Haematopoietic and immune systems
10.1 Diagnostic approach to haematopoietic and immune system diseases
10.2 Diagnostic features of common haematopoietic and immune disorders of horses
10.3 Anaemia secondary to haemorrhage
10.4 Immune-mediated haemolytic anaemia
10.6 Heinz body haemolytic anaemia
10.7 Equine infectious anaemia
10.8 Equine piroplasmosis (babesiosis)
10.9 Anaemia due to rickettsial disease
10.11 Anaemia of chronic disease
10.12 Generalized bone marrow suppression or failure
10.13 Immune-mediated thrombocytopenia
10.14 Disseminated intravascular coagulation
10.16 Hereditary defects of haemostasis
10.20 Severe combined immunodeficiency (SCID)
10.22 Selective IgM deficiency
10.23 Transient hypogammaglobulinaemia
10.24 Failure of passive transfer
10.25 Neonatal isoerythrolysis
10.27 Lymphoma (lymphosarcoma)
10.29 Sporadic lymphangitis (‘monday morning leg’; ‘monday morning disease’)
10.1 Diagnostic approach to haematopoietic and immune system diseases
Primary disorders of the haematopoietic and immune system are relatively rare in the horse. Alterations in peripheral blood parameters are more commonly responses to primary disease in other body systems or reflect local or systemic infection. Clinical signs associated with abnormalities of these systems may therefore be varied and complex. Clinicopathological evaluation of the blood is usually included in the diagnostic plan for investigation of many abnormalities identified on physical examination. Further evaluation of the haematopoietic and immune systems themselves may require specific diagnostic investigation, including ultrasonography, cytological evaluation of effusions and histopathological evaluation of lymph node, tissue or bone marrow biopsies. A detailed history is vital to reach probable differential diagnoses, as is a thorough physical examination.
• Age – congenital abnormalities of the haematopoietic and immune system are most likely to become apparent in horses <1 year of age. Most congenital immunosuppressive disorders present before 3 months.
• Breed – congenital abnormalities are also more likely in purebred horses, although they can occur in mixed-breed animals.
• Specific questioning should be directed towards establishing: Presence of exercise intolerance? Change in demeanour, appetite or resting respiratory rate? Extent and rate of any weight loss. History of epistaxis, bleeding episodes or discolored urine? Appearance of limb swelling or unexplained masses?
• In-contact horses – a history of similar clinical signs in other horses may suggest an infectious or toxic aetiology. A history of Streptococcus equi infection on the premises might suggest purpura haemorrhagica in a horse with vasculitis.
• Season – red maple leaf toxicity is more common in the late summer and autumn.
• Environment – fallen trees or poor pasture management might predispose to ingestion of toxic plants. Tick exposure is required for haemolytic anaemia due to piroplasmosis.
• Routine herd health records – weight loss, ventral oedema, and unexplained recurrent fever in an untested horse might indicate equine infectious anaemia. Recent vaccination against Streptococcus equi might suggest a diagnosis of purpura haemorrhagica in a horse with vasculitis.
• Previous illnesses – repeated incidences of infectious diseases may be suggestive of an immune dysfunction. Cryptosporidium spp., adenovirus infections, Candida spp. and Pneumocystis carinii infections have been diagnosed in horses with immunological disease.
Physical examination: A complete physical examination is required to identify all clinical abnormalities. Specific diagnosis of a primary (rather than secondary) haematopoietic and/or immunological disease is then likely to require further diagnostic investigation.
Mucous membranes: Change in mucous membrane colour is key to detection of disease and all available surfaces should be examined, including scleral colour. Pale mucous membranes accompanied by tachycardia, tachypnoea, and a systolic heart murmur are highly suggestive of severe anaemia. However, marked anaemia is required before changes in mucous membrane colour are readily detected. In contrast, congested mucous membranes with prolonged capillary refill time are consistent with polycythaemia. Petechial or ecchymotic haemorrhages occur with thrombocytopenia or vasculitis. Detection of petechiation in the presence of endotoxaemia may be the first indication of impending disseminated intravascular coagulation (DIC). Icteric sclera may be seen in haemolytic anaemia, but will occur also with prolonged inappetance.
Haemorrhage: Often the source of blood loss is evident and a result of trauma. Intrathoracic haemorrhage (fractured rib/intercostal artery laceration) or intra-abdominal bleeding (splenic rupture, mesenteric tear) may require cytological evaluation of pleural or peritoneal fluid respectively or ultrasound examination. Prolonged bleeding from venipuncture sites, petechial or ecchymotic haemorrhages, or melaena occur with severe thrombocytopenia (usually <20 × 109/L). Haematoma formation following relatively minor trauma, or internal haemorrhage into the thorax or abdomen may be indicative of coagulation factor abnormalities or severe DIC.
Urinalysis is important. Whole red cells may be detected in several different conditions of the urinary tract, including idiopathic renal haematuria, or haemoglobinuria in cases of haemolytic anaemia.
Palpation: Jugular veins should be palpated for patency, thickening, heat or painful foci. Jugular vein thrombosis associated with venipuncture is common in horses with endotoxemia or other conditions that cause blood to be hypercoaguable. Thrombosis may occur secondary to DIC, repeated or unskilled venipuncture or infection.
Pitting oedema of the distal extremities, ventral abdomen or head may be an indication of hypoalbuminaemia, lymph obstruction or stasis, congestive heart failure (rare), vasculitis or lymphangitis. Oedematous swelling that is hot and painful on palpation is likely to result from vasculitis, lymphangitis or cellulitis. Ventral thoracic and sternal oedema may be caused by pleural effusions, often with increased filling of the jugular veins. Similarly, ventral abdominal oedema or hind limb oedema may occur secondary to lymphatic obstruction resulting from ascites or enlarged abdominal lymph nodes. Rectal examination and abdominal ultrasound should therefore be included as part of the diagnostic investigation.
Submandibular and prescapular lymph nodes should be palpated for size, consistency, and pain. Single lymph nodes may be enlarged because of abscessation or in reaction to localized inflammatory processes. Multicentric lymphoma may cause generalized lymphadenopathy but enlargement of superficial lymph nodes due to lymphoma is relatively rare.
Peripheral blood evaluation: A complete blood count (CBC) can give invaluable information about the erythron (red cell mass), the leukon (white cell mass) and platelets. Blood samples should be collected from any patent superficial vein when the horse has been calm for at least 1 hour. Catecholamine-induced contracture of the spleen in response to excitement causes a significant increase in red cell numbers. Catecholamine release also causes physiologic leukocytosis with increased neutrophil and lymphocyte counts. Similarly, samples should not be taken after ingestion of a large concentrate meal, as this will reduce circulating plasma volume. By contrast, sedatives such as acepromazine and xylazine cause splenic relaxation and relative anaemia such that blood samples should always be taken prior to sedation.
Erythrocytes: The RBC count, haemoglobin, haematocrit and packed cell volume (PCV) are the most frequently measured parameters of erythrocyte quantitation. Haemoglobin concentrations should be approximately one-third of the PCV. This will be increased with intravascular haemolysis. Anaemia is a decrease in the circulating red blood cell (RBC) mass (decreased RBC count, haemoglobin and packed cell volume) as a result of decreased rate of erythrocyte production or increased rate of erythrocyte destruction or loss. Polycythaemia is an increase in the circulating RBC mass. Red blood cell indices can be used to characterize anaemia:
• Mean corpuscular volume (MCV) – sometimes increased in horses with regenerative anaemia; decreased with iron deficiency anaemia.
• Mean corpuscular haemoglobin (MCH) – increased with intravascular haemolysis; decreased with iron deficiency anaemia.
• Mean corpuscular haemoglobin concentration (MCHC) – increased with intravascular haemolysis; decreased with iron deficiency anaemia.
• Red cell distribution width (RDW) – may be increased in regenerative anaemia, reflecting anisocytosis.
The appearance of the erythrocytes should be evaluated on a stained blood smear. Examples of abnormal RBC morphology and its interpretation include:
• Poikilocyte – any abnormally shaped erythrocyte.
• Anisocytosis – varying sizes of erythrocytes are observed.
• Polychromasia – varying colours of erythrocytes are observed, usually due to variable haemoglobin and RNA content.
• Spherocyte – spherical erythrocytes occasionally observed in horses with immune-mediated anaemia.
• Howell–Jolly bodies – basophilic nuclear remnants occasionally observed in the cytoplasm of erythrocytes of normal horses.
• Heinz bodies – oxidized precipitated haemoglobin observed as round structures extending from the edge of the RBC membrane. Indicative of oxidative damage to the RBC with subsequent intravascular or extravascular haemolysis.
Horse erythrocytes show prominent rouleaux formation on smears, not to be confused with agglutination. Reticulocytes do not appear in the peripheral circulation. Assessment of regenerative response in anaemia is therefore more dependent on measurement of individual red cell parameters as above, collection of sequential blood samples or evaluation of bone marrow biopsies or aspirates. Erythropoiesis in anaemia is slower than in other species, taking 10 days to reach the maximum rate after haemorrhage. Cell life span is 140–155 days.
A reference range for haematology values in the adult horse is given in Table 10.1.
Table 10.1
Reference range for haematology values in the adult horse
Haematology value | Reference range |
Red cell count (RBC), × 1012/L | 6.5–11.6 |
Packed cell volume (PCV), L/L | 0.30–0.48 (30–48%) |
Haemoglobin, g/dL | 11.3–17.9 |
Mean corpuscular volume (MCV), fl | 39–49 |
Mean cell haemoglobin (MCH), pg | 12.3–17.0 |
Mean corpuscular haemoglobin concentration (MCHC), g/L | 31.0–38.6 |
Red cell distribution width (RDW), % | <20.0 |
Leukocytes (WBC), × 109/L | 5.3–11.0 |
Neutrophils, × 109/L | 2.1–6.0 |
Band neutrophils, × 109/L | 0.0–0.2 |
Lymphocytes, × 109/L | 1.7–5.4 |
Neutrophil : lymphocyte ratio | 1.5 : 1.0 |
Monocytes, × 109/L | 0.0–0.7 |
Eosinophils, × 109/L | 0.0–0.8 |
Basophils, × 109/L | 0.0–0.1 |
Platelets, × 109/L | 100–350 |
‘Hot-blooded’ horses such as Thoroughbreds and Arabians have a greater red cell count and haemoglobin content than ‘cold-blooded’ draught breeds, with pony and donkey values lying between the two. Red cell variables are also affected by age. Neonatal PCV is initially high, returning to adult levels over the course of the first week. Foals have microcytosis and increasing MCV until 3 years old, with variable hypochromasia and anisocytosis. In pregnant mares, PCV, haemoglobin and RBC all increase slightly towards the end of gestation, and then decrease during lactation.
Leukocytes: Both total WBC count and differential WBC counts should be assessed.
• Neutropenia – decreased neutrophil count – most often a result of a shift of neutrophils from the circulating to the marginating pools due to excessive tissue demand. This may occur with endotoxaemia, septicaemia, and a wide variety of acute bacterial, rickettsial or viral disorders. In endotoxaemia the neutrophils develop basophilic cytoplasm, with granulation, vacuolation and presence of dark-staining Dohle inclusion bodies (‘toxic neutrophils’). Chronic neutropenia may occur with severe infectious or inflammatory diseases or bone marrow suppression.
• Band neutrophils – are immature neutrophils released prematurely into the circulation as a response to high tissue demand (‘left shift’). A degenerative left shift occurs when the number of immature cells exceeds the number of mature neutrophils because of an inability of the bone marrow to keep pace with demand. It is considered a poor prognostic indicator.
• Neutrophilia – increased neutrophil count – may occur in response to stress, excitement, exercise, glucocorticoid or adrenaline (epinephrine) administration, or almost any infectious or inflammatory condition. May be accompanied by a regenerative left shift. Often follows neutropenia caused by endotoxaemia or infection.
• Eosinopenia – decreased eosinophil count – may be normal or may occur in response to stress or glucocorticoid administration.
• Eosinophilia – increased eosinophil count – occurs in response to some parasitic infections or hypersensitivity reactions e.g. allergic respiratory disease; allergic dermatitis. It may also occur as a primary myeloproliferative disease, or in response to systemic neoplasia.
• Basophilia – increased basophil count – may be observed in response to allergic, inflammatory, or neoplastic diseases or in association with lipaemia. (Basopenia is not clinically significant.)
• Monocytosis – increased monocyte count – occasionally observed in association with chronic inflammation. (Monocytopenia is not clinically significant.)
• Lymphopenia – decreased lymphocyte count – occurs in response to stress, glucocorticoid administration, many acute viral infections, endotoxaemia and in foals with severe combined immunodeficiency (SCIDs).
• Lymphocytosis – increased lymphocyte count – associated with excitement, exercise, adrenaline (epinephrine) administration, lymphocytic leukaemia, and chronic antigenic stimulation.
Foals are born with a higher neutrophil : lymphocyte ratio than adults. Neutrophil production decreases, and lymphocyte output increases during the first few weeks, such that lymphocyte : neutrophil ratio reaches 1.5 : 1.0 by 2 months.
Platelets: Total counts in the horse are usually less than in other species. Platelets may clump if they have become activated in vivo or in vitro. Clumped platelets may be observed at the feathered edge of a stained blood smear, particularly with EDTA samples. If platelets have clumped, automated counts are not accurate. A repeat sample should be collected in sodium citrate and counted for more accurate data. Below 100 × 109/L thrombocytes is of clinical significance, but frank haemorrhage is unlikely to occur unless platelet numbers fall below 20 × 109/L. Splenic contraction will raise numbers.
• Autoagglutination – agglutination of erythrocytes that is not eliminated by diluting blood in sterile saline is suggestive of immune-mediated haemolytic disease (dilute 1 drop EDTA blood: 4 drops saline).
• Coombs test – direct Coombs test detects antibody and/or complement on the surface of erythrocytes; indirect Coombs test detects the presence of circulating anti-erythrocyte antibodies. Either or both may be positive in immune-mediated haemolytic disease.
• Osmotic fragility test – assay assesses stability of erythrocytes in saline solutions of decreasing tonicity. Increased fragility is associated with immune-mediated haemolytic disease.
• Flow cytometry – flow cytometric detection of platelets with surface-bound IgG can be used to identify cases of immune-mediated thrombocytopenia (IMT). Diagnosis of IMT otherwise depends on elimination of other causes of thrombocytopenia.
• Cross-matching – major cross-match checks for compatibility between blood donor erythrocytes and any alloantibody present in patient serum. The minor cross-match checks for compatibility between donor serum and patient erythrocytes. Both tests are important prior to whole blood transfusion. The minor cross-match is most important prior to plasma transfusion.
• Iron status – may be important in characterization of some anaemias. Serum ferritin concentration is indicative of stored hepatic and splenic iron. Prussian blue stain of bone marrow aspirates also allows assessment of iron stores. Serum iron concentration reflects total quantity of transport iron in the plasma (iron bound to transferrin). Total iron binding capacity (TIBC) is the amount of iron that plasma transferrin could bind if fully saturated. Percentage transferrin saturation is calculated from TIBC and serum iron concentration and should normally be approximately 30%.
Bone marrow evaluation: Bone marrow aspirates and core biopsies may be obtained from the sternebrae on the ventral midline between the forelimbs, from the tuber coxae with the needle directed toward the opposite coxofemoral joint, or from the proximal ribs. The area is clipped and surgically scrubbed. A small amount of local anaesthetic is deposited in the subcutaneous tissues and periosteum. A stab incision is made with a small surgical blade. A 16-gauge or larger bone marrow needle is inserted and forced into the marrow cavity with constant pressure and rotational movement. The stylet is removed and a sterile syringe containing a small amount of EDTA or sodium citrate as anticoagulant is used to aspirate material. Smears should be made immediately and stained for cytological analysis. Core biopsies are similarly obtained using a 10-gauge Jamshidi needle and fixed for routine histopathological analysis.
Bone marrow myeloid to erythroid ratio (M : E ratio) should be 0.5–0.9, lower than in other species. An M : E ratio of <0.5 is indicative of erythrocyte regeneration or myeloid suppression. Polychromatophilic reticulocytes should be seen in the bone marrow if erythropoiesis is ongoing.
Evaluation of haemostatic function: Evaluation of haemostatic function should include a total platelet count, tests to evaluate the function of extrinsic and intrinsic coagulation cascades, and assays to estimate fibrinolytic activity. Samples should be collected in a citrate tube and values compared to a healthy control horse.
Prothrombin time (PT) or one-stage prothrombin time (OSPT) measures the function of the extrinsic and common coagulation cascades.
Activated partial thromboplastin time (APTT) measures the function of the intrinsic and common coagulation pathways. Prolonged PT with a normal APTT indicates a deficiency of factor VII in the extrinsic pathway (e.g. in hepatic failure). Prolongation of both PT and APTT is more likely to occur with poisoning (e.g. vitamin K antagonists) or DIC. Mucous membrane changes in a horse with DIC are shown in Figure 10.3.
Antithrombin III (ATIII) activity is increased in some acute inflammatory disorders or hepatic diseases, but decreased with DIC and protein-losing nephropathy or enteropathy.
Platelet count <10 000/µL is associated with haemorrhage (Figure 10.2). Platelet function may be evaluated with template bleeding time assays or platelet aggregation tests.
• Urinalysis – haemoglobinuria occurs with intravascular haemolysis. Haematuria with erythrocytes in urine sediment occurs with urinary tract haemorrhage and/or inflammation.
• Thoracocentesis – may be useful for identification of intrathoracic haemorrhage. Abnormal lymphocytes observed on cytology is compatible with a diagnosis of thoracic lymphoma.
• Abdominocentesis – may be useful for identification of intra-abdominal haemorrhage.
• Testing of faecal occult blood may help to indicate if gastrointestinal or respiratory tract haemorrhage has occurred but tests are considered to be unreliable.
Immunoglobulin assays.: Screening tests to assess for gross alterations in serum IgG concentration include zinc sulphate turbidity, glutaraldehyde coagulation, latex agglutination, or enzyme linked immunosorbent assay (ELISA). More accurate quantification is available with radial immunodiffusion (RID) techniques. RID test kits are available to accurately quantitate serum IgG, IgM, IgG(T) and IgA. Production of antibody in response to vaccination with a specific antigen may be used to assess responsiveness of the humoral immune system.
Lymphocyte typing.: Peripheral lymphocyte counts are extremely low in Arabian foals with severe combined immunodeficiency (SCID). Dramatically increased counts with abnormal cell morphology may be observed in horses with lymphocytic neoplasias. Monoclonal antibodies are available for identification of lymphocyte subsets (CD4, CD8, pan T cell, B cell) in peripheral blood by flow cytometry. Staining of tumours with antibodies against CD3 and CD79 has been used to classify T-cell and B-cell tumours respectively.
Blood typing.: Several laboratories perform erythrocyte alloantigen typing and alloantibody detection for prevention or diagnosis of neonatal isoerythrolysis (NIE). These laboratories will also perform lymphocyte typing for progeny verification. NIE is more likely to occur in the progeny of horses with blood groups Aa or Qa.
Cellular immune function.: Intradermal skin testing with phytohaemagglutinin (PHA) may be used to assess delayed type hypersensitivity. A crude estimate of T cell function may be obtained with lymphocyte blastogenesis assays using PHA, concanavalin A or pokeweed mitogen. More specific tests may be available in some research laboratories.
10.3 Anaemia secondary to haemorrhage
As in other species, anaemia in the horse is caused by either increased loss of red cells, increased destruction or by decreased production. The former two are the most common causes of severe anaemia in horses. Chronic disease is a relatively common cause of less severe chronic anaemia. The causes of increased loss of cells or increased destruction are multiple, and must be identified in order to treat the condition effectively.
Haemorrhage: aetiology and pathogenesis: Blood loss or haemorrhage may be external or internal, acute or chronic.
• External blood loss is often a result of trauma and easily recognized and diagnosed with a visual examination. An exception to this is haemorrhage into the gastrointestinal tract. Significant haemorrhage into the gastrointestinal tract may occur with gastric ulceration or intestinal neoplasia without evident melaena or haematochezia.
• Internal blood loss, into the thorax or abdomen, allows the body to re-use (autotransfuse) up to two-thirds of the erythrocytes lost. Common causes include severe exercise induced pulmonary haemorrhage (EIPH), post partum rupture of the uterine artery and splenic rupture subsequent to trauma.
• Acute blood loss – often associated with normal RBC parameters because all blood components have been lost in equal volumes. Redistribution of fluid from interstitial tissue into the intravascular space results in a detectable drop in total plasma protein after 6 hours. Anaemia only becomes apparent after 12–24 hours, with the lowest values in PCV occurring at 36–48 hours post haemorrhage. Bone marrow exhibits increased erythropoiesis by 3 days post haemorrhage; maximal by 10 days. Horses do not release reticulocytes into the circulation during a regenerative response. PCV should increase by 0.32–0.42% per day during maximum regeneration. Complete restoration of circulating red cell count may require 1 to 3 months. Weekly assessment of PCV and TP aids monitoring of regeneration process. Serial bone marrow aspirates must be examined if regenerative responses are questionable.
Clinical signs: Signs depend on the source of haemorrhage, plus the rate and total volume of blood loss. Mares with post partum uterine artery rupture for example show signs of colic followed by progressive tachypnoea, tachycardia and pallor. Signs of hypovolaemic shock develop when horses lose more than 30% of circulating blood volume (i.e. 24 mL/kg or 9 L of blood for a 400 kg horse). Signs are more subtle in cases of chronic blood loss due to physiological adaptation, such that vital parameters may only be elevated when the horse is exercised or stressed. Packed cell volume may decrease as far as 8–10 L/L in chronic cases before clinical signs become readily apparent at rest.
A summary of clinical signs is shown in Table 10.3.
Table 10.3
Signs of acute and chronic blood loss
Acute blood loss | Chronic blood loss |
Hypovolaemic shock | May be anorexia and weight loss |
Tachycardia | Decreased exercise tolerance |
Tachypnoea | Pale mucous membranes |
Pale mucous membranes | Weakness, lethargy |
Poor venous distension | Tachycardia when stressed |
Thready pulse | Tachypnoea when stressed |
Pansystolic heart murmur | Vitals may be normal at rest |
Weakness, lethargy | Pansystolic heart murmur |
Renal hypoxia: oliguria | |
Acute renal failure |
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