PFK deficiency in dogs

WHAT IS YOUR DIAGNOSIS? A 5 year, male entire, English Springer spaniel was presented to the R(D)SVS Internal Medicine Service for investigation of intermittent haematuria over the last two months. The dog had been in the owner’s possession for the last three months, having been adopted from a local rescue kennels. Episodes of passing discoloured urine were noted during the 10 days the dog was kennelled. The dog was found from the rescue society as a stray so no prior history was available. During the episode, there was no evidence of pollakiuria or dysuria. The dog had been panting more and had a mild decrease in exercise tolerance. On clinical examination, the dog was bright, alert and response with a body condition score of 4/9. Mucous membranes were moist with mild pallor. The heart rate was 120 bpm with no arrhythmia or pulse deficits. The respiratory rate was 44 breaths/minute, with no adventitious lung sounds. The abdomen was comfortable on palpation. On rectal examination, the prostate gland was intrapelvic, slightly enlarged but symmetrical and not tender. Peripheral lymph nodes were unremarkable and the rectal temperature was 39.0°C. A free catch urine sample was collected and had the appearance shown below. Urinalysis revealed a specific gravity of 1.030. On dipstick analysis there was blood ++++ and pH 7. The rest of the dipstick was unreadable due to discolouration of the urine. page 1 of 5 1) Given the urinalysis results and the above appearance, what are the three main possible causes of this urine discolouration and how would you differentiate them? The red-brown appearance of the urine and a positive for blood on dipstick would be consistent for red blood cells (haematuria), haemoglobin or myoglobin. If red bloods were present they should sediment out on centrifugation and leave a clear yellow supernatant. The red blood cells could also be identified under the microscope on sediment examination. If haemoglobin or myoglobin were present, a centrifuged sample would remain discoloured throughout. If haemoglobin was present, it would be due to marked haemolysis so we would expect anaemia to be present. If myoglobin was present, we might expect to find markedly elevated creatine kinase (CK) and AST. Additionally, if 2.8g of ammonium sulphate is added to 5ml urine and centrifuged, haemoglobin would be expected to precipitate out while for myoglobin the supernatant would remain discoloured. In this case, the urine appearance was due to haemoglobin. To investigate the haemolysis further, blood sample was taken for routine haematology and serum biochemistry (results below). Haematology Parameter Result Reference Range RBC (x 1012/l) 3.32 5.5-8.5 PCV (%) 25 39-55 Hb (g/dl) 8.4 12-18 MCV (fl) 67 60-77 MCHC (%) 34.3 32-36 TWBC (x 109/l) 9.0 6-15 Neutrophils (x 109/l) 7.29 3.6-12 (x 1.26 0.7-4.8 Lymphocytes 109/l) Monocytes (x 109/l) 0.36 0-1.5 Eosinophils (x 109/l) 0.09 0-1 Basophils (x 109/l) 0 0-0.2 Platelets (x 109/l) 471 200-500 Reticulocytes (%) 11.2 0-5 page 2 of 5 Serum biochemistry Parameter Result Reference Range Total protein (g/l) 66.7 58-73 Albumin (g/l) 29.3 26-35 Globulin (g/l) 22 18-37 Bile acids (μmol/l) 4.5 0-7 Total bilirubin (μmol/l) 12 0-6.8 Urea (mmol/l) 5.8 1.7-7.4 Creatinine (μmol/l) 106 40-132 Glucose (mmol/l) 5.1 3-5 AP (iu/l) 60 20-60 ALT (iu/l) 26 21-102 Sodium (mmol/l) 148 139-154 Chloride (mmol/l) 111 99-115 Potassium (mmol/l) 5.1 3.6-5.6 Inorganic (mmol/l) phosphate 1.62 Total calcium (mmol/l) 2.6 0.9-2.0 2.0-3.0 1) Do the above results support the diagnosis of haemolysis? 2) What are your differentials for haemolytic anaemia? 3) What other tests are needed to differentiate the cause of the anaemia? . page 3 of 5 2) Yes, there is good evidence for haemolysis. We have a regenerative anaemia, based on the reticulocytosis (Absolute reticulocytes count= reticulocyte percentage x red blood cell count x 10 = 372 x 109/l where >60 is regenerative). We know a regenerative anaemia is due to haemolysis or haemorrhage. The normal blood proteins and raised bilirubin (due to increased breakdown of haemoglobin) support the diagnosis of haemolysis. 3) Differentials diagnoses for haemolytic anaemia include: a) Immune-mediated (can be primary or secondary to another disease) b) Inherited disorders e.g. phosphofructokinase deficiency, pyruvate kinase deficiency c) Infection e.g. babesiosis d) Chemical induced e.g. onion/garlic toxicity, drugs (e.g. methylene blue, paracetamol in cats, heavy metals (e.g. zinc) e) Metabolic e.g. hypophosphataemia (especially in cats when phosphate <0.5 mmol/l) f) Physical damage (microangiopathic anaemia) e.g. damage by fibrin strands in DIC, damage due to abnormal vasculature such as in haemangiosarcoma 4) Slide agglutination, smear analysis and Coombs test were performed to investigate for immune-mediated haemolytic anaemia (IMHA). There were no spherocytes on smear, and the agglutination and Coombs test were negative making IMHA unlikely. On smear, no schistocytes were present either making microangiopathic anaemia less likely also. There was history of drug or onion ingestion and abdominal radiographs ruled out the presence of a metallic foreign body. Serum phosphate was normal. We did not know the travel history of this dog so infectious disease was still possible. Springer spaniels are predisposed to phosphofructokinase deficiency (PFK). Therefore, a DNA test was performed (can be done using either EDTA blood or buccal swabs). The DNA test confirmed the diagnosis of PFK deficiency. Management In PFK deficiency, haemolytic crises are precipitated due to alkaemia-induced of red blood cells following excessive barking, panting, strenuous exercise and high temperatures. Therefore, the patient was kept calm, cool and rested. On subsequent testing the anaemia resolved but a reticulocytosis remained, indicating that there was ongoing increased red blood cell turnover but that the management regime allowed the dog to compensate. Discussion PFK deficiency is an inherited glycogen storage disease seen in English springer spaniels, American cocker spaniels, whippets, wachtelhunds (German spaniels) and some cross-bred dogs, which causes haemolytic anaemia and an exertional myopathy. It is an autosomal recessive tract which results in muscle cells and erythrocytes not been able to produce page 4 of 5 enough energy for their needs. A hallmark of this disease is intermittent dark coloured urine. Other clinical manifestations are exercise intolerance, lethargy, muscle cramping, anaemia and jaundice. While IMHA is relatively common in dogs, especially in spaniels, consideration should always be given for non-immune mediated causes, such as PFK deficiency. The lack of auto-agglutination, absence of marked spherocytosis and negative Coombs test would steer us away from a diagnosis of immune mediated disease which is important as immunosuppressive therapy would not be appropriate in these cases. If well managed to avoid haemolytic crises, these dogs can lead relatively normal lives, both in terms of quality of life and duration. DNA tests are now available to allow breeders to eradicate the disease from the breed. .References Harvey JW. Pathogenesis, laboratory diagnosis and the clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses. Veterinary Clinical Pathology 2006; 35 (2): 144-156 Hillström A, Tvedten H, Rowe A, Giger U. Hereditary phosphofructokinase deficiency in wachtelhunds. Journal of the American Animal Hospital Association 2011; 47 (2): 145-150 Skibild E, Dahlgaard K, Rajpurohit Y, Smith BF, Giger U. Haemolytic anaemia nad exercise intolerance due to phosphofructokinase deficiency in related springer spaniels. Journal of Small Animal Practice 2001; 42: 298-300 The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336. www.ed.ac.uk/vet/hfsa-int-med page 5 of 5