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Equine vet. J . (1990) 22 (4) 292-294

Case Reports A case of primary autoimmune haemolytic anaemia in a pony D. J. BECK 1297 0 Wild Acres Road, Largo, Florida 34643- 1518, USA.

Introduction AUTOIMMUNE haemolytic anaemia (AIHA) is an uncommon disease of the adult horse (Warner and Moms 1987). The pathophysiology of the condition is hypothesised to be an aberrant production of antibodies targeted at the surface antigens of the red blood cell. Antibody production is thought to occur in one of two ways: (1) the erythrocyte membrane is altered by systemic bacterial or viral infections or neoplastic disorders and antibody production is directed at this perceived antigen: (2) immunocompetent clones develop and direct their products at the red cell membrane. Antibodies may be agglutinating or non-agglutinating and are also temperature dependent with some showing optimum activity at body temperature and others at varying degrees below. They are generally of the IgM or IgG classes, and complement components may be found on the red cell surface. In autoagglutinating AIHA, the diagnosis can be made microscopically, taking care to distinguish agglutination from rouleau formation by diluting the suspension with isotonic saline. The Coombs, an antiglobulin test, is used to confirm the diagnosis in cases of non-agglutinating AIHA. An effective Coombs reagent should contain antisera to IgC, IgM and C3, the third component of complement. Equine infectious anaemia (EIA) is commonly associated with immune-mediated haemolytic anaemia. However, at least 10 cases of AIHA in horses not suffering from EIA have been reported (Schalm and Carkon 1982). Farrelly, Collins and Collins (1966) first reported AIHA in a horse with lymphosarcoma. Since then three more cases have been reported (Reef, Dyson and Beech 1984). Clostridium perfirfringes infections have been reported in association with AIHA in at least two cases (Reef 1983; Weiser, Kohn and Vachon 1983). An infectious agent was suspected in another case of AIHA in a pony (Sutton et a1 1978). Primary or idiopathic AIHA has been reported less commonly. In one documented case the horse responded to continued treatment with glucocorticoids (Anderson 1974). This paper reports primary AIHA in a pony that responded to a 10 week regimen of glucocorticoids after diagnosis was confirmed by a 4+ positive direct 37°C Coombs test. Six months after therapy was discontinued the pony was Coombs negative and asymptomatic.

seven years of ownership, and no drugs had been administered within two months prior to the illness.

Clinical findings Physical examination The patient's temperature was 40.2"C, the heart rate was 60/min and the respiratory rate was 24/min. Mucous membranes were pale to slightly icteric and the capillary refill time was 1 sec. The pony weighed about 180 kg and appeared well fed. Auscultation of the thorax and abdomen revealed no abnormalities. A slight serous discharge from the eyes and nares was noticed, along with slightly enlarged prescapular lymph nodes bilaterally. Clinical pathology A sample for a white blood cell count and packed cell volume (PCV) was taken for analysis at a local veterinary hospital. Blood glucose and blood urea were estimated at 4.4 mmoVlitre and 7.1 mmolflitre respectively, using appropriate labsticks (Chemstrip bG, Boehringer Mannheim Diagnostics Inc; Azostix, Miles Laboratories InC.).

Treatment Fever of unknown origin (FUO) explained all symptoms at this point and non-specific treatment for fever was instituted with methapyrone (Dipyrone 50 per cent Injection,Quality Plus Products Co., Iowa, USA) (22 mgkg bodyweight [bwt]) and sponge baths. The white blood cell count (Table 1) indicated a moderate leucocytosis with a mature neutrophilia (Schalm and Carlson 1982). The PCV was 0.18 litresflitre. No other erythron parameters were measured. A focus of infection was suspected based on test results and clinical signs. Trimethoprim-sulphamethoxazole (Rugby trimeth/sulfa D/S tablets, Chelsea Laboratories, New York) was administered with an initial dose of 30 m@g bwt via nasogastric tube in 5 litres of water. The antimicrobial therapy was continued at 15 mgkg bwt orally twice a day. The following morning the pony's temperature was

Case history

TABLE 1 : Differential white cell counts of a pony with primary AIHA

A 21-year-old grade pony gelding was presented after a one week episode of partial anorexia, lethargy, depression and weight loss. The pony had latterly progressed to complete anorexia. The annual Coggins test was negative. The pony had been vaccinated one year previously for eastern and westem equine encephalomyelitis, influenza and tetanus. The pony was de-wormed with ivermectin two months prior to presentation. According to the owner, the pony had never experienced any type of illness before this episode in

Day

WBC total Neut ("/)'

109/1 1

7 8

18.2 14.3 18.3

10911

Lymph (Yo) Mono ("A)

10911

15.8(87) 1.0(6) 12.7(89) 0.6(4) 15.4(84) 2.2(12)

Eos (O/.)

1 0911

10911

0.5(3) 0.1 (1) 0.7(3)

0.7(4) 0.9 (6) 0.2(1)

WBC: White blood cell count; Neut: neutrophils; Lymph: lymphocytes; Mono: rnonocytes; Eos: eosinophils

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38.6'C and for three consecutive days it was normothermic for May in central Florida, supporting the clinical impression of antimicrobial responsive fever of unknown origin. On Day 7 (5 days after initial antimicrobial therapy) the pony was markedly icteric and anorectic. Its temperature was 38.4"C. The PCV had dropped from 0.18 to 0.10 litresflitre. The blood smear revealed noticeable anisocytosis and macrocytosis. The plasma was icteric in the EDTA tube on collection. The erythrocytes began to lyse in the EDTA tube within 1 h. The total protein was 90 flitre, the total white cell count was 14.3 x l@flitre (Table I , Day 7) and a faecal flotation was negative for endoparasite ova. A tentative diagnosis of AMA was made; supporting evidence included no signs of blood loss, absence of Heinz bodies, no obvious intravascular haemolysis, cells lysing in vitro and elevated total protein. Urine had a normal appearance. Paracentesis was not performed to rule out haemoperitoneum or haemothorax because total protein was increased rather than decreased (Clark 1987) and the patient was icteric suggesting erythrocyte destruction rather than recompartmentalisation. A blood urea within normal range did not indicate hypovolaemia. In Florida, equine babesiosis (piroplasmosis) is considered eradicated and was deemed an unlikely aetiology. The tentative diagnosis did not distinguish between primary and secondary AIHA. The owner was informed of the possibility of AIHA secondary to equine infectious anaemia or neoplasia and the possible side effects of glucocorticoid therapy. The antimicrobials were discontinued and dexamethasone (Bums Veterinary supply Dexamethasone sodium phosphate, Anthony Products, California) therapy initiated (28 mg intravenously [iv]). The following morning (Day 8) the pony's temperature was 3 7 . W and the PCV was still 0.10 litresflitre.

Blood samples were taken for white cell count, Coombs test and Coggins test, after which 24 mg dexamethasone were administered. In the afternoon the pony received another 24 mg of iv dexamethasone and was transfused with erythrocytes from 1.8 litres of whole blood separated by sedimentation, about 650 ml of packed erythrocytes. PCV after the transfusion was still 0.10 litresflitre. Laboratory results (Table 2) were received the following day. The patient had a 4+ direct 37°C Coombs test (range is negative to A), a negative Coggins test, decreased platelet count and a white blood cell count of 18.3 x logflitre (Table l/day 8). Total bilirubin was 68.4 pmolflitre with high indirect bilirubin at 63.3 pmolflitre supporting the assumption of extravascular haemolysis. Total globulins were elevated at 56 flitre. Dexamethasone dosage was increased to 32 mg iv twice on the day the test results were received (Day 9), and reduced to 28 mg iv twice on Day 10. There was significant clinical improvement on Day 11 and the dose was reduced to 24 mg iv dexamethasone, twice daily, on alternate days for seven days. Therapy was then changed to 600 mg oral prednisone (Rugby prednisone U. S. P.50 mg, Heather Drug Co. Inc. New Jersey) every other day for two weeks. A second direct 37°C Coombs test was performed on Day 33 (25 days after the initial test) and the results were still +4. However, PCV was 0.20 litresflitre and total protein 70 flitre. After two weeks, a declining dosage of alternate day therapy was instituted at 300 mg, 150 mg, and then 50 mg over the ensuing 6 weeks. The patient improved clinically over the 10 week course of therapy. When therapy was discontinued PCV was 0.27 litresfiitre and total protein was 65flitre. Six months later the patient was Coombs negative, The PCV increased steadily and 1 year after therapy ended the PCV was 0.38 litresflitreand the pony appeared to be in good health.

TABLE 2: Commerclal laboretory test results of a pony with primary AIHA

Discussion

Test procedure

Hi/lo

Result

Normal range

Red blood cell parameters: RBC Lo MCV Hi McHc Lo McH Hi Lo Haemoglobin PCV Lo Serum chemistries: AST ALT Total bilirubin Hi Direct bilirubin Indirect bilirubin Hi Urea Creatinine Hi Cholesterol Alkaline phosphatase Hi Glucose Hi Phosphate Calcium Total protein Albumin Globulin Hi Sodium Potassium Chloride Hi CK Gamma G.T. Hi

1.07 101211 64 fl 15.9 mmol/l 13.0 mmol/l 2.2 g/dl 0.082 1/1

6.5-1 2.5 32-52 19.2-22.9 7.6-12.2 11.0-19.0 0.24-0.44

287 iu/l 18 iu/l 68.4 pmol/l 5.1 pmol/l 63.3 pmol/l 7.8 mmol/l 176.8 p o l / l 2.7 mmol/l 357 iu/l 8.2 mmol/l 0.65 mmol/l 2.8 mmolil 82 g/l 26 g/l 56 g/l 136 mmol/l 3.5 mmol/l 110 mmolll 66 iu/l 46 iu/l

184-566 5-30 1.71-34.2 0.0-8.6 0-37.6 3.6-8.9 106.1-168.0 1.9-3.9 80-216 3.8-6.8 0.6-1.8 2.5-3.5 57-84 23-38 16-50 130-146 2.5-5.4 93-109 50-350 3-30

Serology: Coombs direct (37'C) 4t Coggins

Positive n-4t Negative +/-

negative negative

Test results from Day 8. 28 mg dexamethasone were administered iv 12 h before blood samples were collected for tests. Comments: Atypical lymphocytes present; Anisocytosis 1+; Hypochromasia 1+; platelet estimation: decreased

The major problems on presentation were anorexia, pyrexia, icterus, anaemia, moderate leucocytosis and lymphadenopathy. The set of symptoms was categorised as fever of unknown origin (FUO). The aetiology of fever is presented by Blood, Radostits and Henderson (1983). A focus of infection could not be determined from physical examination. A blood glucose of 4.4 mmolflitre did not support endotoxaemia (Semrad and Moore 1987). A normal blood urea and aspartate amino transferase (AST) with only a mild increase in gamma glutamyl transferase (GGT) discounted hepatic disease (Divers 1987). A viral infection such as equine influenza was considered because of the fever of 40.2'C (Liu 1983) but clinical signs were lacking. Chronic inflammatory disease such as abdominal abscessation was suspected and could account for the clinical signs. Anorexia would account for the icterus and the anaemia of chronic disease would explain the PCV of 0.18 litresflitre. The initial response to antimicrobial therapy was indeed supportive of such a condition. However, on the fifth day after antimicrobial therapy was instituted, the patient deteriorated. PCV dropped to 0.10 litresnitre and the pony was markedly icteric, supportive of extravascular haemolysis. A blood smear revealed macrocytosis and anisocytosis. In equids, a single reticulocyte or polychromatophilic erythrocyte in peripheral blood in anaemia in remission is extremely rare (Schalm, Jain and Carroll 1975). Equine red cell indices lack the sensitivity of those of other species, although a progressively increasing mean corpuscular volume (often remaining within the normal range) can be observed with regenerative anaemia (Bloom and Roby 1987).Although a bone marrow aspirate examination was not performed, the anaemia was categorised as regenerative based on macrocytosis (MCV 64 fl), anisocytosis and hypochromasia observed in the peripheral blood in this case (Weiser ef a1 1983; Easley 1985). Oxidant induced haemolytic anaemia was deemed unlikely because there was no historical ingestion of Acer ruhrum leaves, wild onion or phenothiazine. Sulpha drug induced oxidative

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haemolytic disease could not be substantiated with the presence of Heinz bodies on the blood smear (Bloom and Roby 1987). An intravascular haemolytic syndrome associated with liver failure (Schalm and Carlson 1982) could not be substantiated biochemically and the serum was icteric and not haemoglobinaemic. The aetiology of the AIHA was initially considered to be either equine infectious anemia (EIA), lymphosarcoma or idiopathic. The primary differential, EIA, was discounted because of a negative Coggins, and leucocytosis with a neutrophilia is not consistent with EIA (Warner and Moms 1987). Lymphosarcoma has been associated with AIHA in horses (Reef et a1 1984). Prescapular lymphadenopathy was present but no other palpable lymph nodes were affected and clinical presentation did not indicate the condition. However, lymphosarcoma could not be ruled out on these clinical observations. The treatment for primary A M (Warner and Moms 1987) and lymphosarcoma (Sweeney 1987) in the horse is glucocorticoids. Published dosages (Carlson and Zinkl 1983) of dexamethasone were used initially. The dosages were then titrated in response to clinical signs and PCV. Due to life threatening anaemia and persistent icterus, glucocorticoid dosage was increased to prevent further clearance of opsonic erthrocytes by the reticuloendothelial system (Frank, Schreiber, Atkinson and Joffe 1977). Clinical improvement occurred on Day 10 and, when oral prednisone therapy began on Day 17, the PCV was 0.14 litresflitre. A transfusion of unmatched red cells was performed because of the severe anaemia. The patient had never received a transfusion before and was on immunosuppressive therapy, which made the possibility of a transfusion reaction unlikely (Becht and Gordon 1987). Spherocyte formation in immune mediated haemolytic disease is mediated by loss of membrane coated with autoantibodies; these cells have increased osmotic fragility (Reef et a1 1984). The blood samples taken from this pony would begin to haemolyse in a short time in an EDTA tube. Spherocytes or erythrophagocytosis were not seen on blood smears. The osmotic fragility test was considered unnecessary because any break in the red cell membrane greater than 64 angstroms will allow direct escape of haemoglobin without osmotic swelling. Red cells injured by the action of complement and antibody may haemolyse in this manner (Jandl 1966). The difference in PCV between the commercial laboratory (0.08 litresflitre) and hospital tests (0.10 litresflitre) may be explained partially by the aforementioned observation because the hospital measured PCV almost immediately whereas samples had to be transported to the commercial laboratory. Erythrophagocytosis, if observed, would have helped support a tentative diagnosis of AIHA. The final diagnosis of primary AIHA was based on two 4+ direct 37'C Coombs test 25 days apart and the lack of any evidence of a concomitant disease process. The pony responded to a 10 week course of glucocorticoid therapy and was Coombs negative six months after diagnosis was made, with a PCV of 0.32 litresflitre and a total protein of 67 flitre. The pony was clinically normal one year after therapy ended. Both immunological laboratories (Veterinary Reference Laboratories, Texas and College of Veterinary Medicine, University of Florida) utilised similar protocols with equine specific Coombs reagents. This is the first reported case of a primary AIHA to go into remission. Anderson (1974) reported a case in the horse but it required continuous glucocorticoid therapy to remain asymptomatic and had a mildly positive Coombs test five months after diagnosis. The clinical course of the present case was similar to a suspected case of AIHA in a pony which became clinically normal with glucocorticoid therapy but the diagnosis was never confirmed with a positive CoOmbs test (Schalm and Carlson 1982). Other recent cases of AIHA reported in conjunction with neoplasia or infectious agents died or were destroyed (Reef 1983; Reef et a/ 1984; Weiser et a1

1983; Sutton et a1 1978). The aetiology of idiopathic AIHA is unknown. In this case, the financial resources were not available to perform exhaustive testing of every organ system to establish an idiopathic diagnosis. However, a negative direct 37°C Coombs, and the continued good health one year after therapy, supports the diagnosis. The remission in this case indicates that AIHA is a treatable disease and there were no untoward side effects of glucocorticoids. However, side effects of glucocorticoid therapy can include laminitis, diarrhoea and gastroduodenal ulceration especially in ponies which are predisposed to the former (Lowe 1983).

Acknowledgements The author thanks Dr Dennis Meyer, for consultation in the case, and Dr Richard Halliwell, who performed the final Coombs test and provided technical assistance.

References Anderson, L. J. (1974)Idiopathic and autc-immune haemolytic anaemia in a horse N 2 i'er .I 22, 102-105.

Becht. J. L.and Gordon, B. J. (1987)Blood and plasma therapy. In: Currenr Theropy in Equine Medicine. 2. Ed: N. E. Robinson, W. B. Saunden. Philadelphia. pp 3 17-322. Blood. D. C.. Radostits, 0. M. and Henderson. J. A. f 19x3)General systemic states. In: Vererinaiy Medicine a Tenthook i f the Diseases i f (brrte, Sheep. P i p . Cimrs and Horses. 6th edn. Bailliere Tindall. London. pp 40-41. Bloom, J. C. and Roby, K. A. W. (1987)Evaluation of the erythron. In: Currenr Therapy in Equine Medicine. 2. Ed: N. E. Robinson, W. B. Saunders. Philadelphia.

pp291-294. Carlson. G. P and Zinkl. J. A. (1983) Hemolytic anemia. In: Current Therapy in Equine Medicine. Ed: N. E. Robinson, W. B. Saunders, Philadelphia. pp 299-303. Clark, E. S. (1987)B l d loss anemia. Currenr Therapy in Equine Medicine. 2. Ed: N. E. Robinson, W. B. Saunders, Philadelphia. pp 300-303. Divers, T. J. (1987)Acute hepatic failure (Theiler's disease)Currenr Therapy in Equine Medicine. 2. Ed: N. E. Robinson. W.B. Saunders. Philadelphia. pp 110-112. Farrelly. B. T., Collins, J. D. and Collins. S. M. (1966) Autoimmune haemolytic anaemia in the horse. Irish wr. J. 20,42-45. Easley. J. R. (1985) Erythrogram and red cell distribution width of equidae with experimentally induced anemia. Am. J. vet Res. 46,2378-2384. Frank. M. M., Schreiber. A. D.. Atkinson, M. D. and Joffe. C. 1. (1977) Pathophysiology of immune hemolytic anemia Ann. inr. Med. 87,2 10.222. Jandl, J. H.(1966)The pathophysiology of hemolytic anemia Am. J. Med. 41,657-665. Liu, I. K. (1983)Fquine influenza. In: Currenr Therapy in Equine Medicine. Ed: N. E. Robinson, W. B. Saunders, Philadelphia. pp 3-4. Lowe. J. E. (1983) Founder. Currenr Therapy in Equine Medicine. 2 . Ed: N . E. Robinson, W. B. Saunders, Philadelphia. pp 104-106. Reef. V. B. (1983)Closrridium perfkingens cellulitis and immune-mediated hemolytic anemia in a horse. J. Ani. ref. nred. Ass. 182,251-254. Reef, V. B.. Dyson. S. S. and Beech. J. (1984)Lymphosarcoma and associated immunemediated hemolytic anemia and thrombocytopenia in horses. J. Am. wr. med. Ass.

ins,313-317. Schalm, 0.W. and Carlson, G. P. (1982)The blood and blood forming organs. Equine Medicine ond Surgeiy. Vol 1 3rd Edn. Eds: R. A. Mansmann, E. S . McAllister and E. S . Pratt, American Veterinary Publications. Santa Barbara. pp 377-414. Schalm, 0. W., Jain, N. C. and Carroll. E.J. (1975)Normal values in blood morphology with comments on species characteristics in response tn disease. Vererinary Hemurology 3rd edn. Lea and Febiger, Philadelphia p180. Semrad, S. D. and Moore, J. N. (1987) Endotoxemia. Cicrrenr Therapy in Equine Medic.ine. 2. Ed: N. E. Robinson, W. B. Saunders, Philadelphia. pp 84. Suton. R. H.,Pearce, H. G., Kelly, C. M., Alley, M. R. and Falconer, G . (1978)Autoimmune haemolytic anaemia in a horse N.Z. ver. J. 26,3 I1-313. Sweeney, R. W. (1987) Lymphoproliferative and myeloproliferative disorders. In: Current Tho-apy in Equine Medicine. 2. Ed: N. E. Robinson, W. B. Saunden. Philadelphia. pp 3 14-317. Warner, A. and Moms, D. D. (1987) Hemolytic anemias. In: Current Therapy in Equine Medicine. 2. Ed: N. E. Robinson. W. B. Saunders, Philadelphia. pp 295300. Weiser. G.. Kohn. C. and Vachon. A. ( 1983) Erythrocyte volume distribution analysis and hematologic changes in two horses with immune-mediated hemolytic anemia. Vet. Parhol. 20.424-433.

Received for publication: 9.2.88 Accepted: 7.12.89

A case of primary autoimmune haemolytic anaemia in a pony.

EQUINE VETERINARY JOURNAL 292 Equine vet. J . (1990) 22 (4) 292-294 Case Reports A case of primary autoimmune haemolytic anaemia in a pony D. J. BE...
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