Blood Group, Blood Transfusion, and Hematological Disorders

3.5.1 Blood Grouping in Animals

The determinants of blood groups are the specific polymor­phic antigens that reside on the surface of erythrocytes (agglutinogens) and the antibodies (agglutinogens) present in the plasma.

1. Naturally occurring antibodies (alloantibodies): They are produced against the isoantigens produced by the other members of same species and destroy the isoantigens. For example: anti-A antibody in the individ­ual with B blood group.

2. Acquired antibodies: They are produced in response to the exposure of a blood group antigen mostly through blood transfusion.

Unlike humans the naturally occurring alloantibodies in animals are less immunogenic and don’t induce severe hemo­lytic reactions. Thus, the first blood donation for an animal can be done from any animals of same species without any hemolytic reactions but thereafter, the donor blood will have to match to prevent possible complications.

4.5.1.1 CanineBloodTypes

The dog erythrocyte antigen (DEA) system: The major determinant of canine blood group is dog erythrocyte antigen (DEA), and there are eight major blood groups in the dog, labeled as DEA 1-8. The different types of blood grouping systems in canines are summarized in Table 4.20.

Dalmatian (Dal) blood types: A new red cell antigen was discovered in 2007 in Dalmatian dogs. This antigen was named as Dal as it was first discovered in Dalmatian dogs. The prevalence of Dal antigen is about 93% dogs in the USA. Dal⁺ phenotype was autosomal dominant. The highest incidence was reported in Dalmatians (85.6-100%) followed by Doberman Pinschers (78.6%). Higher incidence of Dαl^-antigens was identified in Shih Tzus (57.1%).

Kai 1 and Kai 2 blood types: Two new blood groups namely Kai 1 and Kai 2 were reported in the dogs that are mostly found in North America.

Table 4.20 DEA blood group system in dogs

4.5.1.2 FelineBloodTypes

Unlike canines, cats have only one blood group system, the AB system (Table 4.21). In this blood grouping system, three blood types are available namely A, B, and AB which is based on two erythrocyte surface antigens namely antigen A and B. Antigen A is actually N-glycolyl-neuraminic acid which has most common incidence among cats. Antigen B is N-acetyl-neuraminic acid with very rare incidence. The extremely rare AB blood group contains both A and B antigens in equal amount. The naturally occurring alloantibodies are strong agglutinins and hemolysins and can cause potentially life-threatening transfusion reactions. There is no universal donor for cat because of the cat’s naturally occurring alloantibodies.

4.5.1.3 EquineBloodTypes

In equines and donkeys, around 30 erythrocyte antigens are available but, only seven major blood group systems are inter­nationally recognized namely A, C, D, K, P, Q, and U. A unique erythrocyte antigen called Donkey factor not only found in mule and donkey but not found in the horse. It is responsible for neonatal iso-erythrolysis in mule pregnancies. Aa- and Qa-negative horses are the best choice as donors.

Table 4.21 AB blood group system in cats

4.5.1.4 Bovine Blood Types

There are 11 major blood group systems in cattle namely, A, B, C, F, J, L, M, R, S, T, and Z in which A and F are the most common.

4.5.1.5 Ovine and Caprine Blood Types

Seven blood group systems have been identified viz. A, B, C, D, M, R, and X in sheep and five major systems (A, B, C, M, and J) have been identified in goats. The B system is highly polymorphic in nature. R system of blood group is similar with J system in cattle. Heterophilic antibodies in bovine colostrum may lead to neonatal iso-erythrolysis in lambs receiving bovine colostrum.

4.5.1.6 Swine Blood Types

In pigs, eight blood group systems were identified depicted in Table 4.22.

A system has similarities with ABO system in human. A gene encodes enzyme α1 → 3 N-acetyl-D- galactosaminyltransferase which synthesizes A antigens are lacks in pigs with O blood group.

Table 4.22 Blood grouping systems in pigs

3.5.2 Blood Transfusion

Indication for Blood Transfusion

Anemia is the major indication of blood transfusion followed by hypovolemia, hypoproteinemia, and coagulopathies.

3.5.2.1 Collection of Blood

Selection of donor: A donor should be healthy young adults that have never encountered blood transfusion. The donors must be evaluated for routine physical, hematolog­ical, and clinical chemistry and should be screened for blood parasites and other infectious diseases (Ehrlichia spp., Babesia spp., Anaplasma spp., and Mycoplasma hemocanis or Mycoplasma haemofelis). The permissible amount of blood to be collected in different species is summarized in Table 4.24.

Table 4.23 Blood grouping systems in pigs

Table 4.24 Blood collection amount in different species

Methods of blood collection from donors: In cats and dogs, jugular venipuncture is generally practiced for blood col­lection under general anesthesia, but sedation should not be performed with acepromazin as it interferes with the platelet functions. Commercially available human blood collection bags (450 mL capacity) are generally used for dogs and special pediatric collection bags (75 mL capac­ity) are used for cats. To minimize platelet activation during collection, blood flow should be rapid and the venipuncture should be clean. Sometimes mild vacuum pressure should be applied for the ease of collection. To accelerate hemostasis, direct pressure should be applied at the site of collection. Collected blood should be immedi­ately refrigerated at 4 °C till transfusion or component separation. It was recommended to use desmopressin (DDAVP), a synthetic analog of arginine vasopressin to maximize the yield of vWf.

3.5.2.2 Preservation of Blood for Transfusion

The preservatives for blood are so designed to restore the normal metabolism of the corpuscles along with anticoagu­lant activity. The common ingredients for preserving blood along with their functions are summarized in Table 4.25.

Two anticoagulants-preservatives listed below have been preferred to preserve the blood after collection (Table 4.26). Preservation injuries or changes in stored blood: Long-term storage or inappropriate cold chain during transportation and preservation can lead some biochemical, biomechani­cal, and oxidative changes as listed in Table 4.27.

Bringing the blood in the room temperature before trans­fusion can correct 2,3 DPG concentration. The plasma potas­sium level also decreased due to the activation of Na⁺-K⁺- ATPase pump at the room temperature. The oxidative changes are very difficult to correct.

Table 4.25 Blood preservatives and their functions

Table 4.26 Commonly used blood preservatives and their functions

Table 4.27 Preservation injuries of blood

spinal needle. Intraperitoneal administration is generally avoided as the extraction rate is only 40%. A filter in the transfusion set must be used to minimize cellular aggregations and microthrombi that may lead to pulmo­nary edema. Under normal condition, the standard speed is 5-10 mL/kg/h in normovolemia which can be increased a maximum of 20 mL/kg/h under hypovolemia. A decreased flow rate (2 mL/kg/h) is usually recommended in patients with renal failure.

4.5.2.3 Blood Transfusion in the Recipient

Cross matching technique: In cross matching system, the blood samples of both donor and recipient are subjected to separation of plasma, serum, and erythrocytes. In major cross match system, patient serum and donor RBCs are mixed whereas donor serum and patient RBCs are mixed in minor cross match system. No hemolysis after 15-30 min at room temperature indicates compatibility for transfusion. The cross matching technique is described flow diagrammatically in Fig. 4.7.

Calculations of blood volumes to be transferred to the recipient: Following formula is used to calculate the required volume of blood to be transferred to the recipient.

Blood volume to be transfused = k ? Weight (kg) (Required PCV — Recipient PCV)

PCV of donated blood

where k = 90 for dogs and 66 for cats. Required post trans­fusion PCV is 20% in cats and 25-30% in dogs that are sufficient to cope up the complications related to anemia.

Route of administration: Generally, blood is administered through intravenous catheter into a cephalic or jugular vein. Transfusion of blood in the marrow is as efficient as intravenous administration, and in some cases (severe hypotension or pediatric patients) blood can also be administered into proximal femur through a syringe fitted with 18-20 gauge needle or in the trochanteric fossa with 4.5.2.4 Transfusion Hazards

Monitoring of recipient health status is essentially required during transfusion period. Transfusion should be stopped immediately. Commonly occurring transfusion reactions are discussed below.

Hemolysis: Hemolytic reactions are common during mismatched transfusions. In dogs, acute hemolytic reaction with mismatched blood is rare during first transfusion due to presence of less immunogenic alloantibodies but, in cats even a small volume of mismatched blood can be life threatening due to the presence of strong naturally occurring alloantibodies. The normal lifespan of compatible transfused erythrocytes in dogs is approximately 21 days which may decrease to minutes or even up to 12 h after acute hemolytic transfusion reaction and for cat it may decrease to 1 day from normal lifespan of 30-40 days of transfused erythrocytes.

Acute hypersensitivity reactions: Allergic and anaphylactic reactions can also occur during transfusion which may lead to cardiopulmonary arrest. Allergic reactions during transfu­sion can be monitored by observing erythema, pruritus, urti­caria, vomition, and dyspnea. In such cases, corticosteroids, antihistamines, and adrenaline are recommended.

Pyrexia: Increase in the body temperature by 1 °C or more within 3-4 h of the transfusion has been reported. It may

Fig. 4.7 Flow diagram of cross matching technique

be either due to bacterial contamination or reactions of antibodies with platelets and leukocytes that are unde­tected during typing. Non-hemolytic and non-infectious pyrexia usually requires no treatment.

Contamination: It can occur through blood bag contaminated with pathogens. Some pathogens can escape refrigeration and multiply when the blood is brought to room temperature. The signs of contamination are pyrexia, diarrhea, vomition, and abdominal pain. Severe bacterial contamination may lead to shock. In suspected cases, blood bags should be evaluated for culture and sensitivity and accordingly antibiotic therapy can be recommended. Proper screening of donors for FeLV, FIV, and Hfelis is recommended for cats and heartworm, rickettsial diseases, and blood parasites (Babesia canis) are evaluated for canines.

Hypocalcemia: Its occurrence is rare and may happen when large volume of blood with citrate anticoagulant is administered rapidly which chelates body calcium. The signs of hypocalcemia includes tremors, cardiac arrhyth­mia and vomition.

Circulatory overload: It is associated with rapid administra­tion of large volume of blood in the patients with cardiac abnormalities, renal failure, and normovolemia. The clini­cal manifestations of circulatory overload are tachypnea, dyspnea, tachycardia, coughing, and pulmonary edema. Diuretics and oxygen therapy are generally recommended to control the complications.

4.5.3 Hematological Disorders

4.5.3.1 Anemia

It is the qualitative and quantitative decrease in RBC or hemoglobin or both with respect to age and sex of the individual. It interferes with the oxygen carrying capacity of the blood and the clinical manifestations of anemia are due to compensatory mechanism to increase oxygen saturation such as tachypnea, tachycardia, lethargy, and reduced exercise tolerance. Sometimes icterus is also associated with anemia.

4.5.3.1.1 ClassificationofAnemia

4.5.3.1.1.1 Based on Bone Marrow Function

On the basis of bone marrow functions, anemia can be classified as regenerative and non-regenerative anemia.

In regenerative anemia, bone marrow produces erythrocytes normally in response to decreased erythrocytes. The etiology of regenerative anemia is blood loss and destruction/lysis of erythrocytes (hemolytic anemia). Bone marrow regeneration can be evaluated through the reticulo­cyte response.

Hemolytic Anemia

This occurs due to intra- or extravascular hemolysis. Intravas­cular hemolysis is characterized by hemoglobinemia and hemoglobinuria which is absent in extravascular hemolysis. The causes of hemolytic anemia are immune mediated, drug induced (aspirin, benzocaine, propofol, levamisole, sulfonamides), or toxin (dicoumarol, naphthalene, benzene, crude oil) induced, feed borne (onions, oak, red maple), infec­tious (hemoprotozoa, Clostridium spp., Leptospira spp., Myco­plasma spp., equine infectious anemia virus, feline leukemia virus Ehrlichia spp.), or hereditary (pyruvate kinase deficiencies, phosphofructokinase deficiency, and porphyria).

In non-regenerative anemia, the bone marrow is affected and unable to produce sufficient erythrocytes. The main factors which cause this type of anemia are nutritional deficiencies, chronic diseases, renal failure, and the diseases affecting bone marrow.

Nutritional Deficiencies

Deficiency of iron, copper, vitamin Bi₂, vitamin B₆, ribofla­vin, niacin, and vitamin E leads to anemia. The deficiency of iron is the most common form of nutritional deficiency ane­mia affecting piglets and puppies particularly before weaning. Milk is deficient in iron thus the young animals are unable to get adequate iron during suckling. Microcytosis is common in iron deficiency anemia. In ruminants, cobalt deficiency causes normocytic, non-regenerative anemia caused by grazing cobalt deficient soil. Cobalt is required for the synthesis of cobalamin by rumen bacteria.

The deficiency of vitamin B₁₂ leads to pernicious anemia.

Pernicious Anemia

It is characterized by the maturation failure of RBC due to poor absorption of vitamin B₁₂ from the intestine. It occurs due poor secretion of a glycoprotein called intrinsic factor (IF) from parietal cells. It helps in the absorption of vitamin B₁₂ after binding tightly with the vitamin B₁₂ and protects from gastric digestion. Vitamin B₁₂ then internalizes in the brush border membrane of ileum. Lack of intrinsic factor thus leads to poor absorption of vit-B₁₂. Pernicious anamia is very rare in dogs and cats and is usually manifested due to the genetic mutations in the ileal cubam receptor, often occurred in dog breeds like Beagles, Border Collies, Giant Schnauzers, and Australian Shepherds.

Anemia Associated with Chronic Disease

Chronic inflammation, tumor, liver disease, and endocrine disturbances (hyper- or hypo-adrenocorticism and hypothy­roidism) lead to non-regenerative anemia in animals. Chronic inflammatory diseases lead to cytokine production which affect red blood cell survival, bone marrow’s ability to regen­erate and poor iron availability.

Anemia associated with chronic renal failure is common in animals. Chronic kidney disease causes less erythropoietin production which affects erythropoiesis in the bone marrow.

Anemia Associated with Bone Marrow Diseases

Diseases affecting bone marrow not only affect erythrocyte production but also the reduction in the number of all types of blood cells including leukocytes and thrombocytes. The major diseases affecting bone marrow are discussed below:

Aplastic anemia: This type of anemia is associated with decreased bone marrow response to generate erythrocytes. The underlying causes of aplastic anemia are infections (Feline leukemia virus, Feline immunodeficiency virus, Ehrlichia, Mycoplasma), drug therapy, toxins, and total body irradiation.

Myelodysplasia: It is characterized by defective formation of blood cell precursors in the bone marrow. Leukemia and thrombocytopenia are also occurred in addition to anemia in myelodysplasia.

Myelofibrosis: In this condition, normal marrow tissue is replaced by fibrous (scar) tissue. It can be occurred as a result of neoplasia, immune-mediated hemolytic or hered­itary anemias, and whole-body irradiation. Immune sup­pression also occurred in myelofibrosis.

3.5.2.2.1.1 Based on Erythrocyte Volume and Hemoglobin Concentration

Normocytic anemia: In this condition, the size of erythrocyte is normal but the number decreases. Normocytic anemia can be classified into normocytic normochromic anemia in which MCV, MCH, and MCHC are within normal range but, the lifespan of RBC is short. Normocytic hypochro­mic anemia is characterized by decreased MCH and MCHC as erythrocytes are paler than normal indicating less hemoglobin. Normocytic normochromic anemia is often associated with hypothyroidism in the dogs. Blood smear of normocytic normochromic anemia shows polychromasia, anisocytosis, or nucleated red cells.

Microcytic anemia: In microcytic anemia, erythrocyte num­ber is adequate but the size of erythrocyte is smaller than normal and decreased MCV. Iron deficiency is the most common cause of microcytic anemia. Iron deficiency leads to impaired hemoglobin synthesis and the hemoglobin-deficient erythrocyte precursors are

undergoing additional mitosis to achieve normal hemo­globin level, thus generated microcytosis. In microcytic anemia, both MCV and MCH are decreased. Microcytic anemia can also occur due to copper deficiency.

Macrocytic anemia/megaloblastic anemia: Macrocytic ane­mia is characterized by the appearance of macrocytic erythrocytes (erythrocytes with increased size) as evident from increased MCV. Deficiency of cobalamin and folic acid is the prime cause of macrocytic anemia which interferes with DNA synthesis and leads to maturation arrest of erythrocyte precursors. In cats, feline leukemia virus (FeLV) infection and myelodysplastic syndromes lead to macrocytosis. Some poodles also have macrocytic anemia.

Sickle cell anemia: This anemia is hereditary origin and characterized by sickle shaped RBC with increased fragil­ity. It is characterized by malformed β chain of Hb (amino acid glutamic acid at sixth position is replaced by valine) that forms thread-like structure in deoxygenated form and leads to change the shape of RBC-like sickles. In deer, the sickle shaped erythrocytes are common, but the occurrences of sickle cell anemia in other animals are rare.

Spherocytosis In this condition, the RBC loses its bicon­cavity and becomes spherical in shape. It is caused due to the defect in RBC cytoskeleton proteins like spectrin, ankyrin, Band 3, or Protein 4.2 or due to partial phagocytosis of cell surface. Spherocytes are prone to immune-mediated hemolysis.

3.5.2.3 ErythrocytosisZPolycythemia

An increase in the erythrocyte number together with increased hemoglobin concentration and hematocrit value is termed as erythrocytosis. Erythrocytosis and polycythe­mia are often synonymous but in human medicine polycy­themia restricts not only to erythrocytosis but increases in leukocytes and thrombocytes also. Pathologically, erythrocytosis can be classified into two categories namely relative and absolute.

In relative erythrocytosis, increase in the hematocrit value doesn’t associate with increased RBC but due to reduced plasma volume. Severe dehydration due to fluid loss is associated with diarrhea, vomition, or polyuria results in relative erythrocytosis and is most common in dogs and cats. Hemoconcentration due to catecholamine mediated splenic contraction during excitement also leads to mild erythrocytosis in horse and dogs.

Absolute erythrocytosis is characterized by true increase in erythrocyte numbers. It can be classified into primary and secondary erythrocytosis.

Primary erythrocytosis is also called polycythemia vera in which proliferation of erythroid precursors occur indepen­dent of erythropoietin. Splenomegaly, leukocytosis, and thrombocytosis are the common clinical manifestations of polycythemia vera. It is generally occurred in dogs and cats of middle age groups (6-7 years). It is interesting to note that primary erythrocytosis is common among male dogs whereas female cats mostly suffer from it. Secondary erythrocytosis results due to excess production of erythropoietin with sys­temic hypoxia (appropriate secondary erythrocytosis), or without (inappropriate secondary erythrocytosis) systemic hypoxia. Inappropriate secondary erythrocytosis is common in neoplasia and kidney disorders. Endocrinopathies associated secondary erythrocytosis occurs in conditions such as hyperadrenocorticism, hyperthyroidism, and acromegaly.

3.5.2.4 Bleeding Disorders

3.5.2.4.1 Platelet Defects

Disorders of platelets include thrombocytopenia (decreased number of platelets) or having thrombocytopathies (impairment of platelet functions). Both of these disorders can be congenital or acquired.

3.5.2.4.1.1 Congenital Thrombocytopenia

It is actually a hematopoietic disorder which maintains a 12 days cycle. In this condition, all types of blood cells are affected including platelets. It is very fatal as most of the affected dogs die before 6 months of age. Cavalier King Charles Spaniels suffer from benign hereditary macrothrombocytopenia which is characterized by thrombo­cytopenia with giant platelets.

3.5.2.4.1.2 Acquired Thrombocytopenia

The potential causes of acquired thrombocytopenia are

Rickettsial diseases: Mild to moderate degree of thrombocytopathies are seen in Ehrlichia and Anaplasma infections. It is characterized by epistaxis, gum bleeding, and gastrointestinal hemorrhage (black stools).

Immune-mediated thrombocytopenia: Disfunctions in the immune system produces antibodies against platelets or platelet precursors in the bone marrow. Repeated vaccinations of dogs with live adenovirus or paramyxovi­rus vaccines lead to mild thrombocytopenia.

Drug-induced thrombocytopenia: Estrogen and some antibiotics can suppress the production of platelets whereas certain drugs (acetaminophen, aspirin, penicillin) destroy the circulating platelets.

3.5.2.4.1.3 Congenital Thrombocytopathies

It can be classified into several categories

Canine thrombopathia: It is common in Basset Hounds irrespective of normal levels of platelets and von Willebrand’s factor. It can be diagnosed by platelet func­tion testing.

Glanzmann thrombasthenia: This condition is characterized by prolonged bleeding times. Blood smear reveals giant platelets with altered shape. Platelet aggregation during coagulation mechanism is inhibited in this condition. It is seen in Otterhounds and Great Pyrenees dogs.

Von Willebrand disease: Affected individuals are deficient in von Willebrand’s factor. It is one of the most common inherited bleeding disorders in canines with almost all the breeds.

The disease is classified into three categories

• Type 1: Low amount von Willebrand factor with mild to moderate signs.

• Type 2: Low amount of the factor with moderate to severe signs.

• Type 3: Absence of von Willebrand factor with frequent episodes of bleeding. It is most frequent in Shetland Sheepdogs and Scottish Terriers.

3.5.2.4.1.4 Acquired Thrombocytopathies

Acquired thrombocytopathies are consequences of bone mar­row tumor (multiple myeloma) and chronic kidney diseases.

Acquired thrombocytopenia also leads to platelet functional defects. It can also be drug induced.

3.6