• View in gallery
    Figure 1

    Representative color-flow (A) and continuous-wave (B) Doppler echocardiographic views of a Jack Russell Terrier in the study population with very severe congenital pulmonic stenosis (PS) and hemolytic anemia. A—Color-flow Doppler shows an aliased color-flow turbulent systolic jet, markedly narrowed at the level of the pulmonic annulus (yellow arrows). B—Very high peak systolic velocity flow (9.22 m/s) recorded within the right ventricular outflow tract, the pulmonary trunk, and across the pulmonic valve by use of continuous-wave Doppler mode from the right parasternal short-axis view at the level of the aortic valve (peak systolic trans-stenotic pressure gradient [∆P], 340 mm Hg). PT = Pulmonary trunk. RVOT = Right ventricular outflow tract.

  • View in gallery
    Figure 2

    Photomicrographs representative of blood smears from a Jack Russell Terrier presented initially with very severe PS (peak systolic trans-stenotic ∆P, 340 mm Hg; see also Figure 1). Before pulmonary balloon valvuloplasty (A), severe schistocytosis (16.5% RBCs; arrowheads) and polychromasia (asterisk) were noted, suggestive of a regenerative hemolytic anemia due to abnormal blood flow. Schistocytosis totally resolved 2 months after pulmonary balloon valvuloplasty (B). At this time, only discocytes were observed. May-Grünwald-Giemsa stain. Original magnification X 100. Bar = 20 µm.

  • View in gallery
    Figure 3

    Photomicrographs representative of a blood smear from an English Bulldog presented with severe PS (peak systolic trans-stenotic ∆P, 166 mm Hg). Moderate schistocytosis (2.0% RBCs; arrowhead [A]) and moderate acanthocytosis (6.0% RBCs; asterisk [A and B]) were observed with minimal keratocytosis (0.3% RBCs; arrow [B]). May-Grünwald-Giemsa stain. Original magnification X 100. Bar = 20 µm.

  • 1.

    Jacobson RJ, Rath CE, Perloff JK. Intravascular haemolysis and thrombocytopenia in left ventricular outflow obstruction. Br Heart J. 1973;35(8):849854.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Tsuji A, Tanabe M, Onishi K, et al. Intravascular hemolysis in aortic stenosis. Intern Med. 2004;43(10):935938.

  • 3.

    Kawase I, Matsuo T, Sasayama K, Suzuki H, Nishikawa H. Hemolytic anemia with aortic stenosis resolved by urgent aortic valve replacement. Ann Thorac Surg. 2008;86(2):645646.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Sugiura T, Okumiya T, Kubo T, Takeuchi H, Matsumura Y. Evaluation of intravascular hemolysis with erythrocyte creatine in patients with aortic stenosis. Int Heart J. 2016;57(4):430433.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Vahidkhah K, Cordasco D, Abbasi M, et al. Flow-induced damage to blood cells in aortic valve stenosis. Ann Biomed Eng. 2016;44(9):27242736.

  • 6.

    Myhre E, Dale J. Hemolysis in mitral valvular disease and mitral ball valve prostheses. Acta Med Scand. 1971;189(6):547550.

  • 7.

    Febres-Roman PR, Bourg WC, Crone RA, Davis RC Jr, Williams TH. Chronic intravascular hemolysis after aortic valve replacement with Ionescu-Shiley xenograft: comparative study with Bjork-Shiley prosthesis. Am J Cardiol. 1980;46(5):735738.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Magilligan DJ Jr, Fisher E, Alam M. Hemolytic anemia with porcine xenograft aortic and mitral valves. J Thorac Cardiovasc Surg. 1980;79(4):628631.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Chang H, Lin FY, Hung CR, Chu SH. Chronic intravascular hemolysis after valvular surgery. J Formos Med Assoc. 1990;89(10):880886.

  • 10.

    Shim D, Wechsler DS, Lloyd TR, Beekman RH III. Hemolysis following coil embolization of a patent ductus arteriosus. Cathet Cardiovasc Diagn. 1996;39(3):287290.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Ellis JT, Wick TM, Yoganathan AP. Prosthesis-induced hemolysis: mechanisms and quantification of shear stress. J Heart Valve Dis. 1998;7(4):376386.

    • Search Google Scholar
    • Export Citation
  • 12.

    Hijazi ZM. Coil closure of the ductus and hemolysis: strive to achieve complete closure. Cathet Cardiovasc Diagn. 1998;43(1):54. doi: 10.1002/(sici)1097-0304(199801)43:1<54::aid-ccd15>3.0.co;2-t

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Tomita H, Fuse S, Akagi T, et al. Hemolysis complicating coil occlusion of patent ductus arteriosus. Cathet Cardiovasc Diagn. 1998;43(1):5053.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Pedra CA, Pedra SF, Font VF. Hemolysis associated with coil occlusion of the arterial duct. Cardiol Young. 1998;8(1):6366.

  • 15.

    Lee C, Hsieh K, Huang T, Choong C. Spontaneous resolution of hemolysis after partial coil occlusion of ductus arteriosus. Pediatr Cardiol. 1999;20(5):371372.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Uzun O, Veldtman GR, Dickinson DF, Parsons JM, Blackburn ME, Gibbs JL. Hemolysis following implantation of duct occlusion coils. Heart. 1999;81(2):160161.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Birnbaum D, Laczkovics A, Heidt M, et al. Examination of hemolytic potential with the On-X(R) prosthetic heart valve. J Heart Valve Dis. 2000;9(1):142145.

    • Search Google Scholar
    • Export Citation
  • 18.

    Eda K, Ohtsuka S, Seo Y, et al. Conservative treatment of hemolytic complication following coil embolization in two adult cases of patent ductus arteriosus. Jpn Circ J. 2001;65(9):834836.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Pate GE, Al Zubaidi A, Chandavimol M, Thompson CR, Munt BI, Webb JG. Percutaneous closure of prosthetic paravalvular leaks: case series and review. Catheter Cardiovasc Interv. 2006;68(4):528533.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Shapira Y, Vaturi M, Sagie A. Hemolysis associated with prosthetic heart valves: a review. Cardiol Rev. 2009;17(3):121124.

  • 21.

    Shimizu M, Tanaka R, Hirao H, et al. Percutaneous transcatheter coil embolization of a ventricular septal defect in a dog. J Am Vet Med Assoc. 2005;226(1):6972.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Van Israël N, French AT, Wotton PR, Wilson N. Hemolysis associated with patent ductus arteriosus coil embolization in a dog. J Vet Intern Med. 2001;15(2):153156.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Campbell FE, Thomas WP, Miller SJ, Berger D, Kittleson MD. Immediate and late outcomes of transarterial coil occlusion of patent ductus arteriosus in dogs. J Vet Intern Med. 2006;20(1):8396.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Gordon SG, Saunders AB, Achen SE, et al. Transarterial ductal occlusion using the Amplatz Canine Duct Occluder in 40 dogs. J Vet Cardiol. 2010;12(2):8592.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Singh MK, Kittleson MD, Kass PH, Griffiths LG. Occlusion devices and approaches in canine patent ductus arteriosus: comparison of outcomes. J Vet Intern Med. 2012;26(1):8592.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Wang LH, Wang JK, Mullins CE. Eradicating acute hemolysis following transcatheter closure of ductus arteriosus by immediate deployment of a second device. Cathet Cardiovasc Diagn. 1998;43(3):295297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Chetboul V, Athanassiadis N, Concordet D, et al. Observer-dependent variability of quantitative clinical endpoints: the example of canine echocardiography. J Vet Pharmacol Ther. 2004;27(1):4956.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Bussadori C, Amberger C, Le Bobinnec G, Lombard CW. Guidelines for the echocardiographic studies of suspected subaortic and pulmonic stenosis. J Vet Cardiol. 2000;2(2):1522.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Boon JA. Stenotic lesions. In: Boon JA, ed. Manual of Veterinary Echocardiography. 2nd ed. Wiley-Blackwell; 2011:477525.

  • 30.

    Chetboul V, Damoiseaux C, Poissonnier C, et al. Specific features and survival of French Bulldogs with congenital pulmonic stenosis: a prospective cohort study of 66 cases. J Vet Cardiol. 2018;20(6):405414.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Warry E, Bohn A, Emanuelli M, Thamm D, Lana S. Disease distribution in canine patients with acanthocytosis: 123 cases. Vet Clin Pathol. 2013;42(4):465470.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Stockham SL, Scott MA. Erythrocytes. In: Stockham SL, Scott MA, eds. Fundamentals of Veterinary Clinical Pathology. 2nd ed. Blackwell Publishing Professional; 2008:107221.

    • Search Google Scholar
    • Export Citation
  • 33.

    Tidholm A. Retrospective study of congenital heart defects in 151 dogs. J Small Anim Pract. 1997;38(3):9498.

  • 34.

    Oliveira P, Domenech O, Silva J, Vannini S, Bussadori R, Bussadori C. Retrospective review of congenital heart disease in 976 dogs. J Vet Intern Med. 2011;25(3):477483.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Schrope DP. Prevalence of congenital heart disease in 76,301 mixed-breed dogs and 57,025 mixed-breed cats. J Vet Cardiol. 2015;17(3):192202.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Nevaril CG, Lynch EC, Alfrey CP, Hellums JD. Erythrocyte damage and destruction induced by shearing stress. J Lab Clin Med. 1968;71(5):784790.

    • Search Google Scholar
    • Export Citation
  • 37.

    Badylak SF, Van Vleet JF, Herman EH, Ferrans VJ, Myers CE. Poikilocytosis in dogs with chronic doxorubicin toxicosis. Am J Vet Res. 1985;46(2):505508.

    • Search Google Scholar
    • Export Citation
  • 38.

    English RV, Breitschwerdt EB, Grindem CB, Thrall DE, Gainsburg LA. Zollinger-Ellison syndrome and myelofibrosis in a dog. J Am Vet Med Assoc. 1988;192(10):14301434.

    • Search Google Scholar
    • Export Citation
  • 39.

    Weiss DJ, Kristensen A, Papenfuss N. Quantitative evaluation of irregularly spiculated red blood cells in the dog. Vet Clin Pathol. 1993;22(4):117121.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40.

    Mitlyng BL, Chandrashekmhar Y, Furne JK, Levitt MD. Use of breath carbon monoxide to measure the influence of prosthetic heart valves on erythrocyte survival. Am J Cardiol. 2006;97(9):13741376.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Kuzman D, Znidarcic T, Gros M, Vrhovec S, Svetina S, Zeks B. Effect of pH on red blood cell deformability. Pflugers Arch. 2000;440(5 suppl):R193R194.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Gedde MM, Davis DK, Huestis WH. Cytoplasmic pH and human erythrocyte shape. Biophys J. 1997;72(3):12341246.

Advertisement

Red blood cell abnormalities occur in dogs with congenital ventricular outflow tract obstruction

Peggy PassavinUnité de Cardiologie d’Alfort, Centre Hospitalier Universitaire Vétérinaire d’Alfort (CHUVA), École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France

Search for other papers by Peggy Passavin in
Current site
Google Scholar
PubMed
Close
 DVM
,
Valérie ChetboulUnité de Cardiologie d’Alfort, Centre Hospitalier Universitaire Vétérinaire d’Alfort (CHUVA), École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France
Institut Mondor de Recherche Biomédicale, U955 Inserm, École Nationale Vétérinaire d’Alfort, UPEC, Maisons-Alfort, France

Search for other papers by Valérie Chetboul in
Current site
Google Scholar
PubMed
Close
 DVM, PhD
,
Camille PoissonnierUnité de Cardiologie d’Alfort, Centre Hospitalier Universitaire Vétérinaire d’Alfort (CHUVA), École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France

Search for other papers by Camille Poissonnier in
Current site
Google Scholar
PubMed
Close
 DVM
,
Vittorio SaponaroUnité de Cardiologie d’Alfort, Centre Hospitalier Universitaire Vétérinaire d’Alfort (CHUVA), École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France

Search for other papers by Vittorio Saponaro in
Current site
Google Scholar
PubMed
Close
 DVM, PhD
,
Emilie Trehiou-SechiUnité de Cardiologie d’Alfort, Centre Hospitalier Universitaire Vétérinaire d’Alfort (CHUVA), École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France

Search for other papers by Emilie Trehiou-Sechi in
Current site
Google Scholar
PubMed
Close
 DVM
,
Maria-Paz AlvaradoUnité de Cardiologie d’Alfort, Centre Hospitalier Universitaire Vétérinaire d’Alfort (CHUVA), École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France

Search for other papers by Maria-Paz Alvarado in
Current site
Google Scholar
PubMed
Close
 DVM
,
Renaud TissierInstitut Mondor de Recherche Biomédicale, U955 Inserm, École Nationale Vétérinaire d’Alfort, UPEC, Maisons-Alfort, France
Unité de Pharmacie-Toxicologie, École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France

Search for other papers by Renaud Tissier in
Current site
Google Scholar
PubMed
Close
 DVM, PhD
,
Isabelle LagrangeUnité de Biochimie et Biologie Clinique/Laboratoire BioPôle, École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France

Search for other papers by Isabelle Lagrange in
Current site
Google Scholar
PubMed
Close
 DVM
, and
Pierre DeshuillersUnité de Biochimie et Biologie Clinique/Laboratoire BioPôle, École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France
UMR Biologie Moléculaire et Immunologie Parasitaires, École Nationale Vétérinaire d’Alfort, Université Paris-Est, Maisons-Alfort, France

Search for other papers by Pierre Deshuillers in
Current site
Google Scholar
PubMed
Close
 DVM, PhD

Abstract

OBJECTIVE

To document RBC abnormalities in dogs with congenital ventricular outflow tract obstruction.

ANIMALS

62 dogs with pulmonic stenosis (PS) or aortic stenosis (AS) and 20 control dogs were recruited.

PROCEDURES

The proportions of RBCs that were schistocytes, acanthocytes, and keratocytes were assessed. Complete blood cell counts were performed. Tested variables included hemoglobin concentration, hematocrit, and erythrocyte count.

RESULTS

Median (interquartile range [IQR]) peak systolic Doppler-derived trans-stenotic pressure gradient (∆P) values were 161 mm Hg (108 to 215 mm Hg) and 134 mm Hg (125 to 165 mm Hg) for dogs with PS and AS, respectively. Hematologic abnormalities were detected in most dogs with AS or PS (54/62 [87%]) versus 8/20 [40%] in control dogs, with schistocytes found in 40 of 62 (65%; median, 0.1% RBCs; IQR, 0% to 0.3%), acanthocytes in 29 of 62 (47%; median, 0.3% RBCs; IQR, 0% to 0.9%), keratocytes in 39 of 62 (63%; median, 0% RBCs; IQR, 0% to 0.2%), and hemolytic anemia in 4 dogs with PS. No significant association was identified between these abnormalities and ∆P. However, 3 of 4 dogs with anemia had a ∆P > 200 mm Hg (range, 242 to 340 mm Hg). The dog with the highest ∆P value also had the most severe anemia and schistocytosis, and both resolved after balloon valvuloplasty.

CLINICAL RELEVANCE

Poikilocytosis is common in dogs with congenital ventricular outflow tract obstruction, with anemia only observed in few dogs with high ∆P values.

Abstract

OBJECTIVE

To document RBC abnormalities in dogs with congenital ventricular outflow tract obstruction.

ANIMALS

62 dogs with pulmonic stenosis (PS) or aortic stenosis (AS) and 20 control dogs were recruited.

PROCEDURES

The proportions of RBCs that were schistocytes, acanthocytes, and keratocytes were assessed. Complete blood cell counts were performed. Tested variables included hemoglobin concentration, hematocrit, and erythrocyte count.

RESULTS

Median (interquartile range [IQR]) peak systolic Doppler-derived trans-stenotic pressure gradient (∆P) values were 161 mm Hg (108 to 215 mm Hg) and 134 mm Hg (125 to 165 mm Hg) for dogs with PS and AS, respectively. Hematologic abnormalities were detected in most dogs with AS or PS (54/62 [87%]) versus 8/20 [40%] in control dogs, with schistocytes found in 40 of 62 (65%; median, 0.1% RBCs; IQR, 0% to 0.3%), acanthocytes in 29 of 62 (47%; median, 0.3% RBCs; IQR, 0% to 0.9%), keratocytes in 39 of 62 (63%; median, 0% RBCs; IQR, 0% to 0.2%), and hemolytic anemia in 4 dogs with PS. No significant association was identified between these abnormalities and ∆P. However, 3 of 4 dogs with anemia had a ∆P > 200 mm Hg (range, 242 to 340 mm Hg). The dog with the highest ∆P value also had the most severe anemia and schistocytosis, and both resolved after balloon valvuloplasty.

CLINICAL RELEVANCE

Poikilocytosis is common in dogs with congenital ventricular outflow tract obstruction, with anemia only observed in few dogs with high ∆P values.

Introduction

Intravascular hemolysis (IH) has been identified in human patients with moderate to severe aortic stenosis (AS).1,2,3,4,5 In these patients, IH was accompanied by poikilocytes (ie, abnormally shaped erythrocytes) and more precisely RBC fragments (ie, schistocytes).1,2 The underlying mechanism of RBC fragmentation involves shear stress exerted on erythrocytes by high-velocity flows through stenotic orifices.5 In human patients, IH is also a well-described complication of cardiac valve prostheses and vascular devices.6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 In veterinary medicine, IH has been identified as secondary to transarterial coil embolization for ventricular septal defects (VSDs)21 and patent ductus arteriosus occlusions in dogs.22,23,24,25In those cases, IH is often not clinically significant and is likely related to the presence and severity of residual interventricular and ductal flows, respectively.24 Such mechanical hemolysis can resolve spontaneously or when occlusion becomes complete; for example, after a second successful percutaneous procedure.15,24,26 To the best of our knowledge, no data about flow-induced damage to RBCs associated with congenital ventricular outflow tract obstruction (VOTO) in dogs are currently available.

The objectives of this prospective observational study were therefore to document erythrocyte fragmentation injury in dogs with congenital VOTO (ie, pulmonic stenosis [PS] and AS) and determine whether these hematologic abnormalities were associated with peak systolic Doppler-derived trans-stenotic pressure gradients (∆P), as well as features of the stenotic lesions (number and location).

Materials and Methods

Animals

The study sample consisted of client-owned dogs diagnosed with congenital VOTO (PS or AS), based on conventional echocardiographic and Doppler examination, that were prospectively recruited at the Alfort Cardiology Unit (National Veterinary School of Alfort, France) between April 2017 and January 2020. Blood samples for CBC analysis are routinely collected in our unit for dogs with PS and AS at the time of diagnosis or for follow-up purposes. These blood samples were used for the present study, with specific additional hematologic analyses after the owners had given informed consent on this additional free-of-charge blood analysis.

All dogs underwent a complete physical examination as well as conventional echocardiographic (ie, M-mode and 2-D) and standard Doppler examinations with, if possible, 3-D echocardiography. Collected data included signalment (breed, age, sex, and body weight), clinical status, echocardiographic and Doppler findings, and treatment at the time of diagnosis. Dogs with concomitant hemodynamically significant congenital heart diseases were excluded from the study as were dogs with clinical signs suggesting a concomitant systemic or organ-related disease that could result in hematologic alterations.

A population of apparently healthy dogs (control group) was also included during the same period. These dogs were student-owned dogs, free from medication, with unremarkable medical history, no known illness, and normal physical examination findings. Blood was collected upon approval from the dog owners.

Conventional echocardiography and Doppler examination

Standard transthoracic M-mode, 2-D, and Doppler examinations were performed by trained observers on awake, standing dogs by use of ultrasound units (Vivid 7 and Vivid E9; GE Healthcare) as previously described and validated.27 All images were reviewed by a diplomate of the European College of Veterinary Internal Medicine in cardiology (VC).

Pulmonic stenosis—Obstructive abnormalities of the right ventricle outflow tract, pulmonic valve, and pulmonic trunk were diagnosed by use of the right and left parasternal short-axis views at the level of the aortic valve.28,29 The location (valvular, subvalvular, and/or supravalvular obstructions) and number of stenotic lesions were assessed from the same views by use of 2-D, 3-D (if possible), and color-flow Doppler modes.

Continuous-wave Doppler mode was used to assess the peak systolic velocity through the PS. The modified Bernoulli equation was then applied to calculate ∆P (in mm Hg) and thus grade PS severity. Pulmonic stenosis was considered mild for ∆P > 20 mm Hg and ≤ 50 mm Hg (grade 1), moderate for ∆P > 50 and ≤ 80 mm Hg (grade 2), severe for ∆P > 80 mm Hg and ≤ 200 mm Hg (grade 3), and very severe for ∆P > 200 mm Hg (grade 4).29,30

Aortic stenosis—The same echocardiographic protocol was applied for AS diagnosis by use of the right and left parasternal short-axis views at the level of the aortic valve as well as right and left parasternal 5-chamber views. The same classification was used to grade AS severity.

Hematologic examination

Blood samples for this study were gathered in the context of routine clinical assessment. Blood was collected and placed into a plastic tube containing K3EDTA. All samples were entirely processed within 2 hours. A CBC was performed with a hematology analyzer (Sysmex XT 2000i; Kobe) at the BioPôle Laboratory, National Veterinary School of Alfort, France. The tested variables included hemoglobin concentration, hematocrit, and RBC count.

Additionally, for each dog, a blood smear was stained with May-Grünwald-Giemsa stain and reviewed by a diplomate of the American College of Veterinary Pathologists (PD) who was blind to the animal status and ∆P values, and who recorded the number of schistocytes, acanthocytes, and keratocytes among 1,000 erythrocytes using an image processing program (ImageJ version 1.52; NIH). An acanthocyte was considered as an abnormally shaped erythrocyte with between 2 to 20 unevenly distributed thorn-like projections, as defined previously.31,32 A schistocyte was considered as an irregularly shaped RBC fragment and keratocytes as an RBC fragment with only 1 to 2 spicules.32

Statistical analysis

Statistical analyses were performed by computer software (Xlstat-Biomed version 2016; Addinsoft). Data were expressed as proportions (%), median, and interquartile ranges (IQRs).

Continuous variables were compared between groups with a Mann-Whitney test, and a χ2 test (or the Fisher exact test when necessary) was used for categorical variables. The level of significance was set at P < 0.05.

Results

Study population

During the study period, a total of 62 dogs with PS or AS meeting the inclusion criteria were included in the study. The median age was 1.6 years (IQR, 0.7 to 4.1 years) and body weight was 16.4 kg (IQR, 9.8 to 28.8 kg). There were 38 (61%) males and 24 (39%) females (male-to-female ratio, 1.6). The most commonly represented breeds were French Bulldog (n = 13), White Swiss Shepherd Dog (6), Golden Retriever (5), English Bulldog (4), Boxer (4), Bull Terrier (3), Chihuahua (2), Doberman Pinscher (2), and 23 other breeds (1 each). Additionally, 20 healthy dogs (control group) of similar age (P = 0.47) were also included in the study. The median age was 2.0 years (IQR, 1.3 to 3.5 years) and body weight was 26.8 kg (IQR, 22.3 to 30.4 kg). There were 13 (63%) males and 7 (37%) females (male-to-female ratio, 1.9).

Clinical findings at the time of diagnosis of VOTO in dogs

Approximately half of the dogs with VOTO (32/62 [52%]) had at least 1 clinical sign related to congenital stenosis at the time of diagnosis. The most common clinical signs (isolated or combined) were exercise intolerance reported by the owners (26/62 [42%]), syncope (10/62 [16%]), and ascites (2/62 [3%]).

Data regarding cardiac auscultation at the time of diagnosis were available for all dogs with VOTO. A left systolic basilar heart murmur (median grade, 4; IQR, 4 to 5) was found in each dog, with a palpable thrill in 29 of 62 (47%) dogs.

Current medical treatment at the time of diagnosis was known for all dogs with VOTO. Of the 62 dogs, 45 were receiving 1 (20/62 [32%]) or a combination (25/62 [40%]) of the following drugs: atenolol (37/62 [60%]), spironolactone (26/62 [42%]), benazepril (5/62 [8%]), furosemide or torsemide (4/62 [6%]), altizide (2/62 [3%]), clopidogrel (1/62 [2%]), and pimobendan (1/62 [2%]).

Conventional echocardiographic and Doppler findings in dogs with VOTO

Within the diseased population (62 dogs with VOTO), 46 (74%) dogs had PS (Figure 1) and 16 (26%) had AS. The median (IQR) ∆P values were 161 mm Hg (108 to 215 mm Hg) and 134 mm Hg (125 to 165 mm Hg) for dogs with PS and AS, respectively, with ∆P value ≥ 200 mm Hg for 17 of 62 (27%) dogs.

Figure 1
Figure 1

Representative color-flow (A) and continuous-wave (B) Doppler echocardiographic views of a Jack Russell Terrier in the study population with very severe congenital pulmonic stenosis (PS) and hemolytic anemia. A—Color-flow Doppler shows an aliased color-flow turbulent systolic jet, markedly narrowed at the level of the pulmonic annulus (yellow arrows). B—Very high peak systolic velocity flow (9.22 m/s) recorded within the right ventricular outflow tract, the pulmonary trunk, and across the pulmonic valve by use of continuous-wave Doppler mode from the right parasternal short-axis view at the level of the aortic valve (peak systolic trans-stenotic pressure gradient [∆P], 340 mm Hg). PT = Pulmonary trunk. RVOT = Right ventricular outflow tract.

Citation: American Journal of Veterinary Research 83, 3; 10.2460/ajvr.21.11.0188

Various concomitant, isolated or combined, hemodynamically nonsignificant defects were diagnosed, including left persistent cranial vena cava (n = 4 dogs), small atrial septal defects (10), VSD (14; 12 dogs with PS and 2 dogs with AS), patent ductus arteriosus (1), and minor mitral valve insufficiencies (51).

The location of the VOTO was known for each animal. In 40 dogs (40/62 [65%]), only 1 obstructive lesion was observed whereas the remaining 22 (35%) dogs showed 2 (15/62 [24%]) or 3 (7/62 [11%]) concomitant obstructive lesions. The overall distribution of stenotic lesions was as follows: subvalvular stenosis for 27 of 62 dogs (13/46 with PS; 14/16 with AS), valvular stenosis for 45 of 62 dogs (39/46 with PS; 6/16 with AS), and supravalvular stenosis for 16 of 62 dogs (only dogs with PS). Lastly, 12 of 62 (19%) dogs with congenital VOTO had dynamic VOTO owing to subvalvular (infundibular) PS (6/12 dogs) or PS-related marked right ventricular concentric hypertrophy (6/12 dogs).

Hematologic findings

Hematologic analyses were performed on all included dogs (ie, 62 dogs with VOTO and 20 healthy dogs).

Hematologic abnormalities (Figures 2 and 3) were detected in most dogs with AS or PS (54/62 [87%]), with schistocytes found in 40 of 62 dogs (65%; median proportion of RBCs, 0.1%; IQR, 0% to 0.3%), acanthocytes in 39 of 62 dogs (63%; median proportion of RBCs, 0.3%; IQR, 0% to 0.9%), keratocytes in 29 of 62 dogs (47%; median proportion of RBCs, 0%; IQR, 0% to 0.2%), and intravascular hemolytic anemia in 4 dogs with PS (hemoglobin concentrations, 9.8 to 12.3 g/dL; reference range, 12.4 to 19.2 g/dL). The regenerative nature of the anemia, defined by a corrected reticulocyte percentage > 1% by use of CBC and confirmed by blood smear, was identified for 2 of these 4 dogs (1.37% and 4.27%). By comparison, abnormalities were detected in 8 of 20 healthy control dogs (40% vs 87% for the diseased dogs; P = 0.08). Schistocytes and keratocytes were found in only 3 of 20 (15%) healthy dogs in the control group (0.1% RBCs for each), and acanthocytes were noted in 6 of 20 healthy dogs (30%; median proportion of RBCs, 0%; IQR, 0% to 0.1%). All proportions (ie, schistocytes, keratocytes, and acanthocytes) were significantly different between the diseased population and the control group (P < 0.001, P < 0.01, and P < 0.01, respectively). Additionally, none of the healthy control dogs were anemic (median hemoglobin concentration, 16.5 g/dL; IQR, 15.2 to 17.1 g/dL).

Figure 2
Figure 2
Figure 2

Photomicrographs representative of blood smears from a Jack Russell Terrier presented initially with very severe PS (peak systolic trans-stenotic ∆P, 340 mm Hg; see also Figure 1). Before pulmonary balloon valvuloplasty (A), severe schistocytosis (16.5% RBCs; arrowheads) and polychromasia (asterisk) were noted, suggestive of a regenerative hemolytic anemia due to abnormal blood flow. Schistocytosis totally resolved 2 months after pulmonary balloon valvuloplasty (B). At this time, only discocytes were observed. May-Grünwald-Giemsa stain. Original magnification X 100. Bar = 20 µm.

Citation: American Journal of Veterinary Research 83, 3; 10.2460/ajvr.21.11.0188

Figure 3
Figure 3
Figure 3

Photomicrographs representative of a blood smear from an English Bulldog presented with severe PS (peak systolic trans-stenotic ∆P, 166 mm Hg). Moderate schistocytosis (2.0% RBCs; arrowhead [A]) and moderate acanthocytosis (6.0% RBCs; asterisk [A and B]) were observed with minimal keratocytosis (0.3% RBCs; arrow [B]). May-Grünwald-Giemsa stain. Original magnification X 100. Bar = 20 µm.

Citation: American Journal of Veterinary Research 83, 3; 10.2460/ajvr.21.11.0188

Regarding dogs with PS or AS, no significant difference in ∆P values, grade of stenosis severity, location, number of obstructive lesions, and presence or absence of a VSD was observed between dogs with and those without schistocytosis, keratocytosis, and acanthocytosis (Tables 13). Furthermore, no significant differences in schistocyte (P = 0.91), acanthocyte (P = 0.23), or keratocyte (P = 0.96) proportions were found between the PS group (median proportion of RBCs [IQR], 0.1% [0% to 0.2%], 0.3% [0% to 0.1%], and 0% [0% to 0.2%], respectively) and the AS group (0.1% [0% to 0.3%], 0.1% [0% to 0.7%], and 0% [0% to 0.1%], respectively). Similarly, no significant difference in schistocyte (P = 0.14), acanthocyte (P = 0.58), or keratocyte (P = 0.89) proportions of RBCs (IQR) was found between dogs with 1 (0.1% [0% to 0.2%], 0.3% [0% to 1.1%], and 0% [0% to 0.2%], respectively) and several (0.2% [0% to 0.5%], 0.3% [0% to 0.7%], and 0% [0% to 0.2%], respectively) obstructive lesions.

Table 1

Comparative echocardiographic parameters assessed in 62 dogs with congenital ventricular outflow tract obstruction (VOTO; ie, aortic stenosis [AS] or pulmonic stenosis [PS]) with (n = 40) or without (22) schistocytosis.

Variables No. of dogs P values
Schistocytosis (n = 40) No schistocytosis (n = 22)
Median (IQR) ∆P value (mm Hg) 156 (103–214) 138 (122–186) 0.81
Stenosis severity* 0.36
  Grade 1 2 0
  Grade 2 4 1
  Grade 3 21 17
  Grade 4 13 4
Dogs with PS (%) 30/46 (65) 16/46 (35) 1.0
Dogs with AS (%) 10/16 (63) 6/16 (38)
Dogs with 1 stenotic lesion 25 15 1.0
Dogs with > 1 stenotic lesions 15 7
Localization of obstructive lesions 0.96
  Subvalvular 18 9
  Valvular 29 16
  Supravalvular 10 6
VSD present 11 3 0.34
VSD absent 29 19

Pulmonic stenosis was considered mild for ∆P > 20 mm Hg and ≤ 50 mm Hg (grade 1), moderate for ∆P > 50 and ≤ 80 mm Hg (grade 2), severe for ∆P > 80 mm Hg and ≤ 200 mm Hg (grade 3), and very severe for ∆P > 200 mm Hg (grade 4).

VSD = Ventricular septal defect.

Table 2

Comparative echocardiographic parameters assessed in 62 dogs with congenital VOTO (ie, AS or PS) with (n = 29) or without (33) keratocytosis.

Variables No. of dogs P values
Keratocytosis (n = 29) No keratocytosis (n = 33)
Median (IQR) ∆P value (mm Hg) 141 (109–183) 164 (115–219) 0.24
Stenosis severity 0.21
  Grade 1 0 2
  Grade 2 4 1
  Grade 3 19 19
  Grade 4 6 11
Dogs with PS (%) 21/46 (46) 25/46 (54) 1.0
Dogs with AS (%) 8/16 (50) 8/16 (50)
Dogs with 1 stenotic lesion 19 21 1.0
Dogs with > 1 stenotic lesions 10 12
Localization of obstructive lesions 0.74
  Subvalvular 12 15
  Valvular 21 24
  Supravalvular 9 7
VSD present 8 6 0.38
VSD absent 21 27

See Table 1 for key.

Table 3

Comparative echocardiographic parameters assessed in 62 dogs with congenital VOTO (ie, AS or PS) with (n = 39) or without (23) acanthocytosis.

Variables No. of dogs P values
Acanthocytosis (n = 39) No acanthocytosis (n = 23)
Median (IQR) ∆P value (mm Hg) 164 (108–207) 142 (123–192) 0.78
Stenosis severity 0.92
  Grade 1 2 0
  Grade 2 3 2
  Grade 3 23 15
  Grade 4 11 6
Dogs with PS (%) 31/46 (67) 15/46 (33) 1.0
Dogs with AS (%) 8/16 (50) 8/16 (50)
Dogs with 1 stenotic lesion 26 14 0.64
Dogs with > 1 stenotic lesions 13 9
Localization of obstructive lesions 0.71
  Subvalvular 15 12
  Valvular 29 16
  Supravalvular 9 7
VSD present 9 5 1.0
VSD absent 30 18

See Table 1 for key.

Nevertheless, 3 of 4 dogs with anemia had ∆P values > 200 mm Hg. Interestingly, the dog (a Jack Russell Terrier) with the highest ∆P value (340 mm Hg) also had the most severe anemia (hemoglobin concentration of 9.8 g/dL and hematocrit of 27.6%; reference range, 35% to 52%) and schistocytosis (16.5% of RBCs), with no visible acanthocyte or keratocyte. In this dog, anemia resolved 1 week after balloon valvuloplasty (postoperative ∆P, 217 mm Hg vs 340 mm Hg) while schistocytosis progressively decreased over the next month and had disappeared by 2 months postprocedure. The other 3 dogs did not undergo balloon valvuloplasty.

Discussion

Congenital VOTO is among the most common congenital heart diseases in dogs.33,34,35 Nevertheless, to the best of our knowledge, no studies have specifically focused on IH secondary to these defects. In veterinary medicine, most descriptions of mechanical hemolysis have referred to device embolization for patent ductus arteriosus occlusion.22,23,24,25 Similarly, in human patients, hemolysis is a common complication of cardiac valve prostheses and vascular devices20 and is also described in patients with moderate to severe spontaneous AS.1,2,3,4,5 The underlying mechanism is considered as being mediated by high shear forces exerted on the erythrocyte membrane owing to high-velocity flow through stenotic orifices.3,5

The present study provided original data on hematologic abnormalities in 62 dogs with PS or AS by comparison with an age-matched control group. Due to its prospective observational nature, epidemiological, clinical, echocardiographic, and Doppler variables were available for all recruited animals.

Our results confirmed that RBC morphological abnormalities are apparently common in dogs with congenital VOTO, the majority remaining subclinical, as described in dogs with vascular devices.24 These RBC morphological abnormalities, including schistocytosis, acanthocytosis, and keratocytosis, were detected in nearly 90% of affected dogs and were associated with anemia in 6%. This high percentage of dogs with schistocytosis, acanthocytosis and/or keratocytosis may be due, at least in part, to the high proportion of dogs with severe and very severe VOTO, as nearly 90% of the included animals had ∆P values > 80 mm Hg (representing 1 limitation of the present study). In human patients with AS, IH with or without anemia is a common complication that usually develops for ∆P values > 50 mm Hg, corresponding to shear stresses of at least 4,000 dynes/cm2 on RBCs.1,2,3,4,5,36 By comparison, the shear stress exerted on RBCs for a ∆P value of 80 mm Hg can be estimated at 6,400 dynes/cm2.1,36

The high proportion of severe and very severe VOTO in the present study may also have contributed to an unknown extent to the lack of statistical association between erythrocyte abnormalities (incidence of schistocytosis, acanthocytosis, and keratocytosis) and tested Doppler parameters (ie, ∆P values and stenosis severity grade). Similarly, although the number of obstructive lesions might be expected to influence erythrocyte alteration severity, no statistical association was found between the incidence and magnitude of schistocytosis, acanthocytosis, and keratocytosis and the number of stenotic lesions (1 vs > 1).

Intravascular hemolytic anemia is a well-described complication in human patients with AS.2,3,4 Interestingly, in the present study, intravascular hemolytic anemia was observed in only a few dogs (6%), all affected by PS. However, the diagnosis of IH in these 4 dogs was based solely on concomitant erythrocyte morphological alterations (ie, acanthocytosis, schistocytosis, and keratocytosis). Although schistocytosis and keratocytosis usually result from direct physical damage to the erythrocytes secondary to turbulent blood flow or vascular abnormalities such as endothelial membrane alteration, disseminated intravascular coagulation, glomerulonephritis, or hemangiosarcoma, they can also develop in association with other disorders such as myelofibrosis, dyserythropoiesis, chronic doxorubicin toxicosis, or oxidative stress.32,37,38,39 Nevertheless, in the present study, there were no clinical signs suggestive of defective erythropoiesis, no history of previous doxorubicin administration, and no other evidence of oxidative stress. Various disease conditions associated with acanthocytosis have been reported in dogs, including neoplasia and nonneoplastic diseases such as renal failure, gastroenteritis, hepatitis, and immune-mediated diseases.31,39 This lack of specificity of schistocytosis, keratocytosis, and acanthocytosis for IH may be overcome by measuring blood (eg, lactic dehydrogenase activity, haptoglobin, or erythrocyte creatine concentrations) or urinary markers (eg, hemosiderin), which were not used in the present study.4,20 In human patients, diagnosis of the intravascular hemolytic nature of the anemia can also be based on the direct measurement of the erythrocyte life span by use of expiratory carbon monoxide concentration.40 Unfortunately, these tests were not available during the study period.

This study presented several limitations. The majority of diseased dogs were characterized by severe or very severe VOTO. The clear impact of less severe PS and AS on RBCs therefore remains unknown. The control group was composed of apparently healthy dogs, and therefore subclinical diseases associated with RBC abnormalities cannot be excluded with certainty. Exercise testing was not done. Therefore, exercise intolerance was a spontaneous complaint provided by the owner and could have biased the present result on clinical signs. Additionally, nearly three-fourths of the included dogs with VOTO were receiving 1 or more treatments for heart disease, which may have influenced blood pH and thus RBC shape to an unknown extent.41,42 As blood gases were not assessed in the present study, blood pH of the diseased dogs could not be compared to that of the healthy control dogs, representing another limitation of the present study. Lastly, nearly one-fourth of the included dogs with congenital VOTO also had VSD, which might have contributed to an unknown extent to the RBC fragmentation.

In conclusion, erythrocyte damage (ie, acanthocytosis, schistocytosis, and keratocytosis) is common in dogs with AS and PS, with hemolytic anemia in a few cases with severe or very severe PS. Consequently, IH needs to be considered as the cause of anemia in canine patients with both severe or very severe VOTO and anemia. Further prospective studies are needed to better document these findings and elucidate the physical mechanisms underlying such erythrocyte damage in dogs.

Acknowledgments

The authors declare that there were no conflicts of interest.

The authors sincerely acknowledge the Fondation Un Cœur (foundation under the aegis of the Fondation de France), as well as the Vetoquinol and Zoetis companies for having sponsored the research assistant positions of Drs. Camille Poissonnier and Maria-Paz Alvarado via the foundation at the Alfort Cardiology Unit (UCA). The authors would also like to thank Dr. Cyril Tilmant for his help.

References

  • 1.

    Jacobson RJ, Rath CE, Perloff JK. Intravascular haemolysis and thrombocytopenia in left ventricular outflow obstruction. Br Heart J. 1973;35(8):849854.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Tsuji A, Tanabe M, Onishi K, et al. Intravascular hemolysis in aortic stenosis. Intern Med. 2004;43(10):935938.

  • 3.

    Kawase I, Matsuo T, Sasayama K, Suzuki H, Nishikawa H. Hemolytic anemia with aortic stenosis resolved by urgent aortic valve replacement. Ann Thorac Surg. 2008;86(2):645646.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Sugiura T, Okumiya T, Kubo T, Takeuchi H, Matsumura Y. Evaluation of intravascular hemolysis with erythrocyte creatine in patients with aortic stenosis. Int Heart J. 2016;57(4):430433.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Vahidkhah K, Cordasco D, Abbasi M, et al. Flow-induced damage to blood cells in aortic valve stenosis. Ann Biomed Eng. 2016;44(9):27242736.

  • 6.

    Myhre E, Dale J. Hemolysis in mitral valvular disease and mitral ball valve prostheses. Acta Med Scand. 1971;189(6):547550.

  • 7.

    Febres-Roman PR, Bourg WC, Crone RA, Davis RC Jr, Williams TH. Chronic intravascular hemolysis after aortic valve replacement with Ionescu-Shiley xenograft: comparative study with Bjork-Shiley prosthesis. Am J Cardiol. 1980;46(5):735738.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Magilligan DJ Jr, Fisher E, Alam M. Hemolytic anemia with porcine xenograft aortic and mitral valves. J Thorac Cardiovasc Surg. 1980;79(4):628631.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Chang H, Lin FY, Hung CR, Chu SH. Chronic intravascular hemolysis after valvular surgery. J Formos Med Assoc. 1990;89(10):880886.

  • 10.

    Shim D, Wechsler DS, Lloyd TR, Beekman RH III. Hemolysis following coil embolization of a patent ductus arteriosus. Cathet Cardiovasc Diagn. 1996;39(3):287290.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Ellis JT, Wick TM, Yoganathan AP. Prosthesis-induced hemolysis: mechanisms and quantification of shear stress. J Heart Valve Dis. 1998;7(4):376386.

    • Search Google Scholar
    • Export Citation
  • 12.

    Hijazi ZM. Coil closure of the ductus and hemolysis: strive to achieve complete closure. Cathet Cardiovasc Diagn. 1998;43(1):54. doi: 10.1002/(sici)1097-0304(199801)43:1<54::aid-ccd15>3.0.co;2-t

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Tomita H, Fuse S, Akagi T, et al. Hemolysis complicating coil occlusion of patent ductus arteriosus. Cathet Cardiovasc Diagn. 1998;43(1):5053.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Pedra CA, Pedra SF, Font VF. Hemolysis associated with coil occlusion of the arterial duct. Cardiol Young. 1998;8(1):6366.

  • 15.

    Lee C, Hsieh K, Huang T, Choong C. Spontaneous resolution of hemolysis after partial coil occlusion of ductus arteriosus. Pediatr Cardiol. 1999;20(5):371372.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Uzun O, Veldtman GR, Dickinson DF, Parsons JM, Blackburn ME, Gibbs JL. Hemolysis following implantation of duct occlusion coils. Heart. 1999;81(2):160161.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Birnbaum D, Laczkovics A, Heidt M, et al. Examination of hemolytic potential with the On-X(R) prosthetic heart valve. J Heart Valve Dis. 2000;9(1):142145.

    • Search Google Scholar
    • Export Citation
  • 18.

    Eda K, Ohtsuka S, Seo Y, et al. Conservative treatment of hemolytic complication following coil embolization in two adult cases of patent ductus arteriosus. Jpn Circ J. 2001;65(9):834836.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Pate GE, Al Zubaidi A, Chandavimol M, Thompson CR, Munt BI, Webb JG. Percutaneous closure of prosthetic paravalvular leaks: case series and review. Catheter Cardiovasc Interv. 2006;68(4):528533.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Shapira Y, Vaturi M, Sagie A. Hemolysis associated with prosthetic heart valves: a review. Cardiol Rev. 2009;17(3):121124.

  • 21.

    Shimizu M, Tanaka R, Hirao H, et al. Percutaneous transcatheter coil embolization of a ventricular septal defect in a dog. J Am Vet Med Assoc. 2005;226(1):6972.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Van Israël N, French AT, Wotton PR, Wilson N. Hemolysis associated with patent ductus arteriosus coil embolization in a dog. J Vet Intern Med. 2001;15(2):153156.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Campbell FE, Thomas WP, Miller SJ, Berger D, Kittleson MD. Immediate and late outcomes of transarterial coil occlusion of patent ductus arteriosus in dogs. J Vet Intern Med. 2006;20(1):8396.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Gordon SG, Saunders AB, Achen SE, et al. Transarterial ductal occlusion using the Amplatz Canine Duct Occluder in 40 dogs. J Vet Cardiol. 2010;12(2):8592.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Singh MK, Kittleson MD, Kass PH, Griffiths LG. Occlusion devices and approaches in canine patent ductus arteriosus: comparison of outcomes. J Vet Intern Med. 2012;26(1):8592.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Wang LH, Wang JK, Mullins CE. Eradicating acute hemolysis following transcatheter closure of ductus arteriosus by immediate deployment of a second device. Cathet Cardiovasc Diagn. 1998;43(3):295297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Chetboul V, Athanassiadis N, Concordet D, et al. Observer-dependent variability of quantitative clinical endpoints: the example of canine echocardiography. J Vet Pharmacol Ther. 2004;27(1):4956.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Bussadori C, Amberger C, Le Bobinnec G, Lombard CW. Guidelines for the echocardiographic studies of suspected subaortic and pulmonic stenosis. J Vet Cardiol. 2000;2(2):1522.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Boon JA. Stenotic lesions. In: Boon JA, ed. Manual of Veterinary Echocardiography. 2nd ed. Wiley-Blackwell; 2011:477525.

  • 30.

    Chetboul V, Damoiseaux C, Poissonnier C, et al. Specific features and survival of French Bulldogs with congenital pulmonic stenosis: a prospective cohort study of 66 cases. J Vet Cardiol. 2018;20(6):405414.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Warry E, Bohn A, Emanuelli M, Thamm D, Lana S. Disease distribution in canine patients with acanthocytosis: 123 cases. Vet Clin Pathol. 2013;42(4):465470.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Stockham SL, Scott MA. Erythrocytes. In: Stockham SL, Scott MA, eds. Fundamentals of Veterinary Clinical Pathology. 2nd ed. Blackwell Publishing Professional; 2008:107221.

    • Search Google Scholar
    • Export Citation
  • 33.

    Tidholm A. Retrospective study of congenital heart defects in 151 dogs. J Small Anim Pract. 1997;38(3):9498.

  • 34.

    Oliveira P, Domenech O, Silva J, Vannini S, Bussadori R, Bussadori C. Retrospective review of congenital heart disease in 976 dogs. J Vet Intern Med. 2011;25(3):477483.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Schrope DP. Prevalence of congenital heart disease in 76,301 mixed-breed dogs and 57,025 mixed-breed cats. J Vet Cardiol. 2015;17(3):192202.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Nevaril CG, Lynch EC, Alfrey CP, Hellums JD. Erythrocyte damage and destruction induced by shearing stress. J Lab Clin Med. 1968;71(5):784790.

    • Search Google Scholar
    • Export Citation
  • 37.

    Badylak SF, Van Vleet JF, Herman EH, Ferrans VJ, Myers CE. Poikilocytosis in dogs with chronic doxorubicin toxicosis. Am J Vet Res. 1985;46(2):505508.

    • Search Google Scholar
    • Export Citation
  • 38.

    English RV, Breitschwerdt EB, Grindem CB, Thrall DE, Gainsburg LA. Zollinger-Ellison syndrome and myelofibrosis in a dog. J Am Vet Med Assoc. 1988;192(10):14301434.

    • Search Google Scholar
    • Export Citation
  • 39.

    Weiss DJ, Kristensen A, Papenfuss N. Quantitative evaluation of irregularly spiculated red blood cells in the dog. Vet Clin Pathol. 1993;22(4):117121.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40.

    Mitlyng BL, Chandrashekmhar Y, Furne JK, Levitt MD. Use of breath carbon monoxide to measure the influence of prosthetic heart valves on erythrocyte survival. Am J Cardiol. 2006;97(9):13741376.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Kuzman D, Znidarcic T, Gros M, Vrhovec S, Svetina S, Zeks B. Effect of pH on red blood cell deformability. Pflugers Arch. 2000;440(5 suppl):R193R194.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Gedde MM, Davis DK, Huestis WH. Cytoplasmic pH and human erythrocyte shape. Biophys J. 1997;72(3):12341246.

Contributor Notes

Corresponding author: Dr. Passavin (peggy.passavin@vet-alfort.fr)