHIntroduction to FISH FISH processing FISH processing

technicaltechnical

Probes Probes TypesTypes ExamplesExamples

ScoringScoring Troubleshooting and QCTroubleshooting and QC QuestionsQuestions

What is FISH?What is FISH?

FFluorescence luorescence iin n ssituitu hhybridizationybridizationmolecular cytogenetic techniquemolecular cytogenetic technique

FISH looks at specific segments of DNA FISH looks at specific segments of DNA in the context of the whole genomein the context of the whole genome

FISH is a bridge between classical FISH is a bridge between classical cytogenetics and molecular geneticscytogenetics and molecular genetics

FISH provides answers to questions about possible chromosome abnormalities at a higher resolution than standard cytogenetics

…think of FISH as a camera’s zoom lens

FISH Uses DNA technology Applied to Chromosomes

FISHFISH AdvantagesAdvantages

No need for metaphases (for some studies)No need for metaphases (for some studies) Fast- no culturing necessaryFast- no culturing necessary High specificity and sensitivity (can detect minimal High specificity and sensitivity (can detect minimal

residual disease)residual disease) Detection of cryptic abnormalitiesDetection of cryptic abnormalities Large numbers of cells can be analyzed quicklyLarge numbers of cells can be analyzed quickly

DisadvantagesDisadvantages Lower abnormality rates due to normal cell Lower abnormality rates due to normal cell

contamination (for interphase cancer studies)contamination (for interphase cancer studies) Specific to certain gene regionsSpecific to certain gene regions Can not detect point mutations or deletions smaller Can not detect point mutations or deletions smaller

than the probe sequencethan the probe sequence Gives only specific answers to specific questions Gives only specific answers to specific questions

► ► The right question must be The right question must be askedasked

FISH for what?FISH for what? Confirm a clinical diagnosisConfirm a clinical diagnosis

Microdeletion syndromesMicrodeletion syndromes Clarify abnormal chromosome results Clarify abnormal chromosome results

Complex rearrangementsComplex rearrangements Diagnosis and prognosis of cancersDiagnosis and prognosis of cancers

Staging of cancer, type of chemotherapy to Staging of cancer, type of chemotherapy to useuse

Quick, preliminary resultsQuick, preliminary results InSightInSight

Internal laboratory quality controlInternal laboratory quality control ““tube checks”tube checks”

FISH ProcessingFISH Processing

General steps in FISH General steps in FISH processingprocessing

Specimen fixed on slideSpecimen fixed on slide AgingAging

PretreatmentPretreatment Probe applicationProbe application

DenaturationDenaturation HybridizationHybridization

WashWash CounterstainCounterstain

AnalysisAnalysis

Generalized FISH Generalized FISH procedureprocedure

Specimen DNASpecimen DNA

Directlabeledprobe

Denaturation (high temperature)

Hybridization (37°C)

Wash and counterstain

Detection

Aging, pretreatment

Commonly used FISH Commonly used FISH equipmentequipment

ThermobriteThermobrite Allows for co-denaturation of target DNA and Allows for co-denaturation of target DNA and

probesprobes Eliminates manual steps and reduces hands-on Eliminates manual steps and reduces hands-on

time during FISH procedures time during FISH procedures Slides do not need to be fully loaded to maintain Slides do not need to be fully loaded to maintain

temperature accuracy temperature accuracy Slide guide keeps slides in place and allows for Slide guide keeps slides in place and allows for

one hand removal one hand removal Humidity Control Cards inside the lid maintain a Humidity Control Cards inside the lid maintain a

humid environment humid environment VP 2000 Processor VP 2000 Processor

Automated processing of paraffin embedded Automated processing of paraffin embedded tissue slides (deparaffinization and pretreatment tissue slides (deparaffinization and pretreatment steps)steps)

Commonly used FISH Commonly used FISH equipmentequipment

Commonly used FISH Commonly used FISH ReagentsReagents

FormamideFormamide: used as a denaturation reagent in conjunction : used as a denaturation reagent in conjunction with a salt solution. Breaks double stranded DNA into with a salt solution. Breaks double stranded DNA into single strands and is typically done at 73single strands and is typically done at 73°°CC

FormaldehydeFormaldehyde: Typically used in a 1% solution as a post : Typically used in a 1% solution as a post fixative to conserve DNA during the denaturation process. fixative to conserve DNA during the denaturation process. Can be used to improve morphology of metaphase Can be used to improve morphology of metaphase chromosomes.chromosomes.

SSCSSC: a salt solution used to dehydrate (artificially age) the : a salt solution used to dehydrate (artificially age) the DNA. Used in various stringencies as a wash reagent to DNA. Used in various stringencies as a wash reagent to rinse the non-specific hybridizations of labeled DNA probe.rinse the non-specific hybridizations of labeled DNA probe.

EthanolEthanol: Used to dehydrate the specimen. Can also be used : Used to dehydrate the specimen. Can also be used as a fixative for certain types of specimens.as a fixative for certain types of specimens.

PepsinPepsin: Protease used to digest tissue to remove protein : Protease used to digest tissue to remove protein material and non cellular components. Increases probe material and non cellular components. Increases probe accessibility to DNA.accessibility to DNA.

AgingAging

The aging process removes water The aging process removes water from the specimen and makes the from the specimen and makes the cells “harder” and more able to cells “harder” and more able to withstand the abuse they will take withstand the abuse they will take during later stepsduring later steps

2 minutes at 732 minutes at 73°C in a 2xSCC solution°C in a 2xSCC solution

PretreatmentPretreatment

Pretreatment is used to make the DNA Pretreatment is used to make the DNA in the specimen more able to withstand in the specimen more able to withstand the harsh conditions that will exist the harsh conditions that will exist during later processing stepsduring later processing steps

Pretreatment is also used to make the Pretreatment is also used to make the DNA more accessible to the probe by DNA more accessible to the probe by removing cytoplasm and proteins that removing cytoplasm and proteins that can interfere with probe hybridizationcan interfere with probe hybridization

•Drop slides

•2xSCC73°C ↓ 2 min (37°C for 30 min)

•Pepsin37°C ↓ 5 min

•ETOH (70%, 85%, 100%)RT ↓2 min each

Aging/PretreatmentAging/Pretreatment

[Co-denaturation eliminates the need to use formamide]

Co-denaturation of target DNA and probes using Thermobrite system (Abbott Molec)

73°C ↓ 5 min

Hybridization of probes to target DNA37°C~42°C ↓ overnight

Post-hybridization wash72°C ↓ 0.4xSSC/0.3% NP-40

Counterstain with DAPI

Analyze/image under fluorescent scope

Co-Denaturation/Co-Denaturation/Hybridization/WashHybridization/Wash

FISH ProbesFISH Probes

What are What are Probes?Probes?

A “probe” is a short sequence of DNA specifically A “probe” is a short sequence of DNA specifically designed to pair with the complementary sequence designed to pair with the complementary sequence in the patient’s DNA (the “target”).in the patient’s DNA (the “target”).

A fluorescent molecule (fluorochrome) is attached to A fluorescent molecule (fluorochrome) is attached to the DNA sequence. Several different colored probes the DNA sequence. Several different colored probes can be used in one solution.can be used in one solution.

Most probe mixtures have a ratio of 1:2:7-probe to Most probe mixtures have a ratio of 1:2:7-probe to water to buffer. This can vary by company or by water to buffer. This can vary by company or by probe intensity.probe intensity.

A “ready to use” or “working solution” probe A “ready to use” or “working solution” probe contains probe fragments and a hybridization buffer.contains probe fragments and a hybridization buffer.

Types of ProbesTypes of Probes

Whole Chromosome Paints Whole Chromosome Paints (WCPs)(WCPs)

Centromeric (CEPs)Centromeric (CEPs)

Telomere & subtelomereTelomere & subtelomere

Locus-specific (LSI)Locus-specific (LSI) Fusion/fissionFusion/fission

Whole Chromosome Whole Chromosome Paint (WCP)Paint (WCP)

WCPs are made by creating large quantities of small probes WCPs are made by creating large quantities of small probes which hybridize to the unique DNA sequences along the which hybridize to the unique DNA sequences along the entire length of a given chromosome making it one colorentire length of a given chromosome making it one color

Common repetitive DNA sequences that are shared by more Common repetitive DNA sequences that are shared by more than one chromosome are blocked out to prevent cross-than one chromosome are blocked out to prevent cross-hybridizationhybridization

If only part of a chromosome is present, the probes If only part of a chromosome is present, the probes matching that portion of the chromosome will still hybridize matching that portion of the chromosome will still hybridize to itto it

Useful to identify marker chromosomes and cryptic Useful to identify marker chromosomes and cryptic translocationstranslocations

The chromosome in question must be ‘identified’ in order to The chromosome in question must be ‘identified’ in order to apply the correct set of WCPs to the specimenapply the correct set of WCPs to the specimen

Can Can ononlyly be used on metaphase cells be used on metaphase cells

3-red15-green

Centromere/Alpha Satellite Centromere/Alpha Satellite (CEP)(CEP)

Hybridize to highly repetitive human Hybridize to highly repetitive human satellite DNA sequences, usually located satellite DNA sequences, usually located near centromere (Alpha Satellite or near centromere (Alpha Satellite or Satellite II and/or III)Satellite II and/or III)

Used mainly for enumeration or as a Used mainly for enumeration or as a control signal for LSI probescontrol signal for LSI probes

The gain or loss of specific chromosomes is The gain or loss of specific chromosomes is visualized as an increase or decrease in the visualized as an increase or decrease in the number of centromeric FISH signals within number of centromeric FISH signals within a nucleusa nucleus

Centromere probes are larger and typically Centromere probes are larger and typically more diffuse than LSI probesmore diffuse than LSI probes

Useful for identifying marker chromosomes Useful for identifying marker chromosomes and aneuploidyand aneuploidy

TelomereTelomere

Used to detect “cryptic” Used to detect “cryptic” translocations and deletions translocations and deletions

TTAGGG tandem repeats located on TTAGGG tandem repeats located on the terminal ends of all the terminal ends of all chromosomeschromosomes

Requirement:Requirement: metaphase cellsmetaphase cells

Locus Specific (LSI)Locus Specific (LSI) Locus Specific Indicators are smaller Locus Specific Indicators are smaller

probes designed to anneal to ‘specific’ probes designed to anneal to ‘specific’ DNA sequencesDNA sequences

They are used to detect structural They are used to detect structural abnormalities including duplications, abnormalities including duplications, amplifications, deletions and reciprocal amplifications, deletions and reciprocal translocations and inversiosntranslocations and inversiosn

LSI probes usually range in size from LSI probes usually range in size from ~350kb to 1Mb~350kb to 1Mb

Types of Locus Specific Types of Locus Specific ProbesProbes

Enumeration probes i.e. Enumeration probes i.e. p53, ATMp53, ATM FissionFission probes (aka: Break-apart probes) probes (aka: Break-apart probes)

i.e. i.e. MLL, CBFBMLL, CBFB FusionFusion probes: probes:

Dual color Single fusion i.e. Dual color Single fusion i.e. PML/RARAPML/RARA Dual color Extra signal (es) fusion i.e. Dual color Extra signal (es) fusion i.e. Tel-Tel-

AML esAML es Dual color Dual fusion i.e. Dual color Dual fusion i.e. FGFR3/IGHFGFR3/IGH Triple color Dual fusion i.e.Triple color Dual fusion i.e. BCR/ABL+9q34 BCR/ABL+9q34

Enumeration probesEnumeration probes

Enumeration probesEnumeration probesCEP7/D72522 D5S721/EGR1 CEP8

CEP12/D13S319/LAMP1 ATM/p53 CEP7/D72522

Fission or break apart Fission or break apart probesprobes

““Inverse” of fusion probesInverse” of fusion probes

- the normal pattern is two fused - the normal pattern is two fused (~yellow) signals(~yellow) signals

- the abnormal pattern is one - the abnormal pattern is one fused fused (~yellow) signal, one red, (~yellow) signal, one red, and one green signal. and one green signal.

The second normal fused signal has been split into The second normal fused signal has been split into

one red and one green signal.one red and one green signal.

MLL dual color break apart probe

Abnormal has three signals: one fusion, one red and one green

Normal has 2 fusion signals

Designed to detect 11q23 rearrangements of the MLL gene

Fusion ProbesFusion Probes

These probes typically consist of a red These probes typically consist of a red probe on one chromosome and a green probe on one chromosome and a green probe on the other chromosme. When a probe on the other chromosme. When a translocation is present, the red and translocation is present, the red and green signals will become juxtaposed and green signals will become juxtaposed and appears as a fusion ‘yellow’ signal.appears as a fusion ‘yellow’ signal. Single fusion probes have one fusion ‘yellow’ Single fusion probes have one fusion ‘yellow’

signalsignal Dual fusion probes are designed so that both Dual fusion probes are designed so that both

derivative chromosome have a fusion ‘yellow’ derivative chromosome have a fusion ‘yellow’ signalsignal

Single Fusion Single Fusion BCR/ABL ProbeBCR/ABL Probe

Sensitivity: ~5.0-10.0%

Dual Fusion Dual Fusion BCR/ABL ProbeBCR/ABL Probe

Sensitivity: ~0.5-1.0%

InSightInSight®® (Aneuvysion) (Aneuvysion) for Prenatal for Prenatal

DiagnosisDiagnosis PurposePurpose

preliminary diagnosis of numerical preliminary diagnosis of numerical chromosome abnormalities involving chromosome abnormalities involving chromosomes 13,18, 21, X and Ychromosomes 13,18, 21, X and Y

represent 2/3 of chromosome abnormalities at mid-represent 2/3 of chromosome abnormalities at mid-trimester and 85-90% at birth trimester and 85-90% at birth

Useful in cases of abnormal ultrasound Useful in cases of abnormal ultrasound suggesting aneuploidysuggesting aneuploidy

Turn-around TimeTurn-around Time 24-48 hours24-48 hours

Red Probe: Chromosome 21Green Probe: Chromosome 13

InSightInSight® ® BasicsBasics

Specimen is prepared directly without Specimen is prepared directly without culturing, allowing for 24-48 hour TATculturing, allowing for 24-48 hour TAT

Probes for chromosomes 13, 18, 21, X Probes for chromosomes 13, 18, 21, X and Y are examined in interphase nucleiand Y are examined in interphase nuclei

Two technologists analyze the slides Two technologists analyze the slides independentlyindependently

A minimum of 50 nuclei are analyzedA minimum of 50 nuclei are analyzed Statistical analysis of number of signals Statistical analysis of number of signals

per cellper cell

InSight LimitationsInSight Limitations

Does not detect:Does not detect: 1/4 of chromosome abnormalities:1/4 of chromosome abnormalities: aneuploidies other than 13, 18, 21, X, and aneuploidies other than 13, 18, 21, X, and

YY any structural abnormalities any structural abnormalities mosaicisimmosaicisim

► May be performed only as an adjunct to full cytogenetics

InSight 13/21

Key:13-green21-red

InSight: Trisomy 21Key:13 - green21 - red

InSight: 18, X, and Y

Key:X - greenY - red18 - aqua

InSight: Trisomy 18, male

Key:X - greenY - red18 - aqua

What do you think? XXY

Key:X - greenY - red18 - aqua

cIg-FISH for Multiple cIg-FISH for Multiple MyelomaMyeloma

What is cIg FISHWhat is cIg FISH

Dual staining process that allows for Dual staining process that allows for enhanced detection of plasma cells and enhanced detection of plasma cells and genetic abnormalitiesgenetic abnormalities

cIg stain: immunoglobulin-fluorescence cIg stain: immunoglobulin-fluorescence antibody for kappa and lambdaantibody for kappa and lambda

cIg stains identifies plasma cellscIg stains identifies plasma cells cIg staining visualized while scoring cIg staining visualized while scoring

probesprobes Only cIg positive cells scoredOnly cIg positive cells scored

What is the Benefit of cIg What is the Benefit of cIg FISHFISH

BenefitsBenefits

Scoring of probe in plasma cells onlyScoring of probe in plasma cells only Important because often in PCM/MGUS Important because often in PCM/MGUS

percentage of plasma cells is low (can be percentage of plasma cells is low (can be <5%)<5%)

Improves detection of genetic abnormalityImproves detection of genetic abnormality Cells either positive or negative Cells either positive or negative

(generally – there are exceptions) (generally – there are exceptions) Conventional FISH: score 500 cellsConventional FISH: score 500 cells cIg FISH: score 25-100 cellscIg FISH: score 25-100 cells Able to detect abnormality even when just a Able to detect abnormality even when just a

few plasma cells presentfew plasma cells present Performed on bone marrow not bloodPerformed on bone marrow not blood

Examples of cIg FISH Examples of cIg FISH

Plasma cells stained with cIg at 60x magnification

Examples of cIg FISH Examples of cIg FISH Results Results

Plasma cells stained with cIg: positive for t(4;14)

Two fusion signals

StringencyStringencyStringency refers to the conditions that a FISH Stringency refers to the conditions that a FISH

specimen is subjected to during its specimen is subjected to during its processing and analysis.processing and analysis.

Degree to which DNA and probe are Degree to which DNA and probe are disassociated.disassociated.

There are many different conditions that affect There are many different conditions that affect stringency:stringency:

Time and TemperatureTime and Temperature Salt concentrationSalt concentration Formamide concentrationFormamide concentration

Temp StringencyTemp Stringency7373°°C more stringent than 37C more stringent than 37°°CC

SSC concentration StringencySSC concentration Stringency0.4xSSC more stringent than 2xSSC0.4xSSC more stringent than 2xSSC

Formamide Concentration Formamide Concentration StringencyStringency

55% Formamide more stringent than 45% 55% Formamide more stringent than 45% FormamideFormamide

FISH ScoringFISH Scoring

Scoring CriteriaScoring Criteria•Routine FISH evaluation should be scored by two technologists

•Technologists should be familiar with any unique scoring criteria that were established as part of the test validation process, i.e. large gap between probe

•Technologists must also have an understanding of general technical problems that may influence a test result, such as poor slide prep, poor morphology with reduces signal intensity, over-denaturation

•Technologists should be knowledgeable about the reason the test is being performed, i.e PML/RARA probe for patient with history of trisomy 15

MetaphaseMetaphase

Signals should be bright enough to see Signals should be bright enough to see with confidencewith confidence

Choose cells with the least overlap and Choose cells with the least overlap and better morphologybetter morphology

Chromosomes in question should not be Chromosomes in question should not be overlapped, especially near the targetoverlapped, especially near the target

Should have no/minimal debris and/or Should have no/minimal debris and/or background signalbackground signal

Splitting signals should be counted as Splitting signals should be counted as oneone

InterphaseInterphase

Cells should not be overlappedCells should not be overlapped Signals should be clearly Signals should be clearly

distinguishabledistinguishable Should have no/minimal debris and/or Should have no/minimal debris and/or

background signalbackground signal Splitting signals should be counted as Splitting signals should be counted as

oneone Cells should be skipped if they cannot Cells should be skipped if they cannot

be confidently scoredbe confidently scored

Count as one red and one green signal. The red signal is split

Do not count. Nuclei are too overlapping to determine boundaries.

Count as two red and two green signal. One red signal is diffuse.

Count as three red signals and two green signals.

ISCN Nomenclature 2009ISCN Nomenclature 2009

SymbolsSymbols - - absent from a specific chromosomeabsent from a specific chromosome ++ present on a specific chromosomepresent on a specific chromosome xx precedes number of signals seenprecedes number of signals seen ampamp amplified signalsamplified signals concon connected signalsconnected signals dimdim diminished signaldiminished signal enhenh enhanced signal intensityenhanced signal intensity ishish in situ hybridizationin situ hybridization nuc nuc ishish nuclear or interphase in situ nuclear or interphase in situ

hybridizationhybridization wcpwcp whole chromosome paintwhole chromosome paint sepsep separated signalsseparated signals

General Rules of UseGeneral Rules of Use

Interphase (nuclear) in situ hybridization Interphase (nuclear) in situ hybridization (nuc ish)(nuc ish)

nuc ish is followed by, in parentheses, the nuc ish is followed by, in parentheses, the locus designation, a multiplication sign (x) locus designation, a multiplication sign (x) and the number of signal seenand the number of signal seen

For two or more loci, the locus For two or more loci, the locus designations follow one another in a designations follow one another in a single set of parentheses, separated by a single set of parentheses, separated by a coma, and a multiplication sign (x) outside coma, and a multiplication sign (x) outside the parentheses and the number of the parentheses and the number of signals seensignals seen

2009 ISCN examples2009 ISCN examples

nuc ish(D8Z1x2)[500]nuc ish(D8Z1x2)[500] nuc ish(D13S319x2)[500]nuc ish(D13S319x2)[500] nuc ish(D13S319,LAMP1)x2[500]nuc ish(D13S319,LAMP1)x2[500]

nuc ish(NMYCx4~>50)[200]nuc ish(NMYCx4~>50)[200] nuc ish amp(NMYC)[200]nuc ish amp(NMYC)[200] nuc ish(D13S319x1)[72/200]nuc ish(D13S319x1)[72/200] nuc ish(D13S319x0, LAMP1x2)[39/200]nuc ish(D13S319x0, LAMP1x2)[39/200]

nuc ish(D13S319x0, nuc ish(D13S319x0, LAMP1x2)[39/200]/(D13S319x1,LAMP1x2)[72/200]LAMP1x2)[39/200]/(D13S319x1,LAMP1x2)[72/200]

nuc ish(D7S25x1,D7Z1x2)

nuc ish(EGR1x1,D5S721x2)

nuc ish(D8Z1x3)

nuc ish(D12Z3x2,D13S319x1,LAMP1x2)

nuc ish(ATMx1,p53x2)

nuc ish(D72522,D7Z1)x1

2009 ISCN examples2009 ISCN examples

2009 ISCN examples2009 ISCN examples

nuc ish(MLLx2)[500] ornuc ish(MLLx2)[500] ornuc ish(5’MLL,3’MLL,5’MLL con nuc ish(5’MLL,3’MLL,5’MLL con 3’MLL)x2[500]3’MLL)x2[500]

nuc ish(MLLx2),(5’MLL sep 3’MLL x1)[200]nuc ish(MLLx2),(5’MLL sep 3’MLL x1)[200]

MLL dual color break apart probe

TroubleshootingTroubleshooting

Problem: High Problem: High backgroundbackground

Re-washRe-wash Wash was not stringent enoughWash was not stringent enough

ResetReset Slides were dirty leaving a filmSlides were dirty leaving a film

New reagentsNew reagents Possible contamination, pH not correct, Possible contamination, pH not correct,

etcetc Fix pellet and redropFix pellet and redrop

Too much cellular debrisToo much cellular debris

Problem: Diffuse SignalsProblem: Diffuse Signals

Repeat hyb @ lower temp and shorter timeRepeat hyb @ lower temp and shorter time Reset slideReset slide

Problem: Dim/No SignalProblem: Dim/No Signal Repeat hyb @ higher temp and longer Repeat hyb @ higher temp and longer

time-specimen not denatured enoughtime-specimen not denatured enough Reset slideReset slide Make sure probe was added to bufferMake sure probe was added to buffer

Easy MistakesEasy Mistakes

Forgetting to hit start on the Forgetting to hit start on the thermobritethermobrite

Accidentally using straight probeAccidentally using straight probe Forgetting to add probe to the bufferForgetting to add probe to the buffer Air bubbles trapped under the Air bubbles trapped under the

coverslipcoverslip

Quality ControlQuality Control

QC of probesQC of probes

Control ProbesControl Probes

Control samplesControl samples

Establishment of acceptable rangesEstablishment of acceptable ranges

Competency of technologistsCompetency of technologists

Quality ControlQuality ControlValidation of new tests is necessary to ensure safe Validation of new tests is necessary to ensure safe

and effective testing for the specific intended and effective testing for the specific intended clinical purpose.clinical purpose.

Before diagnostic use all probes are assessed for:Before diagnostic use all probes are assessed for: Localization Localization

target accuracy checked on metaphases on normal male slidestarget accuracy checked on metaphases on normal male slides

Specificity Specificity % of FISH signals found at the expected location % of FISH signals found at the expected location

Sensitivity Sensitivity % of targeted chromosomes with the expected signal % of targeted chromosomes with the expected signal

* Both specificity and sensitivity must approach * Both specificity and sensitivity must approach 100% for a probe to be diagnostically useful.100% for a probe to be diagnostically useful.

Probe Inventory Probe Inventory ManagementManagement

FISH probe working inventory FISH probe working inventory spreadsheetspreadsheet Updated as probes are received, QC’d, Updated as probes are received, QC’d,

and usedand used Expired probes shaded gray to indicate Expired probes shaded gray to indicate

status, not used for clinical casesstatus, not used for clinical cases New lots of probe are validated against New lots of probe are validated against

an old lot (calculate specificity and an old lot (calculate specificity and sensitivity)sensitivity)