Erythrocytes: Overview, Morphology, Quantity

Overview

Production

Red blood cells (RBC) are produced in the bone marrow.
Numbers of circulating RBCs are affected by changes in plasma volume, rate of RBC destruction or loss, splenic contraction, erythropoietin (EPO) secretion, and the rate of bone marrow production.
A normal PCV is maintained by an endocrine loop that involves generation and release of erythropoietin (EPO) from the kidney in response to renal hypoxia.

Erythropoietin stimulates platelet production as well as red cell production. However, erythropoietin does not stimulate white blood cell (WBC) production.
Erythropoiesis and RBC numbers are also affected by hormones from the adrenal cortex, thyroid, ovary, testis, and anterior pituitary.

Destruction

Red cells have a finite circulating lifespan. In dogs, the average normal red cell circulates approximately 100 days. In cats, 85 - 90 days.
Since in health circulating red cell numbers remain fairly constant, approximately 1% of the circulating red cells of the dog are replaced daily (slightly higher percentage in the cat). The new cells are young and morphologically distinct (large, polychromatophilic - see morphology section).
Effete red cells are phagocytized and metabolized by the macrophages of spleen, bone marrow, and liver. Iron is preserved for reutilization.

Function

The primary function of the red cell is to carry oxygen to tissue cells and to carry carbon dioxide away.
To facilitate this exchange, red cells consist essentially of gas-carrying soluble protein (hemoglobin) surrounded by a protective cell membrane.
The fluidity of normal red cells allow them to traverse tortuous capillary beds leading to close approximation of red cells with tissue cells. This in turn makes gaseous exchange efficient.

Physiology

Red cell physiology is geared to facilitate function and protect red cell integrity.
The primary red cell metabolic pathway is anaerobic glycolysis. The glycolytic pathway allows the cell to produce energy to maintain membrane stability with minimal utilization of oxygen.
The red cell also has metabolic pathways (hexose monophosphate shunt and methemoglobin reductase) which protect hemoglobin from oxidation. Oxidation of hemoglobin leads to methemoglobinemia and/or Heinz body formation.
Cat hemoglobin is more susceptible to oxidation than dog hemoglobin because it contains a high percentage of sulfur-containing amino acids which are easily oxidized. Therefore, Heinz body formation and Heinz body hemolytic anemia occurs more readily in cats than dogs.

Morphology

Normal Morphology: Dog

Shape: biconcave disc with prominent central pallor (Fig. 4-1)
Figure 4-1. Canine blood, normal erythrocyte morphology. Canine RBCs are all about the same size, shape, and color, and have a prominent area of central pallor (100x).
Size: 6.0 - 7.0 μ
Rouleaux formation (stacking): moderate (Fig. 4-2).

Figure 4-2. Canine blood, rouleaux formation. RBCs are arranged in overlapping chains (60x).

Polychromatophils (bluish-red immature erythrocytes): comprise approximately 1% of total red cell population (Fig. 4-3 and Fig. 4-4).
Polychromatophils correspond to reticulocytes in new methylene blue stained preparations (Fig. 4-5).
Howell-Jolly bodies are deeply staining nuclear remnants found in red cells. These inclusions are rare in normal dogs (Fig. 4-6).

Figure 4-3. Canine blood. Regenerative anemia. Immature RBCs are visible as larger erythrocytes that have blue-grey cytoplasm. These cells are termed macrocytic polychromatophilic erythrocytes (100x).

Figure 4-4. Canine blood. Iron deficiency anemia. Hypochromia and poikilocytosis are evident. Hypochromic RBCs have a large area of central pallor due to reduced hemoglobin content. The polychromatophilic RBC (arrow) in this field has a vacuolated or moth-eaten cytoplasm. Compare these cells with those in Fig. 4-3 (100x).

Figure 4-5. Canine blood. New methylene blue stain. Reticulocytes are visible as pale yellow cells with basophilic precipitates of RNA (100x).

Figure 4-6. Canine blood, Howell Jolly body. Small basophilic round inclusion in the RBC cytoplasm (arrow) is a remnant of a nucleus (100x).

Normal Morphology: Cat

Shape: biconcave disc with minimal central pallor (Fig. 4-7).

Figure 4-7. Feline blood. Normal erythrocyte morphology. Feline RBCs are smaller than dog erythrocytes, exhibit a slight amount of crenation, and have a minimal area of central pallor (100x).

Size: 5.5 - 6.0 μ
Rouleaux formation: marked
Polychromatophils: comprise 1.5 - 2.0% of total red cell population (Fig. 4-8).
Howell-Jolly bodies: occasional; more common than in dogs.

Figure 4-8. Feline blood. Regenerative anemia. Immature RBCs are visible as larger erythrocytes that have blue- grey cytoplasm. These cells are termed macrocytic polychromatophilic erythrocytes. Two NRBCs (arrows) are noted and are smaller than a lymphocyte (100x).

Morphology in Disease

Anisocytosis - variation in red cell size (Fig. 4-9).

• Macrocytes - large red cells
• Microcytes - small red cells

Polychromasia - increased numbers of polychromatophils on the blood film.
Hypochromasia - decreased hemoglobin concentration. Hypochromic red cells have enlarged areas of central pallor (Fig. 4-10).

Figure 4-9. Canine blood. Marked anisocytosis and poikilocytosis are present due to macrocytic cells, spherocytes, schistocytes, acanthocytes and microcytes (100x). See subsequent figures for identification of specific poikilocytes.

Figure 4-10. Canine blood. Numerous poikilocytes, marked hypochromia, and microcytosis are evident. Platelets are numerous and enlarged. These changes are usually associated with iron deficiency secondary to chronic hemorrhage (100x).

Poikilocytosis - the presence of abnormally shaped red cells.

• Acanthocytes - red cells with 2 - 10 blunt elongate finger-like surface projections (Fig. 4-11).
• Spherocytes - small round red cells that stain intensely and lack central pallor (Fig. 4-12).
• Elliptocytes - oval erythrocytes.
• Dacryocytes - tear-drop shaped erythrocytes.
• Stomatocytes - cup-shaped erythrocytes (Fig. 4-12).
• Heinz bodies - precipitates of oxidized hemoglobin. Often recognized as nose-like projections from the red cell surface (Fig. 4-13 and Fig. 4-14).

Figure 4-11. Canine blood. The poikilocytes in this blood smear have irregular membrane projections that have rounded tips. These cells are called acanthocytes (arrows) and represent an in vivo alteration rather than an artifact. Acanthocytes are frequently associated with hemangiosarcoma in the liver. A few spherocytes are also noted. (100x).

Figure 4-12. Canine blood. Regenerative anemia with spherocytes. Anisocytosis is due to macrocytic cells and spherocytes, which are smaller than normal and lack central pallor. Spherocytes are associated with hemolytic anemias due to immune disease or fragmentation. The polychromatophilic RBC with a rod- shaped area of central pallor (arrow) is a stomatocyte (100x).

Figure 4-13. Feline blood. Heinz body hemolytic anemia. Morphologic evidence of regenerative anemia is present as anisocytosis and polychromasia. Smaller RBCs (arrows) have singular rounded membrane projections that are Heinz bodies. These inclusions are caused by oxidative injury due to drugs, toxic plants, certain chemicals, and metabolic diseases. Heinz bodies can be seen in cats that are clinically normal and not anemic (100x).

Figure 4-14. Feline blood. New methylene blue stain. Heinz bodies are noted on the edge of RBCs as turquoise inclusions (arrows). Aggregate and punctate reticulocytes can also be seen. Aggregate reticulocytes have definite clumps of RNA precipitate in the RBC cytoplasm. Punctate reticulocytes left of center have numerous small individual granules of RNA precipitate. When counting reticulocytes for the absolute reticulocyte count, only aggregate reticulocytes are enumerated to assess the regenerative response (100x).

• Eccentrocytes - red cells with hemoglobin concentrated at one pole with an unstained area at the other pole (Fig. 4-15 and Fig. 4-16).
• Schistocytes - irregularly shaped, often roughly triangular, red cell fragments (Fig. 4-17).
• Burr cells - elongated red cells with ruffled margins (also termed echino elliptocytes).

Figure 4-15. Canine blood. Hemolytic disease. RBC with eccentric hemoglobin staining can be seen (eccentrocytes). Very little evidence of anisocytosis or polychromasia can be seen (60x).

Figure 4-16. Canine blood. Hemolytic disease. Numerous eccentrocytes or "blister cells" are seen. These cells have an eccentric displacement of hemoglobin because opposing sides of the RBC membrane have fused due to oxidative injury. Causes of this change are identical to those that produce Heinz bodies (100x).

Figure 4-17. Canine blood. Schistocytes. Fragmentation of RBCs results in the formation of schistocytes. These cells are formed when RBCs are sheared by convoluted vascular channels, intravascular fibrin deposition, or excessive turbulence. The RBCs have sharp cuts in the cell margin that produce membrane tags and cells that have a helmet shape (100x).

Artifacts

Crenation: the presence of red cells covered by short spiky surface projections (Fig. 4-18).

• Crenation is the most common artifactual change seen in blood films.
• Crenation can be confused with acanthocytic change.
• Crenation is more prominent in films made from EDTA blood.
• Crenation is differentiated from true poikilocytosis in that crenation affects all the red cells in a given area of the film whereas true poikilocytosis affects only scattered red cells on the blood film.

Figure 4-18. Short regular membrane projections with sharp points represent an artifact in RBC morphology called crenation. This change can occur due to a delay between collection of blood and smear preparation, or due to a drying artifact (100x).

Refractory "bubbles" on the surface of red cells (Fig. 4-19).

• Refractory areas on the red cell surface are most commonly seen when blood films are stained while still wet.
• These "bubbles" probably represent gas being trapped in the specimen as it escapes from the red cells.

Figure 4-19. Feline blood. Refractile artifact. Air bubbles trapped in the area of central pallor produces a refractile rounded structure that may be confused with an RBC inclusion. These structures "jewel" as the fine focus is moved and can be seen in a plane of focus that is beyond the RBC membrane (100x).

Stain precipitate

• Aggregates of stain precipitate are commonly observed on Romanowsky (Wright’s, Diff Quik) stained slides.
• Stain precipitate along the margins of red cells must be differentiated from Haemobartonella organisms (Fig. 4-20 and Fig. 4-21).
• Stain precipitate must also be differentiated from basophilic stippling (Fig. 4-22).

Figure 4-20. Canine blood. Granular artifact. Stain precipitate can sometimes adhere to RBCs and produce the appearance of a parasite or inclusion. These granules vary in size and do not resemble any of the usual canine RBC parasites. If the focus were adjusted, this material would appear to "jewel" or be refractile (100x)

Figure 4-21. Feline blood. Precipitated stain. Basophilic granular material that obscures the RBCs is stain precipitate. This material is sometimes mistaken for bacteria or RBC parasites (100x).

Figure 4-22. Canine blood. Basophilic stippling. Several of the RBCs have small fine basophilic granules in the cytoplasm (arrows). Basophilic stippling can be seen in dogs and cats with severe regenerative anemias or in dogs with lead poisoning. In the latter disease, stippling is associated with an inappropriate release of NRBCs (100x).

Over-staining of red cells

• Slides which have been exposed to formalin fumes will have an overall greenish discoloration when stained. It will be difficult to recognize polychromasia in such preparation.
• Slides which are exposed too long to the basophilic (blue) component of Romanowsky stains will also be over-stained, making evaluation of polychromasia difficult to impossible.

Scratching the preparation

• Wiping or blotting the surface of a blood film with a tissue or lens paper to dry it or remove oil will leave scratches. This should be avoided.
• Scratches are seen microscopically as linear disruptions of cells, particularly red cells.
• Cells broken in this manner may be mistaken for schistocytes.
• Pseudothrombocytopenia
• Clumping of platelets can falsely decrease the platelet counts especially in cats (Fig. 4-23).

Figure 4-23. Canine blood, platelet clump. Platelet clumping is usually a collection artifact that can produce a false decrease in platelet count. This is especially a problem in cats. Blood smears should be examined for aggregates of platelets when the electronic platelet count is low (100x).

Quantity

Anemia

Decreased red cell mass is anemia.
Anemias are classified as regenerative or non-regenerative based on bone marrow responsiveness.

Regenerative Anemia

Regenerative anemias are anemias where marrow red cell production is increased to the point that red cell mass will eventually be returned to normal.

• Increased polychromatophils (young red cells - see morphology) in the peripheral circulation is the reflection of increased production on routinely stained blood films.
• The first step in differentiating regenerative from nonregenerative anemia is evaluation of the blood film.

Whenever increased polychromasia is seen on routine blood films, reticulocyte counts are warranted.
Reticulocyte counts of greater than 80,000/μl indicate regeneration.
Regenerative anemias are either the result of blood loss or hemolysis.

Regenerative Anemia: Blood Loss / Hemorrhage

History, clinical signs, and physical examination often point to the source of blood loss.
In general, blood loss anemias are less responsive than hemolytic anemia. The main reason is that when blood loss occurs, iron is lost from the body and iron is needed for hemoglobin synthesis.
If blood loss is severe or chronic, iron depletion and/or iron deficiency can occur, resulting in a nonregenerative anemia.
The nonregenerative anemia of iron deficiency is often microcytic (small red cell size) and hypochromic (reduced hemoglobin). A description and illustration of iron deficiency anemia is found in "Maturation Defect Anemias: Cytoplasmic Defect" section.
Since loss of blood involves loss of plasma protein as well as loss of cells, blood loss anemias may also have reduced plasma protein, serum protein, albumin, and globulin levels.
As a general guideline, absolute reticulocyte counts between 80,000/μl and 200,000/μl can indicate either blood loss or hemolysis. As reticulocyte counts elevate above 200,000/μl, hemolysis should be suspected.

Regenerative Anemias: Hemolytic Disease

Hemolytic anemia occurs when the circulating red cell lifespan is reduced due to increased rates of RBC destruction. Hemolysis is the result either of a defect in the red cells (inherent or acquired) or a defect in the microvasculature through which the red cells circulate.

Hemolysis can occur intravascularly (while the red cells are circulating) or extravascularly.

• Extravascular hemolysis involves the destruction and removal of damaged red cells by the macrophages of the spleen and liver.
• Many hemolytic anemias have elements of both intravascular and extravascular lysis.

Hemoglobinemia and/or hemoglobinuria are seen only in severe cases of intravascular hemolysis.
Icterus can be seen in either intravascular or extravascular hemolytic anemia.
Because hemolytic anemias are associated with red cell defects, red cell morphologic alterations often are associated with specific causes of hemolytic anemia. The common hemolytic anemias of the dog and cat are listed, described, and illustrated below.
Hemolytic Disease: Immune-Mediated Hemolytic Anemia (IMHA)

• Occurs in both dogs and cats
• Pathogenesis:

• Red cells become coated with antibodies as they circulate (Fig. 4-24).
• Antibody-coated red cells either lyse intravascularly (due to complement fixation) or are removed by macrophages in the liver and spleen.

Figure 4-24. Canine blood. RBC agglutination. Branching chains of RBCs form clusters in three areas of the field. Agglutination of RBCs occurs when antibodies cause bridging between adjacent cells (100x).

> Antibodies may be directed against the red cells themselves (autoimmune hemolytic anemia) or against foreign antigens.

> Among the conditions which can cause immune-mediated hemolytic disease are:

• Heartworm disease
• Lymphoma
• Lupus erythematosus
• Drug-induced immune mediated hemolysis

> The morphologic hallmark of immune-mediated hemolytic anemia is the presence of significant numbers of spherocytes (Fig. 4-25and Fig. 4-26).

> Spherocytes are red cells with the following characteristics (Fig. 4-26):

• Small
• Round
• Stain intensely
• Lack central pallor

> Ghost cells may also be present (Fig. 4-25 and Fig. 4-27)

> Whenever blood film morphology suggests immune hemolysis (regenerative anemia with spherocytes), a Coomb’s test (direct antiglobulin test) can be run for confirmation.

> The Coomb’s test must be run with species specific Coomb’s reagent or false positives can occur.

> False negative Coomb’s tests are also common; these can be reduced in number by requesting that the laboratory run the test with serial dilutions of Coomb’s reagent.

> The positive endpoint of the Coomb’s test is agglutination.

• Autoagglutination is seen in some cases of immune-mediated hemolysis (Fig. 4-24).
• If autoagglutination is present, the diagnosis of immune hemolysis is confirmed without running the Coomb’s test.

> Autoagglutination on blood films is seen as three-dimensional clumping of erythrocytes. This may be confused with rouleaux formation.

> Autoagglutination can be distinguished from rouleaux by preparing and examining a saline-diluted wet preparation.

• Place 1 drop of EDTA-blood on a slide.
• Add 2 drops of isotonic saline.
• Coverslip and examine. If clumping is present, autoagglutination is confirmed.

Figure 4-25. Canine blood. Spherocytosis. The smaller RBCs that lack central pallor are spherocytes. These cells are frequent in dogs with immune-mediated hemolytic disease. They can also occur in fragmentation hemolysis. A ghost RBC (arrow) is noted in the center of the field and suggests some degree of intravascular lysis (100x).

Figure 4-26. Spherocytosis. If this were a feline blood smear, the RBC morphology would indicate regenerative anemia. The small RBC without central pallor would represent normal feline erythrocytes. However, this is canine blood and the small RBCs without central pallor are spherocytes. This dog has a marked spherocytosis due to immune-mediated hemolytic disease (100x).

Figure 4-27. Canine blood, RBC ghosts. Several RBCs are noted that have almost no hemoglobin within their cytoplasm. Ghost RBCs usually indicate intravascular lysis of RBC which can occur with immune-mediated disease, Heinz body hemolysis, or with fragmentation injury (100x).

Hemolytic Disease: Heinz Body Anemia

> Occurs in dogs and cats

> Pathogenesis:

• Circulating oxidants act on red cell hemoglobin at two primary sites: the sulfhydryl containing amino acids in globin, and the iron moiety.
• Oxidation of globin leads to precipitation and the formation of Heinz bodies.
• Oxidation of iron leads to methemoglobinemia.
• Methemoglobinemia and Heinz bodies may occur in the same patient but one form is generally predominant.
• Heinz body formation is most prevalent and easiest to recognize.

> Most cases of Heinz body hemolytic anemia are the result of ingestion of oxidizing substances such as onions or the action of oxidizing drugs such as acetaminophen (Fig. 4-28).

> Heinz bodies are precipitates of oxidized hemoglobin.

• They often become fixed to the red cell membrane and are recognized as nose-like projections on the red cell surface.
• Cells from which Heinz bodies have been removed (torn away) may also be observed; these are known as bite cells.

Figure 4-28. Canine blood. Heinz bodies. Round membrane projections on most of the RBCs are Heinz bodies. This dog ate onions, which can cause oxidative injury and Heinz body formation (100x).

> Eccentrocytes are also commonly observed in Heinz body hemolytic anemia.

• Eccentrocytes are red cells which lack central pallor.
• All of the hemoglobin is concentrated at one pole of the cell.
• At the other pole is a small area of unstained cytoplasm bound by a distinct cell membrane.

> Eccentrocytes form when oxidation of the red cell membrane occurs. This leads to fusion of opposing sites on the red cell membrane which pushes the hemoglobin peripherally.

Schema.

> In routine blood films, Heinz bodies have the same staining characteristics as hemoglobin.

> In new methylene blue stained films, Heinz bodies are turquoise (Fig. 4-29).

> Cat hemoglobin is more susceptible to oxidant damage than dog hemoglobin.

• Cats are therefore more susceptible to Heinz body hemolysis.
• Furthermore, Heinz bodies can be seen in large numbers in the absence of hemolytic anemia in certain metabolic conditions in cats (eg, diabetes mellitus, liver disease, hyperthyroidism, etc.).

> The diagnosis of Heinz body hemolytic anemia in cats requires the demonstration of large numbers of Heinz bodies and the presence of a significant regenerative anemia.

> In dogs, if Heinz bodies are present, Heinz body hemolytic disease is confirmed.

Hemolytic Disease: Feline Haemobartonellosis

> Caused by Haemobartonella felis

> H. felis organisms are recognized on the red cell surface as either chains of small (1 μ) basophilic rods or ring forms (Fig. 4-30 and Fig. 4-31).

Figure 4-29. Canine blood. New methylene blue stain. Heinz bodies are noted as turquoise inclusions on the edge of the RBC membranes. Reticulocytes are also present (100x).

Figure 4-30. Feline blood. Haemobartonella felis. Blood smear from an anemic cat reveals the rod and coccoid forms of Haemobartonella felis. Ring forms (arrows) can be seen on the RBC surface in small groups (100x).

> Organisms must be differentiated from stain precipitate (Fig. 4-20) and Cytauxzoon felis (Fig. 4-32).

> Feline haemobartonellosis can occur as a primary disease or secondary to other immunosuppressive disorders such as FeLV infection, FIV infection, or FIP infection.

> Primary haemobartonellosis presents as a typical regenerative hemolytic anemia. Organisms appear intermittently in large numbers on peripheral erythrocytes.

> Secondary haemobartonellosis often presents as a severe nonregenerative anemia.

> Both primary and secondary haemobartonellosis may have an immune-mediated component and be Coomb’s positive.

Figure 4-31. Feline blood. Haemobartonella felis. RBC density on the smear is reduced due to severe anemia. In this area of the smear, Haemobartonella organisms can be seen as basophilic rings on the RBC surface. These parasites produce hemolytic anemia by causing RBCs to be phagocytized by macrophages (100x).

Figure 4-32. Feline blood. Cytauxzoon felis. This is an intracellular protozoan parasite of wild and domestic cats. In domestic cats, the parasite causes a nonregenerative anemia with round, oval, or safety pin shaped basophilic parasites in RBCs. (100x).

Hemolytic Disease: Canine Haemobartonellosis

Less common than in cats

Caused by Haemobartonella canis

H. canis organisms form multiple chains on the red cell surface. Individual organisms are larger and easier to see than H. felis (Fig. 4-33).

Canine haemobartonellosis generally presents as a true regenerative hemolytic anemia. However, it usually occurs secondarily to splenectomy or immunosuppressive events such as chemotherapy.

Figure 4-33. Canine blood. Haemobartonella canis. Long chains of small basophilic coccoid organisms are noted on the surface of two RBCs. In one cell, a double chain, or "bow string" form, is present. These organisms cause mild hemolytic anemia in dogs following splenectomy, or in association with immunosupression (100x).

Hemolytic Disease: Canine Babesiosis

> Tick-borne protozoal disease of dogs.

> The causative agent is Babesia canis (Fig. 4-34) or Babesia gibsoni (Fig. 4-35).

> Babesiosis is a true intravascular hemolytic anemia, often characterized by hemoglobinemia and hemoglobinuria.

> Babesiosis may also have an immune-mediated component and be Coomb’s positive.

> Babesia are recognized on peripheral blood films as intra-erythrocytic teardrop shaped organisms measuring approximately 1.5 (B. gibsoni) to 3μ (B. canis) in length. Up to four organisms may be seen in a parasitized cell.

> As with haemobartonella organisms, prevalence of Babesia organisms in the blood film is cyclic.

Figure 4-34. Canine blood. Babesia canis. Pairs of pyriform intracellular protozoal organisms are consistent with Babesia canis organisms. This parasite causes hemolysis by intravascular and extravascular RBC destruction (100x).

Figure 4-35. Canine blood. Babesia gibsoni. Round, oval, or elongate protozoal organisms that vary in size are present in several RBCs. An eccentric nuclear structure is visible in some organisms. This parasite was endemic in parts of Asia and is now present in North America (120x).

Hemolytic Disease: Pyruvate Kinase Deficiency (PK)

> Pyruvate kinase is an enzyme in the glycolytic pathway essential to the production of energy (ATP).

> ATP is essential to maintaining red cell membrane stability; reduced ATP causes a decrease in red cell lifespan.

> PK deficiency is a heritable hemolytic condition described in Beagles and Basenjis. It is present from the time of birth and usually recognized clinically by 3 years of age.

> In its early phases, pyruvate kinase deficiency induced hemolysis may present as compensated hemolysis. The blood film shows marked regeneration but the patient may not be anemic.

> Anemia develops as the patient ages. In late phases of the disease, the bone marrow apparently becomes exhausted and may become scarred (myelofibrosis). At this point, the anemia is non-regenerative and terminal. There is no treatment for this condition.

> PK deficiency hemolysis has been described as nonspherocytic hemolysis (Fig. 4-36).

• In some cases, spheroechinocytes can be found scattered on blood films of affected animals, but this is not a constant finding.
• Spheroechinocytes are smaller than normal, lack central pallor, and are covered by short sharp spiky surface projections.

> Diagnosis can be confirmed by measuring red cell PK levels.

Figure 4-36. Canine blood. PK deficiency. Pyruvate kinase deficiency causes a chronic hemolytic anemia. RBC lifespan is decreased due to a metabolic defect in RBC metabolism. An intense regenerative response is present characterized by marked polychromasia, anisocytosis, macrocytosis, and NRBCs (100x).

Hemolytic Disease: Phosphofructokinase (PFK) Deficiency

> PFK is also a glycolytic enzyme essential to ATP production.

> Reduced ATP in RBC’s leads to shortened red cell lifespan (hemolysis).

> PFK deficiency is a rare heritable hemolytic condition described only in Springer Spaniels.

> There is no distinctive morphologic footprint for PFK deficiency.

> Diagnosis is confirmed through analysis of red cell enzyme levels.

Hemolytic Disease: Mechanical Hemolysis

> There are two distinct mechanisms for mechanical hemolysis:

• Normal red cells are forced to traverse abnormally tortuous capillary beds. This leads to intravascular shearing of red cells and shortened red cell lifespan. This is termed microangiopathic hemolysis. Common causes include:

• Glomerulonephritis
• DIC
• Hemangiosarcoma

• Normal red cells are exposed to turbulent blood flow in large vessels. Examples include:

• Heartworm disease
• Traumatic disruption of red cells in heart disease

> The morphologic footprint of mechanical hemolysis is the schistocyte. Schistocytes are small irregularly shaped red cell fragments.

Non-regenerative Anemia

Non-regenerative anemias are the result of either ineffective erythropoiesis (maturation defect anemias) or reduced production of red cells (hypoproliferative anemias).

Maturation defect anemias can generally be suspected from changes in the peripheral blood.

With a few notable exceptions, hypoproliferative anemias require bone marrow examination for diagnosis.

Maturation Defect Anemias

These anemias have non-regenerative peripheral blood patterns (absence of increased polychromasia/reticulocytosis) but erythroid hyperplasia in the bone marrow.

Red cell production is therefore ineffective in that the increased red cell production in the marrow is not reflected in the peripheral blood.

Maturation defect anemias are classified as either nuclear maturation defect anemias or cytoplasmic maturation defect anemias.

Figure 4-37. Feline blood. Nuclear maturation defect. Three NRBCs are present in the field. Two of the NRBCs have excessive polychromatophilic cytoplasm. The nucleus in one NRBC (arrow) is very large and has a reticulated chromatin pattern. This nucleus should be found in a cytoplasm that is dark blue with very little visible hemoglobin. The nucleus is maturing at a different rate than the cytoplasm indicating a severe defect in development. This cat had a severe anemia with a very high MCV but no increase in reticulocytes (100x).

Maturation Defect Anemias: Nuclear Defect (megaloblastic anemias)

> The principal problem in these anemias is an acquired bone marrow defect in which precursor nuclei of all cell lines fail to mature and divide normally while cytoplasmic maturation proceeds unimpaired (Fig. 4-37 and Fig. 4-38).

> This nuclear/cytoplasmic asynchrony results in the formation of red cells known as megaloblasts.

> Megaloblasts have the following features:

• Large cells
• Immature, pale ("watery") nuclei with irregular chromatin clumps.
• Cytoplasm is too hemoglobinized for the degree of nuclear maturation.

Figure 4-38. Feline blood. Nuclear maturation defect. Two NRBCs are located in the center of the field. The smaller NRBC appears normal. The larger cell has severe nuclear /cytoplasmic maturation defect. The nucleus in this cell is very immature for the degree of cytoplasmic development. This morphology is sometimes referred to as megaloblastic erythropoiesis. FeLV should be considered as a major cause of this abnormality (100x).

> The presence of megaloblasts in the marrow confirms the diagnosis.

> The nuclear defect is the result of abnormal/reduced DNA synthesis.

> The bone marrow is hypercellular; all cell lines are affected and left-shifted.

> While definitive diagnosis depends on bone marrow morphology, peripheral blood findings are suggestive:

• Mild pancytopenia is usual.
• The anemia generally presents with normocytic, normochromic to macrocytic, normochromic red cell indices.
• Occasional megaloblasts may be present on blood films.
• Occasional giant fully hemoglobinized red cells are present.
• Occasional red cells containing bizarre and/or multiple nuclear fragments may also be observed.

> Nuclear maturation defect anemias are far more common in cats than dogs.

> Nuclear maturation defect anemias are most commonly associated with FeLV infection in cats.

> Folic acid deficiency is also a cause.

> Chemotherapy can cause interference with DNA synthesis and nuclear development (methotrexate) resulting in megaloblastic change.

> Toy Poodles sometimes have megaloblastic red cells in the peripheral blood. In this breed, these findings are normal.

Maturation Defect Anemias: Cytoplasmic Defect

> The principal defect in these anemias is failure to form hemoglobin; nuclear maturation of red cell precursors is normal.

> The result is a hypercellular red cell bone marrow with a build-up of small metarubricytes (Fig. 4-39).

• Red cell precursors continue to divide, getting smaller and smaller, because they never acquire a full complement of hemoglobin.
• Normal reticulocytes are produced and released at a much reduced rate.

> Causes include:

• Iron deficiency
• Lead poisoning
• B6 deficiency

> Iron deficiency and lead poisoning are by far the most important and are described in greater detail.

> Iron deficiency

• The end stage of blood loss anemia.
• Blood films reveal small red cells with an increased area of central pallor (microcytic, hypochromic).
• Increased red cell fragility results in marked poikilocytosis and red cell fragmentation.
• Some polychromasia may be observed but it is inadequate to return the red cell mass to normal.

> Lead poisoning

• A true cytoplasmic maturation defect anemia; lead interferes with hemoglobin synthesis at several points.
• Lead also causes marrow stromal damage which results in release of nucleated red cells into circulation.
• The resultant peripheral blood findings are those of normocytic, normochromic, nonregenerative anemia with large numbers of nucleated red cells.

• Large numbers of nucleated red cells in the absence of an even greater number of polychromatophils is termed an inappropriate nucleated red cell response.

• Inappropriate nucleated red cell responses of greater than 10 NRBC/100 WBC should cause the clinician to investigate the possibility of lead poisoning in dogs and FeLV infection in cats!.

Figure 4-39. Canine bone marrow. Chronic hemorrhage. A marked increase in the late members of the erythroid series is present. Erythroid maturation is active up to the rubricyte and metarubricyte stages. Marrow iron stores are decreased due to chronic blood loss. These changes are consistent with iron deficiency anemia in the early stages (40x).

Hypoproliferative Non-regenerative Anemias (Erythroid Marrow Hypoplasia)

These anemias fall into three classes:

> Hypoproliferative anemia with granulocytic hyperplasia.

> Hypoproliferative anemia with selective erythroid hypoplasia.

> Hypoproliferative anemia with generalized marrow hypoplasia.

Hypoproliferative Anemia with Granulocytic Hyperplasia

> This is the anemia of inflammatory disease (Fig. 4-40).

> The anemia of inflammatory disease is the most common form of anemia in domestic animals and occurs with acute or chronic inflammation.

> Features include:

• Normocytic, normochromic anemia
• Anemia is mildly to moderately severe (hematocrits range from 20 - 35%).
• There is an inflammatory leukogram.
• Marrow smears are characterized by erythroid hypoplasia, granulocytic hyperplasia, increased marrow iron in marrow macrophages, and often increased numbers of plasma cells.

> This anemia can be associated with infections, neoplastic processes, and immune disorders.

Figure 4-40. Canine bone marrow. Anemia of inflammation. The marrow is cellular in this dog (PCV = 33, inflammatory leukogram). The M:E ratio is increased due to granulocytic hyperplasia and erythroid hypoplasia. Plasma cells and iron stores are increased. These findings are consistent with the anemia of inflammation (40x).

Anemias with Selective Erythroid Hypoplasia

> Peripheral blood findings include:

• Mild to severe normocytic normochromic anemias
• Normal white cell and platelet counts

> There are three major mechanisms involved in the genesis of selective erythroid hypoplasia.

• Reduced erythropoietin production (renal disease: Fig. 4-41)
• Reduced oxygen demand and utilization by peripheral tissues (reduced basal metabolic rate as in hypothyroidism)
• Selective destruction of red cell precursors by toxic or immune- mediated mechanisms. A complete history which includes enumeration of possible drug or chemical exposure is extremely important.

> Bone marrow evaluation is required for confirmation. Features include:

• Normal granulocyte and platelet production
• Rare red cell precursors
• Increased marrow iron
• Possible erythrophagocytosis by marrow macrophages

Figure 4-41. Feline bone marrow. Anemia of renal disease. Selective depression of erythropoiesis occurs in chronic renal disease due to diminished erythropoietin production. The M:E ratio is increased due to erythroid hypoplasia. Nearly all cells in the field are developing granulocytes (25x).

Anemias with Generalized Marrow Hypoplasia (also known as aplastic anemias)

> Peripheral blood features include:

• Severe anemia
• Severe leukopenia
• Variable platelet counts, often severe thrombocytopenia

> Etiologic mechanisms fall into two major categories:

• Toxic or immune-mediated destruction of precursors in all cell lines (Fig. 4-42)
• Myelophthisic anemias (replacement of marrow space by abnormal cellular elements)

Figure 4-42. Feline bone marrow. Severe marrow hypoplasia. This cat presented with severe pancytopenia. Bone marrow is extremely hypocellular and contains adipose tissue and some connective tissue elements. Normal myeloid cells are rare. This cat had been treated with griseofulvin for a skin fungus. This drug can cause severe marrow hypoplasia in cats (25x).

> Generalized marrow cytotoxicity can be caused by infectious agents, toxic chemicals, ionizing radiation, or immune destruction of marrow stem cells.

• Infectious causes include FeLV and ehrlichiosis.
• Toxic causes includes estrogen and chemotherapeutic agents such as Adriamycin.
• Clinical presentation depends upon the severity of the various cell defects at diagnosis.

• If anemia is the most prevalent, then pale mucous membranes, lethargy, and anorexia are likely to be the presenting signs.
• With profound thrombocytopenia, bleeding will be the presenting problem.
• Where severe leukopenia is the primary problem, animals tend to present with serious infections.

• Confirmation of diagnosis depends on marrow aspiration and core biopsy. Findings vary with the stage of the process.

> Myelophthisic anemias

• May be caused by both neoplastic and non-neoplastic diseases.
• The most common neoplastic causes are the hematopoietic and lymphoid neoplasms including granulocytic leukemia, lymphoid leukemias and lymphosarcoma, and erythremic myelosis (Fig. 4-43).
• The principal non-neoplastic cause is myelofibrosis, the replacement of marrow spaces by connective tissue (Fig. 4-44).

• Myelofibrosis may be the endpoint of previous severe marrow injury (as in the case of estrogen toxicity and ionizing radiation) or it may occur spontaneously.

• Peripheral blood features of myelofibrosis usually include severe nonregenerative anemia, severe leukopenia, and a variable platelet response.

• A striking morphologic feature of many cases of myelofibrosis is the presence of dacryocytes or tear-drop shaped erythrocytes on the blood film.

• Confirmation of the diagnosis depends on marrow core biopsy and histopathology with a demonstration of connective tissue filling marrow space.

Figure 4-43. Canine bone marrow. Myelophthisis. Normal myeloid cells have been replaced by a population of dark round blast cells. Two neutrophils are visible in the entire field. Closer examination of the cells confirmed a diagnosis of lymphoma (40x).

Figure 4-44. Core biopsy of bone marrow. Myelofibrosis. The marrow spaces contain palisading layers of fibrous connective tissue instead of developing granulocytes and erythroid precursors. This dog was pancytopenic, and repeated attempts to aspirate bone marrow were unsuccessful (40x).

Polycythemia (Table 4-1)

Polycythemia is defined as increased circulating red cell mass.

Values for PCV, hemoglobin concentration, and RBC count are higher than reference ranges due to a relative or absolute increase in circulating RBCs.

Reference values for PCV, hemoglobin, and RBC count can vary with geographic location and breed.

> Animals at altitudes >6000 ft have higher values than those at sea level.

> Brachycephalic breeds have higher PCVs than normocephalic breeds.

Severity of clinical signs in polycythemic animals is proportional to the degree of RBC excess.

> At PCV values >65%, hyperviscosity, poor perfusion, and reduced oxygenation of tissues are present.

Polycythemias can be classified as relative, transient, or absolute.

Relative Polycythemia

Relative polycythemia occurs when a decrease in plasma volume, usually due to dehydration, produces a relative increase in circulating RBCs.

> Clinical findings - Dehydration or shift of plasma H2O to interstitium or gastrointestinal lumen.

> Causes - Vomiting, diarrhea, diminished water intake, diuresis, hyperventilation, renal disease.

> Laboratory features: Moderate increase in PCV and total protein concentration in plasma. Prerenal azotemia may also be present.

Transient Polycythemia

Transient polycythemia is caused by splenic contraction which injects concentrated RBC into circulation.

> Occurs in anxious or excitable dogs or cats and subsides within an hour.

> Large excitable breeds are prone to splenic contraction.

> Laboratory features: Increase in PCV with normal hydration and normal total protein concentration in plasma.

Absolute Polycythemias

Absolute polycythemia is characterized by an absolute increase in the circulating RBCs as a result of increased marrow production.

> Classified as either primary, or secondary to increased production of EPO.

> Animals with absolute polycythemia have an expanded blood volume.

> Clinical findings - General lethargy, low exercise tolerance, behavioral change, brick red or cyanotic mucous membranes, sneezing, bilateral epistaxis, increased size and tortuosity of retinal and sublingual vessels, or cardiopulmonary impairment.

Primary absolute polycythemia (polycythemia rubra vera) is a rare myeloproliferative disorder characterized by the uncontrolled but orderly production of excessive numbers of mature RBCs.

> Clinical findings - In addition to above, variable degrees of splenomegaly, hepatomegaly, thrombosis, hemorrhage, and seizure activity.

> Hyperviscosity may result in thrombosis, infarction, or hemorrhage.

> Laboratory features: Marked increase in PCV (values of 65 - 75%) with erythroid hyperplasia in marrow. Clinical evidence of hypoxemia is not apparent. Erythropoietin levels are normal or reduced.

Secondary absolute polycythemia is caused by a physiologically appropriate release of EPO resulting from chronic hypoxemia.

> Clinical findings - Hypoxemia as a result of chronic pulmonary disease, cardiac disease or anomaly with right to left shunting, or hemoglobinopathy.

> Causes - Chronic pulmonary disease, cardiac disease, cardiac anomaly with right to left shunting, high altitude, brachycephalic breeds, methemoglobinemia, and impairment of renal blood supply.

> Laboratory features: Moderate to marked increase in PCV and marrow erythroid hyperplasia with decreased arterial PO2 content. Erythropoietin levels are increased.

Secondary absolute polycythemia is also caused by an inappropriate and excessive production of EPO or an EPO-like substance in an animal with normal arterial oxygen saturation.

> Clinical findings - Signs associated with renal or hepatic neoplasia, space-occupying renal lesion, or endocrine disorder.

> Causes - Renal cyst or tumor, hydronephrosis, hyperadrenocorticism, hyperthyroidism, pheochromocytoma, nasal fibrosarcoma, hepatic neoplasia, hyperandrogenism.

> Laboratory features: Moderate or marked increase in PCV with marrow erythroid hyperplasia and a normal arterial PO2 content. Erythropoietin levels plasma are increased.