Interpretation of Automated Hematology

Transcription

1 Interpretation of Automated Hematology Andrew Loar, DVM, DACVIM (Internal Medicine and Oncology October 13, 2011 Interpretation of Automated Hematology - Omissions, errors and reviews Normal & Not-normal results Instruments: In-House vs Commercial Lab Criteria for slide review Lab tech versus Pathologist Compliance How does primary clinician troubleshoot? Automated Hematology: Instrument limitations Platelets Neutrophils vs bands vs monocytes Unclassified cells Red cell and leukocyte morphology Nucleated red blood cells When, who and how to review slide 1

2 Photos of platelets Normal field for estimate Low numbers in field Big and little clumps Platelets and Automated Hematology NOT IDENTIFIED Presence or absence of clumps Small platelets (particularly feline) Megaplatelets Commercial laboratory platelet comments Examples If count is low - MUST address presence of clumps If count is low WITH clumps - the count isn t low Normal count WITH clumps - were clumps counted What is a manual platelet count Photos of leukocytes Normal field for estimate Neutrophils/bands/myelocytes 2

3 Leukocytes and Automated Hematology NOT identified Bands, myelocytes Counted as segmented neutrophil or monocytes May overcount monocytes Undercounting neutrophils Physiologically significant? May undercount lymphocytes (as neutrophils) How/When to verify Photos of Unclassified Cells Reactive lymphs, lymphoblasts, immature hematopoetic cells & other unidentified blast forms Leukocytes and Automated Hematology Unclassified cells NOT identified Counted as monocytes or lymphocytes Represents: Reactive lymphocytes Immature granulocytes Neoplastic round cells: lymphoma, other leukemias Significant in low proportion Tech Review finding & criteria for Path Review How/When to verify 3

4 Automated Hematology: Instrument Flags Automated Hematology: Instrument Flags Photos of Abnormal Red Cells Hypochromasia, polychromasia, spherocytes, inclusions & reticulocytes 4

5 Cell morphology and Automated Hematology: Red Cells Not identified Hypochromasia, anisocytosis, polychromasia, spherocytosis & inclusions Indices suggest some of these Review of anemic specimens confirms Reflex reticulocyte counts Significance of absence Photos of abnormal leukocytes Toxic changes, left shift (see previous), parasites, Pelger-Huet Cell morphology and Automated Hematology: Leukocytes Toxic changes Left shift Parasites/Inclusions Pelger-Huet syndrome Blast forms 5

6 Photos of NRBCs Examples of NRBCs Nucleated Red Cells (NRBCs) and Automated Hematology NRBCs generally identified as lymphocytes Requires slide review > 10 NRBCs per 100 WBCs requires correction of reported WBC Clinical significance Appropriate response to abrupt anemia Common response to acute hypoxemia Criteria for Slide Review Commercial Lab* PCV < 30% WBC > 20,000 per ul Lymphs > 5,000; Monocytes > 2,000 Platelet count < 150,000 Other parameters Criteria for Path Review In-House instrument recommendations Manual review on all slides 6

7 Feline Herpesvirus Keratoconjunctivitis: Overview and Update Nicole Roybal Veterinary Specialty Hospital of San Diego October 13, 2011 Introduction Virology and Epidemiology Clinical signs Differential Diagnoses Sequelae and associated conditions Diagnostic testing Treatment Virology Feline Herpesvirus type 1 (FHV 1) Highly species specific Double stranded DNA virus Subfamily alphaherpesvirinae Acute cytolytic disease followed by latency 1 Largest subfamily and includes most clinically important diseases Herpes simplex type 1 and 2, Varicella Zoster Virus

8 Virology Targets epithelial cells of upper respiratory tract and conjunctiva Less tropism for corneal epithelium Migrates up sensory nerves FHV 1 establishes latency in trigeminal ganglion Recrudescence Viral reactivation and migration back to epithelial cells resulting in clinical signs 2 Virology Spread via direct, aerosol, fomite transmission Persists for 1 hour in fluorescein and proparacaine solutions but 5 days in saline eye wash. 3 Relatively unstable in environment 18 h in moist conditions, less when dry 1 Susceptible to routine disinfection Epidemiology 97% of cats are seropositive 80% of infected cats develop latency 45% shed virus spontaneously 4 Variably associated with clinical signs 70% shed virus after steroid administration 5

9 Primary Infection Naïve cats, usually kittens 8 12 weeks old as maternal antibodies wane day incubation period 6 Associated with transient viremia 7 Clinical signs more severe vs. recrudescence Fever, malaise, sneezing, nasal and ocular discharge, conjunctivitis Vaccination reduces severity of primary infection Does not prevent latency, reactivation or shedding 6,8 Recrudescent Disease Clinical signs vary in severity Mild serous discharge to severe keratoconjunctivitis Unilateral or bilateral Unilateral cases tend to recur in the same eye 6 Concurrent mild upper respiratory signs uncommon Clinical Signs Most common manifestation is conjunctivitis, keratitis/corneal ulceration is second Targets epithelium = superficial ulceration Cell lysis allows viral spread to adjacent cells Dendritic ulceration seen early Pathognomonic for feline herpesvirus 6 Prominent neutrophil response Purulent discharge even without secondary bacterial infection

10 Differential Diagnoses Chlamydophila felis Upper respiratory component mild to absent Severe chemosis common, no corneal involvement Rule out via cytology Feline Calicivirus Minor conjunctiva pathogen, no corneal involvement 2 Sequelae and Related Conditions Confirmed Symblepharon Stromal keratitis Tear film deficiencies Spastic entropion Unconfirmed Corneal sequestrum Eosinophilic keratitis Symblepharon Conjunctival and corneoconjunctival adhesions Result of severe ulceration and subsequent fibrosis More likely with primary infection Treatment Early lesions can be manually disrupted with topical anesthesia and cotton tipped applicator 6 Mature lesions require surgical intervention if significantly obstructing vision Technically complex and prone to recurrence

11 Stromal Keratitis Uncommon, immune mediated Lymphocytic infiltration of corneal stroma with fibrosis and vascularisation Viral antigen enters stroma during periods of epithelial ulceration and is ineffectually cleared. Can progress to blindness 2, 6 Treatment requires both antiviral and judicious anti inflammatory therapy. 2 Tear Film Deficiencies Qualitative tear film deficiency Inadequate mucin production Mucin promotes smooth, cohesive tear film Normally produced by conjunctival goblet cells Conjunctivitis destroys goblet cells Reduced tear film break up time (TFBUT) Persisted for > 1 month post inoculation 11 Clinical application: mucinomimetic tear replacements can be beneficial for corneal health and comfort Quantitative tear film deficiency Reduced Schirmer Tear Test values Viral destruction of lacrimal gland? Stricture of lacrimal ductules? Reported, uncommon 6 Spastic Entropion Secondary to chronic blepharospasm Most common type of entropion in cats Not specific to herpesvirus Self perpetuating Physical irritation of entropion exacerbates blepharospasm Surgical correction often indicated Conservative approach with analgesia and therapeutic soft contact lens may help

12 Corneal Sequestrum Plaque of necrotic corneal stroma Starts amber, progresses to brown black Composition of pigment uncertain Can progress to full thickness and perforation Not specific to herpes 10 Secondary to chronic superficial ulceration and exposure of anterior stroma Breed predisposition in brachycephalic cats 6 Spontaneous resolution can occur with sufficient vascular response Keratectomy usually required to speed healing and prevent progression Eosinophilic Keratitis Immune mediated, proliferative white pink plaques on corneal and conjunctival surfaces Characteristic cytology Eosinophils, mast cells Possibly herpes related 45/59 (76%) cats PCR (+) vs. 6% of normal cats 12 Unilateral or bilateral Often require long term anti inflammatory (steroids or cyclosporine) with antiviral Diagnostic Testing Results can be difficult to interpret for the individual clinical patient False Positives: Stress/Illness induced viral shedding Subclinical virus harbored in conjunctiva and cornea 50% of normal cats harbor herpesvirus DNA in their corneas 9, 10 Cross reaction with vaccine virus False Negatives: Intermittent viral shedding Inadequate sample collection or handling Reduced test sensitivity 1,2,4,6

13 Diagnostic Testing More informative in research and epidemiology settings Tracking trends in larger populations Presumptive diagnosis often based on signalment, history, clinical signs, response to treatment 1,2,6 Diagnostic Testing Fluorescent Antibody Testing Low sensitivity and specificity, affected by fluorescein stain, limited clinical value 2 Serum Antibody Titers Does not differentiate between vaccine and wildtype virus No difference in seroprevalence between affected and clinically normal cats 12 Diagnostic Testing Virus Isolation Considered gold standard Meticulous sample handling requirements Impractical for clinical settings Lower sensitivity 2 Cytology Non specific: neutrophils and epithelial cells Intranuclear viral inclusion bodies difficult to see Primarily to rule out C. felis

14 Diagnostic Testing Polymerase Chain Reaction Highly sensitive, specific for FHV Possibly too sensitive? Coincidence? Consequence? Cause? 1,2 Sample handling less strict More clinically applicable (and sensitive) than VI 14 Different transit times and temperatures made no difference in ability to identify DNA 15 Treatment Variables to consider Age, immune status, primary vs. recrudescent, level of discomfort 1,2,6,16 Stress of treatment can exacerbate signs Treatment needs vary between patients and within individuals Benign neglect vs. topical antiviral vs. topical and systemic antivirals and antibiotics Antiviral Therapy No veterinary antiviral drugs available Nucleoside Analogues Interfere with viral DNA replication In vitro FHV 1 efficacy studies: Trifluridine >>idoxuridine = ganciclovir>> cidofovir = penciclovir > vidarabine >> acyclovir 17, 18 Complicated metabolism Phosphorylation by virus, host or both to reach active form Systemic treatment often limited by toxicity Virustatic Require frequent application (q1 4h) for optimum efficacy, one week beyond resolution of clinical signs 6

15 Topical Antiviral Medications Trifluridine 1% solution (Viroptic) Only commercially available product evaluated in cats Effective but cost and common topical irritation limit use Idoxuridine 0.1% solution, 2.5% ointment Available through compounding pharmacies Well tolerated, reasonably effective, most common Vidarabine 3% ointment Available through compounding pharmacies Similar clinical efficacy to idoxuridine 1,2,16 Topical Antiviral Medications Ganciclovir 0.15% gel (Virgan) Relatively new for humans, not yet evaluated in cats Cidofovir 0.5% solution Compounded Long half life results in BID dosing Recent study showed improved clinical signs and reduced viral shedding 19 Interferons Cytokines that induce antiviral effects and stimulate immunologic defenses, may be synergistic with antiviral drugs In vitro studies promising 20 but in vivo studies less convincing 21 Systemic Antiviral Medications Acyclovir (Zovirax) Previously the only systemic antiviral evaluated in cats Limited bioavailability and efficacy against FHV 1 Risk of bone marrow suppression Pro drug valacyclovir (Valtrex) better bioavailability but more toxic Fatal liver and kidney necrosis 1,6,16

16 Systemic Antiviral Medications Famcyclovir (Famvir) Complicated non linear pharmacokinetics Optimum dosing schedule not yet determined Wide published range: 62.5 mg/cat q24h 22 to 90 mg/kg PO TID mg PO TID didn t achieve adequate plasma levels for viral inhibition 24 but 90 mg/kg was sufficient 23 Minimal systemic side effects reported L Lysine Amino acid, available OTC Reduces viral replication by competitive inhibition with arginine 500 mg PO BID: reduced clinical signs post inoculation mg PO q24h: reduced viral shedding with latently infection 26 However No effect noted in large population of shelter cats 27 Shelter cats fed diet supplemented with lysine had more severe clinical signs and more frequent viral shedding 28, 29 Bottom line No severe reported side effects or controlled study in client owned cats, may be effective when given in bolus form, safe for long term use in chronically affected cats 16 Conclusions Feline herpesvirus is ubiquitous and a common cause of ocular disease in cats Recognizing typical history, signalment and clinical signs may be more helpful than diagnostic testing Treatment needs vary with each patient Owner education important Recognizing potential for disease recurrence Minimizing stress whenever possible in affected pets

17 Questions? San Marcos: Wednesday Friday Sorrento Valley: Saturday Sources 1. Gould D. Feline herpesvirus 1 ocular manifestations, diagnosis and treatment options. J Feline Medicine and Surgery 2011; 13: Maggs DJ. Update on pathogenesis, diagnosis and treatment of feline herpesvirus type 1. Clin Tech Sm Anim Pract 2005; 20: Storey ES, Gerding PA, Scherba G, et al. Survival of equine herpesvirus 4, feline herpesvirus 1, and feline calicivirus in multidose ophthalmic solutions. Vet Ophthalmol 2002; 5: Maggs DJ, Lappin MR, Reif JS, et al. Evaluation of serologic and viral detection methods for diagnosing feline herpesvirus 1 infection in cats with acute respiratory tract or chronic ocular disease. J Am Vet Med Assoc 1999;214: Gaskell RM, Povey RC. Experimental induction of feline viral rhinotracheitis virus re excretion in FVR recovered cats. Vet Rec 1977; 100: Stiles J, Townsend WM. Feline Ophthalmology. In: Gelatt KN, ed. Veterinary ophthalmology. 4th ed. Ames, Iowa: Blackwell Publishing, 2007; Westermeyer HD, Thomasy SM, Kado Fong H. Assessment of viremia associated with experimental primary feline herpesvirus infection or presumed herpetic recrudescence in cats. Am J Vet Res 2009; 70: Scott FW, Geissinger CM. Long term immunity in cats vaccinated with an inactivated trivalent vaccine. Am J Vet Res 1999; 60: Townsend WM, Stiles J, Guptill Yoran L, et al. Development of a reverse transcriptase polymerase chain reaction assay to detect feline herpesvirus 1 latency associated transcripts in the trigeminal ganglia and corneas of cats that did not have clinical signs of ocular disease. Am J Vet Res 2004; 65: Stiles J, McDermott M, Bigsby D, et al. Use of nested polymerase chain reaction to identify feline herpesvirus in ocular tissue from clinically normal cats and cats with cornea sequestra or conjunctivitis. Am J Vet Res 1997; 58: Sources 11. Lim, CC, Reilly CM, Thomasy SM, et al. Effects of feline herpesvirus type 1 on tear film break up time, Schirmer tear test results, and conjunctival goblet cell density in experimentally infected cats. Am J Vet Res 2009; 70: Nasisse MP, Glover TL, Moore CP, et al. Detection of feline herpesvirus 1 DNA in corneas of cats with eosinophilic keratitis or corneal sequestration. Am J Vet Res 1998: 59; Maggs DJ, Lappin MR, Reif JS, et al. Evaluation of serologic and viral detection methods for diagnosing feline herpesvirus 1 infection in cats with acute respiratory tract or chronic ocular disease. J Am Vet Med Assoc 1999;214: Sykes JE, Browning GF, Anderson G, et al. Differential sensitivity of culture and the polymerase chain reaction for detection of feline herpesvirus 1 in vaccinated and unvaccinated cats. Arch Virol 1997; 142: Clarke HE, Kado Fong H, Maggs J. Effects of temperature and time in transit on polymerase chain reaction detection of feline herpesvirus DNA. J Vet Diagn Invest 2006; 18: Maggs DJ. Antiviral therapy for feline herpesvirus infections. Vet Clin Small Anim 2010; 40: Nasisse MP, Guy JS, Davidson MG, et al. In vitro susceptibility of feline herpesvirus 1 to vidarabine, idoxuridine, trifluridine, acyclovir, or bromovinyldeoxyuridine. Am J Vet Res 1989; 50: Maggs DJ, Clarke HE. In vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type 1. Am J Vet Res 2004; 58: Fontanelle JP, Powell CC, Vier JK. Effect of topical ophthalmic application of cidovir on experimentally induced primary ocular feline herpesvirus 1 infection in cats. Am J Vet Res 2008; 69: Sandmeyer LS, Keller CB, Bienzle D. Effects of interferon alpha on cytopathic changes and titers for feline herpesvirus 1 in primary cultures of feline corneal epithelial cells. Am J Vet Res 2005; 66:

18 Sources 21. Haid C, Kaps S, Gonczi E, et al. Pretreatment with feline interferon omega and the course of subsequent infection with feline herpesvirus in cats. Vet Ophthalmol 2007; 10: Malik R, Lessels NS, Webb S, et al. Treatment of feline herpesvirus 1 associated disease in cats with famciclovir and related drugs. J Feline Med Surg 2009; 11: Evaluation of orally administered famciclovir in cats experimentally infected with feline herpesvirus type 1. Am J Vet Res 2011; 72: Thomasy SM, Maggs DJ, Moulin NK, et al. Pharmacokinetics and safety of penciclovir following oral administration of famciclovir in cats. Am J Vet Res 2007; 68: Stiles J, Townsend WM, Rogers QR, et al. Effect of oral administration of L Lysine on conjunctivitis caused by feline herpesvirus in cats. Am J Vet Res 2002; 63: Maggs DJ, Nasisse MP, Kass PH, et al. Efficacy of oral supplementation with L lysine in cats latently infected with feline herpesvirus. Am J Vet Res 2003; 64: Rees Tim, Lubinski JL. Oral supplementation with L Lysine did not prevent upper respiratory infection in a shelter population of cats. J Feline Med Surg 2008; 10: Maggs DJ, Sykes JE, Clarke HE, et al. Effects of dietary lysine supplementation in cats with enzootic upper respiratory disease. J Feline Med Surg 2007; 9: Drazenovich TL, Fascetti AJ, Westermeyer HD, et al. Effects of dietary lysine supplementation on upper respiratory and ocular disease and detection of infectious organisms in cats within an animal shelter. Am J Vet Res 2009; 70: Image Sources Eosinophilic Keratitis Symblepharon: articles ocular herpes kittens.html Sequestrum Entropion Stiles J, Townsend WM. Feline Ophthalmology. In: Gelatt KN, ed. Veterinary Ophthalmology. 4th ed. Ames, Iowa: Blackwell Publishing, 2007; Dendritic ulcer Stromal Keratitis, Chlamydophila cytology Stiles J, Townsend WM. Feline Ophthalmology. In: Gelatt KN, ed. Veterinary ophthalmology. 4th ed. Ames, Iowa: Blackwell Publishing, 2007;

19 Objectives What s New: A review of recently approved drugs Onsior, Incurin, Trifexis, Propoflo 28 and more! Margo Karriker, PharmD, FSVHP Review new FDA approvals for small animal products Discuss these products place in therapy Review where to find information on these products Robenacoxib Brand name: Onsior Sponsor: Novartis Approval date: March 8, 2011 Release date: 2012 Therapeutic class: Non steroidal anti inflammatory drug (NSAID) Presentation: 6mg, non scored tablets Label indications: Control of postoperative pain and inflammation associated with orthopedic surgery, ovariohysterectomy and castration in cats > 5.5 lbs (2.5 kg) and > 6 months of age; for up to a maximum of 3 days Dosing: 1 mg/kg orally once daily, for a maximum of three days. Preoperatively: Administer dose approximately 30 minutes prior to surgery. May be given with or without food. Tablets are not scored and should not be broken. Place in Therapy: Highly Cox 2 selective NSAID approved for cats Shown to be non inferior to ketoprofen in cats with signs and symptoms of acute pain and inflammation in musculoskeletal disorders Extra label use in dogs has been studied Shown to be non inferior to carprofen in dogs with OA in a 12 week study Healthy cats: 10month old, 2x and 5x for 3 days did not produce toxicity Healthy dogs: 10mg/kg/day for 6 months, no toxicity Estriol Brand name: Incurin Sponsor: Merck Animal Health (Intervet, Inc) Approval Date: July 15, 2011 Release date: 2012 (per Merck Animal Health Technical Services) Therapeutic class: Hormone Presentation: Single scored, 1mg tablets Label indications: For the control of estrogen responsive urinary incontinence in ovariohysterectomized female dogs Dosing:

20 Initial dose of 2mg (2 tabs) orally once daily for a minimum of 14 days. After incontinence is controlled, the lowest effective dose should be determined in a step wise fashion. Dose is not dependant on body weight. Minimum of 7 days between adjustments. Max dose of 2mg per day. User Safety: Women who are of child bearing age or those who are breastfeeding should use caution when administering INCURIN Tablets. Wash your hands with soap and water after administration to avoid exposure to the drug. Place in Therapy: An approved product with a similar efficacy profile to DES Canine approved, commercially available Estrogenic effects seen in 5 9% of dogs at 2mg every 24 hours Spinosad and Milbemycin oxime Brand name: Trifexis Sponsor: Elanco Approval Date: January 4, 2011 Release Date: 2011 Therapeutic class: Antiparasitic Presentation: chewable tablets range of sizes, 6pk Label indications: Prevention of heartworm disease (Dirofilaria immitis); kill fleas; the prevention and treatment of flea infestations (Ctenocephalides felis), and the treatment and control of adult hookworm (Ancylostoma caninum), adult roundworm (Toxocara canis and Toxascaris leonina) and adult whipworm (Trichuris vulpis) infections in dogs and puppies 8 weeks of age or older and 5 pounds of body weight or greater. Place in Therapy: Combination parasiticide; appropriate for year round heartworm prevention Oral option Combination did not cause neurotoxicity in collie dogs when administered above the labeled dose Propofol multi dose (with benzyl alcohol) Brand name: Propoflo 28 Sponsor: Abbott Animal Health Approval date: February 4, 2011 (supplemental approval) Release date: 2011 Therapeutic class: intravenous anesthetic Presentation: 10mg/mL, 20ml multi dose vials, 5 vial pack Label indications: Induction of anesthesia; maintenance of general anesthesia by intermittent bolus injections for short procedures; induction of general anesthesia where maintenance is provided by inhalant anesthetics Place in therapy

21 Multi dose product Safety and efficacy previously established Extra label use in cats Benzyl alcohol content 20mg/mL Toxicity not seen at these levels Orbifloxacin oral suspension Brand name: Orbax Sponsor: Intervet (Merck) Approval date: March 25, 2010 (supplemental approval) Therapeutic class: Antimicrobial quinolone Presentation: 30mg/mL oral suspension, 20mL bottle, 6 pack Label indications: Cats: treatment of skin infections (wounds and abscesses) caused by susceptible strains of Staphylococcus aureus, Escherichia coli, and Pasteurella multocida Dogs: treatment of UTIs in dogs caused by susceptible strains of Staph. pseudintermedius, Proteus mirabilis, E. coli and Enterococcus faecalis skin and soft tissue infections caused by susceptible strains of Staph. pseudintermedius, Staph. aureus, coagulase positive staph., Pasteurella multocida, Proteus mirabilis, Pseudomonas spp., Klebsiella pneumoniae, E. coli, Enterobacter spp., Citrobacter spp., Enterococcus faecalis, Beta hemolytic strep.(group G) and Strep. equisimilis. Place in therapy: Safety and efficacy previously established for oral tablets Only approved oral suspension Improved palatability Discard 30 days after opening, does not require refrigeration Propofol microemulsion Brand name: PropoClear Sponsor: Pfizer Approval date: May 21, 2010 Release date: Not released in US (Approved in 2009 in UK and EU). February 2011 distribution stopped. Therapeutic class: intravenous anesthetic Presentation: 10mg/mL, multi dose, 20mL; 50mL; 100mL vial Label indications: Induction and maintenance of anesthesia and for induction followed by maintenance with an inhalant anesthetic, in cats and dogs. References: Freedom of Information Summaries: > Animal & Veterinary > Products > FOIA Drug Summaries

22 Onsior: King, J N, Hotz, R, Reagan, E L, et al. (2011). Safety of oral robenacoxib in the cat. JVPT, July King, J N, Arnaud, J P, Goldenthal, E I, et al. (2011). Robenacoxib in the dog: target species safety in relation to extent and duration of inhibition of COX 1 and COX 2. Journal of veterinary pharmacology and therapeutics, 34(3), Giraudel, J M, Gruet, P, Alexander, D G, et al. (2010). Evaluation of orally administered robenacoxib versus ketoprofen for treatment of acute pain and inflammation associated with musculoskeletal disorders in cats. American journal of veterinary research, 71(7), Pelligand, L, King, J N, Toutain, P L, et al. (2011). Pharmacokinetic/pharmacodynamic modelling of robenacoxib in a feline tissue cage model of inflammation. JVPT, July Reymond, N, Speranza, C, Gruet, P, et al. (2011). Robenacoxib vs. carprofen for the treatment of canine osteoarthritis; a randomized, noninferiority clinical trial. JVPT, April Gruet, P, Seewald, W, & King, J N. (2011). Evaluation of subcutaneous and oral administration of robenacoxib and meloxicam for the treatment of acute pain and inflammation associated with orthopedic surgery in dogs. American journal of veterinary research, 72(2), Incurin Hamaide, A J, Grand, J, Farnir, F, et al. (2006). Urodynamic and morphologic changes in the lower portion of the urogenital tract after administration of estriol alone and in combination with phenylpropanolamine in sexually intact and spayed female dogs. American journal of veterinary research, 67(5), Mandigers, R J, & Nell, T. (2001). Treatment of bitches with acquired urinary incontinence with oestriol. Veterinary record, 149(25), Trifexis Holmstrom, S D, Totten, M L, Newhall, K B, et al. (2011). Pharmacokinetics of spinosad and milbemycin oxime administered in combination and separately per os to dogs. Journal of veterinary pharmacology and therapeutics, September Snyder, D E, Wiseman, S, Bowman, D, et al. (2011). Assessment of the effectiveness of a combination product of spinosad and milbemycin oxime on the prophylaxis of canine heartworm infection. Veterinary parasitology, 180(3 4), Sherman, J G, Paul, A J, & Firkins, L D. (2010). Evaluation of the safety of spinosad and milbemycin 5 oxime orally administered to Collies with the MDR1 gene mutation. American journal of veterinary research, 71(1), PropoClear

23 Dyer, F. (2011). PropoClear 10 mg/ml emulsion for injection for cats and dogs. Veterinary record, 168(6), 166. Hill, R J, & Williams, C. (2011). PropoClear 10 mg/ml emulsion for injection for cats and dogs. Veterinary record, 168(7), 194.