Methemoglobinemia

Hemoglobin is a protein consisting of four polypeptide globin chains, each of which contains a heme prosthetic group within a hydrophobic pocket. Heme is composed of a tetrapyrrole with a central iron molecule that must be maintained in the ferrous (+2) state to reversibly bind oxygen. Methemoglobin differs from hemoglobin only in that the iron molecule of the heme group has been oxidized to the ferric (+3) state and is no longer able to bind oxygen.²⁸ The presence of large amounts of deoxyhemoglobin accounts for the dark bluish color of normal venous blood samples. Methemoglobinemia may not be recognized in venous blood samples because the brownish color of methemoglobin is not readily apparent when methemoglobin is mixed with deoxyhemoglobin (Fig. 2-3, A). When deoxyhemoglobin binds oxygen to form oxyhemoglobin, it becomes bright red; consequently the brownish coloration of methemoglobin becomes more apparent in the oxygenated samples (Fig. 2-3, B). A simple spot test provides a rapid way to oxygenate a venous blood sample and to determine whether clinically significant levels of methemoglobin are present. One drop of blood from the patient is placed on a piece of absorbent white paper and a drop of normal control blood is placed next to it. If the methemoglobin content is 10% or greater, the patient’s blood will have a noticeably brown color compared with the bright red color of control blood (Fig. 2-4).²⁸ Accurate determination of methemoglobin content requires that blood be submitted to a laboratory that has this test available. Methemoglobinemia results from either increased production of methemoglobin by oxidants or decreased reduction of methemoglobin resulting from a hereditary deficiency in the erythrocyte cytochrome-b₅ reductase enzyme (see Chapter 4).²⁷

FIGURE 2-3 Gross appearance of mixtures of oxyhemoglobin, deoxyhemoglobin, and methemoglobin. A, Venous blood sample from a cat with 28% methemoglobin (left sample) compared with blood from a normal cat with less than 1% methemoglobin (right sample). Both samples also contain a mixture of oxyhemoglobin and deoxyhemoglobin. B, Oxygenated blood sample from a cat with 28% methemoglobin (left sample) compared with blood from a normal cat with less than 1% methemoglobin (right sample). The sample on the left contains a mixture of oxyhemoglobin and methemoglobin; the one on the right contains almost exclusively oxyhemoglobin.

FIGURE 2-4 Methemoglobin spot test. A drop of blood from a methemoglobin reductase-deficient cat with 50% methemoglobin (left) is placed on absorbent white paper next to a drop of blood from a normal cat with less than 1% methemoglobin.

Erythrocyte Agglutination

The appearance of red granules in a well-mixed blood sample (Fig. 2-5) suggests the presence of erythrocyte autoagglutination. However, it is important to differentiate agglutination (aggregation of erythrocytes together in clusters) from rouleaux (adherence of erythrocytes together like a stack of coins), which can be seen in the blood from healthy horses and cats (Fig. 2-6). Rouleaux formation is eliminated by washing erythrocytes in physiologic saline, but agglutination is not. This differentiation requires centrifugation of blood, removal of plasma, and resuspension of erythrocytes in saline. A rapid way to differentiate rouleaux from agglutination is to mix five drops of physiologic saline with a drop of anticoagulated blood on a glass slide and examine it as a wet mount using a microscope. This dilution reduces rouleaux, but agglutination is not affected. The microscopic appearance of agglutination in a sample of washed erythrocytes is shown (Fig. 2-7). The presence of agglutination indicates that the erythrocytes have increased surface-bound immunoglobulins. These immunoglobulins are usually of the IgM type when agglutination is present, because the greater distance between binding sites on IgM molecules compared with IgG molecules makes it easier for IgM molecules to overcome normal repulsive forces between negatively charged erythrocytes.⁶⁷ In addition, there are 10 antigen-binding sites per IgM molecule compared with 2 binding sites per IgG molecule (Fig. 2-8). A direct antiglobulin test is not needed to identify the presence of immunoglobulin bound to erythrocytes if agglutination is present in saline solution-washed erythrocyte samples.

FIGURE 2-5 Grossly visible agglutination in blood from a dog with immune-mediated hemolytic anemia.

FIGURE 2-6 Microscopic rouleaux in an unstained wet mount preparation of normal cat blood.

FIGURE 2-7 Microscopic agglutination in an unstained wet mount preparation of saline-washed erythrocytes from a foal with neonatal isoerythrolysis.

FIGURE 2-8 Antierythrocyte IgM antibodies causing erythrocyte agglutination.

Packed Cells

The PCV is measured after centrifugation by determining the fraction of total blood volume in a microhematocrit tube that is occupied by erythrocytes. Leukocytes and platelets are primarily located within the buffy coat, although certain leukocyte types may be present in the top portion of the packed erythrocyte column in some species (e.g., neutrophils in cattle). The width of the buffy coat generally correlates directly with the total leukocyte count. A large buffy coat suggests leukocytosis (Fig. 2-9, A) or thrombocytosis, and a small buffy coat suggests that low numbers of these cells may be present. The buffy coat may appear reddish owing to the presence of a marked reticulocytosis.

FIGURE 2-9 Gross appearance of microhematocrit tubes demonstrating leukocytosis, icterus, hemolysis, and lipemia. A, Microhematocrit tube from a cat with a large buffy coat resulting from a chronic lymphocytic leukemia. B, Microhematocrit tube from an anemic cat, with icteric plasma secondary to hepatic lipidosis. C, Microhematocrit tube with evidence of hemolysis in plasma from a cat with acetaminophen-induced Heinz body hemolytic anemia. Less dense erythrocyte ghosts can be seen above the packed intact erythrocytes. D, Microhematocrit tube with evidence of hemolysis in plasma from a horse with intravascular hemolysis induced by the inadvertent intravenous and intraperitoneal administration of hypotonic fluid. Less dense erythrocyte ghosts can be seen above the buffy coat. E, Microhematocrit tube with marked lipemia in plasma from a dog with hypothyroidism that was also being treated with prednisone for an allergic dermatitis. It was unclear from the medical record if this was a fasting blood sample.

A, Courtesy of Heather Wamsley.

The Appearance of Plasma

Plasma is normally clear in all species. It is nearly colorless in small animals, pigs, and sheep but light yellow in horses because they naturally have higher bilirubin concentrations. Plasma varies from colorless to light yellow (carotenoid pigments) in cattle, depending on their diet.⁶² Increased yellow coloration usually indicates increased bilirubin concentration. This increase often occurs secondarily to anorexia (fasting hyperbilirubinemia) in horses owing to reduced removal of unconjugated bilirubin by the liver.¹³ Failure of the liver to remove unconjugated bilirubin from blood has also been reported to cause hyperbilirubinemia in one-fourth of the sick (often anorectic) cattle in one study.⁴⁰ In other species, yellow plasma with a normal HCT suggests hyperbilirubinemia secondary to liver disease. Hyperbilirubinemia associated with a marked decrease in the HCT suggests an increased destruction of erythrocytes; however, the concomitant occurrence of liver disease and a nonhemolytic anemia could produce a similar finding (Fig. 2-9, B).

Red discoloration of plasma indicates the presence of free hemoglobin. This discoloration may represent either true hemoglobinemia, resulting from intravascular hemolysis, or hemolysis occurring after sample collection due to such causes as rough handling, fragile cells, lipemia, or prolonged storage (Fig. 2-9, C,D). The HCT value may help differentiate between these two possibilities, with red plasma and a normal HCT suggesting in vitro hemolysis. The concomitant occurrence of hemoglobinuria indicates that intravascular hemolysis has occurred. Plasma will also appear red following treatment with cross-linked hemoglobin blood substitute solutions.

Lipemia is recognized as a white opaque appearance caused by chylomicrons and very low density lipoproteins (VLDLs). The presence of chylomicrons may also result in a white layer at the top of the plasma column (Fig. 2-9, E). The presence of lipemia is frequently the result of a recent meal (postprandial lipemia), but diseases including diabetes mellitus, pancreatitis, hypothyroidism, hyperadrenocorticism, protein-losing nephropathy, cholestasis, obesity, and starvation may also contribute to the development of lipemia in dogs and cats.^23,72 Transient lipemia and accompanying anemia has been described as a syndrome in kittens, but a direct link between hyperlipidemia and anemia has not been clearly documented.²³ Hereditary causes of lipemia include lipoprotein lipase deficiency in cats and idiopathic hyperlipidemia in miniature schnauzer dogs.^20,73

Equids of any age and either sex may develop lipemia, but obese ponies, miniature horses, and miniature donkeys are most susceptible. Lipemia has been associated with a broad range of diseases, but it is more prevalent in association with pregnancy, lactation, and/or anorexia.⁶⁵ Lipemia has also been reported in llamas and alpacas with severe systemic diseases. It is not associated with age, sex, or reproductive status in these camelids.⁶⁴ The pathogenesis of marked secondary hyperlipidemia is not always clear. It often appears to result from a mobilization of unesterified fatty acids from adipose tissue and the subsequent overproduction of VLDLs by the liver, but it may also result from ineffective clearance of VLDLs by tissues or a combination of both, as occurs in diabetes mellitus.

Plasma Protein Determination

After the PCV is measured and the appearance of the plasma and buffy coat is noted, the microhematocrit capillary tube is broken just above the buffy coat and the plasma is placed in a refractometer for plasma protein determination. Plasma protein concentrations in newborn animals (approximately 4.5 to 5.5 g/dL) are lower than adult values and increase to within the adult range by 3 to 4 months of age.³³ The presence of lipemia or hemolysis will falsely increase the measured plasma protein value. Maximum information can be gained by interpretation of the HCT and plasma protein concentrations simultaneously (see Chapter 4).

Fibrinogen Determination

Fibrinogen can be measured in a hematocrit tube because it readily precipitates from plasma when heated to 56°C to 58^oC for 3 minutes. The difference between the total protein of the plasma and the total protein of the defibrinogenated (heated) plasma gives an estimate of the fibrinogen concentration in the plasma.³² This method is useful in identifying high fibrinogen concentrations, but it is not accurate in identifying low fibrinogen concentrations.^4,11

Manual Cell Counting

Manual erythrocyte counts are not accurate enough to be useful. Manual total leukocyte counts and platelet counts can be performed using commercially available reservoirs and pipettes to dilute the sample and lyse erythrocytes prior to the microscopic counting of leukocytes and platelets using a hemacytometer chamber. Manual leukocyte counts and platelet counts are done when errors are suspected in cell counts generated by automated cell counters. Manual cell counts may also be done in an emergency situation when automated cell counts are not available.

All blood cell types in birds and reptiles are nucleated, making accurate separation and counting difficult or impossible with automated cell counters. Consequently manual leukocyte counts are generally required in nonmammalian species. Thrombocytes can be estimated based on the number present in stained blood films.⁴⁷

If properly maintained, automated blood cell counters are more precise and accurate than manual techniques in mammalian species. Various technologies—including quantitative buffy coat analysis, impedance measurements, laser flow cytometry, and cytochemistry—are utilized to generate these cell counts.

Automated Cell Counting

Quantitative buffy coat analysis (QBC VetAutoread Hematology System, IDEXX, Inc., Westbrook, ME) depends on variations in cell density to separate cell types. Cells are not actually counted; instead the widths of the various layers of cell types that form are measured and the cell “count” is generated assuming a standard cell size for the cell type in question.³⁵

Impedance counters such as the Heska CBC-Diff (Heska Corporation, Loveland, CO) and Abaxis VetScan HMII (Abaxis, Union City, CA) depend on the principle that cells are poor electrical conductors. Blood is diluted in an electrically conductive solution and a precise volume of this diluted suspension is drawn through a small aperture between two electrodes. Each cell produces a change in electrical impedance, resulting in a change in voltage that is proportional to the size of the cell counted. Several thousand cells per second can be counted and sized. Erythrocytes and platelets can be differentiated by size alone in many species using impedance counters, but not in cats, because their platelets are large and their erythrocytes relatively small. Leukocytes are counted as free nuclei following lysis of erythrocytes and platelets.

The development of laser flow cytometry for use in instruments such as the CELL-DYN 3500R (Abbott Laboratories, Abbott Park, IL), the Advia 120 (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY), the Sysmex XT-2000iV (Sysmex Corporation, Kobe, Japan), and the LaserCyte (IDEXX, Inc., Westbrook, ME) has allowed for cells to be characterized in greater detail. Individual cells pass through a laser beam, absorbing and scattering light. Interruptions in light are used to count cells and light scatter is used to determine size and internal complexity or density. The Advia 120 also utilizes a peroxidase channel to aid in determining specific leukocyte types. When properly calibrated, laser flow analysis allows for a more complete and accurate differential leukocyte count than can be done using cell size alone. Laser flow cytometry also permits the development of automated reticulocyte counts and reticulated platelet counts^36,44 as well as the development of new erythrocyte and reticulocyte parameters based on the simultaneous measurement of size and hemoglobin concentration within individual cells.¹⁷

Nucleated erythrocytes are counted as leukocytes in most electronic cell counters; consequently total leukocyte counts must usually be corrected for the presence of nucleated erythrocytes when present.

Errors in Blood Cell Counting and Sizing

The accuracy of blood cell counting depends on the quality and characteristics of the blood sample as well as the accuracy of the analytic methods used. Storage of blood samples for more than a few hours can result in sample deterioration and inaccurate cell counts. The presence of even small clots in the blood tube invalidates all cell counts. Even without clot formation, platelet aggregation may occur if platelets become activated during blood collection, as can happen when specimens are collected with a syringe and then transferred to an anticoagulant tube. Heparin is generally not used as an anticoagulant because it does not prevent platelet clumping, and leukocyte staining is poor on blood films. EDTA is the preferred anticoagulant for CBC determinations in most species, but EDTA may induce platelet, leukocyte, and erythrocyte aggregate formation in some individuals with antibodies bound to the surfaces of these respective cell types.^{10,29,54,71,75} When this occurs, collection of blood into citrate may prevent the problem. Cell aggregation tends to be more pronounced as blood is cooled and stored; consequently processing samples as rapidly as possible after collection may minimize the formation of leukocyte and/or platelet aggregates. EDTA can cause marked hemolysis in some species of birds (ostriches, emus, and jays),^25,35 reptiles (turtles and tortoises),^25,42 and fish (carp and brown trout).^43,66 Heparin is used as an anticoagulant in these species. Both lysis and clumping can result in reduced cell counts of the cell types involved.

Platelet clumping can not only result in an erroneously decreased platelet count⁵⁹ but also in a falsely increased mean platelet volume (MPV) and occasionally a falsely increased total leukocyte count.^35,75 Autoagglutination of erythrocytes in the blood sample can result in a spuriously increased mean cell volume (MCV) and mean cell hemoglobin concentration (MCHC) and a decreased HCT and total erythrocyte count.⁴⁸ As indicated earlier, with the use of most hematology analyzers, the presence of nucleated erythrocytes can result in a falsely increased total leukocyte count. Residual erythrocyte stroma from inadequate lysing of erythrocytes may also result in spuriously increased total leukocyte counts.^35,76

The presence of severe lipemia can result in spuriously increased hemoglobin and MCHC values and possibly even increased platelet and total leukocyte counts.⁷⁵ The presence of in vitro or in vivo hemolysis in the sample or prior treatment with a cross-linked hemoglobin solution can result in an erroneously increased MCHC value.³⁸ The presence of numerous Heinz bodies in erythrocytes can also result in spuriously increased hemoglobin and MCHC values, increased automated reticulocyte counts,^14,52 and sometimes increased total leukocyte counts.⁶⁹ The precipitation of an IgM paraprotein in blood from a dog by the lysing reagent used in the CELL-DYN 3500 falsely increased the spectrophotometric measurement of hemoglobin (Hb), which falsely increased the calculated MCHC.⁸

Laboratory errors may occur as a result of mistakes made by operators. These operator errors include a lack of knowledge or the skills for the test being done, improper labeling of samples, dilution errors, use of outdated reagents, inadequate quality control measures, or improper calibration of equipment. The instruments being used must be optimized and validated for the species being tested.

Erythrocytes and platelets vary considerably in volume among animal species, and instruments must be adjustable to accurately count and size these blood cells for the species being assayed. Cats naturally have large platelets and moderately small erythrocytes. The resultant overlap of erythrocyte and platelet size makes separation of cat platelets and erythrocytes unreliable with the use of impedance counters.⁷⁷ Consequently cat platelet counts are spuriously decreased when measured with impedance counters. This inclusion of platelets in the erythrocyte measurements can result in increased erythrocyte counts and HCT values and reduced MCV and MCHC values, but the ratio of platelets to erythrocytes is usually not large enough to have appreciable effects on these parameters (exceptions include cats with severe anemia and/or marked thrombocytosis).⁷⁶ Leukocytes are generally included in erythrocyte measurements, but the ratio of leukocytes to erythrocytes is usually not large enough to have appreciable effects on erythrocyte parameters (exceptions include animals with severe anemia and marked leukocytosis). In these instances the inclusion of leukocytes in erythrocyte measurements can result in increased erythrocyte counts, HCT and MCV values, and reduced MCHC values because leukocytes are larger than erythrocytes.⁷⁶ These errors resulting from difficulties in separating cell type by size alone should be minimized in automated cell counters that separate cells not only by size but also by internal complexity.

With the advent of new laser flow cytometry techniques, it is now possible to perform automated differential leukocyte counts; however, most instruments cannot accurately count basophils. For most instruments, a high basophil count is an error,³⁵ as has been noted in old blood samples.⁶³ These flow cytometers must be specifically calibrated for each species, and they work best when leukocyte morphology is normal. More reliable flags are needed to identify the presence of left shifts and neoplastic cells.³⁵

The examination of a stained blood film is essential as a quality control measure regardless of the technology used to count blood cells. In addition to verifying the accuracy of leukocyte and platelet counts, a number of other evaluations are made. Examples include determining whether erythrocyte polychromasia, erythrocyte shape abnormalities, neutrophilic left shifts, neutrophil toxicity, reactive lymphocytes, blast cells, mast cells, and/or blood parasites are present.