Interpreting Laboratory Results

Key Points

Accurate differential diagnoses can be made from a systematic study of the laboratory profiles of patients in a large majority of cases.
There are basically four types of anemia: iron deficiency, anemia of chronic disease, hemolytic anemia, and macrocytic/nutritionally deficient anemia. These can be readily distinguished from one another both by the hematologic profile and simple laboratory testing.
By examining the urinary sodium, potassium, and osmolarity, the causes of hyponatremia and hypernatremia can be readily determined.
Liver function tests can distinguish among six different diseases of the liver: hepatitis, cirrhosis, biliary disease, space-occupying lesions of the liver, passive congestion, and fulminant hepatic failure.
Renal failure can be readily diagnosed by observing elevated blood urea nitrogen and creatinine; it is possible to pinpoint the site of renal failure—that is, glomerular or tubular—from the ratio of serum to urine osmolality.
Blood gas results allow determination of the causes of metabolic versus respiratory acidosis or alkalosis; there is a critical relationship between the partial pressure of oxygen and carbon dioxide such that, in respiratory diseases, high levels of carbon dioxide block oxygenation of venous blood, leading to respiratory crisis.
Elevation of cardiac troponin in serum, in the proper clinical context, is diagnostic of myocardial infarction.
Elevations of serum C-reactive protein indicate inflammatory disease.
Elevations of serum amylase and lipase point to acute pancreatitis.
Two types of endocrine disease are discussed: thyroid and adrenal. Serum levels of T ₄ (or, better, free T ₄ ) and thyroid-stimulating hormone (TSH) can be used to diagnose primary or secondary hypothyroidism or hyperthyroidism; serum levels of cortisol and adrenocorticotropic hormone (ACTH) can be used to diagnose primary or secondary hypoadrenalism or hyperadrenalism.

Interpreting and Correlating Abnormal Laboratory Values

General Considerations

The major purpose of performing analyte determinations in the clinical laboratory is to aid in the diagnosis and management of disease and in health assessment. In this regard, the clinical pathologist is often called on as a consultant to explain abnormal laboratory values, especially those that do not seem to correlate with one another, and to recommend or even to order laboratory tests that may lead to the correct diagnosis in the workup of patients for particular medical problems. In addition, evaluation of laboratory test results on individual patients by the clinical pathologist can not only reveal the (infrequent) occurrence of laboratory errors but can help in the selection of appropriate, cost-effective tests from a wide variety of increasingly complex test choices ( ; ; ).

For evaluation of test results, the laboratory computer is an invaluable aid. Virtually all such systems perform daily checks for patient values that lie significantly outside of their established reference intervals or that have undergone large changes over a 24-hour period. These are often reported as “failed delta checks.” Thus, patients with significant laboratory findings can be identified.

This chapter presents an approach to the interpretation of laboratory values that will enable laboratorians to aid in the establishment of clinical diagnoses and to assist in clinical management. This discussion is by no means comprehensive and cannot possibly cover every conceivable illness afflicting patients. Rather, this presentation is concerned with general approaches to interpreting abnormal values and discussion of the most common causes of such findings so that the reader has a framework for interpreting abnormal values.

The reader may prefer to complete the Clinical Chemistry (Part 2) and Hematology and Transfusion Medicine (Part 4) sections of this book before reading this chapter, which gives an overview of both of these vital diagnostic areas. Alternatively, the reader may decide to read this chapter before the several chapters on chemistry and hematology to benefit from the overview presented here.

Fundamental Principles in Interpretation of Values

Before embarking on a discussion of specific conditions giving rise to abnormal values, certain precepts should always be followed:

1.
Never rely on a single out-of-reference range value to make a diagnosis. It is vital to establish a trend in values. A single sodium value of, for example, 130 mEq/L does not necessarily indicate hyponatremia. This single abnormal value may be spurious and may reflect such factors as improper phlebotomy technique, laboratory variability, and so on. Rather, a series of low sodium values in successive serum samples from a given patient does indicate this condition. Thus, it is vital to follow trends in particular values.
2.
Osler’s rule. Especially if the patient is under the age of 60 years, try to attribute all abnormal laboratory findings to a single cause. Only if there is no possible way to correlate all abnormal findings should the possibility of multiple diagnoses be entertained.

Abnormalities in the Hematology Profile

Often, in laboratory reports, the first section contains the hematology profile, including the complete blood count (CBC). Comprehensive discussions of clinical hematopathology are given in Part 4. Here, we discuss very basic patterns of abnormalities to provide an overall reference frame for interpreting values and for the ordering of further examinations. Though this part of the book is concerned with clinical chemistry or chemical pathology, we discuss the hematology profile because the interpretation of hematopathologic results often depends on the results of quantitative determinations performed in clinical chemistry.

Anemias

Anemia, a common hematologic disorder, is defined pathophysiologically as a decrease in the oxygen-carrying capacity of the blood. All oxygen-carrying capacity of the blood is due to the binding of oxygen to hemoglobin contained uniquely in red blood cells. Because anemia can cause tissue hypoxia, it often produces such symptoms as fainting, fatigue, pallor, and difficulty in breathing.

Practically, the best indicator for this condition is a low red blood cell count or number of red blood cells per volume of whole blood. Although the reference range for the red cell count varies with age, sex, and population, it encompasses values from around 4 to 6 × 10 ⁶ red blood cells per cubic millimeter (cu mm) or microliter. This range may change somewhat depending on population. Red blood cell counts below the lower limit of the reference range suggest the presence of anemia. In addition, red blood cells occupy a well-defined range in terms of the percent of the volume that they occupy of whole blood or the hematocrit. Generally, normal adult hematocrit values range from about 36% to 45% (normal values for females are generally slightly lower than those for males). In addition, the concentration of hemoglobin in whole blood is about 12 to 15 g/dL or approximately 33 to 36 g/dL in red blood cells—that is, the mean corpuscular hemoglobin concentration. Normal values are also dependent on patient age and altitude of residence. Normally, the hematocrit is about three times the value of the hemoglobin concentration, which, in turn, is about three times the value of the red blood cell count.

If anemia has been diagnosed, it is then mandatory to determine the cause of the anemia. An excellent history and physical examination are required for appropriate test selection, diagnosis, and the best possible patient care and treatment. In addition, a review of the peripheral blood film with respect to red and white cell morphology is often helpful.

To narrow further the differential diagnosis and to facilitate appropriate test selection, a number of classification schemes for anemia have been developed, with no single ideal scheme available. A particularly useful approach utilizes the common red cell indices of mean corpuscular volume (MCV), measured in femtoliters (fL), or 1 × 10 ⁻¹⁵ L, in conjunction with the red cell distribution width (RDW) and the reticulocyte count (percent reticulocytosis) or reticulocyte production index (RPI). A further aid in diagnosis is the chromicity of the red cells—that is, the intensity of the red color of the cells due to intracellular hemoglobin. The chromicity is measured quantitatively by the mean corpuscular hemoglobin concentration (MCHC). Taken together, these indices help to form a working hypothesis for the underlying cause of the anemia.

Electronic determination of MCV directly from red cell distribution data allows for classification on the basis of red blood cell size as macrocytic (MCV generally >100 fL), microcytic (MCV generally <80 fL), or normocytic (MCV generally between 80 and 100 fL). The sizes (volumes) of red cells vary within a certain range in which the number of cells of particular volumes form a bell-shaped or Gaussian distribution (see Chapter 10 ). The standard deviation of the cell volumes divided by the mean cell volume gives what is called the red cell distribution width, or RDW, measured as a percent. As it happens, the RDW is a parameter that helps to further classify an anemia because it reflects the variation of red blood cell size. The RDW generally varies between about 12 and 17 and is dependent on the patient’s age, sex, and ethnic subgroup. It can be helpful in differentiating causes of microcytosis, because moderate to severe iron deficiency anemia is associated with an increased RDW, whereas thalassemia and anemia of chronic disease (ACD) are associated with a normal RDW.

Peripheral blood reticulocytosis is a measure of bone marrow response in the face of anemia. A similar measure, the reticulocyte proliferation index (RPI) corrects the reticulocyte count with respect to the proportion of reticulocytes present in a patient without anemia and the premature release of reticulocytes into the peripheral circulation. Bone marrow response to anemia may be appropriate (hyperproliferative), with an RPI over 3 generally indicating marrow red cell hyperproliferation. However, the anemia may be due to defective red blood cell production or marrow failure (hypoproliferative), which is generally indicated by an RPI less than 2. Thus, although these red cell indices are not pathognomonic of the cause of a particular type of anemia, the combination of MCV, RDW, and RPI examined together will often significantly narrow the differential diagnosis and facilitate further test selection. Table 9.1 illustrates common examples of anemia and their diagnostic workup using these red cell indices as well as other helpful analytic abnormalities.

TABLE 9.1

Common Types of Anemias and Their Diagnostic Workups ^a

Anemia	Cause	Common Analyte Abnormality
1. Hypoproliferative, microcytic	Iron deficiency	Low ferritin Increased IBC Decreased serum iron Reduced Fe/TIBC ratio Generally increased RDW
2. Hypoproliferative, microcytic	Anemia of chronic disease	Generally high ferritin Normal IBC Decreased serum iron Normal Fe/TIBC ratio Generally normal RDW
3. Hyperproliferative, normocytic	Hemolytic anemia	Schistocytosis Increased reticulocytes Low haptoglobin Elevated carboxyhemoglobin Elevated LD and potassium Elevated indirect bilirubin Generally increased RDW
4. Hypoproliferative, normocytic	Aplastic anemia	Leukopenia Thrombocytopenia Hypocellular bone marrow Generally normal RDW
5. Hypoproliferative, normocytic	Renal failure	Elevated BUN and creatinine Low erythropoietin Burr cells may be present Generally normal RDW
6. Hypoproliferative, macrocytic
A. Megaloblastic	Vitamin B ₁₂ and/or folate deficiency	Low vitamin B ₁₂ and/or folate Hyperlobulated polymorphonuclear leukocytes Macro-ovalocytes Increased RDW
B. Nonmegaloblastic	Hypothyroidism	Elevated TSH Normal RDW

BUN, Blood urea nitrogen; Fe, iron; IBC, iron-binding capacity; LD, lactate dehydrogenase; RDW, red cell distribution width; TIBC, total iron-binding capacity; TSH, thyroid-stimulating hormone.

a Low is equivalent to depressed, and high is equivalent to elevated. Ferritin, haptoglobin, LD, bilirubin, BUN, creatinine, erythropoietin, TSH, and T ₄ are all expressed as concentrations. All of these analytes are measured in serum.

Microcytic Anemia

Common microcytic anemias include iron deficiency anemia (IDA) and the thalassemias. Some hemoglobinopathies and ACD may also be microcytic. In our discussion, we focus on IDA and ACD, a common differential diagnosis for patients with microcytic anemia. Both anemias appear to be disorders involving iron metabolism.

In IDA, there is a primary deficiency of iron available to the red cell (usually due to blood loss, but other causes include dietary deficiency, malabsorption, and pregnancy); chronic blood loss should always lead to further investigation because it is commonly associated with malignancy. ACD, however, appears to be due to defective iron utilization/metabolism and is associated with chronic nonhematologic disorders such as chronic infections, connective tissue disorders, malignancy, and renal, thyroid, and pituitary disorders. Because iron levels in red cells are low, hemoglobin levels are also low in both of these conditions. Thus, the MCHC mentioned earlier tends to be low, giving rise to what is termed a hypochromic (low red color in red cells or low MCHC), microcytic (low MCV) anemia.

To distinguish between IDA and ACD, a number of different laboratory measurements are very useful in addition to the RDW. The diagnosis is typically made using additional serum or whole blood laboratory tests. However, because IDA is always accompanied by loss of iron that is stored bound to the protein ferritin in bone marrow macrophages, the diagnosis can always in principle be made with a bone marrow biopsy with an iron stain—that is, nitroprusside—that shows the absence of marrow iron. This procedure is, of course, invasive and should be performed only as a last resort.

Serum Ferritin Levels

Normally, there is an equilibrium between intracellular and extracellular ferritin. The lower the stored iron becomes, the lower the intracellular ferritin is, and, consequently, the lower the extracellular ferritin becomes. The level of extracellular ferritin can be directly measured by determining the serum ferritin level, which is readily and accurately performed on serum aliquots, using enzyme-linked immunosorbent assay (ELISA) techniques, described in Chapter 45 . Overall, therefore, serum ferritin levels give an excellent measure of available iron stores noninvasively. Because, in ACD, iron stores are abundant, serum ferritin levels are characteristically normal to elevated. In contrast, in IDA, in which iron stores become depleted, serum ferritin levels are characteristically decreased. Thus, serum ferritin level is one assay that can be used in differentiating IDA from ACD.

One caveat in using serum ferritin values to provide this distinction is the fact that ferritin also happens to be an acute-phase reactant. Acute-phase reactants are proteins (discussed in Chapter 20 ) that rise in response to an acute process, usually an acute inflammatory condition. Thus, if a patient has an acute infection, the serum ferritin level may be spuriously elevated. The net effect may be a ferritin in the reference interval. Usually, in IDA, accompanied by an acute process, this level is in the low reference range.

Use of Serum Iron and Iron-Binding Capacity

In addition to ferritin levels, serum iron and serum iron-binding capacity (IBC) can be measured as discussed in Chapter 24, Chapter 28 ). On average, serum iron is, of course, reduced in IDA and normal or sometimes low in ACD. The IBC is a direct measure of the protein transferrin, which transports iron from the gut to iron storage sites in bone marrow. In IDA, the serum iron is reduced, and the IBC increases.

However, both serum iron and transferrin are subject to wide fluctuations because of such factors as diet, and they do not always reliably reflect iron stores. Also, transferrin is a beta-protein—that is, it migrates in the beta-region in serum protein electrophoresis (discussed in Chapter 20 )—and is an acute-phase reactant—that is, its serum levels change (usually decrease, as a so-called “negative acute-phase reactant”) in inflammatory conditions. There is considerable overlap between serum levels of iron and IBC in IDA and ACD. A somewhat more reliable discriminating measure of IDA is the ratio of serum iron to the IBC, known as the transferrin saturation ( TS ). This ratio is around 1:3 for normal individuals, while in IDA it is significantly reduced to values of around 1:5 or lower. Again, there is considerable overlap even in this ratio for patients with IDA and ACD; thus, the values should always be interpreted with care.

Use of Red Blood Cell Distribution Width

Finally, use of automated procedures for determination of cell counts and indices enables us to obtain average erythrocyte sizes and size distributions. In IDA, there is a marked dispersion in cell volumes (sizes) so that the RDW increases, whereas it generally remains within normal limits in ACD. Normal RDWs occur in the range of 12% to 17%. Unfortunately, the standard deviations for normal subjects and for patients with IDA or ACD can overlap significantly, tending to limit the validity of using the RDW exclusively for distinguishing between these conditions.

The major laboratory findings that distinguish IDA from ACD are summarized as entries 1 and 2 in Table 9.1 . Note that most of the major tests used to distinguish these two conditions are performed in the clinical chemistry laboratory. This emphasizes the strong interdependence of both of these services in obtaining definitive diagnoses through laboratory measurements.

Normocytic Anemia

In these anemias, the red cells show normal MCVs and MCHCs—that is, they are normocytic and normochromic. Common causes of normocytic anemia include acute hemorrhage, hemolytic anemia, marrow hypoplasia, renal disease, and ACD. It may seem paradoxical that acute hemorrhage presents as normocytic anemia because it involves major blood loss that is associated with loss of iron stores. However, iron depletion requires time to develop; acutely, major blood loss presents as normocytic anemia.

A very useful measurement in determining the cause of normocytic anemia is the reticulocyte count. Reticulocytes are newly formed red blood cells that have recently lost their nuclei but retain high levels of cytosolic mRNA dedicated to the synthesis of hemoglobin. This RNA reacts with the dye, methylene blue, to give a bright-green color, making it possible to perform reticulocyte counts. In addition, reticulocytes and other red blood cell precursors react with the Wright stain to give dark-staining cytoplasm, in contrast to the cytoplasm of mature red blood cells. This darker staining of the cytoplasm of precursors is termed polychromasia . In cases, prominently in hemolytic anemia, where there is a loss of red blood cells due to peripheral destruction of these cells, there is an increased synthesis in bone marrow of red blood cells and an early release of red blood cell precursors, especially reticulocytes.

Hyperproliferative Normocytic Anemias

Hyperproliferative normocytic anemias, associated with an increased reticulocyte count, include both hemolytic anemia and the anemia associated with acute blood loss, while hypoproliferative anemias, associated with a decreased reticulocyte count, include such causes as renal disease, ACD, and, variably, bone marrow aplasia/hypoplasia. Renal disease can affect juxtaglomerular cells that are involved in the synthesis of erythropoietin, the growth factor that induces differentiation of bone marrow hematopoietic stem cells into the red cell line. Bone marrow aplasia/hypoplasia can give rise to low reticulocyte counts if caused by agents, such as chemotherapeutic agents, that kill bone marrow hematopoietic stem cells. In such conditions as myelofibrosis, clonal expansion of cancer cells such as occurs in leukemia, lymphoma, myeloma, and metastatic cancer, there may be a release of early red and white blood cell precursors from bone marrow (the leukoerythroblastic picture discussed later), leading to increased nucleated red blood cell and reticulocyte counts.

As mentioned earlier, a history and a complete physical, as well as an examination of the patient’s peripheral blood film, are helpful in establishing a differential diagnosis. In this section, we focus on the most common causes of normocytic anemia: hemolytic anemia, aplastic anemia, and the anemia associated with renal disease.

Hemolytic Anemia

Hemolysis is defined as the destruction of the red cell membrane, causing hemoglobin release. This may occur slowly as a normal physiologic process or may be accelerated in pathologic states. Many different underlying causes exist for the decrease in survival/increase in destruction of red blood cells. These include membrane defects (e.g., hereditary spherocytosis), enzyme defects (e.g., glucose-6-phosphate dehydrogenase [G6PD] deficiency), hemoglobinopathies (e.g., sickle cell disease or beta-thalassemia), immune destruction (e.g., autoimmune hemolytic anemia or hemolytic transfusion reaction), and nonimmune destruction. Immune and nonimmune destruction include destruction due to infectious agents, toxic agents/drugs, physical agents, hypersplenism, and microangiopathic hemolytic anemias. Microangiopathic hemolytic anemias are caused by the mechanical destruction of red blood cells, mainly in bone marrow where they are synthesized, from such factors as fibrin deposition in the blood vessels of the bone marrow microvasculature, fibrosis, or malignancies such as leukemia, lymphoma, and metastatic cancer. In addition, mechanical destruction can result from extramedullary causes such as prosthetic heart valves.

Hemolytic anemia can be recognized from hemoglobin in plasma/serum and breakdown products of heme. Specific laboratory measurements that readily confirm the diagnosis of hemolytic anemia are based on the natural events that occur subsequent to hemolysis. After erythrocyte membrane breakdown, hemoglobin is extruded. Thus, plasma and urine may contain free hemoglobin or its degradation products. Free hemoglobin may be present acutely in the plasma (hemoglobinemia) or urine (hemoglobinuria), while hemosiderin may be present in the urine (hemosiderinuria) in more chronic episodes of hemolysis. Extruded hemoglobin becomes bound to the alpha-2 fraction protein, haptoglobin. The hemoglobin–haptoglobin complex becomes catabolized by macrophages that engulf these complexes by receptor-mediated endocytosis. Thus, an excellent indicator for hemolytic anemia is a low haptoglobin value . Extremely sensitive and rapid ELISA tests for haptoglobin are available for this purpose.

Because the red cell contents are extruded into the plasma, besides hemoglobin, there are other indicators of red cell damage: high serum potassium because intracellular potassium concentration is much higher in red cells than in the extracellular fluid and serum elevations of the enzyme lactate dehydrogenase (LD). As discussed in Chapters 19 and 21 , this enzyme has five major isozymes, labeled LD1 through LD5. Peculiarly, the predominant isozyme of LD in red cells is LD-1, which occurs predominantly in cardiac tissue.

Carbon monoxide (CO) and unconjugated bilirubin become elevated in blood in hemolytic anemia. When hemoglobin is extruded, large amounts of it become oxidized to methemoglobin. The heme portion dissociates and then becomes oxidized ultimately to bilirubin. The first step in this process is the oxidative opening of the porphyrin ring of heme with the attendant liberation of CO. CO may be measured easily by gas chromatographic techniques, or even more conveniently by co-oximetry, based on spectrophotometry (see Chapter 4 ), as carboxyhemoglobin (see Chapter 31 ). Elevated CO levels in normochromic, normocytic anemias are an excellent indicator of hemolytic anemia.

Because there is an increased production of bilirubin, which is unconjugated (see Chapter 22 ), there will be at least a transient elevation of serum indirect bilirubin. This elevation, in the presence of normal liver function, will be modest, usually in the range of about 2 to 2.5 mg/dL. (The upper limit of normal is around 1.2 mg/dL.)

Hemolytic anemia is almost always accompanied by an increased reticulocyte count and by evidence of red cell damage. As mentioned earlier, the reticulocyte count will be elevated (increase in polychromasia on the blood film), with erythroid hyperplasia present in the bone marrow, indicative of increased red cell production. In addition, the peripheral blood film may show evidence of the particular type of red cell damage associated with the particular type of hemolytic anemia (e.g., sickle cells in sickle cell disease or schistocytes/helmet cells in microangiopathic hemolytic anemia). There is also a noticeable difference in red cell size (anisocytosis) and shape (poikilocytosis) due to the presence of damaged and/or young cells. Because of the often marked changes in size and/or shape, the RDW is usually elevated. A number of nucleated red blood cells may also be identified.

Other findings in hemolytic anemia can identify the cause. A direct antiglobulin test (DAT)/direct Coombs test can be used to detect immunoglobulin attached to the red cell surface that identifies immune hemolytic anemia as the cause of red cell destruction. In this case, red cells are incubated with antihuman immunoglobulin. If the red cells are coated with antibody, they will agglutinate in the presence of the antihuman immunoglobulin, but if not coated with antibody, they will not agglutinate. A positive test suggests that an autoantibody or alloantibody may be responsible for the anemia. A positive screening test for G6PD deficiency will identify this enzyme deficiency as the cause of the hemolytic anemia. The selection of these tests will be dependent on the clinical evaluation as well as the preliminary laboratory data.

Laboratory findings that are diagnostic of hemolytic anemia are summarized in entry 3 in Table 9.1 . Note that virtually all of the quantitative diagnostic testing for hemolytic anemia—that is, serum and urine hemoglobin, haptoglobin, carboxyhemoglobin, indirect bilirubin, and LD—are performed in the clinical chemistry laboratory, again emphasizing the strong interdependence of the clinical chemistry and hematology laboratories.

Microangiopathic Hemolytic Anemia

As previously mentioned, red cell fragments (schistocytes) may be present on peripheral blood films due to mechanical (e.g., prosthetic heart valve) or thermal (severe burns) destruction. Mechanical rupture of red cells within the microvasculature may also occur by physical damage to red cells in the microvasculature of bone marrow. This may be due to space-occupying lesions, such as metastatic tumors or leukemia or lymphoma, to myelofibrosis, or to the intravascular deposition of fibrin strands on endothelial cell surfaces. Because red blood cells are damaged and destroyed, this process leads to so-called microangiopathic (i.e., lesions in the microvasculature) hemolytic anemia (MHA).

Besides space-occupying lesions, other causes of this type of anemia include disease states in which fibrin is deposited on endothelial surfaces, also resulting in the shearing and fragmentation of newly synthesized red blood cells, as in disseminated intravascular coagulopathy (DIC). In this condition, there is an abnormal activation of the coagulation process in which fibrin-platelet clots form intravascularly and embolize to virtually any tissue. These clots block the microvasculature of tissues, including that of bone marrow, resulting in the destruction of newly synthesized red cells. In addition, other disease states may give rise to MHA, in which there may be an immunologic component—that is, antibodies to determinants on endothelial cells or on other structures in the microvasculature, resulting in immune complex deposits with or without fibrin deposits.

In thrombotic thrombocytopenic purpura (TTP), the microvascular disease is caused by platelet thrombi. A prominent cause of this condition is dysfunction due to the absence of a critical chymotrypsin-like protease known as ADAMTS13 (A Disintegrin And Metalloproteinase with a Thrombospondin type 1 motif, member 13) that hydrolyzes large multimer forms of von Willebrand factor (vWF) into lower molecular mass forms; the high molecular mass forms promote platelet aggregation, leading to this condition. However, this condition can also arise, rarely, in patients with no known abnormality of ADAMTS13 in cases of acquired TTP. Because the microvasculature is blocked in this condition, there is multiple system pathology—prominently renal failure—evidenced by high serum levels of creatinine and BUN (blood urea nitrogen), as described in the Abnormalities in Clinical Chemistry section, neural signs and symptoms due to cerebral infarctions and thrombocytopenia, and widespread purpura from local hemorrhaging.

In the closely related condition called the hemolytic anemic/uremic syndrome (HUS), which occurs mainly in children, bacterial infection by certain toxin-producing strains of organisms such as E. coli , S. pneumoniae , Mycoplasma, and Shigella induce platelet aggregation. This is believed to arise in part from toxin-induced inhibition of ADAMST13, although the full pathophysiology of this condition still remains unexplained. Here, as in TTP, there is renal failure anemia, and, though less prominent than in TTP, neural symptoms.

MHA may also occur with other immune-mediated disorders—for example, connective tissue disorders such as disseminated lupus erythematosus, where endothelial damage from the attachment of immune complexes and complement produces fibrin deposition on endothelial surfaces. More important, because MHA results from traumatic destruction of newly formed red blood cells in the microvasculature where both red and white blood cell precursors are being formed, often both red and white blood cell precursors are released into the circulation. Thus, all of the findings of hemolytic anemia are present, in addition to which a significant number of precursor cells are seen in the peripheral blood, such as nucleated red blood cells, myelocytes, and metamyelocytes, a pattern termed the leukoerythroblastic picture .

As discussed later, in DIC and occasionally in TTP, laboratory findings include thrombocytopenia and increased prothrombin time, activated partial thromboplastin time, thrombin time, fibrin degradation products, and D-dimer levels (described later); fibrinogen levels, however, are low. As discussed earlier, BUN and creatinine are also elevated.

Hypoproliferative Normocytic Anemias

Bone Marrow Hypoplasia/Aplastic Anemia

This is a hypoproliferative anemia, with MCV and RDW usually within normal limits, that typically affects all peripheral blood elements (red cells, white cells, and platelets; see later discussion). Immature white cells and red cells are not usually present on peripheral blood films. Bone marrow biopsy is commonly performed to obtain the diagnosis and typically shows severely hypoplastic/aplastic marrow with severe depletion of all hematopoietic marrow precursors. Aplastic anemia may be primary/inherited or secondary/acquired, with the latter due to chemotherapy, chemical toxins, infection, radiation, or immune dysfunction. Serum iron may be elevated due to lack of erythropoiesis. The typical hematologic findings in this condition are summarized in entry 4 in Table 9.1 . More important, none of the quantitative serum diagnostic tests for hemolytic anemia, such as haptoglobin, carboxyhemoglobin, and indirect bilirubin elevation, are positive in this condition.

Myelodysplastic Syndrome

Another less common condition, but nonetheless an important cause of hypoproliferative normocytic anemia, is myelodysplastic syndrome (MDS). This syndrome, which often presents as a normocytic anemia, although it can also on occasion present as a mildly macrocytic or microcytic anemia, is refractory to treatment such as transfusions of packed red blood cells. It may present simply as a refractory anemia in its early stages and is thought to progress to refractory anemia with ringed sideroblasts and eventually to so-called preleukemic stages, in particular refractory anemia with an excess of blasts in bone marrow (generally in the myeloid or lymphoid lines) and an excess of blasts in transformation. The condition may also present initially as a refractory cytopenia that involves all three (erythroid, granulocytic, megakaryocytic) hematopoietic cell lines. As might be surmised from this latter observation, MDS appears to be a clonal stem cell disorder that is characterized by ineffective hematopoiesis. Further discussion of this fascinating disease can be found in Chapter 33 .

Anemia of Renal Failure

Another normocytic, hypoproliferative anemia is the anemia of chronic renal failure. Loss of the kidneys’ excretory function produces an increase in BUN and creatinine (discussed later) as well as a buildup of metabolic byproducts. The resulting uremia appears to be responsible for changes in red cell shape, with burr cells (echinocytes) and ellipsoidal cells commonly present on peripheral blood films. Identification of burr cells on peripheral blood films during the course of illness may signal the development of renal dysfunction. In addition to a decreased excretory function, there is a decrease in the kidneys’ ability to produce erythropoietin, resulting in impaired erythropoiesis, such that the marrow’s response to hypoxia becomes inadequate. Distinguishing this condition from aplastic anemia (entry 4 in Table 9.1 ), white cell and platelet counts usually remain within normal limits. The typical findings in this condition are summarized in entry 5 in Table 9.1 . Again, as in bone marrow hypoplasia/aplastic anemia (entry 4, Table 9.1 ), none of the quantitative serum diagnostic tests for hemolytic anemia (e.g., haptoglobin, carboxyhemoglobin, indirect bilirubin elevation), are positive in this condition.

Macrocytic Anemia

Macrocytic anemia can be diagnosed from the hemogram from a low red blood cell count and a high MCV, often exceeding 100 fL. By far the most common cause of macrocytic anemia is nutritional deficiency, such as vitamin B ₁₂ and/or folate deficiency. Lack of either factor is thought to disrupt DNA synthesis but not RNA synthesis, such that the nucleus and cytoplasm of the cell no longer mature in synchrony. Morphologically, the cell cytoplasm matures, while the nucleus remains immature, and the cell appears megaloblastic. This lack of synchrony produces hypersegmented neutrophils (five-lobed nuclei in more than 5% of the neutrophils or any neutrophil with six or more lobes) and large, oval-shaped red cells termed macroovalocytes , both of which are present on blood films of patients with megaloblastic anemia. In addition, the RDW is typically increased, and the reticulocyte count is decreased.

If macrocytic anemia is diagnosed, the first serum analytes whose concentrations should be determined are B ₁₂ and folate, for which rapid and accurate ELISA tests are performed. If these analytes are both found to be within the reference range, assays for thyroid function should be performed because hypothyroidism is a cause of macrocytosis. As discussed later in the Endocrine Function Testing section, elevated thyroid-stimulating hormone (TSH) with low or normal thyroxine (T ₄ ) serum levels confirm the diagnosis of primary hypothyroidism. Because certain therapeutic drugs—particularly azidothymidine (AZT), used in the treatment of retroviral disease—are known also to induce macrocytic anemia, it is important to ascertain whether a patient is being treated with such drugs.

In this era of the automated CBC, it is possible also that red cell precursor forms, such as nucleated red blood cells, may be counted as mature erythrocytes. Therefore, in a patient with a “macrocytic” anemia with normal B ₁₂ , folate, and thyroid hormone levels, it is important to check the reticulocyte and nucleated red blood cell count to determine whether these are significantly elevated. If so, the possibility of a hemolytic anemia should be considered. Thus, the diagnostic workup for hemolytic anemia described in the preceding section should be instituted.

Other possible causes of macrocytic anemia include posthemorrhagic states (differentiated by an elevation of the reticulocyte count and polychromasia), alcoholism (associated with folate deficiency), liver disease, and myelodysplasia. Note again that the definitive tests for determining the cause of macrocytic anemia—that is, B ₁₂ , folate, and thyroid function tests—are often performed in the clinical chemistry section.

Major clinical laboratory findings for macrocytic anemia are summarized as entries 6A and B in Table 9.1 . Note that macrocytic anemias are divided into megaloblastic (entry 6A), typical of B ₁₂ and folate deficiency, and nonmegaloblastic (entry 6B), typical of hypothyroidism. Whether the macrocytic anemia is megaloblastic can be determined only by bone marrow biopsy. This procedure is not necessary in most cases because the cause of the macrocytosis can be determined by the assays described earlier.

Table 9.1 summarizes some of the pertinent findings and specific determinations used to distinguish and diagnose common anemias previously discussed. Note that this table is a guide as to what specific tests should be ordered and, by implication, what tests need not be ordered. For example, a microcytic anemia should be worked up with orders for ferritin levels, IBC, and Fe/IBC ratios, but generally there is no need to order B ₁₂ and folate levels. Conversely, there is no need to order ferritin, IBC, and so on for a macrocytic anemia, for which B ₁₂ and folate levels should be ordered.

Quantitative White Blood Cell Abnormalities

The white blood cell (WBC) count encompasses several types of commonly circulating nucleated cells: granulocytes (principally, mature neutrophils, basophils, and eosinophils), lymphocytes, and monocytes. It should be noted that absolute concentrations (and not percentages) of these cells are significant in interpreting the WBC count. An increase above the normal physiologic level in the WBC count, termed leukocytosis , may primarily involve any of these white cells, depending on which cell type is chiefly elevated (i.e., neutrophilia, basophilia, eosinophilia, monocytosis, and lymphocytosis). Occasionally, plasma cells may be found in the peripheral blood. Likewise, a decrease in the WBC count, termed leukopenia , may also center on a single cell series (i.e., neutropenia, monocytopenia, and lymphocytopenia). Absolute decreases in the eosinophils and basophils are difficult to identify because of the low numbers present normally. Certain differential diagnoses are commonly associated with certain WBC changes (e.g., infection and/or inflammation with neutrophilia; allergic reactions and parasitic infections with eosinophilia). In addition, elevations may be due to a benign process (e.g., infection) or a malignant process (e.g., leukemia). Here, we note several quantitative patterns and their associations that correlate with abnormal chemical findings.

Again, clinical history and physical findings are important in diagnosis and management of the patient. In addition, the CBC and white cell differential are important laboratory findings used, in conjunction with the clinical impression, to formulate the differential diagnosis. In adults, the reference range for the WBC count is approximately 4000 to 7000 white cells/μL; approximately two thirds of the white cells are neutrophils, and slightly less than one third are lymphocytes.

Infection

Infection is the most common cause of an elevated WBC count. An elevated WBC count of between about 10,000 and 20,000/μL commonly points to an infectious/reactive process. In general, neutrophilia is associated with infection (bacterial, fungal, viral), inflammatory states (trauma, surgery), certain drugs (e.g., corticosteroids), and myeloproliferative conditions. Exceptions to the neutrophilia seen in bacterial infections are tuberculosis; brucellosis; pertussis, in which the predominant cells are lymphocytes; and infections, mainly in newborns, with Listeria monocytogenes , for which a monocytic response is predominant.

Eosinophilia is commonly associated with allergic reactions, parasitic infections, and hematologic malignancies ( ; ; ; ; ). Basophilia is also commonly associated with hematologic malignancies (e.g., chronic myelogenous leukemia [CML]), but it may be seen in some inflammatory states and allergic reactions. Lymphocytosis is commonly associated with acute viral infections, such as infectious mononucleosis (Epstein-Barr virus infection); chronic infections, such as tuberculosis, brucellosis, and pertussis; and hematologic disease and immune stimulation. Monocytosis is commonly associated with hematologic disease, such as acute monocytic leukemia (M5 by the French-American-British, or FAB, classification system), acute myelomonocytic leukemia (M4, in the FAB system), or chronic myelomonocytic leukemia, as well as with some infectious processes, such as tuberculosis, rickettsia, and listeria.

Elevated White Blood Cell Count Due to Leukemoid Reaction

In patients who do not have leukemia, very high white blood cell counts (generally >50 × 10 ³ /μL) may produce a peripheral blood film appearance similar to leukemia. This is termed a leukemoid reaction . The more common type of leukemoid reaction is granulocytic, although lymphocytic reactions may also occur. The granulocytic type usually reveals reactive neutrophils present in the peripheral blood film, with a left shift in the neutrophil series (i.e., immature forms such as bands, metamyelocytes, and myelocytes). Changes in the cells’ cytoplasmic appearance, such as toxic granulation and Döhle body production (see Chapter 33 ), are also commonly present. Causes of the granulocytic type of reaction include bacterial infection (e.g., diphtheria), malignancy (Hodgkin disease), and reactive conditions such as rebound granulocytosis. Although these changes are helpful, C-reactive protein (CRP), an acute-phase plasma protein, rapidly rises and falls with the onset and resolution of inflammation. CRP appears to be an earlier and more sensitive indicator of acute inflammation and infection ( ; ; ) and can now be quickly measured using present-day analyzers. Leukemoid reaction must be distinguished from CML and other myeloproliferative conditions. More important, the enzyme neutrophil alkaline phosphatase will be normal or elevated in a granulocytic leukemoid reaction but is decreased in CML.

Elevated White Blood Cell Count Due to Chronic Myelogenous Leukemia

At present, definitive diagnosis of CML rests on the demonstration of the Philadelphia chromosome (i.e., the BCR/c-abl translocation between chromosomes #9 and #22) by either cytogenetics or molecular techniques (e.g., see ; ; ; ; ; ; also, see Chapters 34 , 70 , and 77 ). Detection of molecular or cytogenetic abnormalities is also of diagnostic (and can be of prognostic) significance in other hematologic diseases, including acute myeloid leukemia, acute lymphoblastic leukemia, T cell leukemia/lymphoma, and myelodysplasia ( ). Molecular techniques are also currently utilized to detect very early stages of disease as well as to detect minimal residual disease—that is, disease that may only be apparent at the molecular level. For example, quantitative polymerase chain reaction can be used to monitor BCR/c-abl translocation levels in CML patients being treated with the kinase inhibitor imatinib (Gleevec) ( ). An important clue for diagnosing CML is an increase, in peripheral blood, of the basophil count often also accompanied by an increased eosinophil count (El-Ghammaza, 2015). Basophilia is an independent adverse prognostic factor in patients with CML.

Elevated White Blood Cell Count Due to Chronic Lymphocytic Leukemia

When the lymphocytes appear normal but are significantly increased in number in an older individual, the possibility of chronic lymphocytic leukemia (CLL) must be considered. Again, molecular techniques, such as flow cytometric, immunophenotypic, and cytogenetic/fluorescence in situ hybridization analysis ( ; ; ) can help establish the diagnosis. In CLL, the neoplastic B lymphocytes will be found to express an unusual (but characteristic) human leukocyte differentiation antigen designated CD5, which is typical for this disease. Other CD antigens can also be detected by flow cytometry and have become useful in resolving other hematologic diagnostic problems.

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