Clinical Use of Cardiac Biomarkers

Clinical Use of Cardiac Biomarkers
**Name**, DVM, DACVIM-Cardiology **Date**

Cardiac Biomarkers: Outline
Cardiac Troponins (cTnI) Natriuretic Peptides (BNP and ANP) Physiology Assays Clinical Uses

What are biomarkers? Characteristic objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.

Common Cardiac Biomarkers
Blood Sample Measurements Blood Pressure ECG Radiographs Echocardiogram

Cardiac Biomarkers Leakage Markers Troponin I Troponin T
Troponin C Myoglobin Creatine Kinase LDH AST Myosin light & heavy chain Functional Markers BNP C-reactive Protein ANP Endothelin-1 Epinephrine Norepinephrine Renin Angiotensin II Aldosterone Arginine vasopressin TNF-alpha Many biomarkers are not used readily to assess cardiac disease because of the lack of sens/spec and short T 1/2

Cardiac Troponins:Physiology
Regulatory proteins Part of contractile apparatus of skeletal and cardiac muscle tissue Part of the thin filament (with actin and tropomyosin) Essential for calcium-mediated regulation of muscle contraction

Cardiac Troponins: Physiology
3 Interacting & Functionally Distinct Proteins Troponin C 2 isoforms (cardiac and slow-twitch muscle) Homology between isoforms limits cardiac specificity and diagnostic usefulness Binds calcium to initiate muscle contraction

Troponin T Multiple isoforms in skeletal tissue 4 isoforms in human cardiac tissue (1 adult, 3 fetal tissue) Fetal isoforms may be re-expressed during heart failure or in damaged skeletal muscle Attaches the troponin complex to tropomyosin and actin Troponin I 3 isoforms (2 skeletal, 1 cardiac) Not expressed in fetal tissue or after damage/regeneration in adult tissue Inhibits actomyosin ATPase, preventing the structural interaction of myosin with actin-binding sites Calcium binds cTnC  cTnI displaced  tropomyosin conformational change (no longer interferes with myosin/actin)  muscle contraction occurs

Proteins exists in 2 populations within the cells Structurally bound within thin filaments of contractile apparatus Free in cytosol (2-4% cTnI, 6-8% cTnT) Leakage Markers Damage to cardiac myocytes results in loss of membrane integrity  release of cTn

Release Kinetics Acute cardiac injury release of cytosolic pool early rise blood levels  slow release of bound protein  sustained blood elevations T1/2 in circulation = 2 hours Humans with acute MI: levels rise 4-12hr after infarction, peak 12-48hr; remain elevated 7-10d (cTnI) and 10-14d (cTnT) Dog MI model: peak 10-16hr (more rapid development of necrosis?) Mechanism of elimination unknown Due to release kinetics, it is possible that patients presenting w/in hours of an acute MI may not have elevated cTn levels Take samples at admission, 6-9hr, 12-24hr post-admission

Cardiac Troponin I Unique to heart
Healthy animals may have low, but detectable levels Time to initial elevation = 3-8 hours Time to peak elevation = hours Time to return to baseline = 5-10 days

Indicate myocardial damage Evidence to suggest cTn release associated with irreversible injury AND reversible injury Do NOT identify cause of damage or mechanism of injury

Cardiac Troponins: Assays
Levels determined with ELISA Using antibodies specific for cTn I and T cTnI No gold standard assay Can be modified or complexed to other proteins (ex. cTnC) Assay antibodies may have differing specificity for each circulating form Can not compare results from different assays False Positives Rheumatoid factor, excess fibrin, hemolysis, lipemia, ALP, etc. cTnI more sensitive than cTnT The unique amino acid sequence of cTnI and cTnT allows the production of monoclonal antibodies and immunassays

Cardiac Troponins: Assays
Human Assays Can be used for most species Homology between canine (95%) and feline (96%) genes and humans Region targeted by assays differs by 1 amino acid Most normal animals have levels below threshold Normals Dog: < ng/mL (median 0.02 ng/mL) Cat: < ng/mL

Cardiac Troponins: Prognosis in Humans
Magnitude of troponin level elevation correlates with risk of future cardiac events or death Aids in indentification of patients with greater disease severity who may benefit from more aggressive therapy Size of infarcted area may be predicted based on cTnI or cTnT levels at 72hr

Cardiac Troponins: Causes of Elevation
Breed Greyhound, Boxer Direct Trauma Secondary to mechanical injury Ex. cardiac sx, catheterization procedures, myocardial contusions from blunt trauma, electrical injuries (cardioversion, defibrillation)

Infection, Inflammation Myocardial dysfunction common during sepsis Due to:  MvO2 Septic microemboli Toxic effects of bacterial endotoxins Cardiac depressant effects from cytokines Ischemia, reperfusion injury In humans, LV dysfunction common (hence cTn elevations) Study in foals - cTnT elevated in septic foals compares to healthy, but no difference in levels comparing non-survivors and survivors

Infection, Inflammation Canine babesiosis Due to: Epi- and endocardial hemorrhage Inflammatory cell infiltrate Myocardial fiber necrosis Pericardial effusion Changes thought to be related to SIRS and anemic hypoxia cTnI elevations can be used to determine cardiac involvement Association between cTnI levels and clinical severity and mortality

Cardiac Troponins: Cause of Elevation
Infection, Inflammation Canine ehrlichiosis 44% of infected dogs had cTnI elevations without clinically apparent heart disease, suggesting myocardial involvement All dogs with severe ECG changes were infected with E. canis 33% of infected dogs had evidence of LV systolic dysfunction Severity of anemia and SIRS may contribute to pathophysiology of myocardial damage Mutlisystemic disease - Reported assoc w/ cardiomyopathy Cardiac Evaluation in Ehrlichia Study in Brazil, n=194 w/ clinical and lab abnorm consistemt with E cani infection Mild Abnorms = sinus bradycardia, sinus tachycardia (HR bpm), 1st or 2nd AVB Mod Abn = persistent sinus tach (HR >180), increased T wave amplitude (> 1/3 R- wave, but not biphasic), ST elevation or depression, single monomorphic VPC, supraventricular premature complex, transient RBBB or LBBB Severe Abn = freq VPC or SVPC, couplets, triplets, polymorphic VPC Echo (changes in 33% E. canis dogs, 27% other org infection, 34% PCR-neg sick dogs) LVIDd, LVIDs, LVWd, shortening fraction, ejection fraction LV systolic dysfunction = fractional shortening <26% BP

Primary Heart Disease HCM Due to: Intramural coronary artery disease  areas of ischemia Myocardial hypertrophy with cellular necrosis Levels  with moderate-severe disease Se 85%/Sp 97% for differentiating HCM from normal HCM + CHF had significantly high levels Use to differentiate cardiac vs. non-cardiac cause of dyspnea in cats? Mitral Valve Disease Subaortic Stenosis 3rd Degree AVB Fibrosis or inflammation Hypoxic damage (secondary to  perfusion) HCM - cTnI levels may be normal in cats with mild disease HCM - weak correlation b/w ventriclar thickness and cTnI level; LA:Ao ratio MVD, SAS, DCM --> level may correlate with severity and prognosis

Cardiac Troponins: Causes of Elevation Primary Heart Disease Cont.
ARVC (“Boxer Cardiomyopathy”) Characterization: ventricular tachycardia, fibrofatty replacement of the right +/- left ventricles, interventricular septum Screening: 24-hr Holter monitor ARVC (>1,000 VPCs/24h) Median VPCs/24h = 2,375 Median Grade of Ventricular Arrhythmia = 3 Median cTnI = ng/mL Significant correlation between cTnI concentration and number of VPCs/24hr and grade of ventricular arrhythmia Clinical Signs: syncope, signs of CHF, sudden death Ventricular Grade (using Lead II) 0 = no VPCs 1 = only single monomorphic VPCs 2 = single VPCs (in a bigeminal or trigeminal pattern) or multiform complexes 3 = couplets or triplets 4 = runs of Vtach (4 beats or longer) or an R-on-T phenomenon Increases in BNP have not been shown to be assoc w/ presence of disease

Heart Failure Elevated cTnI and cTnT due to LV dysfunction Levels often return to normal after successful treatment of acute episode Class IV CHF due to MVD 6/15 (40%) had detectable cTnI (median 0.24 ng/mL) 1/15 (7%) had detectable cTnT (0.02ng/mL) No statistical difference in survival to discharge between dogs with detectable and non-detectable cTn levels Dogs with detectable cTn levels had overall shorter survival times (MST 67.5d, compared to 390d) HF 2nd most common cause of death in dogs in US (Morris Animal Foundation study in 1990’s) MVD is the most common cause of acquired heart disease in dogs Mortality rate in severe CHF is 1year, even with medical Tx Prospective study, 15 dogs, Class IV CHF d/t MVD - as designated by the NY Heart Assoc Described as dyspnea and coughing that are present at rest and these signs are exaggerated by any physical activity 14 dogs survived to discharge 13 died/euth related to heart disease (1 non-cardiac euth); 12/13 euth d/t recurrence of CHF 1 died 2d after presentation d/t refractory CHF --> dog had detectable cTnI ISACHC (International SA Cardiac Health Council) - dogs w/ Class II and IIIA HF had sig. higher levels of cTnI than normal dogs and dogs with class IA and IB HF Using cTnI cutoff off ng/mL, study identified dogs with class II or worse HF w/ 96% sens. And 88% spec. International Small Animal Cardiac Health Council (ISACHC) - classify dogs with DCM and MVD Ia = subclinical heart dz, w/o CXR evidence of heart enlg II-compensated = CHF controlled w/ meds for >12 months II-active = mild signs of CHF ( inc RR/RE, exercise intol, cough); serum sample for NT-proBNP collected while clin signs and CXR abnorm consistent w/ pulm edema or eff evident IIIa = over, severe signs of CHF (resp distress, profound weakness, syncope); patients treated as OP IIIb = severe signs of CHF that require hospitalization

Pericardial Disease Due to: Release of cTn secondary to epicardial inflammation Se/Sp marker for myocardial ischemia and necrosis  coronary perfusion during tamponade Changes tend to be more severe in dogs with hemangiosarcoma May be used to differentiate idiopathic pericarditis and pericardial effusion from HAS Study - 37 dogs with pericardial eff and 5 normal dogs cTnT 0.00 in pericardial effusion dogs and <0.01 in normal dogs (I.e. not detectable levels) No definitive dx in 11, or remaining 26 18 HAS - median cTnI 2.77 ng/dL 6 idiopathic - median cTnI 0.05 ng/dL 1 heart base tumor 1 mesothelioma

Pericardial Disease – differentiation of HSA vs HBT vs idiopathic vs other Comparison of plasma cTnI among dogs w/ cardiac HSA, non-cardiac HSA, other neoplasms, PCE of non-HSA origin (JAVMA 2010;237(7): ) cTnI >0.25 ng/mL - used to ID PCE due to cardiac HSA with 81% Se and 100% Sp Utility of cTnI in differentiating b/w underlying etiologies of PCE in K9 patient (ACVIM Forum 2011 Abstract) HSA vs HBT vs Idiopathic cTnI >0.78 ng/mL 67% Se/95% Sp for HSA Not reliable to DDx HBT vs. Idiopathic - Cardiac HSA has significantly higher levels compared to other causes of other causes - Measurement of cTnI in PCE fluid does not improve Se of test is distinguishing HSA from other neoplastic or non-neoplastic causes 57 dogs = 18 with confirmed [5] or suspected [13] cardiac HSA, 14 with confirmed HSA involving sites other than the heart [non-cardiac HSA], 10 with pericardial effusion not caused by HSA, and 15 with non-cardiac non-HSA neoplasms cTnI is a sensitive and specific marker for myocardial ischemia and necrosis and dogs with pericardial effusion frequently have these changes. Because these changes tend to be more severe in dogs with hemangiosarcoma, they have been shown to have higher cTnI concentrations than dogs with idiopathic pericardial effusion. Lack of standardization b/w assays – each company uses a different target on the cTnI protein for their assay Values obtained on one analyzer can not be directly compared to another cTnI value that is a cutoff in a certain study will only have meaning if the same assay is used in clinical practice

Chemotherapy Anthracyclines (Doxorubicin) Cardiomyopathy irreversible, typically fatal Result of cumulative dose Retrospective study of 44 dogs showed an elevated cTnI in 32 during doxorubicin therapy for LSA and OSA Elevations did NOT predict development of cardiac disease (based 1’ on PE and TxR) In dogs w/ cTnI elevations, the elevation preceded recognition of heart disease

Exercise PTE and Pulmonary Hypertension Due to:  pulmonary arterial resistance   RV pressure   myocardial perfusion and O2 supply PTE associated with RV infarction Patients with elevated cTn have worse prognosis cTnI and cTnT elevated as well as having echo abnormalities in some participants at the 1994 Hawaii Ironman - not determined if temporary or long-term cardiac damage, although believed to be transient b/c other studies f Ironman competitors have shown abn echos returning to normal within 48hr AND athletes continues to compete Alaskan Sled Dogs study showed elevated cTnI w/ highest levels attained after running 160km cTnI elevations noted in endurance hrses

Feline Hyperthyroidism CV abnormalities associated with disease: Tachycardia Myocardial hypertrophy Hypertension Arrhythmias CHF Concurrent primary cardiomyopathy Release suspected due to: Myocardial cell damage associated with intramural coronary artery ischemia Physiological effects of excess thyroid hormone With treatment (I-131), myocyte damage may resolve Study of 23 hyperT4 cats --> 11 had elevated cTnI 6 months after Tx, only 3 cats still had cTnI elevations

GDV CV complications common Shock Ischemia and reperfusion injury Arrhythmias (develop in 40-70%) Myocardial degeneration and necrosis Elevations Due to: Subendocardial necrosis Ventricular arrhythmias cTnI - 87%, 93% Peak levels 48-72hr after surgery 24.9 ng/mL (dogs died vs ng/mL dogs survived) cTnT - 51%, 57% 0.18ng/mL (dogs died vs. <0.01 ng/mL dogs survived) Study of 85 dogs with GDV --> ALL dogs that died (19%) had cTnI elevations (all dogs that died in 2nd study [21%] had cTnI elevations) Causes: decreased CO, MAP, coronary blood flow --> subendocardial necrosis, ventricular arrhythmias

Renal Failure Chronic elevations in 50% patients cTnT more commonly associated (82%, cTnI 6%) Proposed Mechanisms: Silent myocardial necrosis or ischemia Ventricular hypertrophy Inpaired renal clearance Indicator of mortality in humans with renal disease or on hemodialysis Cats --> study of 14, 11 had elevated cTnI levels Dogs --> study of 36, 29 had elevated cTnI levels 2nd Study 31 dogs  RF = Cr >3mg/dL, USG (9/31 ARF) RF cTnI 0.35mg/mL (normal/healthy 0.2ng/mL) BP significantly higher in RF dogs Humans 7-18% with RF w/o primary cardiac disease have high serum cTnI

Cardiac Troponins: Clinical Uses
Means to differentiate cardiac and non-cardiac causes of dyspnea in cats D-CHF median cTnI 1.59 ng/mL D-NCC median cTnI ng/mL Reliable to diagnose dyspnea caused by CHF? cTnI > 0.2 ng/mL Se 100% / Sp 58% cTnI > 1.42 ng/mL Se 58% / Sp 100% Using 100% Se and Sp, cTnI would discriminate cause of dyspnea in ~50% of cats Recent study in JAVMA of 43 cats with dyspnea attributable to CHF (n= HCM, 8 unclass CM, 7 DCM, 2 hyperT4, 1 TV dysplasia) or Noncardiac cause (n= asthma, neoplastic pl eff, 2 idiopathic pl eff, 1 lung tumor, 1 laryngeal tumor, 1 undiagnosed) Cats w/ dyspnea relate to CHF had significantly greater cTnI concentration Based on results, measurement of cTnI may help r/o CHF in large portion of dyspneic cats - if cTnI is within or near RR, dyspnea can be ruled non-cardiac and indicate a need to pursue other causes Similarly, if cTnI >1.42ng/mL, most likely CHF is causing dyspnea --> may spare patient of potentially compromising diagnostic testing (ex. TxR) and/or treatment (ex. inappropriate fluid admin) So why would cats with D-NCC have elevated cTnI? --> myocardial hypoxemia secondary to severe pulmonary dz? Increased myocardial wall strss and injury secondary to pulonary hypertension assoc w/ primary pulmonary dz? Septic myocardial injury from pneumonia? Nonspecific inflammatory nyocardial injury by inflamm mediators involved in pulmonary dz? Scatterplot - N=31 w/ D-CHF N=12 w/ D-NCC N=19 w/ heart dz but not CHF Shaded area = ref interval in healthy cats Dashed line 100% 0.2 ng/mL; dotted line 100% 1.42 ng/mL 14/31 w/ D-CHF had cTnI < 1.42 6/21 w/ D-NCC has cTnI > 0.2 Therfore. Bt use of the 100% sens and spec thresholds, measurement of plasma cTnI conc would discriminate b/w cardiac and NCC cause of dyspnea in ~50% of cats 5/19 w/ ht dz but no CHF has cTnI > 1.42

Cardiac Troponins: Clinical Uses
Detection of occult DCM 3 Phases of Disease 1: Dogs with genetic predisposition to disease w/o detectable morphologic or ECG signs 2: Dogs with detectable abnormalities on ECG or ECHO that do not have overt clinical signs 3: Dogs with ECG and ECHO abnormalities as well as clinical signs 1: DCM and ARVC are caused b specific genetic defects - ID of mutant gene or genes repsponsible and subsequent genomic testing would ID such dogs 2: Occult disease Currently detected using combo of ECG and ECHO exams Relatively well-defined criteria for occult disease have been established for Dobermans, Irish Wolfhounds, and Boxers and depend on evidence of ventricular or atrial arrhythmias detected by serial ECG or a 24hr Holter monitor and ECHO eval of LV dimension and function Blood-based testing would provide a minimally-invasive technique, quantitative results, would be cost efficient way to diagnose the disease earlier in it’s course hopefully allowing for a more favorable prognosis 3: Clinical signs - CHF, exercise intolerance, syncope; b/c of advanced nature of disease in this phase, clinical outcome is poor and many of these dogs have already served as breeding stock

Cardac Troponins: Clinical Uses
Cardiac Troponin I to detect occult DVM (JVIM 2010;24: ) cTnI in DP with DCM 447 cTnI measurements from 264 healthy DP 206 cTnI measurements from 75 DCM DP Control group – 264 samples Age was the only variable that had a signiﬁcant association on cTnI in healthy Doberman Pinschers Systolic and diastolic M-mode measurements, weight, and sex were not associated with cTnI values in the control group. VPC group (>100 VPC/24hr, normal echo) – 88 samples Echo group – 19 samples VPC and echo group – 56 samples More males compared to other groups Clinical group – 20 samples Last normal/incipient group – 23 samples cTnI values of dogs in all disease groups, including the ‘‘incipient’’ and ‘‘VPC group’’, were signiﬁcantly higher than the control group No significant difference of cTnI conc between occult disease groups Clinical group cTnI conc significantly higher than occult disease and control group A cut-off value of 0.22ng/mL had a Se 79.5%, Sp 84.4% to detect all forms of cardiomyopathy.

Cardiac Troponins: Clinical Use
Prognosis in dogs with DCM cTnI >0.2 ng/mL  MST 112d (<0.2 ng/mL  MST 357d) Correlation between heart chamber size and cTnI level

Natriuretic Peptides Green mamba snake; D Type is Dendroapsis NP and is only found thus far in the venom of the green mamba snake…

Brain (or b-type) Natriuretic Peptide (BNP): Physiology
Cardiac neurohormone specifically secreted from the ventricles in response to increased wall stress (pressure or volume) Released as prohormone which is then cleaved into active hormone BNP and NT-proBNP Functions Diuretic Natriuretic Vascular relaxation Regulating BP and fluid balance Inhibition of RAAS Enhances diastolic function Decreased filling pressures in the heart BNP elaborated early in course of disease and in proportion to severity of disease Conditions assoc w/ high circ conc of BNP Systolic dysfunction Diastolic dysfunction Volume overload Pulmonary hypertension

BNP Release Kinetics Not stored
Regulation of synthesis and excretion occurs at the level of gene expression (transcription)  BNP requires  synthesis Production is rapidly up-regulated when cardiomyocytes are stimulated Secretion requires longer term stimulus and is regulated by myocardial wall stretch and wall tension/stretch and hypoxia Although its production is up-regulated in situations of volume overload, the diuretic and natriuretic properties of BNP in (patho)physiological concentrations are still insufficient to retain euvolemia

BNP vs. NT-proBNP BNP NT-proBNP Short T1/2 (90 sec; humans 22 min.)
Requires a small amount of blood Does not require specialized collection or handling Longer T1/2 (exact unknown; 15x longer BNP in sheep; humans 120 min.) Typically present in higher concentrations Quick turn-around time Affordable Lack of homology b/w humand and K9 BNP - share 45% (87% ANP) Introduces difficulty in utilization of human assays

BNP: Humans Strongest predictor of concentration is end-diastolic wall stress Discriminate between cardiac and non-cardiac cause of disease Stratify severity of disease Guide medical treatment Predict outcome Detect patients with occult LV dysfunction Evaluate at-risk populations for asymptomatic disease In humans, the use of BNP reduced the need for hospitalization in the ICU, the median time to discharge by 3 days, and the total cost of care by $3000 when used in the ER to evalaute patients presenting for acute dyspnea. Framingham Study - people with increased concentrations of BNP and ANP had a sig. higher risk for HF, major CV event, Afib, and death during the subsequent 5yr period Women - BNP >23.3pg/mL, ANP >0.599nmol/L Men - BNP >20pg/mL, ANP 0.485nmol/L These levels were all well below the threshold thr dx of clinicall apaprent heart diseae (BNP >100 pg/mL) indicating that results within the red range still have clinical meaning Risk for adverse outcome was correlated with the magnitude of change in BNP conc. For each 1 SD increase in BNP conc., risk for HF increased 77% and death increased 94%

BNP: Veterinary Use Cardiac vs. Primary Respiratory disease causing clinical signs Screening for occult disease Predicting first episode of CHF

Three “New” NT-proBNP Assays
Canine Cardiopet ® proBNP Feline Cardiopet ® proBNP SNAP ® Feline proBNP Sample Plasma (no PI tube) Range 250-10,000 pmol/L Total CV <10% Indications Clinical signs (resp/chf) MMVD ODCM in Dobes Sample Plasma or Serum (no PI tube) Range pmol/L Total CV <15% Indications Clinical signs (resp/chf) Asymptomatic “at risk” Adult/senior panel Sample Plasma or Serum Range Normal Abnormal (> pmol/L) Indications Asymptomatic “at risk” Adult/senior panel Clinical signs ? No longer need a PI tube; snap test is available, rec. use plasma for dog, lab will accept serum but you will get a disclaimer as if not kept cold, may be inaccurate; still rec. icepack particularly in the summer time; even chems should be on ice; new range is up to 10000, variability is acceptable now and is fine to use for monitoring; in monitoring—50% change for a dog with a murmur is significant

NT-proBNP in Feline Patients
Most commonly diagnosed heart disease is HCM One study found that close to 15% of the general population of cats have LV hypertrophy Prevalence of cardiomyopathy in apparently healthy cats. Paige CF, Abbott JA, Elvinger F, et al. JAVMA 234: , 2009. High percentage with no clinical signs Murmurs and gallops may be soft or absent Over 30% of cats with arterial thromboembolism have a history of a normal cardiac physical examination prior to presentation Radiographs – variable Echocardiography definitive, but requires specialized equipment and training

Diagnosis of Feline Cardiac Disease: NT-proBNP for Screening
Screening for Cardiomyopathy - Elevated in any degree of occult feline CM - Weak, but positive correlation with severity – may not be accurate or consistent compared to echo findings - Significantly higher in CHF vs. healthy controls Asymptomatic HCM Tx w/ Atenolol vs un-Tx – not significantly different levels *Maine Coon cats with MYPBC heterogeneous mutation had higher NT-proBNP than those without. **Patients with hypertrophic obstructive disease have significantly higher NT-proBNP than those with HCM and no outflow obstruction

Diagnosis of Feline Cardiac Disease: Idexx proBNP

Diagnosis of Feline Cardiac Disease: NT-proBNP for Screening

SNAP® Feline proBNP New green conjugate 5 drops: g-r-e-e-n
Serum or EDTA plasma 9 months stability refrigerated Warm for 30 minutes SNAP Feline proBNP is unique from other SNAP tests. It uses a green-colored conjugate instead of blue and uses 5 drops of conjugate instead of 4.

Pricing, Packaging and Stability
9 month stability with refrigeration List Price 5/box $11.95/test 10/box $9.95/test 12 months Is coming

SNAP Performance Demonstrates Good Correlation with Cardiopet® proBNP
SSDx cut-off (spots look equal) Abnormal Normal Transition from normal to abnormal on SNAP is between 150 and 200pmol/L. Line represents where snapshot calls it abnormally, circles are abnormal via visual, Pluses are visual as normal; line for snapshot cutoff—sample and referecnce spots are equal in color; even if you are in question about equal, it is high SSDX—Snapshot DX sample to ref. spot ratio Cut off is Not sure—it is still As it gets darker it is greater Cardiopet proBNP (pmol/L)

Interpreting SNAP Feline proBNP Results
From the SNAP Feline proBNP Diagnostic Update Field Trial Data

From the SNAP Feline proBNP Diagnostic Update
“We’ve started to use [proBNP tests] at one of the hospitals I work at, more regularly. And we use it to convince owners. If we pick up a murmur on a cat that we haven’t heard before. And so they’ll do a proBNP on it and if it’s high, now we have an argument—go to the cardiologist.”

Diagnosis of Feline Cardiac Disease: NT-proBNP Limitations
CKD and Systemic Hypertension Hyperthyroidism CKD and SHT - Higher in azotemic cats w/ SHT compared to azotemic cats w/ normal BP - NT-proBNP may be useful in distinguishing b/w white coat syndrome and true hypertension - Higher in cats w/ severe CKD (normotensive) vs mild-mod CKD and normal cats Hyperthyroidism Following successful Tx – total T4, HR, systolic BP, and PCV decreased; BW and creatinine conc. Increased; NTproBNP concentration declined (but not proANP) HyperT4 leads to increased HR (adrenergic hyper-responsiveness) and decreased SVR  resulting in increased CO Stimulus for decreased SVR is NO-induced vasodilation and direct relaxant effect of T3 on vascular smooth muscle cells These stimuli result in arterial underfilling  in turn result in chronic stimulation of the RAAS  leading to increased vascular volume, myocardial stretch, and NP release.

Summary of NT-proBNP in Feline Patients
Prevalence of cardiac disease in the general population of feline patients is estimated to be about 15% With a prevalence of 15% and Sens 90%/Spec 85% PPV is 53% and NPV is 93% A fair number of false positives <10% chance of a false negative Echocardiography is the gold standard for the diagnosis of feline myocardial disease

NT-proBNP in K9 Patients
The most common heart disease in dogs is degenerative mitral valve disease Accounts for 75% of acquired cardiac disease in dogs Estimates that over 60% of older small breeds of dogs are affected many will show signs of congestive heart failure Physical examination findings of a murmur make screening relatively easy Long asymptomatic phase Occult disease is more common with dilated cardiomyopathy Doberman Pinscher incidence of 45-62%

NT-proBNP: Uses Identification of cardiac disease and assessing disease severity Cross-sectional study of 119 dogs evaluted the utility if NT-proBNP in assessing cardiac disease - found that the nt=proBNP could be used to distinguish cardiac disease from control dogs with 83% sens and 90% spec; higher the classificiation of cardiac disease based on ISACHC, the higher the levels --- although a RR was not established for categorizing patients based on NT-proBNP concentrations Study also evaluated type of heart disease and level of NT-proBNP - found that type of heart disease does effect levels; DCM had greatest median concentration International Small Animal Cardiac Health Council (ISACHC) - classify dogs with DCM and MVD Ia = subclinical heart dz, w/o CXR evidence of heart enlg II-compensated = CHF controlled w/ meds for >12 months II-active = mild signs of CHF ( inc RR/RE, exercise intol, cough); serum sample for NT-proBNP collected while clin signs and CXR abnorm consistent w/ pulm edema or eff evident IIIa = over, severe signs of CHF (resp distress, profound weakness, syncope); patients treated as OP IIIb = severe signs of CHF that require hospitalization Cardiac disease MVD (n=118) DCM (n=18) Control/Healthy K9 (n=40) NT-proBNP > 445 pmol/L (Control median NT-proBNP 290 pmol/L) Sensitivity 83.2% Specificity 90% PPV 96.6% NPV 61% Used to differentiate dogs w/ and w/o CXR evidence of cardiomegaly and dogs w/ and w/o CHF Median NT-proBNP conc MVD 1188 pmol/L DCM 1748 pmol/L Conc > in dogs with Gr 5/6 murmur (2233 pmol/L) >> 3-4/6 (1010) >> 1-2/6 (646) Conc > in dogs w/ Class II or III dz (2252/2279) >> I dz (618) --> therefore could be used to ddx dogs w/ CHF and w/o CHF (median conc in CHF dogs 3x that of non-CHF) Gray zone = Median conc sig higher in pure-bred control dogs than mixed breed --> therefore breed may influence conc Humans - conc correlated w/ age, sex, ethnicity, renal function (dogs w/ BUN >70 excluded form study - weak correlation b/w NT-proBNP and renal function)

NT-proBNP: Stage II, Occult DCM
NTproBNP (AJVR 2011;72(5): ) Evaluation of NTproBNP as a diagnostics marker for various stages of DCM in DP Control group – 196 healthy DP Age = only variable with significant influence on NTproBNP conc; increased in dogs >8yr of age Significantly younger overall compared to other groups VPC group (>100 VPC/24hr, normal echo) – 42 DP with occult DCM Echo group – 16 DP with occult DCM VPC and echo group – 36 DP with occult DCM More males compared with remaining groups Clinical group (Overt DCM -ie CS of disease, syncope, ex intol, CHF) DP Last normal/incipient group – initially in control group, but Dx with DCM in following 1.5 yr Groups, age, and LVIDs significantly assoc w/ plasma NTproBNP concentration NTproBNP siginificantly higher in all groups that had or developed DCM compared to control No significant difference in occult groups Overt DCM NTproBNP conc. Significantly higher than occult and control groups Se/Sp of use of NTproBNP to detect all stages of DCM >400 pmol/L – 81.1% Se, 75.0% Sp; AUC 0.84 Se/Sp of NTproBNP to predict echo abnormalities >400 pmol/L – 90.0% Se, 75% Sp; AUC 0.92 >550 pmol/L – 78.6% Se, 90.4% Sp; AUC 0.92

Stage II, Occult: Diagnostics
NTproBNP + Holter (JVIM 2012;26: ) Prospective evaluation of NTproBNP assay to detect ODCM and predict survival in DP 155 asymp DP presented for ODCM screening Healthy avg age 6yr; ODCM avg age 6.7yr Echo, 24hr Holter, NTproBNP performed Dx ODCM = inc LVIDs, >50 VPCs, or both 73/155 DP Dx ODCM 31/73 – Holter criteria met 17/73 – echo criteria met 25/73 – both criteria met Sensitivity of NTproBNP >457 pmol/L to detect groups Holter – 45.2% Echo – 76.5% Both – 96% Combo NTproBNP + Holter to detect ODCM – Se 94.5%, Sp 87.7%, Accuracy 91% Follow-up data available for 78/155 DP MST >50 VPCs (469d), NTproBNP >900 (284), ODCM (474d) significantly shorter than <50 VPCs (1743d), NTproBNP <900 (1743d), without disease (1743d)

Summary of NT-proBNP in Dogs
Helps differentiate between respiratory symptoms secondary to heart vs. pulmonary disease Concentrations correlate with the severity of disease May be predictive of impending congestive heart failure and used in combination with clinical status may predict outcome No studies yet evaluating relation to therapy and response to treatment

CHF Challenges Timely and accurate diagnosis can be challenging
Hx and PE findings not specific TXR - considered to be high yield diagnostic test, can be difficult to attain and interpret Diuretics and HF medication can be detrimental to a mis-diagnosed patient Hx and PE --> HF, cancer, resp disease? Difficult to attain and interpret --> life-threatening resp distress; wide variation in chest conformation, cardiac silhouette, presence of severe pulm edema or effusion

NT-Pro-BNP over 265 pmol/L Sensitivity 90% Specificity 88%
NT-Pro-BNP in Cats with Respiratory Distress Fox et al; JVC 2009, 11, S51-S61 Cats with CHF Cats with Respiratory Number 101 66 Murmur 61% 36% Gallop 42% 8% VHS 9.2 7.7 BNP: Median (IQR) 745( ) 76.5 (24-180) Another cat respiratory distress study Murmur not great Gallop, VHS Almost 10 fold difference in median IQR, some overlap in full range More studies needed to firmly establish cut-offs, great potential (cage-side, 1 drop ear-prick) NT-Pro-BNP over 265 pmol/L Sensitivity 90% Specificity 88%

Diagnosis of Feline Cardiac Disease: NT-proBNP for CHF
Significantly higher in cats with respiratory distress due to heart failure Significantly higher in cats with cardiogenic pleural effusion Level of confidence Respiratory Distress - Significantly higher in CHF - NT-proBP + CXR = improved diagnostic accuracy (69% to 87%) of CHF and improved clinician confidence in Dx of CHF (6 to 8, scale of 1-10) 10 cats with respiratory signs – GP provided Hx/PE/CXR/ECG/Labs; determine if CHF or RD and level of confidence; then given NTproBNP Data showed overall low accuracy and level of confidence in Dx of respiratory signs – NTproBNP is helpful, but not diagnostic in itself Healthy vs HD-no CHF vs HD-CHF - Conc positively correlated with LA:Ao, E:E’ - Conc higher in HD cats vs. control - NT-proANP and NT-proBNP were found to be significantly different between all three groups and the NT-proBNP assay appeared to have the greater discriminating power.

NT-proBNP: Clinical Uses
Dyspnea and coughing: CHF or primary pulmonary disease? CHF Mean 2,554 pmol/L Primary Pulmonary Disease Mean 357 pmol/L In animals, resp distress develops secondary to a myriad of causes w/ CHF and PPD being among the 2 most common etiologies - differentiating the cause of respiratory distress can be challenging, just as it is in cats N=46 of dogs presenting with cough and/or resp distress 25 CHF (18 MVD/16 w/ LHF/2 w/ bi-vent failure; 4 DCM all w/ afib and LHF; 2 TVD w/ RHF/ 1was syncopal; 1 PDA w/ LHF) 21 PPD (11 non-septic inflamm, 5 neut, 3 eos, 3 mixed; 7 bacterial infection, ecoli, klebsiella, strp, pseudomonas, peptostreptococcus; 3 neoplasia, 2 lymphoma, 1 cacinoma) Although lowest values for CHF and highest values for PPD were similar, there was no overlap Cardiac disease defined as - m or a during PE; VHS >11; evidence of LHF on CXR --> patients had Echo PPD - CXR evidence --> patients had BAL **Some of the highest values are found in dogs with PHT**

The PREDICT Study NT Pro BNP along with the LVIDd:AO ratio predicts first onset heart failure in about 70% of patients by their next visit Independent risks factors for first onset of CHF NT ProBNP >1500 LVIDd:AO >3

Breed Variations in BNP J Vet Intern Med 2014;28:451–457, K
Breed Variations in BNP J Vet Intern Med 2014;28:451–457, K. Sjostrand, et al

Clinical Guidance for NT-proBNP in Dogs
<900 pmol/L The likelihood that clinical signs are due to heart failure is low- consider other differentials to determine the cause of symptoms pmol/L Results in this range do not allow differentiation between clinical signs due to heart failure vs those from other causes >1800 pmol/L The likelihood that clinical signs are due to heart failure is high. Further cardiac workup or cardiac consultation is recommended

NTproBNP Limitations Study – 38 healthy dogs, 28 dogs with cardiac disease (14 w/ CHF, 14 w/o CHF), 81 dogs with primary noncardiac disease Dogs with CHF had signiﬁcantly higher plasma cBNP concentrations than dogs with subclinical cardiac disease, apparently healthy dogs, or dogs with primary noncardiac disease. However, 21% (28/133) of dogs without CHF (including healthy dogs, dogs with primary noncardiac disease, and dogs with subclinical cardiac disease) had cBNP concentrations above previously identiﬁed diagnostic thresholds for CHF, reiterating the importance of reestablishing new diagnostic cutoffs when considering comorbidities affecting B-type natriuretic peptide levels. PNDSD = primary nondyspneic systemic disease HD = subclinical heart disease

Atrial Natriuretic Peptide (ANP)
Physiology Cardiac neurohormone released by atria in response to increased atrial wall stress Function To counter increases in BP and volume caused by RAAS Indicator of prognosis in dogs with cardiac disease Group 1 (mild-moderate heart disease) Mean ANP 64 pg/mL No significant difference in ANP at baseline compared to 6mo or 9mo MST 1095d Group 2 (severe heart disease) Mean ANP 328 pg/mL MST 58d (only 1/13 survived to 6mo recheck) N= 23, Prospective 12 month study w/ 3yr follow-up Mild-mod heart disease, n=10 (6 MI, 3 DCM, 1 TI) Severe heart disease, n=13 (5 MI, 5 DCM, 3 MI and TI) MST statistically significant w/ cut-off 95 pg/mL Less stable than other NP Similar results to BNP CHF – higher compared to controls Dyspnea – higher in CHF compared to other causes w/ variable accuracy b/w studies Feline CM – elevated compared to controls; higher in CHF vs asymp, but no significantly MVD – CHF higher compared to asymptomatic Positively correlated to VHS, FS, LA:Ao (sig. overlap b/w CHF and non-CHF makes determining clinically useful cutoff difficult) CKD – higher in cats w/ severe CKD-normotensive vs normal cats and cats w/ mild-mod CKD and CKD-SHT Although ANP has been shown to be an indicator of prognosis in cardiac disease, it’s clinical utility is limited by it’s short T1/2 and lack of availability to practicing veterinarians

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