Nuances of blood films: interpreting abnormal WBC and red cell morphology (Proceedings)

The first part of this session will discuss increased or atypical WBC morphology and describe clues to determine whether the process is leukemic or reactive in origin.

Leukemia = the presence of neoplastic cells in circulation. Leukemia can arise independently of, or concurrently with tissue neoplasia. The opposite is also true. A hematopoietic cell neoplasm can be localized to the BM or tissue without being seen in circulation.

Lymphoproliferative disease is the general term to describe an abnormal expansion (often neoplastic, but occasionally reactive) of the lymphoid line.  Lymphoma or lymphosarcoma describes a solid, sarcomatous lymphoid tumor which may involve a variety of organs, such as lymph nodes, liver, spleen, kidney, and intestines.  Bone marrow is involved in a small percentage of cases.

Myeloproliferative disease (MPD) is the proliferation of any bone marrow origin cell and includes the following: undifferentiated MPD or blastic form, granulocytic, eosinophilic, basophilic, monocyticmyelomonocytic (neutrophils and monocytes), egakaryocytic, erythemic myelosis (RBC), and erythroleukemia (RBC and WBC).Myelofibrosis is a poorly understood entity characterized by proliferation of hemic cells, as well as diffuse fibrosis in the bone marrow.  Extramedullary hematopoiesis accompanies these changes.  Myelofibrosis is a form of MPD and may also be a terminal stage of other forms of MPD.

Leukemia is suspected when the white cell count is high with increased numbers of blasts cells and/or atypical cells. The bone marrow should be sampled when the blood is not definitive or when trying to identify the specific type of leukemia. A bone marrow aspirate provides more information on cell morphology. A bone marrow core biopsy provides more information regarding bone marrow architecture.  This is best used to identify focal lesions, fibrosis, and cellularity. If the bone marrow contains greater than 30% blast cells and lack orderly maturation, it is consistent with myeloproliferative disease.  If less than 30% blast, the process is classified as dysmyelopoiesis.

Acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) are relatively easy to diagnose due to the presence of significant numbers of blasts or other atypical cells.  In the dog, acute leukemia appears slightly more likely to be of myeloid than lymphoid origin. However, it may be difficult to differentiate AML from ALL through examination of hematologic or cytologic features. In addition, AML in dogs and cats is difficult to diagnose through flow cytometry immunophenotyping as the available antibody panels do not provide lineage specific myeloid markers. There is also no PCR test for clonality for AML. CD34 (stem cell marker) can be found on both AML and ALL. One of the best markers for AML is myeloperoxidase. Currently, this is detected by most labs through cytochemistry on cytology or histology slides. Subtle signs that are often used to differentiate AML from ALL on the blood film include the presence of granules or vacuoles in the cytoplasm, a finer nuclear chromatin pattern and as slightly more centralized nuclear placement.  If there is even a slight continuum to more differentiated cells, this can be helpful but many blastic or intermediate leukemias are poorly differentiated. 

Well-differentiated neoplastic cells are easily identified.  The problem is differentiating between a reactive versus a neoplastic process.  For example:

• Neutrophilic left shifts can (rarely) go back to blasts. Frequently they extend to myelocytes and sometimes progranulocytes with many inflammatory and infectious diseases. Thus, the presence of immature cells in circulation does not necessarily equal leukemia.

• A chronic lymphocytosis can be seen as a reactive process against Ehrlichia and other agents and the range of lymphocyte counts reported overlaps that seen for leukemias.

• Eosinophilia can be seen as a reactive process against parasites or other neoplastic processes, can be part of the syndrome of hypereosinophilic disorders, or can represent an eosinophilic leukemia.

• Significant monocytosis can be seen with some immune mediated diseases (e.g. IMHA) and chronic inflammatory diseases as well as part of monocytic and myelomonocytic leukemias.

• Basophilia tends to be seen with eosinophilia, but when it occurs alone, suspect altered plasma lipoprotein metabolism (hypothyroidism, hyperadrenocorticism, chronic liver disease, diabetes mellitus, protein?losing renal disease and genetic hyperlipoproteinemias).

• Mastocytosis can be seen as part of a malignant, disseminated mast cell neoplasm. But has also been reported with several acute inflammatory disorders (canine parvoviral enteritis, skin diseases).

Changes that raise the index of suspicion for myeloid leukemia

• Persistent and/or increasing numbers despite treatment

• Irregular nuclear membrane (particularly for monocytic precursors)

• Giant or bizarrely segmented neutrophils

• Lack of differentiation to mature neutrophils

• A more blue grey cytoplasm as opposed to the deeply basophilic cytoplasm of lymphoblasts

• Vacuoles

• A dusting of azurophilic granules

• In cats, dark basophilic (versus lilac) granules within basophils and basophilic precursors

• Marked thrombocytosis

• Hemoglobinization of cells 

• Asynchrony of nuclear and cytoplasmic maturation

• Cytoplasmic blebs in circulation

• Mitotic figures in circulation

Chronic lymphocytic leukemia

In dogs, CLL appears to be primarily a T-cell disease although B-cell CLL has also been reported (3:1 ratio of T:B). The cytologic morphology of T-cell CLL in dogs is typically of granular lymphocytes. B-cell CLL and T-cell CLL appear to have somewhat different patterns of disease progression.  B-cell CLL affects the bone marrow early in disease.  T-cell CLL typically does not affect the bone marrow until late in the disease and may spread from the marrow after splenic involvement. In dogs, a chronic lymphocytosis comprised of intermediate sized lymphocytes with small azurophilic granules has been reported in association with Ehrlichiosis.  These cells are also CD8+ and must be differentiated from CLL.

Feline CLL is primarily a T-cell disease. However, unlike that seen in dogs, feline CLL is primarily a result of CD4 or helper T-cell proliferation although cases of CD8+, CD4CD8 double positive, and CD4CD8 double negative CLL have also been reported.  These cells may contain granules however they are more often small than the large granules seen with large granular lymphocyte neoplasia.

Non-neoplastic WBC changes

•    Left shift indicates the presence of immature granulocytes (usually neutrophils) in the blood. Regenerative left shift: mature cells outnumber immature neutrophils (and WBC is usually increased). Degenerative left shift:  immature neutrophils outnumber mature cells.

•     Toxic neutrophils? basophilic cytoplasm, vacuolated (foamy) cytoplasm, basophilic granulation, and Döhle bodies (bluish angular cytoplasmic inclusions) are characteristics of toxic neutrophils.  Toxic neutrophils indicate inflammation.

•     Degenerate neutrophils - is a term reserved for neutrophils outside the blood system (e.g. cytology). Characterized by plump smooth nucleus and nuclear breakdown.

•     Barr bodies – female sex chromatin, appears as drumstick appendage on some neutrophil nuclei.

•     Hypersegmented neutrophils ? neutrophils with 5 or more distinct nuclear lobes.  Indication of prolonged transit time (glucocorticoids) or maturation defect (B?12 or folic acid deficiency).

Disease syndromes associated with WBC changes

• Leukocyte adhesion protein deficiency (LAD) reported primarily in Holstein cattle, but also in Irish setter dogs.  A deficiency of the surface glycoprotein adhesion molecule (CD11/CD18) prevents cells from leaving circulation.

• Pelger-Huet anomaly is a hereditary disorder characterized by failure of the granulocyte nucleus to undergo normal segmentation. These cells have normal appearing cytoplasm, clumped chromatin, and apparently normal function in dogs, cats, and humans. However, the homozygous form in rabbits is lethal.  The concern is not to mistake these cells for a left shift inflammatory process.

• Chediak?Higashi syndrome (reported in albino mink, mice, cattle, cats, and killer whales) is an autosomal recessive disease characterized by recurrent infections and abnormal, giant phagocytic lysosomal granules, and defective microbiocidal activity.

• Cyclic neutropenia or Grey collie syndrome.  This is a stem cell defect resulting in cyclic myelopoiesis every 11 days.  All cell lines are affected, but the clinical signs are usually referable to cyclic neutropenia.

• Storage diseases. A diverse group of genetic diseases characterized by storage of incompletely degraded material in lysozymes of macrophages, neurons, and other parenchymal cells.  Frequently, granules and/or vacuoles will be seen in leukocytes. These have been described in most species.

• Abnormal neutrophil granulation of Birman cats.  The granules may appear similar to those of certain storage diseases, but the cells appear to function normally and the cats are clinically normal.

• The second part of this session will discuss cases where changes in erythrocyte morphology are critical to identifying the underlying cause.  For example, signs of iron deficiency or iron sequestration can be suspected by finding keratocytes and ‘apple stem cells' while hemangiosarcoma is suggested by finding acanthocytes in the dog. The diagnostic criteria to be illustrated during the session are described below.

• Fragmentation (microangiopathic hemolytic anemias, DIC) - Red cells are injured by abnormal vasculature (microangiopathy) or fibrin deposited in the vessels (DIC).  Thus fragmentation anemia is a special type of intravascular hemolytic anemia.  Acanthocytes and fragmented red cells (schistocytes) and occasionally keratocytes can be observed on the blood smear.  Platelets may be decreased.  Causes are variable and include DIC, neoplasia (hemangiosarcoma), vasculitis, burns, heart valve damage and heart worm disease (vena cavae syndrome).

• Acanthocyte (spur cell) ? spiculated red cell with single or multiple, blunt, rounded projections.  These are associated with microangiopathy and fragmentation anemia (dogs especially) and liver disease (more in cats).  The latter is due to excess cholesterol:phospholipids ratio in the RBC membrane.

• Keratocyte (helmet cell) - a spiculated RBC with one or 2 pointed projections.  Also in iron deficiency.

• Schistocyte (schizocyte)? fragmented red cell. 

• Differentiated echinocytes from acanthocytes. Echinocyte - spiculated red cell with numerous short, evenly spaced surface projections. Type 3 has a very prickly spine appearance and is associated with snake bite envenomation in dogs. Types 1 and 2 are more blunt, may be due to electrolyte abnormalities, dehydration, or crenation.

• Immune mediated destruction - Immune mediated implies that RBC destruction is due to antibody, complement, or both.  It does not imply the underlying cause.  Immune mediated can be primary i.e. directed against the animals own red cells (autoimmune) or secondary as the result of an immune response against infectious agents, drugs, viral infections, hormonal imbalance, genetic influences, stress, pregnancy, and vaccines.  Antibodies coat erythrocytesleading to primarily extravascular hemolysis and, less commonly, intravascular hemolysis. Spherocytosis and agglutination are the hallmark changes of immune mediated destruction, however agglutination is not seen in all cases and depends on the amount and type of the antibody directed against the patient's RBCs. 

• Agglutination ? grape like aggregation of red cells, will not disperse with saline dilution. 

• Ghost cells – Indistinct erythrocytes that have undergone lysis but still retain some membrane.

• Spherocytes ? small dense cells lacking central pallor.  Result from excessive loss of surface membrane without the same loss of cellular contents.  Cells will appear smaller on the blood film but have a normal MCV.  Spherocytes are easiest to see in dogs.  Occasionally small numbers of spherocytes can be seen as the end stage of any process that results in loss of surface membrane, therefore the presence of a few spherocytes should not be considered diagnostic for immune mediated destruction.

• Howell?Jolly bodies ? basophilic nuclear remnants usually near edge of red cell.  May be seen with accelerated erythrocyte production.  *Seen with Romanowsky stains (don't confuse with Heinz bodies).

• Hemoglobinemia - results from intravascular hemolysis.

• Oxidative damage - Heinz bodies and eccentrocytes can form as a result of denaturation of hemoglobin (specifically to the globin moiety) and leads to destruction of the erythrocytes by both intravascular and extravascular mechanisms.  Membrane proteins can also undergo oxidation.  Finally, oxidation of hemoglobin iron results in the formation of methemoglobin which interferes with oxygen transport but does not cause anemia. Heinz body and eccentrocyte formation may occur simultaneously or independent of methemoglobinemia.  In most cases the oxidant is due to a drug or diet derived compound.  Enzyme deficiencies (G6PD, glutathione reductase) are uncommon.  Oxidants include phenothiazine, onions, naphthalene, methylene blue, acetaminophen, phenyl compounds, propylene glycol in semi moist cat food, crude oil in birds, and ketoacids especially in cats.

• Heinz bodies (erythrocyte refractile bodies) ? round structures (representing denatured hemoglobin) on the internal RBC membrane.  They do not stain with Romanowsky stains but are visible as light blue structures with reticulocyte stains such as new methylene blue stain. Cats are more susceptible to Heinz body formation because of the large number of sulfur containing amino acids in feline hemoglobin which are readily oxidized. Cat spleens are also not as sinusoidal, resulting in less efficient Heinz body removal.  Healthy cats may have some (usually small) Heinz bodies present in their blood.  Diagnosis of Heinz body anemia requires finding larger numbers of usually large Heinz bodies. Unstable hemoglobin and increased numbers of Heinz bodies are seen in cats with diabetes mellitus especially with ketoacidosis, hyperthyroidism, lymphoma.

• Eccentrocyte (pyknocyte) - red cell with condensed hemoglobin in one area of the cell.  These occur due to oxidative damage to the hemoglobin.

• Iron deficiency or iron sequestration - The unavailability of iron can be due to deficiency (e.g. dietary insufficiency or blood loss) or the inability to appropriately use iron due to sequestration from inflammatory or chronic disease or portovascular anomalies. Classically, iron deficiency anemia is microcytic and hypochromic while sequestration types of anemia start as normocytic normochromic, but both can appear similar.  The inability to obtain adequate iron for normal hemoglobin maturation results in cells that have cytoplasmic fragility. Morphologic changes progress from a ‘blister' to a keratocyte to ‘apple stem cells'.  Thrombocytosis is commonly associated with iron deficiency anemia and iron sequestration disorders due to chronic inflammation (but is not reported in portovascular anomalies).

• Keratocyte (helmet cell) - a spiculated red cell with one or 2 pointed projections.  It results from the rupture of a vacuole formed near the cell surface and are frequently seen, in association with other fragments, in iron deficiency anemia but can also be seen in other processes of mechanical damage.

• FeLV associated anemias - FeLV infected cats may have a non-regenerative normocytic or macrocytic anemia.  Macrocytosis without signs of regeneration should raise the index of suspicion form FeLV, however only a subset of FeLV infected cats will have macrocytosis.

• Lead poisoning - The anemia is usually mild (can be more severe in chronic poisoning) and is characterized by an inappropriate number of nRBCs and (sometimes) basophilic stippling. Basophilic stippling (small blue dots within the RBCs on Romanowsky stains) is most consistent in dogs.       

• Nucleated red cell (nRBC) ? immature red cell with a nucleus in peripheral blood.  These can be seen during periods of intense marrow stimulation, with bone marrow injury, and after splenectomy.  Presence of nucleated RBC without progression to polychromasia suggest lead poisoning.

• B12, cobalamin, folic acid deficiency - Macrocytes are usually immature cells (reticulocytes) seen in regenerative anemias.  Macrocytes (large cells with an increased MCV) can be seen in the absence of a regenerative anemia with B12, cobalamin and/or folic acid deficiency.  Cobalamin deficiency has been reported in dogs (Giant Schnauzers, Border Collies, Beagles, and Australian Shepherds) and cats.

Macrocytic/microcytic RBCs

Mild macrocytic, hypochromic anemia has been observed in Alaskan Malamutes with hereditary chondrodysplasia and stomatocytosis. Miniature and toy poodles occasionally are recognized with macrocytosis.  These dogs may have other changes including hypersegmented neutrophils, multiple Howell Jolly bodies or nRBCs with fragmented nuclei but tend not to have clinical signs (e.g. anemia) associated with these changes.

• Microcyte ? small cells, decreased MCV.  These are commonly seen in iron deficiency or in anemia of chronic or inflammatory disease.  Microcytosis is often present in portosystemic shunts.  Microcytosis is normal in Akita and Japanese Shiba dogs

• Stomatocytes ? red cells with mouth shaped (oval) areas of central pallor. Hereditary stomatocytosis has been described in Alaskan malamutes & schnauzers. Can be artifacts in the thick area of the smear.