Laboratory tests are performed on body fluids or tissue samples.

Laboratory tests can be classified as follows:

Routine laboratory tests are most often performed on blood and urine, but on occasion the same tests can be performed on other body fluids such as the cerebrospinal fluid; joint fluid; or effusions in the abdominal, thoracic, or other cavity.

Blood tests are performed usually in batteries, or panels, known as sequential multiple analysis (SMA-6, SMA-7, or SMA-12) (Table 1-1).

Table 1-1 Common Laboratory Panels

Blood may be submitted in tubes that either contain or do not contain anticoagulants. The tops of these tubes are color-coded to avoid confusion. Blood collected into a tube that does not contain an anticoagulant (red tubes) will clot, and the red blood cells, leukocytes, and platelets will separate from the serum (Fig. 1-1). The separation of blood cells from serum can be accelerated by collecting the blood in tubes that are not coated with anticoagulant. Such tubes contain a gel at the bottom that activates clotting and promotes the separation of the clot from the serum (“red/green,” also known as “tiger-top tubes” or “light green-mint tubes”). These tubes are used in the laboratories that perform automated testing. Serum contains all the normal minerals, enzymes, immunoglobulins, and proteins besides the coagulation factors, and is thus used for most routine laboratory tests.

Figure 1-1 Test tubes with color-coded tops. Red, no anticoagulant; lavender, ethylenediaminetetraacetic acid (EDTA); green, lithium heparin; gray, fluoride oxalate.

If the blood is collected into a tube that contains anticoagulants, such as lavender-top tubes containing ethylenediaminetetraacetic acid (EDTA) or green-top tubes with lithium heparin, the red cells, leukocytes, and platelets remain in suspension inside the tube; the blood cells can be separated from the plasma by centrifugation. Plasma contains fibrinogen and all other coagulation factors and is used for specialized tests, such as measurement of the concentration of coagulation factors, lipids and lipoproteins, or folic acid. For the measurement of lactic acid or the optimal measurement of glucose, the blood is collected in gray-top tubes containing fluoride oxalate (an inhibitor of glycolysis), which does not interfere with the measurement of these analytes.

Specialized tests are not performed in all hospital laboratories but are sent to highly specialized reference laboratories. Specialized tests often require that the specimens be collected in a specified manner or under highly controlled circumstances. Most of these tests are performed in specialized reference laboratories or manually by specially trained technicians. This category of tests includes the testing of rare oligominerals such as copper or selenium, certain hormones such as vasoactive intestinal polypeptide (VIP) or parathyroid hormone-related protein (PTHrP), certain drugs and toxins, and DNA analysis. Many tests that were considered specialized in the past have become routine. One example is the troponin test, which is today routinely performed on the serum of patients thought to have suffered a myocardial infarction. Testing for human immunodeficiency virus (HIV) or hepatitis C virus was initially performed only in specialized laboratories but is routine in most hospitals today.

The samples for specialized tests are often collected in a unique manner. For example diurnal variation of hormones can be measured on several specimens collected at predetermined times. The sweat test for cystic fibrosis requires stimulation of sweat production with pilocarpine. Enzyme deficiency, which is the hallmark of some inborn errors of metabolism, requires tissue samples containing cells. DNA analysis requires samples containing nucleated cells from a buccal smear or white blood cells.

Emergency tests are performed on samples obtained during surgery or as part of the work-up on critically ill patients. Intraoperative testing for parathyroid hormone (PTH) may help the surgeon determine whether a PTH-secreting adenoma was removed. Analysis for drugs such as salicylic acid, acetaminophen, or digitalis may be important in diagnosing possible drug toxicity. Blood gasses and electrolytes are measured on an emergency basis in patients with acid–base disorders.

Good laboratory tests must be precise (reliable) and accurate and have high sensitivity and specificity.

In the United States the results for most analytes are expressed as concentrations in grams (g), milligrams (mg), micrograms (μg), or nanograms (ng) or as moles (mol) or millimoles (mmol) per liter (L), deciliter (dL), or milliliter (mL) of fluid. The activity of enzymes is expressed in activity units per liter (U/L). In other countries, laboratory data are reported according to the Système International (SI), using metric units. In many major U.S. hospitals, both systems are used in parallel.

Modern laboratories are highly automated, and the performance of tests is controlled at several levels to ensure high precision and accuracy.

The precision, or reliability, of a test can be determined by repeating the same test on the same sample. Ideally the same result should always be obtained, but in practice this does not always occur. So the standard deviation (SD) from the mean must be calculated for lab results. A highly precise test has a low SD, meaning that the results occur within a narrow range. In most instances the reference range is determined by adding 2 SD to the mean, which means that 95% of the results fall in that range, and only 1:20 (5%) fall outside it.

The accuracy of a test reflects how close the measured value is to the true value. An ideal test should have high precision and high accuracy. Like the target-shooting analogy shown in Figure 1-2, the outcome of each measurement should be as close as possible to the bull’s eye (“true value”) and the results should be as close together as possible (low scatter). These results can be graphically presented and typically have a bell-shaped (Gaussian) distribution with a low standard deviation from the mean. A test is unacceptable if it has high precision but does not reflect the true value (i.e., low accuracy). A test could have low precision but, if repeated several times, could have a statistically acceptable accuracy. Such tests have a wide standard deviation. Tests that have low precision and low accuracy are not used in clinical laboratories. Such tests may have a Gaussian distribution with a very high standard deviation or do not form a bell-shaped curve.

Figure 1-2 Precision and accuracy of tests. A, Ideal test has high precision and high accuracy. B, This test has high precision but low accuracy. C, This test has high low accuracy but overall it has high precision. D, This test has low precision and low accuracy.

Good laboratory tests must have high sensitivity and specificity.

The diagnostic value of each laboratory test is expressed in terms of its specificity, sensitivity, predictive value, and efficiency as follows:

Specificity measures the incidence of “true negative” (TN) results; that is, normal values occur in all tested persons who do not have a disease—those who presented as “true negatives” or “false positives” (FP) (Fig. 1-3). Specificity is calculated according to the following formula:

Figure 1-3 A, Sensitivity and specificity of laboratory tests. An average laboratory test will detect most persons who have a disease (“true positives”—TP), but some persons with the disease will not be detected (“false negatives”—FN). Some healthy persons will be also positive (“false positives”—FP), whereas most of those who are healthy will be negative (“true negatives”—TN). B, Raising the cut-off point increases specificity (“negativity in health”). Lowering the cut-off point increases the sensitivity of the test (“positivity in disease”).

Sensitivity measures the incidence of “true positive” (TP) results among all persons who have the disease irrespective of whether they tested positively or negatively, thus representing a sum of “true positives” plus “false negatives” (FN). Sensitivity is calculated according to the following formula:

The specificity and the sensitivity of a test can be adjusted by raising or lowering the cut-off point for a positive result (see Fig. 1-3). A test with high specificity will be always negative in health; that is, it will be negative for both FP and TN. Tests with a high sensitivity will be positive for all those who have a disease and will include all TP and FN.

Predictive value. The positive predictive value of a test predicts how many persons with a disease will have a positive result, whereas a negative predictive test predicts how many persons who do not have a disease will have a negative result. These predictive values are calculated according to the following formulas:

For example, if a test has a positive predictive value of 90%, it will be positive in 90% of persons with the disease, whereas 10% will have false negative results and will not be detected with this test. In a test with a 90% negative predictive value, 90% of all persons who do not have the disease will have a negative result, but 10% will have a false positive result.

Efficiency relates to the number of all correct results and is calculated according to the following formula:

Sodium is the principal electrolyte in the extracellular volume.

The body of an adult man weighing 70 kg contains approximately 4200 mmol of Na+. Approximately 50% of this is in the extracellular fluid, 40% is in the bone, and 10% is inside the cells. All the Na⁺ in the extracellular and intracellular fluid and approximately 50% in bone form the so-called exchangeable sodium, which is in constant intercompartmental flux. Serum concentration of Na⁺ is in the range of 135 to 145 mmol/L.

The concentration of sodium in the body depends on the amount of water and is controlled by kidneys and several hormones.

A regular diet contains more Na⁺ than the body needs, and the intestines can absorb unlimited amounts of this mineral. Thus intake of sodium is almost never a problem, and the body can adjust even if the exogenous salt is dramatically reduced. Most of the Na⁺ is excreted in urine. Small amounts of Na⁺ are lost in feces and sweat. Hence the excretion and loss equal the total intake (Fig. 1-5).

Figure 1-5 Sodium intake and excretion. ECF, extracellular fluid; ICF, intracellular fluid.

Sodium is in balance with body water content. A 70-kg adult male requires approximately 2.5 L of water, 2.1 L of which is derived from food and drinks and 400 mL from intermediate metabolism. Most of the water is excreted in urine, and the rest in feces and by insensible loss in exhaled air or perspiration.

Sodium balance is under the control of several physiologic mechanisms:

Sodium plays a role in forming the electric charges across the cell membrane and in acid–base regulation and is the most important electrolyte regulating the osmolality of the extracellular fluid.

Even though Na⁺ represents the most abundant ECV cation, its functions are not fully understood. It is known that Na⁺ participates in several physiologic processes, the most important of which are as follows:

Sodium concentration is the primary determinant of serum osmolality.

Osmolality of the serum depends mostly on the concentration of Na+, and to a lesser degree of glucose and urea (expressed as BUN, blood urea nitrogen). The contribution of anions is less than 10% of the total value of serum osmolality, and thus a formula for calculating serum osmolality takes into account only Na+, glucose, and BUN as follows:

Serum osmolality is measured to determine the water content of ECF and exclude the possibility of overhydration or dehydration. In such cases the measured osmolality corresponds to the calculated osmolality, and the difference is usually less than 10 mOsm/kg. When an osmolal gap (>10 mOsm/kg) occurs, the discrepancy between the measured and calculated osmolality is usually caused by the presence of a low-molecular-weight substance (e.g., alcohol, ethylene glycol) in the blood. The study of the osmolal gap is especially useful in patients who are comatose or suspected of intoxication. The most common causes of increased serum osmolality are listed in Table 1-2.

Table 1-2 Causes of Altered Serum Osmolality

Hyponatremia may be classified as dilutional or depletional.

Hyponatremia is a common condition defined as a serum concentration of Na⁺ below 136 mmol/L. Mild hyponatremia is found in 2 to 3% of all hospitalized patients. In most instances it is just a sign of illness, reflecting the inability of cells to maintain the normal gradient and flux of electrolytes across the cell membranes (“sick cell syndrome”). Severe hyponatremia is usually a consequence of water intoxication.

Pathogenetically, hyponatremia may be classified as dilutional or depletional (Fig. 1-6).

Figure 1-6 Pathogenesis of hyponatremia. It can be classified as dilutional, due to water excess, or depletional, due to a net loss of sodium. ECV, extracellular volume; GI, gastrointestinal; SIADH, syndrome of inappropriate antidiuretic hormone secretion.

Dilutional hyponatremia occurs under the following conditions:

Depletional hyponatremia occurs whenever a net loss of Na⁺ occurs. Sodium may be lost through the gastrointestinal (GI) system or the kidneys, most often under the following conditions:

Gastrointestinal loss

Vomiting

Diarrhea

Sequestration of fluid in the intestine (e.g., paralytic ileus)

Gastrointestinal fistulas

Renal loss

Glycosuria of diabetes mellitus

Hypercalcemia associated with polyuria

Salt-wasting kidney diseases. This may occur in any chronic renal disease resulting in a loss of nephron function. The remaining nephrons become overloaded with urea, causing an osmotic diuresis and consequent depletion of Na⁺ from ECV.

Diuretics, especially osmotic, or any form of diuretic overdose

Addison’s disease, characterized by aldosterone deficiency. A lack of cortisol, which is necessary to excrete the water load, leads also to water retention. Thus, the hyponatremia in these patients is in part dilutional.

Dermal loss

Burns

Symptoms of hyponatremia depend on the concentration of sodium and the way it has developed.

Hyponatremia is usually mild and, thus, asymptomatic. Slowly evolving hyponatremia, especially if dilutional, is well tolerated, and even if the Na⁺ concentration drops to 100 mmol/L it may cause few if any symptoms. On the other hand rapidly developing hyponatremia of 120 or even 125 mmol/L may cause significant neurologic symptoms.

Symptoms are mostly related to depressed transmission of neural or neuromuscular signals and include muscle weakness, somnolence, and, in severe cases, coma.

Death, if it occurs, is usually related to the underlying disease rather than hyponatremia itself.

Hyponatremia may be associated with increased total body water levels, relative increase in extracellular water, loss of Na⁺ in excess relative to body water or increase in total body water in excess to increased Na⁺ (Table 1-3).

Table 1-3 Causes of Hyponatremia

When the plasma volume is reduced by 5% in chronic hyponatremia, ADH is released in spite of the osmoreceptor-generated inhibitory effects on its secretion. Due to the effects of ADH in chronic hyponatremia the volume of urine is reduced and it will be concentrated.

Treatment of hyponatremia should be directed at its causes and the correction of sodium–water balance. Water restriction will suffice in mild cases of dilutional hyponatremia. Severe hyponatremia may cause brain injury, but likewise, too rapid correction of hyponatremia with infusion of salts can cause central pontine myelinolysis.

Hypernatremia is clinically almost always a consequence of dehydration.

Hypernatremia is defined as serum concentration of Na⁺ over 150 mmol/L. It is much less common than hyponatremia, but if it occurs, it has more severe clinical repercussions. Most affected patients are either very young or very old, critically ill, or neurologically impaired (e.g., patients in coma).

Theoretically, hypernatremia may result from a loss of water or a gain of Na+. These changes lead to hyperosmolality, to which the body responds by either increasing water intake or conserving water excretion through the kidneys. The second compensatory mechanism includes the release of ADH, thereby reducing urinary excretion of water.

Drinking of water corrects hypernatremia in most instances. Clinically significant hypernatremia develops only in persons who have no access to water or are unconscious and cannot drink (e.g., comatose patients, persons in deep anesthesia, very old immobile persons, or infants). Hypothalamic injury may affect adversely the thirst center and thus cause hypernatremia. It also may lead to diabetes insipidus and excessive loss of water in urine due to ADH deficiency.

Water loss can occur through the kidneys, GI tract, or the skin:

Excessive sodium intake or retention. Sodium retention occurs under the following conditions:

Clinical findings depend on the nature of hypernatremia.

Clinically, hypernatremia must be always evaluated in the context of hydration of the body. If hypovolemia is present, urinary or intestinal water loss must be suspected. In euvolemic patients central or nephrogenic diabetes insipidus, dermal or respiratory loss of water, or inadequate intake of water (hypodipsia) must be considered. In hypervolemic patients an excess of mineralocorticoids, such as occurs in primary hyperaldosteronism, is a possibility (Fig. 1-7).

Figure 1-7 Pathogenesis of hypernatremia.

Symptoms depend on the degree of hypernatremia and usually appear when the Na⁺ concentration reaches 155 to 160 mmol/L. The rate and the time over which hypernatremia develops are also important. Typically the affected person is thirsty, but many are either unconscious or too young to say so. There is oliguria. Neurologic symptoms, such as irritability, restlessness, confusion, and agitation, predominate. In some cases generalized muscle weakness is present. Low-grade fever from dehydration is present, and the skin appears flushed and dry. The plasma volume depletion in hypovolemic hypernatremia may adversely affect the function of the heart and the kidneys. It presents with hypotension, tachycardia, and renal failure due to hypoperfusion. Ultimately the patient becomes lethargic and comatose.

Treatment should include water replacement. Water should be first given by mouth, and if it must be given intravenously it should be administered as a 5% dextrose solution.

Chloride is the major extracellular anion and it is tightly linked to intake, excretion and metabolism of sodium.

Chloride (Cl⁻) is the major anion of plasma and interstitial fluids, accounting with bicarbonate (HCO₃⁻) for most of the anionic charge of plasma. As such its functions include maintenance of hydration, osmotic pressure, and electrolyte balance. The reference range for chloride is 98 to 106 mmol/L.

Chloride is mostly bound to Na+, forming the common salt (NaCl). Accordingly, its metabolism follows that of Na+. The only exceptions occur in certain forms of metabolic acidosis and alkalosis.

Potassium balance is maintained primarily by the kidneys.

The normal diet contains enough K⁺ and thus on average 100 mmol of K⁺ is absorbed daily, 90 mmol is excreted by the kidneys, and the remaining 10 mmol is lost in feces and sweat (Fig. 1-8).

Figure 1-8 Potassium intake and excretion. Intake in the food is balanced by excretion in the urine and, to a smaller extent, in the feces and sweat (not shown here). Most of the K⁺ is stored in the intracellular volume (ICV), and only 2% is found in the extracellular fluid volume (ECF). Only 0.4% of total K⁺ is in the plasma. Most of the intracellular fluid (ICF) contains exchangeable K+, but 8% is tightly bound in tissues. The balance of K⁺ critically depends on the function of the kidneys. The modulators of K⁺ excretion in the kidneys are the Na⁺ and K⁺ concentration in the plasma and inside the kidney compartments, aldosterone, acid–base balance, and the glomerular filtration rate (GFR).

The kidneys can regulate the excretion of K+, although approximately 30 mmol/day is excreted as obligatory renal loss. Potassium is filtered through the glomeruli but is almost completely absorbed in the proximal tubules (Fig. 1-9). The concentration of K⁺ in the blood and the glomerular filtration rate (GFR) regulate K⁺ excretion, because these two factors determine the amount of K⁺ available in the interstitial fluid of the kidneys for the exchange that occurs in the distal tubule and the collecting duct.

Figure 1-9 Potassium excretion in the kidney.

Potassium reenters from the interstitial fluid into the cells of the distal tubules and collecting duct. A small part of it is actively secreted into the lumen, but mostly K⁺ enters passively through diffusion. This diffusion occurs in response to the active reabsorption of Na⁺ from the lumen of tubular and ductal cells. Active Na⁺ reabsorption generates an electric membrane gradient, and K⁺ and hydrogen ions (H+) cross the membranes to neutralize these electric charges. Aldosterone promotes secretion of K⁺ in the distal tubule but also promotes, reabsorption of Na+, thus stimulating the sodium–potassium exchange (i.e., the excretion of K⁺ in urine). Remember that aldosterone secretion is stimulated by the renin–angiotensin system, but can also be increased directly by a high intake of K+.

The excretion of K⁺ in urine depends also on the amount of Na⁺ absorbed in the distal nephron. If Na⁺ reabsorption is stimulated by diuretics, more K⁺ will be needed to replace the reabsorbed Na+.

Potassium and hydrogen ions are in a balance, and the excretion of K⁺ in urine depends on the availability of H⁺. In acidosis, the excess of H⁺ favors the exchange of Na⁺ for H⁺. The urinary excretion of K⁺ is reduced, and renal acidosis is therefore associated with hyperkalemia. The reverse happens in alkalosis, when less H⁺ is available for exchange; higher excretion of K⁺ results in hypokalemia, which is typical of renal alkalosis. The reverse is also true: Since hypokalemia of any origin makes less K⁺ available for the exchange with Na⁺ in the distal nephron, hypokalemia tends to produce alkalosis.

Intercellular potassium concentration depends on the transmembrane exchange between the extracellular and intracellular pool of potassium.

The concentration of K⁺ inside the cells is an order of magnitude (approximately 25 times) higher than in the interstitial fluid. Such a huge gradient can be maintained only by the constant work of the sodium–potassium adenosine triphosphatase (Na+/K+ ATPase), which requires a huge expenditure of energy. Cell injury caused by ischemia or toxins may reduce the energy supply or impair the function of ATPase, resulting in a net increase of the efflux of K⁺ from the cells (Fig. 1-10).

Figure 1-10 Potassium flux across the cell membrane. A, In the normal cell the intracellular potassium (K+) concentration is several times higher than in the interstitial fluid. The gradient is maintained by the Na/K-ATPase in the cell membrane. B, Cell injury leads to a leakage of K⁺ from the cytoplasm into the interstitial fluid. C, In acidosis the hydrogen ions (H⁺) enter the cells from the interstitial fluid, displacing K+, which is translocated into the interstitial fluid (hyperkalemia). Lack of insulin in diabetes mellitus favors efflux of K⁺ from cells. D, In alkalosis the intracellular H⁺ enters the interstitial fluid and the K⁺ enters the cells, causing hypokalemia. Insulin also favors the entry of K⁺ into the cells.

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