Emerging And Problem Analytes In The Clinical...

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Clinical Pathology Update: Emerging and Problem Analytes

in the Clinical Laboratory

Jay L. Bock, MD, PhD, FCAPSheldon M. Campbell, MD, PhD, FCAP

Peter L. Perrotta, MD, FCAP© College of American Pathologists 2004. Materials are used with the permission

of the faculty.

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Disclosure

(no commercial relationships to disclose)

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Agenda

11:50Summary and Closing10:50Microbiology Dr. Campbell

8:30Opening/ Introductions Dr. Bock

10:20Chemistry 2 – hCG Dr. Bock10:00Break9:30Chemistry 1 – CRP Dr. Bock8:35Hematology Dr. Perrotta

TimeTopic

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“Emerging Analytes”

Hematology & Coagulation Laboratories

Peter L. Perrotta, MDAssistant Professor, Pathology

Stony Brook University

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Coagulation Sessions

Wed @ 1:30

Tue @ 10:30

Sun @ 1:30

Sun @ 10:30

Date

Hemostasis, Lupus anticoagulantsThe Tipping Point to Thrombosis

vWD, TTP, Anticoagulants, INR

Recent Advances in Hemostasis/Thrombosis

$$$Use & Abuse of FVIIa concentrates

POC CoagCoagulation for Surgical Procedures

TopicsTitle

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Session Objectives

• Describe newer methods that effectively screen patients for acquired & congenital platelet function defects

• Recognize clinical & laboratory features of heparin-induced thrombocytopenia

• Design & implement a Thrombophilia (hypercoagulable) test “panel”

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Qualitative Platelet Defects

• Drug effects:ASA, NSAID, clopidogrel

• Congenital defects:GT, BS, SPD

• vWD:vWF essential for platelet adhesion

• Acquired platelet function defect:Uremia, FSP, ET, cardiac surgery

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Lab Detection of Qualitative Platelet Defects

• Bleeding time• Platelet-rich plasma optical aggregation• Whole-blood impedance aggregation (Chronolog)• Platelet function analyzer (PFA-100®, Dade Behring)• Rapid Platelet Function Assay (Ultegra®, Accumetrics)• Thromboelastography (TEG, Haemoscope)• Multiple platelet function analyzer (Multiplate®)• Plateletworks®

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Bleeding Time

• Described by Milian (1901)• Introduced by Duke (1910)• Platelet-vessel wall interaction• Influenced by platelet count

and hematocrit• Some sensitivity in acquired

and congenital platelet defects, no specificity

• No correlation between BT and surgical bleeding

Duke WW. JAMA 1910

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Performing Bleeding Times

40 mmHg

Uremia, hepatic function, skin edema, hypothermia, vasculitis, DM, hyperlipidemia, Ehlers-Danlos

Medications (ASA, NSAID, platelet inhibitors, antibiotics, valproic acid, ethanol, garlic, ginkgo, ginger, ginseng

Platelet disorders, anemia, vWD, factor deficiencies (VIII, IX, V), hypofibrinogenemia, MPD, paraproteinemias

Blot q 15s

Normal < 7 min.

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Impact of Discontinuing Bleeding Times

Clinicians report• No change in pre-procedure work-ups• No postponing of invasive procedures• No increase in bleeding complications

Objective measurements• No increase in blood product use• No increase in platelet aggregation studies• No increase in DDAVP administration• No increase in post-procedure bleeding

complications (major surgical risk cases and renal biopsies)

University of Utah, Clin Chem, 2001;47:1204

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Platelet Function Analyzer (PFA-100)

• In vitro test of platelet function using anticoagulated whole blood

• Based on principle of Thrombostat 4000 (Germany)

• Prototype field-tested in 1995 (Sem ThrombHem 1995:21:113)

• Uses 0.45um nitrocellulose filtration membrane coated with 2 ug collagen (CEPI) and either 10ug epinephrine or 50ug ADP (CADP)

• Subjected to shear rate of 5000-6000s-1

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How the PFA-100 WorksCol-Epi

Col-ADP

Closure time (sec)

147um

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Preferable Lab Tests

24/7LimitedAvailability

LowHighTechnical difficulty

YesNoAutomated/Rapid

SoonNo (no sample)Proficiency

Electronic/?LiquidNoControls

Yes (CV<10%)Resident: No

Lab: ? CV 20-25%Reproducible

PFA-100Bleeding Time

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Reference Range Determination and Validation of PFA

Method• Confirmed manufacturer’s suggested reference

range using 20 individuals• Confirmed response following 325 mg ASA• NO back-to-back testing with bleeding time• Limited back-to-back with PRP aggregation

Collagen/EPI [82-150 sec]Collagen/ADP [62-100 sec]

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Historical back-to-back comparisons with bleeding time

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

BTPFA

Receiver/operator characteristic (ROC) curves which reflect combined sensitivity and specificity:

-99 “Normals” by consensus

-70 Abnormals (24 ASA, others)

Sem Thromb Hem 1995:21(2)

AUC BT = 0.698

AUC PFA = 0.89

1-Specificity

Sens

itivi

ty

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Historical back-to-back comparisons with PRP aggregation

88

100 (A.A)

80%

NA

Sensitivity PRP Agg

NT95-98%127ASA

NT94176All Abnormals

5

44

206

n

100%

96%

NA

Sensitivity CEPI PFA

100%

59%

NA

Sensitivity BT

Glanzmann

vWD

Normal

Condition

Sem Thromb Hem 1998;24:2

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Interpreting PFA Results

↑↑NormalNormalCADP↑↑↑NormalCEPI

Cong. PFD*vWDASANormal

*CEPI increased in:- Glanzmann’s (15 of 15) - Bernard Soulier (1 of 2)- Storage pool disease (6 of 6)- Hermansky Pudlak (6 of 6)

(Blood Coag Fib 1999;10:25-31 and others)

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PFA in vWD• Majority of patients with vWD will have abnormal CEPI and

CADP• If BT is long in vWD patient, CEPI will be long• CEPI could be normal in very mild type I or type 2N patients

with normal BT (Thromb Res 1999;96:487)• Probably useful in monitoring response of type I vWD to

DDAVP (Br J Haem 1999;106:777)

Blood 1998;91:1325

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PFA in Cirrhosis & Uremia

• Study of 20 controls, 21 uremics, 20 cirrhotics

• Test subjects had lower Hcts(26%) and platelet counts (140K) and abnormal PRP aggregation

• CTs straddled upper limit of normal

• Fell into normal range when Hct was elevated by adding RBCs to sample

Haematologica 1999;84:1999

Interpret! Don’t just look at the flag

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PFA in Neonates/Pediatrics

• No difference between 21- and 23-gauge needles or between vacutainer and syringe draws

• Children (age 2-17): Normal range similar to adults (? 5-10% lower CEPI CT)

• Newborns: Using cord blood results 25-35% shorter than adults, CEPI 61-108 sec CADP 48-65 sec (SemThromb Hem 1998;24:523)

Related to higher Hcts, WBCs, vWF, increased high weight multimers in neonates

• Not affected by factor VIII and IX levels

We run both CEPI and CADP cartridges in children

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Monitoring for ASA “Resistance”

• Anti-platelet effects of ASA may vary in patients with ischemic stroke (Helgason et al. Stroke 1994)

• ASA resistance associated with increased risk of MI or CVI (Gum et al. J Am Coll Cad 2003)

• ASA resistance associated with increased risk myonecrosis after non-urgent PCI (Chen et al. J Am CollCard 2004)

• PFA can Identify “ASA resistant” patients (Am J Cardiol2001;88:230)

• Ultegra® Rapid Platelet Function Assay-ASA can detect ASA resistance (accumetrics.com)

• RPFA can be used to monitor abciximab therapy in PIC (POC setting)

Do you need to monitor ASA therapy ?Do you need to monitor ASA therapy ?

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QA for PFA

• Each cartridge is self-contained

• Control donor in duplicate with each new cartridge lot or when performance is suspect

• If this donor is out of reference range, check another donor

• If 2nd also outside range, send instrument back

• Electronic instrument check before each run• Unexpected results reviewed by medical personnel• Check platelet count, platelet histogram, Hg if sample

integrity is suspect

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PFA Tidbits

• PFA not influenced by heparin• PPACK O.K. as anticoagulant• Slightly or not prolonged in afibrinogenemia• Group O patients with lower vWF:Ag have

slightly longer (10-20%) CEPI closure times• Decreased when ESR is high (Sepsis)• Pregnant women: CT 20% shorter, shortest in

2nd trimester• Otherwise independent of gender, OC use,

smoking, or fibrinogen levels

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PFA Tidbits, continued• Can detect δ-storage pool disease with CEPI CT

despite normal PRP aggregation (Thromb Res1999;96:213)

• Cannot detect GP Ia 807 C/C polymorphisms associated with low collagen receptor density butmild Type I vWD and polymorph have longer CT (Thromb Res 2001;103:123)

• 807 T/T with high collagen receptor density have shortest CT and respond poorly to ASA

• Check functionality of platelets from repeat platelet donors (Thromb Haem 2001;86:880)

• Decreased CEPI CT in trauma patients with activated platelets (J Trauma 2001;51:63)

• COX-2 inhibitors that inhibit TxB2 production < COX-1 inhibitors increase CT only 10%

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Algorithm for Platelet Function Screening

Bleeding History*

Pre-surgery with Fam Hx PFD

Drug History

Vitamins

Health Foods

vWD Testing

PRP Aggregation

+/- Flow cytometry

PFA CEPI

PFA CADP

STOPNo

Normal

Normal

AdultSTOP

Abnormal

Abnormal

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PFA Illustrative Case?

• 70 yoM post-MI and post-stenting• Meds: ASA & heparin• Labs: WBC = 14 K/uL Plt = 40 K/uL Hct = 31%,

PT= 13.3 sec, aPTT = 60 sec• Needs surgery: Order PFA to check platelet function:

PFA Collagen/EPI = >300 sec [82 – 150 sec]PFA Collagen/ADP = 151 sec [62 – 100 sec]

What is your interpretation?

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Heparin Started

PFA Orders

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Heparin Induced Thrombocytopenia (HIT)

• First described? Natelson et al, Ann Intern Med 1969• Common: 1% to 5% of patients exposed to heparin• Estimated 1/3 of patients with HIT develop thrombosis (HITTS),

of which 1/3 will have amputation or die

Clinically - Thrombocytopenia with or without:• DVT, PE, cerebral venous thrombosis• Coumarin-induced venous limb gangrene• Arterial thrombosis: Lower-limb, cerebral, MI, etc.• Skin lesions at heparin injection• Skin necrosis, erythrematous plaques• DIC• Acute systemic reaction post heparin bolus

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Type I -vs- Type II HIT

Type I HIT• Mild thrombocytopenia (100-

150 x 109/L• Rapid onset (1-2 days)• Resolves despite

continuation of heparin• Asymptomatic• Non-immune mechanism ?

Via GPIIb/IIIa and ADP

Type II HIT• Severe thrombocytopenia

(<100 x 109/L)• Delayed onset (4-14 days)• Persists until heparin is

discontinued

• Thromboembolic complications

• Immune mechanism

Chong et al, 2003

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Platelet Nadir in HIT (n=127, serologic confirmed)

Warkentin & Kelton, Am J Med 1996

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Laboratory Testing for HITFUNCTIONAL TESTS• Serotonin release assay (SRA)• Heparin-induced platelet aggregation (HIPA)• ATP release by lumiaggregation• Microparticle release by flow cytometry• Annexin V binding & P-selectin expression by flow

cytometry

IMMUNOASSAYS• Solid-phase anti-PF4/heparin ELISA• PF4-polyvinylsulfonate antigen ELISA• Fluid-phase anti-PF4/heparin EIA• Particle gel immunoassay (Diamed)

Adapted from Chong, ISTH, 2003

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Structure of Heparin“BIG & negatively charged”

• Molecular weight: 5,000 to 30,000 daltons• Structure: Long, unbranched polysaccharide

chains of repeating disacharide units, highly sulfated

• Source: Bovine lung more likely to cause HIT than porcine mucosa

• Low molecular weight heparin (LMWH): Prepared from unfractionated heparin, MW about 5,000 daltons

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Heparin-PF4 Interaction with Platelets

Heparin-PF4-Antibody Complex

Thromboxane Synthesis

Platelet Granule Release

Microparticle Formation

Platelet aggregation

Activated Platelet

Adapted from Chong & Eisbacher, 1998

FcγRII

*Thrombin GenerationResting

Platelet

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Morphological and immunohistochemical characteristics of heparin-induced thrombi and thrombi of other origin

Adapted from Hermanns et al, Virch Arch 1998

White to grey to grey-redWhitishColor

(+)++ to +++IgG/IgM content

+++(+)Fibrin Content

Thrombocytes, RBCs, leukocytes, few lymphs

ThrombocytesCell Content

Fibrin webbingWide-meshed patternLayerlike structure

Structure

Other thrombiHIT ThrombiFeature

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Pre-test Probability of HIT

Platelet count starts increasing 2 days after stopping heparin

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SRA to detect HIT Antibodies“Gold Standard” in HIT antibody detection credited to

Sheridan et al, Blood 1986

• Based on immune-mediated 14C-serotonin release from platelet dense granules with activation

• Procedure1. Donor PRP is prepared, washed, and incubated with 14C-serotonin2. Incubate platelets with heat-treated patient serum and two concentrations heparin (0.1-0.4 U/mL and 100 U/mL)3. Spin to pellet platelets, measure radioactivity in supernatant4. Positive when >20% release with low dose heparin and <20% release with high dose heparin

• Disadvantages- Time-consuming- Biohazardous radioisotopes- Dependent on donor platelets like the HIPA

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PF4-Heparin ELISA Is Easier

*GTI: PF4 from outdated platelets and polyvinyl sulphonate

*Asserachrom, Stago: recombinant PF4 and heparin

PF4 Heparin

HIT Antibody (IgG, IgA, IgM

Enzyme linked Goat anti-Human IgG,A,M

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Sensitivities of Various HIT Assays

High>90%ELISA

High50-80%HIPA

High>90%SRA

SpecificitySensitivityAssay

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Local validation of HIT ELISA Assay

• 4 positive by SRA & ELISA: Good Hx• 11 negative by SRA & ELISA: Poor Hx (includes ITP

and lupus anticoagulant)• 4 positive by ELISA but negative by SRA: All had

good Hx including (a) Pt dropped plt count on heparin re-exposure, (b) 2 had clinical HIT per hematologists, (c) one had a leg amputation

• 2 cases negative by ELISA but positive by SRA: One had good Hx and other not HIT

Can we institute this assay?

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Reporting of HIT ELISA Results

Other test outcomes• Positive: Consistent with heparin-associated antibody in

appropriate clinical setting • Negative: No evidence of heparin-associated antibody by ELISA

technique. Reactivity in presence of excess heparin more consistent with anti-platelet factor 4 antibody

• Indeterminate result: Cannot demonstrate or exclude a heparin-associated antibody. Suggest repeat testing in 2-3 days or testing by another technique if clinically indicated.

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Our Strategy for HIT Testing

• Clinically suspected HIT: HIT ELISA is performed as the first-line assay

• Expect to detect > 90% of HIT antibodies• If ELISA negative but clinical suspicion high, then

repeat ELISA in 24-72 hours• If repeat negative and suspicion high, then

perform serotonin release assay• Performed weekdays• Positives are “Critical Values”• Pharmacy informed of positives: “Allergy”

*Negative HIT test does not exclude HIT**Negative HIT test does not exclude HIT*

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Treating HIT (stopping heparin may not be enough)

• LMW Heparin: Not recommended because of high cross-reactivity(80-90%) in vitro

• Danaparoid (Orgaran): Heparinoid compound related to LMW heparin. Approx. 10-20% HIT cross-reactive

• Anti-platelet agents: No proven role• High dose IVIG: 3 of 3 patients responded, consider in severe

cases• Plasma exchange: ??

• Direct thrombin inhibitors: Neutralize excess thrombin generated in HIT- Lepirudin (Refludan, recombinant hirudin): - Argatroban (small-molecule, direct thrombin inhibitor)

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Direct Thrombin Inhibitors

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HIT & Cardiac Surgery

(IIb/IIIa)

(prostacyclin)

Warkentin, Ann Thorac Surg 2003

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Is this a HIT Case?

• 40 yoM post CVI• Meds: Received heparin & abciximab (Reopro)• 8 hours after CVI, platelet count was 2 K/uL and

patient bleeding at venous site

• Request for STAT HIT ELISA denied• Responded to platelet transfusions

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Questions in Thrombophilic Testing?

• Why test?• Who should be “screened”?• When should tests be performed?• What are physicians doing at your

hospital?• What tests should comprise a

hypercoagulable “panel”?

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Stages of Laboratory TestingPre-analytical• Patient selection (pre-test probability of disease)• Test selection (order, order entry, Clinicians want

help!)• Sample collection (anticoagulants, line draws, active

thrombosis)Analytical• Method (limitations)Post-analytical• Result reporting• Result interpretation• Action taken based on result (bias)

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Why Test for Thrombophilia?

• Conditions have been identified that carry an increased risk for thrombosis

• Tests are more readily available (Bias toward ordering tests)

• Test results influence decisions on1) Therapy (anticoagulation choice, duration, intensity)2) Prevention (prophylaxis during surgery, pregnancy, etc.)3) Relative risk of thrombosis (counseling)

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Conditions Associated with Venous Thromboembolism

Congenital• AT deficiency• PC deficiency• PS deficiency• FV Leiden• PT G20210A• Hyperhomocyteinemia• Dysfibrinogenemia

Acquired/Mixed• Anti-phospholipid

antibodies• Hyperhomocyteinemia 20

vitamin deficiency• APC resistance• Increased factor VIII (IX,

XI, fibrinogen, TAFI)

Rare, but strong associationCommon, but weak association

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Hereditary Predisposing Conditions

20 – 505 (Caucasian)APC resistance

10 – 255 - 10Hyperhomocysteinemia

62PT G20210A mutation

2 – 80.7PS deficiency

3 - 90.14 - 0.50PC deficiency

1 - 50.17AT deficiency

Prevalence venous thrombosis (%)

Prevalence general population (%)

Disorder

Hematol Oncol Clin North Am, 1998

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Who Should be “Screened”?

• Screening general population not justified (cost to avoid 1 thrombotic event too high)

• Unexplained thromboembolism at any age

• Age <50 with or without predisposing conditions (surgery, immobilization, trauma, malignancy, OCs, pregnancy, etc.

• First-degree relatives of patients with thrombosis & documented abnormality

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When Should Thrombophila Testing be Performed?

• Test results typically don’t affect initial management• Acute thrombosis +/- anticoagulation therapy affect

several plasma-based assays- AT assays affected by heparin- PC and PS assays affected by Coumadin- AT, PC, PS affected by acute thrombosis

IdeallyWait 2 weeks once anticoagulants stopped to run these testsPerform tests 6 months after acute episode (inflammation and fibrinolysis subsides)

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Tests Performed at SB

24Send out to ARUPLp(a)

178178

DNA (Molecular Diagnostics)DNA

FV LeidenPT gene mutation

30 (70)134

13, 83

RVVCT (Phospholipid dependence)ELISA (Immunology)Coag, dilute PT

LA screen (confirm)ACA (IgG + IgM)PT, TTI

110Clotting using FVIII def. plasmaFactor VIII

144Serum immunoassay (Chemistry)Homocysteine

1948282

APC cofactor activity of PC (PT based)ELISAELISA

PS activityPS total AgPS free Ag

22467

Amidolytic using venom activatorELISA

PC activityPC antigen

11073

Heparin cofactor Xa inactivationELISA

AT activityAT antigen

Cost ($)MethodTest

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Review of Thrombophilia Evaluations at SB (n=30)

< 30 mg/dL16Lp(a)5-15 uM/L017Homocysteine

00

1754

FVLPT gene

Both

IgG<15, IgM<12.52

1/31320

LACSACA (IgG/IgM)

82-151% (F=64-138%)Free: 67-151% (F=51-131%)Total: 72-152% (F=60-136%)

33 free0 total

2013

9

PS activityPS free+total

Both

65-122%76-158%

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22139

PC activityPC antigen

Both

85-130%191-369%

00

2075

AT activityAT antigen

Both

Reference rangeAbnormalPerformedTest

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Hypercoagulable Test Panel

Potential benefits• Standardize test ordering practices• Facilitate ordering in LIS• Maximize chance of identifying an abnormality• Detect combined abnormalities• Decrease need to redraw for additional tests• Algorithms not helpful as in bleeding patient

Drawbacks• Will increase overall costs• Questionable cost/benefit ratio• Label patients inappropriately as “clotters”• Regulatory aspects of “bundling” tests

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What Panels do Popular Reference Labs Offer?

ARUP• Acquired: PT, PTT, mix, dRVVT, ACA, d-dimer• Inherited: PTT, FVIII, Homocyst, PVL, APCR, PT gene• Uncommon: PT, PTT, PC activity, AT activity, PS

(free+total)

MGH• Hypercoag: APCR, FVL, ACA, AT, CRP, Homocyst,

Lp(a), plasminogen, PAI-1, platelet hyperaggregation, PC, PS, PT gene, reptilase time, thrombin time

Tricore• Hypercoag: AT, PC activ, PS activ, lupus panel, ACA,

homocyst, FVL, PT gene

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Reference Lab Offerings (continued)

University Vermont• On warfarin: PT, PTT, dRVVT, ACA, TT, AT, FVL, homocyst• No warfarin: dRVVT, ACA, TT, AT, APCR (FVL if abnormal),

homocyst, PC activity (Ag if low), PS activ (Ags if low)

University of Florida• If PC low, reflex to FVII to exclude liver disease, vit. K def.• If PS low, reflex to FVIII to exclude acute phase reactant• If homocyst high, reflex to MTHFR gene mutation

Blood Center Milwaukee• Thrombosis: AT activ., PC activ, PS activ, lupus panel, ACA, FVL,

PT gene• On warfarin: ACA, AT activity, FVL, LA, PC Ag + FX Ag, Free+total

PS Ag, PT gene

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Our Thrombophilia Panel• Antithrombin activity (reflex to antigen if low)• Protein C activity (reflex to antigen if low)• Protein S activity (reflex to free/total antigen if low)

• Lupus anticoagulant screen (confirmatory step in positive)

• Anticardiolipin antibodies (IgG & IgM)

• Factor V Leiden R506Q mutation• Prothrombin nucleotide 20210A mutation

• Homocysteine (total after 12 hour fast) TOTAL

Should requests for hypercoag panels be screened?Should an interpretive report be provided?

Need better screening test for overall thrombotic potential?

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Pearls of Pathology (Summary)

• There are in vitro laboratory tests and POC instruments that are effective alternatives to the bleeding time

• Laboratory assays are helpful in confirming a clinical diagnosis of HIT

• “Panels” provide a more comprehensive approach to thrombophilia testing

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C-reactive protein in coronary risk assessment—a better test than cholesterol?

Jay L. Bock, MD, PhDDepartment of PathologyStony Brook University

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Objectives

• Consider the characteristics that may make a test useful for the evaluation of cardiac risk

• Summarize the biology of CRP and the evidence relating it to coronary heart disease

• Evaluate high-sensitivity CRP as a test for cardiac risk

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Study Says a Protein May Be Better Than Cholesterol in Predicting Heart Disease RiskBy DENISE GRADY

n inexpensive blood test for a protein linked to artery disease may be better than a cholesterol test at predicting a person's risk for a heart attack or stroke, researchers are reporting today.

New York Times, November 14, 2002

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“Supportive but not conclusive research shows that eating 1.5 ounces per day of walnuts as part of a diet low in saturated

fat and cholesterol may reduce the risk of heart

disease.”New York Times, March 28, 2004

Physical InactivityPhysical Inactivity

AgeGender

Family History

Smoking

Hypertension

Diabetes MellitusObesity

CholesterolHDL

LDL

TriglycerideLp(a)

Homocyst(e)ineB12

B6

folate

Fibrinogen

PlateletsFactors II, V, ...

IronOxidants

Anti-oxidantsRed wine

Flavonoids

Alcohol

AggressivenessDepression

InsulinTWARCMV

CRP

Fiber

VegetablesSat. fat

Total fat

Unsat. fat

trans-FA

fish oil

apolipoproteins

vit. Cvit. E

carotenoids

Anxiety Cortisol

IL6aspirin

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What is a risk factor?

• How do you define risk?• Is high risk a disease?• How do you know if a risk assessment

is accurate?• What do you do about it?

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Risk factors may be ...

• Causes• Consequences• Associations

Clinical reasons for measuring risk factors

• Allow individuals to know their risk• Encourage lifestyle improvements• Undertake and monitor preventive measures

– before clinical disease (primary prevention)– after clinical disease (secondary prevention)

• Make decisions regarding acute treatment

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Lines of evidence

• Involvement in the disease mechanism• Animal studies• Uniform occurrence of the disease

when the risk factor is very high• Epidemiology studies• Controlled intervention studies

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The classic example

• Involvement in the disease mechanism

• Animal studies • Uniform occurrence of the disease when

the risk factor is very high • Epidemiology studies • Controlled intervention studies

Serum cholesterol (or LDL cholesterol)

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A change in paradigm

• Old paradigm: coronary risk relates to the degree of obstruction– Gold standard test: coronary angiography– “Cure:” bypass, angioplasty, stent

• New paradigm: risk relates to plaque vulnerability– Acute coronary syndromes are due to erosion or

rupture of unstable plaques– How to test?– How to cure?

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C-Reactive Protein (CRP)

• First described in 1930 - fraction of plasma protein from infected patients that reacted with the C-polysaccharide of pneumococcus

• Also found in patients with other acute illnesses (including acute coronary syndrome) - hence the term “acute phase reactant.”

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Biochemistry• Pentameric protein: five protomers, each of

206 amino acids, arranged in cyclic symmetry (pentraxin family)

• Binds various proteins and phospholipids, particularly phosphocholine. Ca2+ ions ligate the phosphate group and a hydrophobic pocket accommodates the methyl groups of phosphocholine.

• Opsonizes particles and also activates complement via the classical pathway. Actual biological function unknown.

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Pathophysiology

• Normal plasma levels: median ~1 mg/L, 99th percentile is ~10 mg/L

• Synthesized in hepatocytes. In acute illness, cytokines stimulate hepatic productions, levels rise rapidly to as high as 300 mg/L in 24 hr.

• Increased in many disorders, including bacterial (but usually not viral) infections

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Epidemiology Studies

• Study of 22,000 healthy male physicians to examine effect of beta-carotene and aspirin. 543 who developed MI or stroke were compared to 543 controls.

• CRP higher in those who had MI (1.51 mg/L) or stroke (1.26) versus control (1.13)

• Three-fold higher risk of MI in highest quartile. Risk was stable over time and independent of other factors.

• Aspirin reduced risk of those in the highest quartile.

Ridker et al., NEJM 1997;336:973-9

PhysiciansHealth Study

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0

1

2

3

4

5

Rel

ativ

e R

isk

of M

I

1 2 3 4Quartile of CRP

PlaceboAspirin

Ridker et al., NEJM 1997;336:973-9

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[PM Ridker, Circulation 2003;107:363-9; NEJM 2002; 347:1557]

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80


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Risk of recurrent events

0

0.5

1

1.5

2

<1.2 1.2-2.0 2.1-3.7 3.8-6.6 >6.6CRP, mg/L

0

0.5

1

1.5

2

<1.7 1.7-2.3 2.4-3.4 3.5-5.9 >5.9SAA, mg/L

Ridker, Circulation 1998;98:839-844

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HEALTH & FITNESS | April 6, 2004, Tuesday

Heart Study Challenges Protein's Predictive Power

By ANAHAD O'CONNOR (NYT) words Late Edition - Final , Section F , Page 8 , Column 5

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[Danesh et al., C-Reactive Protein and Other Circulating Markers of Inflammation in the Prediction of Coronary Heart Disease, NEJM 2004; 350:1387-97].

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85


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Other lines of evidence

• Is CRP involved in the atherosclerotic process?– Inflammation appears to play an important role in CHD– CRP has been identified in coronary plaque– But why is there the association between CRP and

CHD?• CHD may cause an inflammatory response which causes CRP

elevation• A chronic state of inflammation may cause or exacerbate CHD• CRP itself could cause or exacerbate the atherosclerotic

process

87

[Lagrand, Circulation 100:96, 1999]

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Summary of CRP evidence

• Involvement in the disease mechanism • Animal studies ±• Uniform occurrence of the disease when the

risk factor is very high ±• Epidemiology studies • Controlled intervention studies

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Clinical Guidelines

• Cholesterol/lipids– 1988 – ATP-I– 1993 – ATP-II– 2001 – ATP-III

• CRP– 2003 – AHA/CDC Scientific Statement

(Pearson et al., Circulation 2003;107:499-511)

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2003 AHA/CDC Scientific Statement

• Current evidence supports the use of hs-CRP as the analyte of choice [if inflammatory markers are to be used].

• The hs-CRP assay should be performed in a metabolically stable person without obvious inflammatory or infectious conditions. Two assays, optimally 2 weeks apart, are recommended.

• Patients without known CHD and at intermediate risk (ie, 10-20% over 10 years) may benefit from measurement of hs-CRP.

• The entire adult population should not be screened.

91

Risk stratification

• Low: <1.0 mg/L• Average: 1.0 – 3.0 mg/L• High: >3.0 mg/L

93

Summary / Pearls of Pathology

• Epidemiology studies have consistently shown an association of CRP with coronary risk, independent of other established risk factors

• The magnitude of the risk association is moderate• The role of CRP in coronary disease is unclear• Treatment specifically directed to high CRP is not

available• Current guidelines suggest testing is most useful in

those at intermediate risk based on established factors

94


“‘It’s not clear exactly yet which patients need CRP screening,’ says Dr. Robert Bonow, president of the American Heart Association.”

Newsweek June 16, 2003, p. 50

95

False-positive, “discordant”, “phantom”, “low-level ‘real’ ”, and other confusing results of hCG assays

Jay L. Bock, MD, PhDDepartment of PathologyStony Brook University

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Objectives

• Many instances are known where incorrect or misleading results of hCG assays have led to patient harm. – Understand the causes– Know the solutions

97

Case 1 - 1982

A 23-year-old woman presented with amenorrhea and had a positive, qualitative serum hCG test. The test was done by a competitive-binding, radiolabeled procedure (RIA) and was reproducible on subsequent specimens. She eventually had both uterine curettage and an exploratory laparotomy, with no fetal or placental tissue found. The positive hCG test persisted after these procedures. A different brand of hCG RIA test was performed, and it gave a negative result.

98

Case 1 - explanation

The RIA method that gave a positive result used a goat anti-hCG antibody, whereas the kit that gave a negative result used a rabbit antibody. It was shown that the patient’s serum contained antibodies that bound to goat IgG but not rabbit IgG.AO Vladutiu, JM Sulewski, KA Pudlak, CG Stull. Heterophilic antibodies interfering with radioimmunoassay: a false-postivie pregnancy test. JAMA 1982;248:2489-90

Polyclonal Ab

Protein analyte

Labeled protein

Heterophilic Ab

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Case 2 - 1985A 28-year-old woman presented to the emergency room with abdominal pain. A quantitative serum hCG test, performed using the newly released Hybritech Tandem-E® double-monoclonal immunoenzymometric assay (IEMA) gave a positive result of 32 mIU/mL. Uterine curettage and laparoscopy were eventually performed, with no fetal or placental tissue found. The quantitative hCG result persisted in the range of 32-55 mIU/mL. When specimens were tested using an older β-subunit RIA, results were negative.

102

Case 2 - explanationThe positive result in the Tandem-E assay was diminished by adding goat anti-(human IgM) to the specimen, and was abolished by adding nonspecific mouse IgG to the specimen.It was therefore attributed to heterophilic antibodies (HAMA) in the patient’s serum.

JL Bock, J Furgiuele, B Wenz. False positive immunometric assays caused by anti-immunoglobulin antibodies: a case report. Clin ChimActa 1985;147:241-6.

solid phase

Capture Ab (monoclonal conjugated to solid phase)

Detector Ab (labeled monoclonal)

Protein analyte

solid phase

HAMA

105

Case 3 - 2001A 22-year-old went to her doctor because of irregular menses. A serum hCG test, done by an IEMA method on the Abbot Axsym® analyzer, was positive. There was no other evidence of pregnancy, but the hCG test was consistently positive. She was referred to an oncologist who instituted chemotherapy to cover the possibility of a trophoblastic tumor. The elevated hCG level persisted. She eventually had a hysterectomy and additional surgery before it was found that her hCG level, using other brands of test, was negative.

106

[see: http://abcnews.go.com/sections/primetime/2020/PRIMETIME_010726_abbott_feature.html]

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Myth

• A “positive” hCG test means the patient is pregnant—if it’s not an intrauterine pregnancy, it’s likely an ectopic pregnancy, and if it’s not either of those—it’s likely cancer!

108

Reality

• hCG tests can be positive for a number of reasons. Some simple follow-up measures can often rule out pregnancy or cancer as the cause.

109

Basic facts

• Choriogonadotropin (CG, or hCG) is a member of the glycoprotein hormone (GPH) family (other members are the pituitary hormones LH, FSH, and TSH).

• The GPH’s are heterodimers, each sharing the same α-subunit and having a distinctive β-subunit.

• Plasma hCG rises exponentially in the early first trimester of pregnancy, peaks at 8-10 weeks, then gradually declines

110

hCG assays

• Modern hCG assays are all immunoassays• They have progressed from:

– competitive-binding assays using polyclonal Ab’sagainst the whole molecule

– to competitive assays using Ab’s specific for β-subunit

– to immunometric assays using monoclonals that can have varying molecular specificities.The term “β-subunit assay” is obsolete and should not be used.

• Results generally reported in mIU/mL, relative to the 3rd/4th IS, which is a relatively pure preparation of intact hCG

111

Causes of false-positive, “discordant”, “phantom”,

“low-level ‘real’ ”, and other confusing results of hCG

assays

112

Causes of hCG results that are misleading, discrepant, spurious, …

• 10. Laboratory error– Pre- or post-analytical (specimen mix-up,

etc.)– Analytical

• Reading difficulty with a qualitative test• Specimen carryover• Hook effect

113


• 9. Cross-reactivity– Other GPH’s, especially LH, have similar

structure– But with modern assays this problem is

usually insignificant.

114


• 8. Molecular heterogeneity

Less of a problem now that standard materials are highly purified. But different commercial kits will react differently with the different forms of CG, which include:

– Intact dimer– Free subunits– Cleaved forms (β-core fragment)– Nicked– Hypo- or hyperglycosylated

116


7. Persistent levels from an earlier pregnancy

Half-time for disappearance is fairly long (~24 h), so measurable levels can persist for weeks in the absence of complications

117


6. Early (“micro”) abortion

118

[see: Wilcox, NEJM 1988; 319:189-94]

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5. Administration of hCG as a drug

see Karoshi, Lancet January 4, 2004; 363:362. (“I recently learnt a lesson that I will never forget …”)

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4. An ectopic pregnancy may secrete little or no measurable hCG

see: Lonky NM, Sauer MV. Ectopic pregnancy with shock and undetectable β-human chorionic gonadotropin: a case report. J Reprod Med 1987; 32:559-560. Taylor RN, Padula C, Goldsmith PC. Pitfall in the diagnosis of ectopic pregnancy: immunocytochemical evaluation in a patient with false-negative serum β-hCG levels. Obstet Gynecol 1988; 71:1035-1038.

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3. A finite amount of hCG is normally present in male and female serum

Probably derives mainly from the pituitary

122

[see: Odell & Griffin, NEJM 1987; 317:1688-91.]

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[see: Odell & Griffin, NEJM 1987; 317:1688-91.]

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2. Interfering antibodiesHeterophilic Ab’s may arise due to animal exposure, treatment with mAbdrugs, or for no apparent causeA problem for all immunoassays

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[see: Cole, Gynecologic Oncology, 1998;71:325-329; Lancet 2000; 355:712-15, Clinical Biochemistry 2004; 37:344-349]

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• 1. “Low-level real” hCG

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[see: Kohorn, Gynecologic Oncology 2002; 85:315-20; Cole, Am J Obstet Gynecol2003; 188:1254-9]

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[see: Kohorn, Gynecologic Oncology 2002; 85:315-20; Cole, Am J Obstet Gynecol2003; 188:1254-9]

129

“Low-level real” hCG

• Persistent low-level results, usually <50 mIU/mL, that are verified to be real hCG

• No pregnancy, no GTD, no tumor• History of pregnancy or mole• hCG levels respond poorly to surgery or

chemotherapy• GTD, with surge in hCG, may occur later• Quiescent GTD ?

131

Further hCG testing

• Dilution study (best to use diluent containing animal Ig)

• Different brand of test• Urine test• Refer to hCG Reference Service (Laurence

Cole) at University of New Mexico (www.hcglab.com)

• Test at a later time

132

Block or detect heterophilic Ab’s

• Add nonspecific mouse Ig• Other species work, too—eg, bovine Ig

(commercial assays include blocking reagents, but they are not 100% effective)

• Remove Ig, eg, with protein A column• Heat-inactivate Ig• Assay for HAMA

133

Improve ligand-binding assays

• Use engineered Ab fragments or other binders

• But interfering Ab’s may be anti-idiotypic; there is probably no perfect solution

• Move to other type of assay?

134


• A steady, low-level hCG result should be treated judiciously!– It might be a dangerous ectopic pregnancy—but

consider history, symptoms, imaging findings– It is unlikely to be a cancer for which treatment,

before definitive diagnosis, is essential

135

Emerging Analytes in Microbiology

Sheldon Campbell M.D., Ph.D. Yale School of Medicine

VA Connecticut Healthcare System

136

Disclosure

• No conflicts of interest. • I could probably be bought, but

nobody’s tried that hard.

137

Course Objectives

• Emerging Antibiotic Resistance• Emerging Infections & Bioterrorism• Emerging Technologies

138

Emerging Antibiotic Resistance

• The dreaded Staph. aureus– MRSA detection– Staph with reduced glycopeptide

susceptibility – VISA/VRSA– Inducible clindamycin resistance

139

Staphylococcus aureus• Major nosocomial and community-acquired

pathogen– Responsible for >20% of bacteremia in US/Canada– Transmissible nosocomially and in the community.

• Antibiotic resistance -- lots– Methicillin (oxacillin) resistance

• Nosocomial• Community-acquired

– Vancomycin resistance• Relative resistance (VISA)• High-level resistance (VRSA)

– Macrolide resistance• The D-test

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MRSA• First described in 1961; first penicillinase-

resistant semisynthetic menicillin introduced in 1960.

• Acquisition of the mecA gene. – Codes for altered PBP; PBP2a– Variable expression

• Steadily increasing in nosocomial populations– Multi-resistant

• Community-acquired strains– Tend to be non-multi-resistant– Outbreak and sporadic– Skin & soft tissue infections

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010203040506070

1989 1991 1993 1995 1997 1999 2001 2003

Year

Perc

ent R

esis

tanc

eProportion of S. aureus Nosocomial

Infections Resistant to Oxacillin (MRSA) Among Intensive Care Unit Patients,

1989-2003*

*CDC Slide: Source: NNIS System, data for 2003 are incomplete

Rise of MRSA

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Detection of MRSA

• NO completely satisfactory method• Hetero- and inducible- resistance

– Oxacillin screening agar– Cefoxitin disk test– PBP2a antigen

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Oxicillin Screening Agar

• 6 µg/ml oxacillin• Mueller-Hinton agar supplemented with

4% NaCl• Incubate 24h before reading (-)• Requires a separate plate• 24h reporting delay if not done on all

isolates

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Cefoxitin disk screen

• Normal (30 µg) cefoxitin disk• Report zone ≤19mm as oxacillinR

• Usable also for coagulase(-) staph, zones ≤24mm

• Relatively inexpensive• Requires a separate plate, but may be

combined with D-test and others• 24h reporting delay if not performed on

all isolates

145

PBP2a Antigen

• Rapid antigen test for PBP2a; gene product of mecA

• Rapid but expensive• May require overnight induction for best

sensitivity• Alternatively, confirm negatives by

alternate method or induction

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VISA/VRSA

• Both rare resistance patterns• 3 VRSA (MIC >8) reported; probably

others missed, though• 11 VISAs (MIC=8) reported• ~3 dozen near-VISAs (MIC=4)• No spread to HCW, family, other

patients– Good infection control practices– Requires detection – the Micro Lab

147

VISA

• Vancomycin Intermediate S. aureus• Accumulated changes associated with

decreased fitness– Thickened cell wall by EM– Mixed large & small colony morphotypes

on plates

148

VRSA

• MIC >8• Acquisition of vanA cluster from

Enterococcus• Typically very high MICs, no loss of

fitness– Michigan strain: vanA cluster on a Staph

conjugal plasmid, MIC>1000– PA strain MIC=32 (?loss of cluster or ↓expression)

– NY strain MIC=64

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VRSA (cont.)

• Neither Microscan nor Vitek detected all 3 VRSA strains

• Agar diffusion methods require careful examination –inhibition present in zone.

• Vancomycin Screening Agar is required for sensitive detection

Image from Image from TenoverTenover FC et al FC et al AntimicrobAntimicrob. . Agents & Chemo. 48:275Agents & Chemo. 48:275--80 (2004)80 (2004)

150

VRSA and Automated Methods

≤1, ≤1, ≤1≤0.5, 1, 1≤2, ≤2, ≤2, ATCC 29213

8, 8, 81, 16, 16≤2, ≤2, 4PA-VRSA (induced)

2, 2, 22, 1, 44, ≤2, ≤2, PA-VRSA (uninduced)

Vitek2VitekMicroScan

MICs (ug/ml) by methodS. aureusStrain

Pennsylvania strain; from Pennsylvania strain; from TenoverTenover et al et al AntimicrobAntimicrob. Agents. Chemo. 48:275. Agents. Chemo. 48:275--280 (2004) 280 (2004)

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AcceptablePrimary Test Methods

Include: Disk diffusion2 plus VA screen plate(BHIA with 6 µg/ml of VA)

VA MIC <2 µg/mlAnd NO growth on

VA screen plate

VA MIC <2 µg/mlAND GROWTH onVA screen plate

(rare)

VA MIC >4 µg/mlAND GROWTH onVA screen plate

VA zone <14 mmAND GROWTH on VA screen plate

VA zone >14 mm AND GROWTH onVA screen plate

Report as VSSA3Report as VSSA3 Possible VISA/VRSA

CHECK purity

SAVE ISOLATE

NOTIFY infection control, physician, local health department and CDC5 of “possible VISA/VRSA”

MIC method1 plus VA screen plate(BHIA with 6 µg/ml of VA)

Possible VISA/VRSA

VA zone >14 mmand NO growth on

VA screen plate

CONFIRM isolate ID

RETEST using non-automated MIC method4

SEND to reference laboratory for confirmation

Important Footnotes1Laboratories using automated susceptibility test methods should add a commercial vancomycin agar screen plate. 2Disk diffusion alone is not sufficient to detect VISA.3If a laboratory is concerned about a result based on a patient’s history, MIC testing can be performed at CDC.4 Non-automated methods: reference broth microdilution, agar dilution, agar gradient diffusion (Etest; use a 0.5 McFarland inoculum and Mueller-Hinton agar).5Report to CDC by email: [email protected]

April 2004

More VISA/VRSA info: www.cdc.gov/ncidod/hip/vanco/vanco.htm

Algorithm for Testing S. aureus with Vancomycin (VA)

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S. aureus -- Inducible Clindamycin Resistance

• Clindamycin resistance inducible via cross-resistance with macrolides– erm-mediated; methylation of 23S rRNA

• ‘D-test’ now recommended per Jan. 2002 NCCLS standards for erythromycin-resistant clindamycin-susceptible strains

• Alternatives:– Do D-test routinely– Do only on request; in that case, don’t

report clinda for erythro-R strains

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A positive D-test

• 2 µg clindamycin disk

• 15 mm to 26 mm from edge of 15 µg erythromycin disk

• The erythromycininduces resistanceto the clindamycin

• QC strain TBA

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15 15 -- 26 mm26 mm

Photos courtesy Photos courtesy FiebelkornFiebelkorn KR & Jorgensen JHKR & Jorgensen JH::FiebelkornFiebelkorn KR et al KR et al JClinJClin. Micro. 41(10):4740. Micro. 41(10):4740--4, 2003 Oct4, 2003 Oct ..

Another positive…

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Negative D-test

• NO induction; round zone

• (msrA; efflux-mediated erythromycin resistance)

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The ‘N Disks’ strategy

• A scheme for providing rapid susceptibility to common drugs– Cefoxitin– Erythromycin– Clindamycin– + others…

• The D-test spacing corresponds to the inner ring of a disk dispenser.

• Same inoculum can be used on Vancomycin Screening Agar

• Some laboratories are contemplating going back to K-B for S. aureus pending ability of automated systems to detect resistance

157

Discussion Question 1

• What other information would you want before reporting final susceptibilities on the following S. aureus isolate?

• Microscan results:– Vanco <2 (S)– Oxacillin <2 (S)– Erythro <.5 (S)– Clinda <.5 (S)

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Answer 1

• You’d need to know the results of: – Vancomycin screening agar

• Automated methods are insensitive for vancomycin resistance

– Cefoxitin disk OR oxacillin screening agar OR PBP2a testing• Automated methods don’t pick up hetero-and

inducible MRSA

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Discussion Question 2

• What other information would you want before reporting final susceptibilities on the following S. aureus isolate?

• Microscan results:– Vanco <2 (S)– Oxacillin >8 (R)– Erythro >8 (R)– Clinda <.5 (S)

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Answer 2

• A D-test to look for inducible clindamycin resistance

161

Emerging Infections

• Laboratory role in emerging infections• Example

– West Nile• Biological Weapons and Sentinel

Laboratories– Responsibilities– Resources

162

The Laboratory in Emerging Infections

• Recognition• Laboratory safety• Diagnostic information resource• Transfusion medicine implications• Diagnostic testing

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West Nile Virus

• The largest outbreak of arboviral meningoencephalitis in the Western Hemisphere.

• A flavivirus, related to Japanese encephalitis, St. Louis encephalitis, and HCV.

• Maintained via a bird-mosquito cycle, mammals probably incidental hosts

164

WNV: Epidemiology• Spread in

US from 1999-2004

• 2003 –spread of human infections in 45/48 lower US states

• 49 (!) vector mosquito species

200120012001

200220022002

200320032003

165

WNV: Clinical Illness

• Asymptomatic seroconversion (80%)• West Nile Fever (20%)• West Nile Meningitis or Encephalitis

(roughly 1/150)• Incubation: 3-14d• Sequelae common in CNS disease• Prolonged syptoms (~30d) in WNF, with

headache lasting >10d• Flaccid paralysis

166

WNV: Laboratory• WN CNS disease

– CSF pleiocytosis, <100 cells, usually lymphs– Elevated total WBC with lymphopenia– Serology most useful tool

• IgM (+) by symptom onset in 60% of cases, >95% by day 7

– Virus detection• Culture• RNA virus, RT-PCR• Viremia low in humans (1-130 pfu/ml), rare to isolate from

blood in patients with CNS disease• Viremia is short-lived: 2 Taqman(+) samples / 100 IgM(+)• Persistent virus in CSF

167

WNV Serology

• Commercial EIA kits becoming available• Cross-reaction with other flaviviruses;

SLE, dengue. • Confirmation with plaque neutralization

testing at a reference lab. • Patients may remain IgM(+) 6 months

or more after infection.

168

WNV: Laboratory Safety

• Virus is present in blood, serum, and CSF of affected patients

• Lab-associated infections documented, primarily research labs

• BSL-2 for primary specimens• BSL-3 for cultures and research in

animals/vectors

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WNV: Novel Routes of Transmission

• Transplacental– Of 8 women with proven infections during gestation,

one affected infant with severe CNS damage• Breast milk

– Well-documented for other flaviviruses and HCV– One case in 2002, child had asymptomatic

seroconversion• Solid organ transplantation• Blood transfusion

– WNV is the only flavivirus other than HCV and HGV with documented transfusion-transmission

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WNV: Transfusion

• Transfusion-related cases in the 2002 North American outbreak – prior to NAT– 61 investigations– 23 confirmed cases (1 pt. got 2 infected units)– 16 donors– 19 inconclusive (some probably real)– 19 no evidence for TT WNV

• Estimated risk 5-20/10,000 donations

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WNV: Donors

• Donors in 2002 TT Cases– 18-72 y/o– 14/16 lived in counties with active WNV

transmission– At least 5 asymptomatic– All IgM (-) at donation– Viral titers <80 pfu/ml, higher in

symptomatic donors

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WNV: FDA Guidelines for 2003• Reporting

– Patients with diagnosed WNV who received blood or organs within 4 weeks of onset

– Donors with diagnosed WNV with onset within 2 weeks of donation• Donor screening

– Defer for 28d from onset or 14d from end of symptoms• Diagnosis of WNV• Postdonation febrile illness with suggestive symptoms

– Defer for 28d• Potential donors who respond positively to “In the past week, have you

had fever with headache?”• Donors whose product is associated with a transfusion-related WNV

transmission– NO deferral

• (+) IgM asymptomatic within last 2 weeks

• NAT Testing of minipools

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WNV: 2003 Testing Guidelines

• Minipool NAT testing from pools of 6 or 16 donors began June 2003. – Reactives retested by individual donor

testing– Selected blood banks in high-transmission

areas implemented individual donor testing during epidemic season

– Follow-up of positives with IgM serology as well as NAT; 28d deferral after last positive NAT and (+) antibody

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WNV: 2003 NAT Positives• NAT positive

donors detected from Jun-Dec.

• At least 818 viremic donations removed from supply.

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WNV: Transfusion-Transmitted Cases in 2003• 23 suspected cases

– 15 from public health system– 8 in follow-up of single-donor retrospective testing

• Investigation– Six cases probable or confirmed– 11 non-cases– 3 inconclusive

• Probable & confirmed– All were minipool NAT-negative

• Viral titers .06-.5 pfu/ml– 13-82 y/o recipients– 4 w/ WNV encephalitis, 1 w/ WN fever, 1 critically ill

pt. with no attributable symptoms

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Lessons from West Nile

• Emerging infections can appear anywhere, any time. – Everything bad comes from New York?

• PCR isn’t all-powerful. • Emerging pathogens can enter the

blood supply.

177

Bioterrorism and the Clinical Lab

• Low probability – high impact• Priorities

– Protect the lab– Rule-out– Refer

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Class A Biological Agents

• Variola major (Smallpox)• Bacillus anthracis (Anthrax)• Yersinia pestis (Plague)• Francisella tularensis (Tularemia)• Botulinum toxin (Botulism)• Filoviruses and Arenaviruses (Viral

hemorrhagic fevers)• ALL suspected or confirmed cases

should be reported to health authorities immediately

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Disaster Management

•Disaster Occurs

•First Responders•Identify Problem, Limit Spread, Rescue Victims

•Disaster Management•Rescue / Transport victims, Evidence Gathering, Site Assessment

•Recovery Operations

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First Responders

• Natural disasters, conventional terrorism– Fire Services– Police Services

• Bioterrorism– ER & Primary Care

Physicians– Clinical Laboratories

& Radiologists– Hazmat Teams– Fire & Police

(sometimes)

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Disaster Management

• Conventional– Fire Services– Police Services– Federal Disaster

Mgmt. Teams– FBI & Investigatory

Agencies– Etc.

• Bioterrorism– Public Health

Authorities• Federal• State• Local

– FBI & Investigatory Agencies

– Physicians– Clinical Laboratories

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The Laboratory Response Network

USUS

183

The Role of the Laboratory

• Recognition– ‘Rule-out’ BT pathogens

• Notification– Clinical care team– Public Health authorities; local, state– Hospital authorities

• Referral– Transporting specimens– Continuing workup of suspected cases

184

Laboratory Preparedness

• Plan in place• Sentinel lab protocols

– Rule-out or refer– Protect laboratory staff

• Transporting specimens– Chain of custody– Shipping regulations

• Train and Maintain– Protocols up to date

185

Emerging Pathogens and BT Resources

• Emerging Pathogens– CDC notification listserve

• http://www2a.cdc.gov/ncidod/hip/rns/hip_rns_subscribe.html

– ProMed mail• www.promedmail.org

• CDC BT Protocols & resources– http://www.bt.cdc.gov/labissues/index.asp– Also from ASM; Google ‘Sentinel Lab

Protocols’ (it’s a strange URL)

186

Emerging Technologies

• Molecular microbiology– MRSA by RT-PCR; – The face of the future?

187

MRSA by RT-PCR

• There’s no completely satisfactory screening method for MRSA carriage.

• Multiple mecA types and competition with coag-negative staph makes this a challenging problem.

• Newly FDA-approved method using Real Time PCR on the Cepheid SmartCycler.

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Design of the MRSA Assay

• A multiplex PCR scheme which requires 5 left-side primers (for various mecA cassette types) and one right-side primer.

• Detection by three molecular beacon probes (for various orfX variants)• Internal amplification control, detected by a fourth probe.

• Huletsky et al J. Clin. Microbiol. 42:1875-1884 (2004)

189

MRSA detection

• Method identified 1,636/1,657 (98.7%) of known MRSA strains.

• Mis-detected 26/569 (4.6%) MSSA strains. • Detected 0/62 non-staph, 0/286 CoNS (212

of them methicillin-resistant). • Detected ~25 organisms into MRSa-negative

nasal specimens. • Detected 97% of MRSA detected by culture in

epidemiologic surveillance program.

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MRSA Product

• Roughly 2h TAT• Manual sample prep• More sensitive than a single culture plate, but

with some false positives

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The Face of the Future?

192

One More Scary(?) Thought

• The major patents on PCR expire in Spring of 2005.

• Will there be an explosion of new tests?

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Discussion Question #3• Dr. Welby-Price from the ER calls you about a patient.

– A 57 year old man. – Jaundiced, thrombocytopenic, in DIC. – Dripping green slime from fingernails and conjunctivae.

• You’ve been reading in Promed about an outbreak.– It resembles Crimean-Congo hemorrhagic fever, except the

victims drip green slime. – Began in Turkmenistan after a strange meteorite impact.

• Dr. W-P asks you what specimens to send. What do you tell him? <hint: at least part of the answer is ‘let me look a few things up and get back to you?>

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Answers #3• Things I’d address:

– Who’s the patient? Lab needs to quarantine the specimens and not perform any further testing until biosafety issues are clarified.

– Does the patient have risk factors (e.g. travel to the endemic area)?

– Does the patient have clinical signs/symptoms or risk factors for more common, similar syndromes (sepsis, rickettsial or Ehrlichial disease, acute leukemia…)

195

Question #4• After some research, you get more

information on Crimean-Congo hemorrhagic fever. – It’s a Level A BT agent; hemorrhagic fever virus. – There’s usually detectable viremia during the

acute phase. – There’s a serologic response 5-14d after onset.

• Who do you contact? • What specimens do you suggest to Dr.

Welby-Price• What other actions might you take?

196

Answers #4

• What, you think I’ve got all the answers?

• Discussion…

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Summary• Microbes are smart. Individually, their IQ is ~10-9, but

there are lots of them. • Emerging antibiotic resistance patterns will require

labs to keep up with continuing changes in methodology and interpretation.

• Emerging pathogens will challenge laboratories to provide diagnostic support and consultation, to protect lab workers, provide safe blod products, and to develop new procedures.

• Emerging technology will challenge labs to answer new clinical questions and to answer old questions more rapidly and accurately.

• Pathologists need to play an active role in keeping their laboratories up to date and assisting the front-line clinicians in managing emerging infectious problems.

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Pearls of Pathology• Emerging Antimicrobial Resistance

– Keep up with NCCLS susceptibility guidelines; even if you can’t implement them immediately

– Be aware of emerging mechanisms• Emerging pathogens

– Use listserves to stay aware of emerging threats and information resources

– Be aware of emerging infections– Protect the lab– Be a resource for patient care

• Prepare for the impact of emerging technologies

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