Articles on Medical Diseases and Conditions

Tag: Urinalysis

  • Azotemia (Elevated Blood Urea Nitrogen Level) and Renal Failure

    Many use the term “uremia” as a synonym for azotemia, although uremia is a syndrome and should be defined in clinical terms. A BUN level of approximately 100 mg/100 ml is usually considered to separate the general category of acute reversible prerenal azotemias from the more prolonged acute episodes and chronic uremias. In general, this is an accurate distinction, but there is a small but important minority of cases that do not follow this rule. Thus, some uremics seem to stabilize at a lower BUN level until their terminal episode, whereas a few persons with acute transient azotemia may have a BUN level close to 100 mg/100 ml (36 mmol/L) and, rarely, more than 125 mg/100 ml (45 mmol/L), which rapidly falls to normal levels after treatment of the primary systemic condition. It must be admitted that easily correctable prerenal azotemia with an appreciably elevated BUN level is almost always superimposed on previous subclinical renal damage or function loss. Although the previous damage may not have been severe enough to cause symptoms, the functional reserve of these kidneys has been eliminated by aging changes, pyelonephritis, or similar conditions. The same is usually true for azotemia of mild levels (30-50 mg/100 ml [11-18 mmol/L]) occurring with dehydration, high protein intake, cardiac failure, and other important but not life-threatening situations. After the BUN level has returned to normal levels, a creatinine clearance determination will allow adequate evaluation of the patient’s renal status.

    The onset of oliguria always raises the question of possible renal failure. For a long time oliguria was considered a constant clinical sign of acute renal failure (also called acute tubular necrosis); however, it is now recognized that 30%-50% of patients with acute renal failure do not have oliguria (literature range, 20%-88%). Normal adult urine volume is 500-1,600 ml/24 hours (upper limit depends on fluid intake); a volume less than 400 ml/24 hours is considered to represent oliguria if all urine produced has actually been collected. Incomplete collection or leakage around a catheter may give a false impression of oliguria. Occasionally a patient develops oliguria and progressive azotemia, and it becomes necessary to differentiate between prerenal azotemia, which is correctable by improving that patient’s circulation, and acute renal tubular necrosis. This problem most frequently occurs after a hypotensive episode or after surgery.

    The differential diagnosis of azotemia includes prerenal azotemia, acute renal failure (acute tubular necrosis), and chronic renal failure. Two tests, filtered fraction of sodium and the free water clearance, under certain conditions, are usually capable of differentiating prerenal azotemia from renal failure. In general, if the serum creatinine level as well as the BUN level is elevated, this is more suggestive of renal failure than prerenal azotemia. However, this assumes that the BUN elevation is discovered very early after onset. Under most conditions the differentiation between prerenal azotemia and acute tubular necrosis by means of the serum creatinine level is not sufficiently reliable. A number of other tests have been proposed for the same purpose (Chapter 37). These tests have been found useful by some investigators but not useful in a sufficient number of cases by others. Part of the difference of opinion is based on the test criteria each investigator uses to differentiate prerenal azotemia from acute tubular necrosis. In general, when the test criteria are structured simply to provide best statistical separation for all cases of prerenal azotemia and acute tubular necrosis, there is considerable overlap between the two groups. When the criteria are deliberately structured to separate out either the cases of prerenal azotemia or those of acute tubular necrosis and the diagnosis is made only on the remaining cases, the test becomes insufficiently sensitive. A good example is the test using urine excretion of sodium. If one uses the urine sodium cutoff point of less than 15 mEq/L (mmol/L), the test has excellent reliability in ruling out acute tubular necrosis. However, one investigator found that 67% of patients with acute tubular necrosis and 63% of patients with prerenal azotemia had urine sodium values between 15 and 40 mEq/L (mmol/L). Therefore, if one wishes to use less than 15 mEq/L (mmol/L) as a diagnostic limit to avoid the overlap, one excludes 63% of this investigator’s prerenal azotemia cases, and the test becomes poorly sensitive for prerenal azotemia, despite excellent specificity created by ruling out nearly all cases of acute tubular necrosis.

    Part of the problem is the fact that acute tubular necrosis may be oliguric or nonoliguric. Some test criteria that enable good separation of prerenal azotemia and oliguric acute tubular necrosis are not as good in distinguishing prerenal azotemia from nonoliguric acute tubular necrosis. If the test cutoff points are restructured to include both oliguric and nonoliguric acute tubular necrosis, the overall accuracy of the test may suffer.

    In occasional cases, initial tests indicate that some renal function still remains, but the patient then develops acute tubular necrosis due to progression of the underlying disease or superimposition of some other factor.

    Urinalysis may provide useful information in patients with acute tubular necrosis. Red blood cell (RBC) or hemoglobin casts suggest glomerulonephritis, subacute bacterial endocarditis, transfusion reaction, and collagen disease. Uric acid crystals may provide a clue to uric acid nephropathy. Strongly positive urine chemical tests for hemoglobin without significant microscopic RBC raises the possibility of myoglobin.

    Differentiation of acute tubular necrosis and chronic renal failure is extremely difficult without clinical evidence of acute onset. If the patient does not have an elevated serum creatinine level when first investigated, or if the creatinine level is only mildly elevated and there is no anemia or other evidence of uremia, the evidence is more in favor of acute tubular necrosis than chronic renal failure. Even more difficulty exists when a patient develops acute tubular necrosis and the question is raised as to whether the patient’s renal function is likely to recover. Radioisotope studies with a scintillation camera may be helpful. Bilateral poor uptake, poor cortex-pelvis transit, and decreased blood flow suggest chronic diffuse renal disease. Good cortex uptake, poor cortex-pelvis transit, and good blood flow are more indicative of acute tubular necrosis or prerenal azotemia. Isotope techniques can demonstrate postrenal obstruction and frequently can visualize the kidneys when the IVP cannot.

    In a patient with uremia (chronic azotemia), there is no good way to determine prognosis by laboratory tests. The degree of azotemia does not correlate well with the clinical course in uremia except in a very general way.

    Other diagnostic tests

    Addis count. This procedure is used in suspected subclinical cases of chronic glomerulonephritis to demonstrate 12-hour abnormally increased rates of RBCs and cast excretion too small to exceed the normal range in ordinary random specimen microscopic examinations. If the random urine specimen already shows hematuria or pyuria, and if contamination can be ruled out by catheter or clean catch collection technique, the Addis count cannot do anything more than show the same abnormality. Addis counts are very rarely necessary with a good history and adequate workup.

    Renal biopsy. This procedure is now widely used. It may be the only way to find which disease is present, but it should be reserved for patients whose treatment depends on exact diagnosis. The general category of most renal disease entities can be inferred using routine methods. Because of the random sample nature of renal biopsies, ordinary light microscopy more often than not shows only nonspecific changes reflecting the result rather than the etiology of the disease process, and frequently renal biopsies are requested only for academic rather than for practical diagnostic or therapeutic reasons. In children, electron microscopy and immunofluorescent techniques are more useful than light microscopy.

  • Urinalysis in Miscellaneous Diseases

    Fever. Fever is the most common cause of proteinuria (up to 75% of febrile patients). If severe, it may be associated with an increase in hyaline casts (etiology unknown, possibly dehydration).

    Cystitis-urethritis. Cystitis and urethritis are lower urinary tract infections, often hard to differentiate from renal infection. Clumping of WBCs is suggestive of pyelonephritis but only WBC casts provide absolute specificity. Necrotizing cystitis may cause hematuria. The two-glass urine test helps to differentiate urethritis from cystitis. After cleansing the genitalia, the patient voids about 10-20 ml into container number 1 and the remainder into container number 2. A significant increase in the WBC count of container number 1 over that of container number 2 suggests urethral origin.

    Genitourinary tract obstruction. Neuromuscular disorders of the bladder, congenital urethral strictures and valves, intrinsic or extrinsic ureteral mechanical compressions, and intraluminal calculi produce no specific urinary changes but predispose to stasis and infection. Obstruction, partial or complete, is a frequent etiology for recurrent genitourinary tract infections.

    Amyloidosis. Renal involvement usually leads to proteinuria. In a minority of cases, when the process severely affects the kidney there may be high proteinuria and sediment typical of the nephrotic syndrome. The urinary sediment, however, is not specific, and RBC casts are not present. Renal amyloidosis is usually associated with chronic disease, such as long-standing osteomyelitis or infection, or multiple sclerosis.

    Urinary calculi. Urinary calculi often cause hematuria of varying degree and, depending on the composition of the stone, may be associated with excess excretion of calcium, uric acid, cystine, phosphates, or urates in the urine, even when calculi are not clinically evident. Frequent complications are infections or obstruction, and infection may occur even in the absence of definite obstruction. Ureteral stone passage produces hematuria, often gross. Intravenous pyelography is the best means of diagnosis. Some types of calculi are radiopaque, and others may be localized by finding a site of ureteral obstruction.

    Sickle cell anemia. Hematuria frequently occurs due to kidney lesions produced by intra-capillary RBC plugs, leading to congestion, small thromboses, and microinfarctions. Hematuria is also frequent at times of hematologic crises. Hematuria may be present even without crises in sickle cell disease or sickle cell variants. Sickle cell patients may lose urine-concentrating ability for unknown reasons. This happens even with sickle cell variants but is less common.

    Chronic passive congestion. One cause of renal congestion is inferior vena cava obstruction. It produces mild diffuse tubular atrophy and hyperemia, leads to proteinuria (usually mild to moderate) and hyaline casts, and sometimes also elicits epithelial casts and a few RBCs. Occasionally, but not commonly, severe chronic passive congestion (CPC) may simulate the nephrotic syndrome to some extent, including desquamated epithelial cells containing fat plus many casts of the epithelial series. In CPC of strictly cardiac origin without significant previous renal damage, there is decreased urine volume but usually retained urine-concentrating ability. No anemia is present unless it is due to some other systemic etiology.

    Benign arteriosclerosis. Benign arteriosclerosis involves the renal parenchyma secondarily to decreased blood supply. In most cases in the earlier stages, there are few urinary findings, if any; later, there is often mild proteinuria (0.1-0.5 gm/24 hours) and a variable urine sediment, which may contain a few hyaline casts, epithelial cells, and perhaps occasional RBCs. If the condition eventually progresses to renal failure, there will be significant proteinuria and renal failure sediment with impaired renal function tests.

    Weil’s disease. Weil’s disease is leptospiral infection (Chapter 15) and clinically presents with hepatitis and hematuria. Characteristically, there are also high fever and severe muscle aching, and there may be associated symptoms of meningitis.

    Infectious mononucleosis. Renal involvement with hematuria occurs in 5%-6% of cases.

    Purpura and hemorrhagic diseases. These diseases should be recognized as causes of hematuria, either by itself or in association with glomerular lesions. The Henoch-Schцnlein syndrome (anaphylactoid purpura) is a rare condition featuring gastrointestinal bleeding (Henoch) or skin purpura (Sch?nlein) that often is concurrent with hematuria and nephritis.

    Hypersensitivities. Hypersensitivities may lead to proteinuria (usually slight) with hematuria and perhaps a moderate increase in casts. Kidney involvement may occur due to hypersensitivity to mercurials, sulfas, or other substances.

    Fat embolism. Fat embolism commonly occurs after trauma, especially fractures. Cerebral or respiratory symptoms develop the second or third day after injury, usually associated with a significant drop in hemoglobin values. Fat in the urine is found in about 50% of patients. Unfortunately, a physician’s order for a test for fat in the urine will usually result in microscopic examination of the sediment. Whereas this is the correct procedure to detect fat in the nephrotic syndrome, in which fat is located in renal epithelial cells and casts, it is worthless for a diagnosis of fat embolism, in which free fat droplets must be identified. Since free fat tends to float, a simple procedure is to fill an Erlenmeyer (thin-neck) flask with urine up into the thin neck, agitate gently to allow fat to reach the surface, skim the surface with a bacteriologic loop, place the loop contents on a slide, and stain with a fat stain such as Sudan IV.

    Hemochromatosis. This condition is suggested by hepatomegaly, gray skin pigmentation, and proteinuria in a diabetic patient. Proteinuria may exceed 1 gm/24 hours, but sediment may be scanty and fat is absent. In severe cases yellow-brown coarse granules of hemosiderin are seen in cells, in casts, and lying free. Prussian blue (iron) stains in this material are positive. Distal convoluted tubules are the areas primarily involved. Since hemochromatosis does not invariably involve the kidney until late, a negative urine result does not rule out the diagnosis. False positive results (other types of urine siderosis) may occur in pernicious anemia, hemolytic jaundice, and in patients who have received many transfusions.

    Thyroid dysfunction.

    Myxedema. Proteinuria is said to occur without other renal disease. Its incidence is uncertain, especially since some reports state that proteinuria is actually not common and usually persists after treatment.

    Hyperthyroidism. The kidney may lose its concentrating ability so that specific gravity may remain low even in dehydration; this is reversible with treatment and a return to a euthyroid condition. Occasionally, glucosuria occurs in patients.

  • The Kidney in Disease

    Primary glomerular renal disease for a long time was subdivided into glomerulonephritis (acute, subacute, chronic) and the nephrotic syndrome, based on clinical and light microscopic findings. With the advent of renal biopsy, electron microscopy, and immunoglobulin fluorescent staining of tissue sections, the clinical categories are being reclassified on the basis of ultrastructure and immunologic characteristics (see Table 37-5). Diseases in some of the immunohistopathologic subdivisions have different prognoses (and, in some cases, different responses to certain therapeutic agents) and therefore could logically be regarded as separate entities. Nevertheless, I have chosen to describe laboratory findings in terms of the original clinical syndromes, since this is the way most clinicians encounter primary renal disease. A morphologic classification of glomerular disease is given in Table 37-5.

    Glomerulonephritis

    Acute glomerulonephritis. Classic acute glomerulonephritis (AGN) corresponds to a subcategory of proliferative glomerulonephritis that is considered a hypersensitivity reaction, usually associated with concurrent or recent infection. The most common organism incriminated is the beta-hemolytic Lancefield group A Streptococcus. Only a relatively small number of specific group A strains are known to cause AGN in contrast to the large number that initiate acute rheumatic fever.

    Clinically, onset of the disease is frequently manifested by gross hematuria. The urine may be red or may be the color of coffee grounds (due to breakdown of hemoglobin to brown acid hematin). In mild cases, gross hematuria may be less evident, or the hematuria may be microscopic only. Varying degrees of peripheral edema, especially of the upper eyelids, are often present. Hypertension of varying degree is a frequent initial finding.

    Laboratory features usually include an elevated erythrocyte sedimentation rate and frequently a mild to moderate normocytic-normochromic (or slightly hypochromic) anemia. There is mild to moderate proteinuria (0.5-3.0 gm/24 hours). The urinary sediment reflects varying degrees of hematuria, often with WBCs also present. RBC casts are characteristic and are the most diagnostic laboratory finding. They may be present only intermittently, may be few in number, and may be degenerated enough to make recognition difficult. Although RBC casts are not specific for AGN, relatively few diseases are consistently associated with RBC casts. These conditions include AGN, subacute and occasionally chronic glomerulonephritis, subacute bacterial endocarditis, some of the collagen diseases (especially systemic lupus), and hemoglobinuric acute tubular necrosis.

    Renal function tests. Prolonged azotemia is not common in poststreptococcal AGN (5%-10% of cases), despite hypertension, although as many as 50% of affected persons have some BUN elevation initially. Renal function tests are said to be essentially normal in nearly 50% of patients; the rest have varying degrees of impairment for varying time intervals, and a small percentage show renal insufficiency with uremia. Urine concentrating ability is generally maintained for the first few days; in some patients, it may then be impaired for a considerable time. Function tests in general tend to reflect (although not exclusively) the primarily glomerular lesion found in AGN, manifested on light microscopy by increased glomerular cellularity and swelling and proliferation of capillary endothelial cells and on electron microscopy by subepithelial “humps.”

    Antistreptococcal antibodies. In addition to urinalysis, the antistreptolysin-O (ASL or ASO) titer may be helpful, since a significant titer (>200 Todd units) suggests recent or relatively recent group A streptococcal infection. However, since up to 20% of AGN patients have ASO titers in the normal range, a normal ASO titer does not rule out the diagnosis, nor does a high titer guarantee that the condition is indeed AGN (the group A streptococcal infection may be unrelated to the renal disease). Measurement of other streptococcal enzyme antibodies, such as anti-deoxyribonuclease B (ADN-B), in addition to ASO, will improve sensitivity of the test. Several commercial kits have combined reagents active against several of the antistreptococcal antibodies (Chapter 23). The third component (C3) of serum complement is nearly always depressed in streptococcal AGN and returns to normal in 6-8 weeks. Consistently normal early C3 levels are evidence against streptococcal etiology, and failure of C3 to become normal in 8 weeks also suggests a different etiology.

    Acute glomerulonephritis is a relatively benign disease in childhood, since mortality is only about 1%, and an even smaller percentage suffer permanent damage. In adults, the incidence of the disease is much lower, but 25%-50% of adult patients develop chronic renal disease.

    Rapidly progressive glomerulonephritis. Rapidly progressive glomerulonephritis may follow the acute stage of AGN but much more commonly appears without any previous clinical or serologic evidence of AGN. It is more common in older children and adults. The original term “subacute glomerulonephritis” was misleading; originally it referred to the duration of the clinical course, longer than that of AGN in the average patient but much shorter than that of chronic glomerulonephritis. Histologically, the glomeruli show epithelial cell proliferation with resultant filling in of the space between Bowman’s capsule and the glomerular tuft (epithelial crescent). The urine sediment includes many casts of hyaline and epithelial series; RBCs and often WBCs are present in varying numbers, often with a few RBC casts. There is moderately severe to marked proteinuria, and both the degree of proteinuria and the urinary sediment may sometimes be indistinguishable from similar findings in the nephrotic syndrome, even with fatty casts present. Clinically, rapidly progressive glomerulonephritis behaves as a more severe form of AGN and generally leads to death in weeks or months. It is not the same process as the nephrotic episodes that may form part of chronic glomerulonephritis. In addition to urinary findings, anemia is usually present. Renal function tests demonstrate both glomerular and tubule destruction, although clinically there is usually little additional information gained by extensive renal function studies. Serum complement C3 is temporarily depressed in cases of poststreptococcal origin but otherwise is usually normal.

    Chronic glomerulonephritis. Chronic glomerulonephritis infrequently is preceded by AGN, but usually there is no antecedent clinical illness or etiology. It most often runs a slowly progressive or intermittent course over many years. During the latent phases there may be very few urinary abnormalities, but RBCs are generally present in varying small numbers in the sediment. There is almost always proteinuria, generally of mild degree, and rather infrequent casts of the epithelial series. Disease progression is documented by a slowly decreasing ability to concentrate the urine, followed by deterioration in creatinine clearance. Intercurrent streptococcal upper respiratory tract infection or other infections may occasionally set off an acute exacerbation. There may be one or more episodes of the nephrotic syndrome, usually without much, if any, hematuria. The terminal or azotemic stage produces the clinical and laboratory picture of renal failure. Finely granular and waxy casts predominate, and broad casts are often present. There is moderate proteinuria.

    Nephrotic syndrome

    The criteria for diagnosis of the nephrotic syndrome include high proteinuria (>3.5 gm/24 hours), edema, hypercholesterolemia, and hypoalbuminemia. However, one or occasionally even more of these criteria may be absent. The level of proteinuria is said to be the most consistent criterion. In addition, patients with the nephrotic syndrome often have a characteristic serum protein electrophoretic pattern, consisting of greatly decreased albumin and considerably increased alpha-2 globulin. However, in some cases the pattern is not marked enough to be characteristic. The nephrotic syndrome is one of a relatively few diseases in which the serum cholesterol level may substantially contribute toward establishing the diagnosis, especially in borderline cases.

    The nephrotic syndrome has nothing in common with the entity formerly called hemoglobinuric nephrosis (or lower nephron nephrosis), despite the unfortunate similarity in names. The term hemoglobinuric nephrosis has generally been discarded, since it is only a subdivision of acute tubular necrosis, due to renal tubule damage from hemoglobin derived from marked intravascular hemolysis. Even the term “nephrotic syndrome” as it is currently used is actually a misnomer and dates from the time when proteinuria was thought primarily to be due to a disorder of renal tubules. The word “nephrosis” was then used to characterize such a situation. It is now recognized that various glomerular lesions form the actual basis for proteinuria in the nephrotic syndrome, either of the primary or the secondary type. The nephrotic syndrome as a term is also confusing because it may be of two clinical types, described in the following section.

    Primary (or lipoid) nephrosis. Primary nephrosis is the idiopathic form and is usually found in childhood. The etiology of primary (idiopathic or lipoid) nephrosis is still not definitely settled. Renal biopsy has shown various glomerular abnormalities, classified most easily into basement membrane and focal sclerosis varieties. In most children the basement membrane changes may be so slight (null lesion) as to be certified only by electron microscopy (manifested by fusion of the footplates of epithelial cells applied to the basement membrane). The null lesion is associated with excellent response to steroids, a great tendency to relapse, and eventually relatively good prognosis. Focal sclerosis most often is steroid resistant and has a poor prognosis.

    In lipoid nephrosis, the urine contains mostly protein. The sediment may contain relatively small numbers of fatty and granular casts, and there may be small numbers of RBCs. Greater hematuria or cylindruria suggests greater severity but not necessarily a worse prognosis. Renal function tests are normal in most patients; the remainder have various degrees of impairment.

    Nephrotic syndrome. Although lipoid nephrosis may be found in adults, the nephrotic syndrome is more common and may be either idiopathic or secondary to a variety of diseases. The most common idiopathic lesions include a diffuse light microscope “wire loop” basement membrane thickening, which has been termed membranous glomerulonephritis, and a type that has been called membranoproliferative. Prognosis in these is worse than in childhood lipoid nephrosis.

    The most common etiologies of secondary nephrotic syndrome are chronic glomerulonephritis, Kimmelstiel-Wilson syndrome, systemic lupus, amyloid and renal vein thrombosis. In the urine, fat is the most characteristic element, appearing in oval fat bodies and fatty casts. Also present are variable numbers of epithelial and hyaline series casts. Urine RBCs are variable; usually only few, but sometimes many. Significant hematuria suggests lupus; the presence of diabetes and hypertension suggests Kimmelstiel-Wilson syndrome; a history of previous proteinuria or hematuria suggests chronic glomerulonephritis; and the presence of chronic long-standing infection suggests an amyloid etiology. About 50% of cases are associated with chronic glomerulonephritis. Renal function tests in the nephrotic syndrome secondary to lupus, Kimmelstiel-Wilson syndrome, and amyloid generally show diffuse renal damage. The same is true of chronic glomerulonephritis in the later stages; however, if the nephrotic syndrome occurs relatively early in the course of this disease, test abnormalities may be minimal, reflected only in impaired concentrating ability, Histologically, renal glomeruli in the nephrotic syndrome exhibit lesions that vary according to the particular disease responsible.

    Membranoproliferative glomerulonephritis occurs in older children and teenagers and displays some features of AGN as well as nephrotic syndrome. Hematuria and complement C3 decrease occur, but the C3 decrease usually is prolonged beyond 8 weeks (60% or more cases). However, C3 levels may fluctuate during the course of the disease.

    Malignant hypertension (accelerated arteriolar nephrosclerosis)

    Malignant hypertension is most common in middle age, with most patients aged 30-60 years. There is a tendency toward males and an increased incidence in blacks. The majority of patients have a history of preceding mild or benign hypertension, most often for 2-6 years, although the disease can begin abruptly. The syndrome may also be secondary to severe chronic renal disease of several varieties. Clinical features are markedly elevated systolic and diastolic blood pressures, papilledema, and evidence of renal damage. Laboratory tests show anemia to be present in most cases, even in relatively early stages. Urinalysis in the early stages most often shows a moderate proteinuria and hematuria, usually without RBC casts. The sediment thus mimics to some extent the sediment of AGN. Later the sediment may show more evidence of tubular damage. There usually develops a moderate to high proteinuria (which uncommonly may reach 5-10 gm/24 hours) accompanied by considerable microscopic hematuria and often many casts, including all those of the hyaline and epithelial series—even fatty casts occasionally. In the terminal stages, late granular or waxy casts and broad renal failure casts predominate. The disease produces rapid deterioration of renal function, and most cases terminate in azotemia. Nocturia and polyuria are common owing to the progressive renal damage. If congestive heart failure is superimposed, there may be a decreased urine volume plus loss of ability to concentrate urine.

    Pyelonephritis (renal infection)

    Acute pyelonephritis often elicits a characteristic syndrome (spiking high fever, costovertebral angle tenderness, dysuria, back pain, etc.). Proteinuria is mild, rarely exceeding 2 gm/24 hours. Pyuria (and often bacteriuria) develops. The presence of WBC casts is diagnostic, although they may have to be carefully searched for or may be absent. Urine culture may establish the diagnosis of urinary tract infection but cannot localize the area involved. Hematogenous spread of infection to the kidney tends to localize in the renal cortex and may give fewer initial urinary findings; retrograde ascending infection from the lower urinary tract reaches renal medulla areas first and shows early pyuria.

    In chronic low-grade pyelonephritis, the urine may not be grossly pyuric, and sediment may be scanty. In some cases, urine cultures may contain fewer than 100,000 organisms/mm3 (100 Ч 109/L) or may even be negative. Very frequently, however, there is a significant increase in pus cells; they often, but not invariably, occur in clumps when the process is more severe. Casts other than the WBC type are usually few or absent in pyelonephritis until the late or terminal stages, and WBC casts themselves may be absent.

    A urine specimen should be obtained for culture in all cases of suspected urinary tract infection to isolate the organism responsible and determine antibiotic sensitivity (Chapter 14).

    Tuberculosis is a special type of renal infection. It involves the kidney in possibly 25% of patients with chronic or severe pulmonary tuberculosis, although the incidence of clinical disease is much less. Hematuria is frequent; it may be gross or only microscopic. Pyuria is also common. Characteristically, pyuria is present without demonstrable bacteriuria (of ordinary bacterial varieties), but this is not reliable due to a considerable frequency of superinfection by ordinary bacteria in genitourinary tuberculosis. Dysuria is also present in many patients. If hematuria (with or without pyuria) is found in a patient with tuberculosis, genitourinary tract tuberculosis should be suspected. Urine cultures are said to be positive in about 7% of patients with significant degrees of active pulmonary tuberculosis. At least three specimens, one obtained each day for 3 days, should be secured, each one collected in a sterile container. A fresh early morning specimen has been recommended rather than 24-hour collections. Acid-fast urine smears are rarely helpful. If suspicion of renal tuberculosis is strong, intravenous pyelography should be done to assess the extent of involvement.

    Renal papillary necrosis is a possible complication of acute pyelonephritis, particularly in diabetics.

    Renal papillary necrosis (necrotizing papillitis)

    As the name suggests, this condition results from necrosis of a focal area in one or more renal pyramids. Papillary necrosis is most frequently associated with infection but may occur without known cause. It is much more frequent in diabetics. A small minority of cases are associated with sickle cell hemoglobin diseases or phenacetin toxicity. The disease usually is of an acute nature, although some patients may have relatively minor symptoms or symptoms overshadowed by other complications or disease. The patients are usually severely ill and manifest pyuria, hematuria, and azotemia, especially when renal papillary necrosis is associated with infection. Drip-infusion intravenous (IV) pyelography is the diagnostic test of choice. Naturally, urine culture should be performed.

    Renal embolism and thrombosis

    Renal artery occlusion or embolism most often affects the smaller renal arteries or the arterioles. Such involvement produces renal infarction in that vessel’s distribution, usually manifested by hematuria and proteinuria. Casts of the epithelial series may also appear. Renal infarction frequently produces elevation of serum lactic dehydrogenase (LDH), with the LDH-1 isoenzyme typically greater than LDH-2 (Chapter 21). Aspartate aminotransferase (serum glutamic oxaloacetic transaminase) may also be increased but less frequently. Alkaline phosphatase is temporarily increased in some patients after 5-10 days (range 3-15 days), possibly related to the granulation tissue healing process.

    Acute tubular necrosis

    This syndrome may result from acute or sudden renal failure of any cause, most often secondary to hypotension, although intravascular hemolysis from blood transfusion reactions is probably the most famous cause. Acute tubular necrosis begins with a period of oliguria or near anuria and manifests subsequent diuresis if recovery ensues. Urinalysis demonstrates considerable proteinuria with desquamated epithelial cells and epithelial hyaline casts. There are usually some RBCs (occasionally many) and often large numbers of broad and waxy casts (indicative of severe urinary stasis in the renal parenchyma). Hemoglobin casts are usually present in cases due to intravascular hemolysis. Specific gravity is characteristically fixed at 1.010 after the first hours, and the BUN level begins rising shortly after onset. In cases of acute tubular necrosis not due to intravascular hemolysis, the pathogenesis is that of generalized tubular necrosis, most often anoxic.

    Congenital renal disease

    Polycystic kidney. There are two clinical forms of polycystic kidney, one fatal in early infancy and the other (adult type) usually asymptomatic until the third or fourth decade. The urinary sediment is highly variable; microscopic intermittent hematuria is common, and gross hematuria may occasionally take place. Cysts may become infected and produce symptoms of pyelonephritis. In general, the rate of proteinuria is minimal or mild but may occasionally be higher. Symptoms may be those of hypertension (50%-60% of cases) or renal failure. If the condition does progress to renal failure, the urinary sediment is nonspecific, reflecting only the presence of end-stage kidneys of any etiology. Diagnosis may be suggested by family history and the presence of bilaterally palpable abdominal masses and is confirmed by radiologic procedures, such as IV pyelography. Ultrasound can also be useful.

    Renal developmental anomalies. This category includes horseshoe kidney, solitary cysts, reduplication of a ureter, renal ptosis, and so forth. There may be no urinary findings or, sometimes, a slight proteinuria. In children, urinary tract anomalies often predispose to repeated urinary tract infection. Recurrent urinary tract infection, especially in children, should always be investigated for the possibility of either urinary tract obstruction or anomalies. Diagnosis is by IV pyelography.

    Renal neoplasia

    The most common sign of carcinoma anywhere in the urinary tract is hematuria, which is present in 60%-80% of patients with primary renal adenocarcinoma and (according to one report) in about 95% of bladder, ureter, and renal pelvis carcinoma. In renal cell carcinoma, hematuria is most often visible grossly and is intermittent. In persons over age 40 a neoplasm should be suspected if increased urine RBCs are not explained by other conditions known to produce hematuria. Even if such diseases are present, this does not rule out genitourinary carcinoma. The workup of a patient with hematuria is discussed in Chapter 13. Methods for detecting renal cell carcinoma are described in Chapter 33.

    Lupus erythematosus or polyarteritis nodosa

    About two thirds of lupus patients have renal involvement. Generally, there is microscopic hematuria; otherwise there may be a varying picture. In the classic case of lupus (much less often in polyarteritis), one finds a “telescoped sediment,” that is, a sediment containing the characteristic elements of all three stages of glomerulonephritis (acute, subacute, and chronic) manifest by fatty, late granular, and RBC casts. Usually, hematuria is predominant, especially in polyarteritis. In lupus, RBC casts are more commonly found. Up to one third of lupus patients develop the nephrotic syndrome. Complement C3 levels are frequently decreased in active lupus nephritis.

    Embolic glomerulonephritis

    Embolic glomerulonephritis is most commonly associated with subacute bacterial endocarditis. Scattered small focal areas of necrosis are present in glomerular capillaries. There is some uncertainty whether the lesions are embolic, infectious, or allergic in origin. Since the glomerular lesions are sharply focal, there usually is not much pyuria. Hematuria is usually present and may be pronounced. If localized tubular stasis occurs in addition, RBC casts may appear, with resultant simulation of latent glomerulonephritis or AGN. The rate of proteinuria often remains relatively small, frequently not more than 1 gm/24 hours.

    Diabetes

    The kidney may be affected by several unrelated disorders, including (1) a high incidence of pyelonephritis, sometimes renal papillary necrosis; (2) a high incidence of arteriosclerosis with hypertension; and (3) Kimmelstiel-Wilson syndrome (intercapillary glomerulosclerosis). The nephrotic syndrome may occur in the late stages of the Kimmelstiel-Wilson syndrome. Otherwise, only varying degrees of proteinuria are manifest, perhaps with a few granular casts. Diabetic microalbuminuria, a stage that precedes overt diabetic renal disease, was discussed in the earlier section on urine protein.

    Pregnancy

    Several abnormal urinary findings are associated with pregnancy.

    Benign proteinuria. Proteinuria may appear in up to 30% of otherwise normal pregnancies during labor but surpasses 100 mg/100 ml in only about 3% of these cases. It is unclear whether proteinuria must be considered pathologic if it occurs in uncomplicated pregnancy before labor. Some authorities believe that proteinuria is not found in normal pregnancy; others report an incidence of up to 20%, which is ascribed to abdominal venous compression.

    Eclampsia. This condition, also known as toxemia of pregnancy, denotes a syndrome of severe edema, proteinuria, hypertension, and convulsions associated with pregnancy. This syndrome without convulsions is called preeclampsia. In most cases, onset occurs either in the last trimester or during labor, although uncommonly the toxemic syndrome may develop after delivery. The etiology is unknown, despite the fact that delivery usually terminates the signs and symptoms. Pronounced proteinuria is the rule; the most severe cases may have oval fat bodies and fatty casts. Other laboratory abnormalities include principally an elevated serum uric acid level in 60%-70% of cases and a metabolic acidosis. The BUN level is usually normal. Diagnosis at present depends more on physical examination, including ophthalmoscopic observation of spasm in the retinal arteries and blood pressure changes, than on laboratory tests, except tests for proteinuria. Gradual onset of eclampsia may be confusing, since some degree of edema is common in pregnancy, and proteinuria (although only slight or mild) may appear during labor.

    Glucosuria. Glucosuria occurs in 5%-35% of pregnancies, mainly in the last trimester. Occasional reports state an even higher frequency. It is not completely clear whether this is due to increased glucose filtration resulting from an increased GFR, a decreased renal tubular transport maximum (reabsorptive) capacity for glucose, a combination of the two, or some other factor. Lactosuria may also occur in the last trimester and may be mistaken for glucosuria when using copper sulfate reducing tests for urine glucose.

    Renal function tests. The glomerular filtration rate is increased during pregnancy. Because of this, the BUN level may be somewhat decreased, and clearance tests are somewhat increased. Renal concentration may appear falsely decreased because of edema fluid excretion that takes place during sleep.

    Infection. Bacteriuria has been reported in 4%-7% of pregnant patients, whereas the incidence in nonpregnant healthy women is approximately 0.5%. It is believed that untreated bacteriuria strongly predisposes to postpartum pyelonephritis.

  • Appearance

    The appearance of the specimen is usually reported only if it is abnormal.

    Red: Blood; porphyria; occasionally urates, phenolphthalein, or dihydroxyanthraquinone (Dorbane) (laxative use)
    Brown: Blood (acid hematin); alkaptonuria (urine turns brownish on standing); melanin (may be brown and turn black on standing)
    Dark orange: Bile, pyridium (a urinary tract disinfectant)

    pH

    Normal urine pH is 5.0-6.0 (4.5-8.0); it is over 6.0 in alkalosis or, rarely, if the acidifying mechanism of the kidney fails (renal acidosis syndrome) or is poisoned (carbonic anhydrase inhibitors). Proteus infections typically will alkalinize. Finally, urine standing at room temperature will often slowly become alkaline due to bacterial growth. A pH determination is of minor importance except in a few circumstances.

    Specific gravity

    Specific gravity will be discussed in greater length in Chapter 13 (renal function). It is important for several reasons: (1) it is one parameter of renal tubular function; (2) inability to concentrate may accompany certain diseases with otherwise relatively normal tubular function, such as diabetes insipidus or, occasionally, severe hyperthyroidism and sickle cell anemia; and (3) it affects other urine tests; a concentrated specimen may give higher results for substances such as protein than a dilute specimen, although the amount of substance excreted per 24 hours may be the same in both cases. That is, the same amount of substance assayed in a small quantity of fluid (high specific gravity) may appear more than if present in a large dilute urine volume (low specific gravity).

    Specific gravity is an estimate of relative density (for fluids, the proportion of dissolved solids in a defined volume of solvent) and can be measured in various ways. The oldest was by hydrometer (weighted flotation device), whose reproducibility was poor and which was relatively inaccurate. Most laboratories use some version of a refractometer (e.g., a total solids meter), which is fairly reproducible and accurate, but which is affected by moderate amounts of protein, glucose, or x-ray contrast media. More recently, dipstick specific gravity tests have become available based on changes in urine ion content that produce a pH change of the polyelectrolyte content of the reagent pad, which, in turn, induces color changes of an indicator (bromthymol blue). This method is read in increments of 0.005 units. Several evaluations suggest that the dipstick results will agree within ±0.005 units of total solids meter values about 85% (range 72%-90%) of the time. This suggests that the dipstick method is somewhat less accurate than the total solids meter. Whether the dipstick method is useful depends on whether a difference of 0.004-0.005 from the total solids meter value is considered clinically acceptable. The dipstick is affected by moderate changes in pH or protein but not by glucose or x-ray contrast media.

    Protein

    In health the glomerular membrane prevents most of the relatively large protein molecules of the blood from escaping into the urine. A small amount does filter through, of which a small fraction is reabsorbed by the tubules, and the remainder (up to 0.1 gm/24 hours) is excreted. These amounts are normally not detected by routine clinical methods. One of the first responses of the kidney to a great variety of clinical situations is an alteration in the glomerular filtrate. To a lesser extent, a mere increase in glomerular filtration rate (GFR) may increase normal protein excretion. Since albumin has a relatively small molecular size, it tends to become the dominant constituent in proteinuria but rarely so completely as to justify the term “albuminuria” in a pure sense. Depending on the clinical situation, the constituents of proteinuria may or may not approach the constituents of plasma. Besides an excess of normal plasma proteins, at times abnormal proteins may appear in the urine. The most important of these is Bence Jones protein, which is excreted in up to 50% of patients with multiple myeloma.

    Etiologies of proteinuria. To interpret proteinuria, one must know not only the major conditions responsible but also the reason for production of excess urine protein. A system such as the following one is very helpful in this respect; proteinuria is classified according to the relationship of its etiology to the kidney and also the mechanism involved.

    A. Functional: not associated with easily demonstrable systemic or renal damage.
    1. Severe muscular exertion. This cause is becoming more important with the current popularity of sports and jogging.
    2. Pregnancy.
    3. Orthostatic proteinuria. (This term designates slight to mild proteinuria associated only with the upright position. The exact etiology is poorly understood, with usual explanations implicating local factors that cause renal passive congestion when the patient is in an upright position. Orthostatic proteinuria is easily diagnosed by comparing a specimen of early morning urine, produced entirely while the patient is asleep, with one obtained later when the patient has been upright for some time. This is probably the most common cause of “functional” proteinuria; some reports indicate that it occurs in 15%-20% of healthy young men who have proteinuria on routine urinalysis.)
    B. Organic: associated with demonstrable systemic disease or renal pathology.
    1. Prerenal proteinuria: not due to primary renal disease.
    a. Fever or a variety of toxic conditions. (This is the most common etiology for “organic” proteinuria and is quite frequent.)
    b. Venous congestion. (This is most often produced by chronic passive congestion due to heart failure. It is occasionally produced by intraabdominal compression of the renal veins.)
    c. Renal hypoxia. Renal hypoxia may be produced by severe dehydration, shock, severe acidosis, acute cardiac decompensation, or severe anemias, all of which lead to a decrease in renal blood flow and sometimes hypoxic renal changes. Very severe hypoxia, especially if acute, may lead to renal tubular necrosis.
    d. Hypertension (moderate or severe chronic hypertension, malignant hypertension or eclampsia)
    e. Myxedema.
    f. Bence Jones protein.
    2. Renal proteinuria: primarily kidney disease.
    a. Glomerulonephritis.
    b. Nephrotic syndrome, primary or secondary.
    c. Destructive parenchymal lesions (tumor, infection, infarct).
    3. Postrenal proteinuria: protein added to the urine at some point farther down the urinary tract from the renal parenchyma.
    a. Infection of the renal pelvis or ureter.
    b. Cystitis.
    c. Urethritis or prostatitis.
    d. Contamination with vaginal secretions. This cause may be suggested by presence of moderate or large numbers of squamous epithelial cells in the urine sediment.

    Protein tests are not specific for serum protein. Test results demonstrating protein detect filtered serum protein but may also result from test reaction with WBCs or RBCs, regardless of their origin, if they are present in sufficient number. However, intrinsic renal disease usually (not always) is associated with proteinuria whether abnormal cells are present or not. Thus, by testing the supernate of a centrifuged specimen for protein, one can tentatively assume that WBCs or RBCs originated mainly in the lower urinary tract if pyuria or hematuria occurs in the absence of proteinuria.

    Urine protein test methods. There are several clinically acceptable methods for semiquantitative protein testing. The older methods are heat with acetic acid and sulfosalicylic acid (3%, 10%, or 20%). Sulfosalicylic acid (SSA) is slightly more sensitive than the heat-acetic acid method. Sulfosalicylic acid false positive reactions may occur, notably with tolbutamide (Orinase), urates, hemoglobin (RBCs or hemolysis), WBCs, massive penicillin doses, dextran, occasionally with salicylates, and with various radiopaque x-ray substances. Bence Jones protein also gives a positive reaction. Results are expressed as 1+ to 4+; this correlates very roughly with the amount of protein present (Table 12-2).

    Relationship of qualitative and quantitative urine protein results

    Table 12-2 Relationship of qualitative and quantitative urine protein results

    Kingsbury-Clark procedure. This is a quantitative sulfosalicylic acid method using a series of permanent standards, each representing a different but measured amount of protein. After sulfosalicylic acid is added to the unknown specimen, a precipitate is formed that is compared with the standards and reported as the amount in the particular standard that it matches.

    Dipstick methods. Most laboratories now use dipstick methods, usually with multiple tests on the same paper strip. The sulfosalicylic acid method will detect as little as 10 mg/100 ml of protein, whereas the dipsticks are not reliable for less than 20-30 mg/100 ml. The dipsticks will react with hemoglobin but not with the other substances previously listed which might produce false positive results with sulfosalicylic acid. Dipstick behavior with Bence Jones protein is erratic; in many cases the results are positive, but a significant number do not react. Dipsticks are said to give false positive protein results when the urine has a very alkaline pH, although my experience has been that no change occurs even at pH 8.5 (a value that is very rarely exceeded). False negative results may occur if contact of the dipstick with urine is greatly prolonged, since this could wash out the buffer in the protein detection zone on the test strip. In addition, protein readings are only semiquantitative, and it is often difficult to obtain uniform results from the same specimens read by different individuals. This happens because the result depends on a color change corresponding to a change from one quantity (or level) of protein to the next; the color change may not always be clear cut, and interpretation of any color change is always partially subjective. There tends to be a fairly wide variation in results when specimens containing large amounts of protein are tested.

    24-hour measurement. Because individuals often vary in their interpretations, and also because the degree of urine concentration may greatly influence results on random urine specimens, it is sometimes useful to determine 24-hour urine protein excretion. A 24-hour specimen may also be affected by degree of concentration but to a lesser extent. However, 24-hour specimens also have their problems. One of these is incomplete specimen collection. Another is variation in results using different test methods or reagents, most of which have relatively poor reproducibility (coefficient of variation exceeding 10%) and react differently with different proteins. In some reports this occasionally led to very substantial differences in results of different methods on the same specimen.

    Several studies indicate that the protein/creatinine ratio in single-voided specimens produces an adequate estimation of normality or degree of abnormality compared with 24-hour urine specimens. Since the recumbent and the upright protein excretion rate is often different, first-morning urine specimens are not recommended for this test.

    Microalbuminuria. Recently, several studies have suggested that a latent period exists in renal disease associated with insulin-dependent diabetics. In this phase there is on-going glomerular damage that is manifest by increased albumin excretion but in quantities too small to be detectable by standard laboratory protein tests, either dipstick or usual 24-hour procedures. One current definition of diabetic microalbuminuria is albumin excretion of 20-200 µg/minute (30-300 mg/24 hours) found in at least two of three specimens collected within 6 months. Dipsticks will begin detecting proteinuria slightly above the microalbuminuria range upper limit. Multiple samples are required because diabetics with or without microalbuminuria may have day-to-day variation in albumin excretion as high as 50%. Besides diabetics, nondiabetic persons with hypertension (even mild) have increased incidence of microalbuminuria and dipstick-detectable proteinuria (in one study, the mean protein value for hypertensive person was 3 times that of nonhypertensives); and one report indicates increased incidence of proteinuria in patients with heart failure (see also discussion in Chapter 28).

    In summary, not all proteinuria is pathologic; even if so, it may be transient. Often the kidney is only indirectly involved, and occasionally protein may be added to the urine beyond the kidney. Individual interpretations of turbidity tests vary significantly and so do various assay methods for 24-hour urine protein.

    Glucose

    The most common and important glucosuria occurs in diabetes mellitus. The normal renal threshold is usually about 180 mg/100 ml (literature range 165-200 mg/100 ml) serum glucose level; above that, enough glucose is filtered to exceed the usual tubular transfer maximum for glucose reabsorption, and the surplus remains in the urine. However, individuals vary in their tubular transfer reabsorptive capacities. If the capacity happens to be low, the individual may spill glucose at lower blood levels than the average person (“renal glucosuria”). Certain uncommon conditions may elevate blood glucose levels in nondiabetic persons. A partial list of the more important conditions associated with glucosuria includes the following (discussed more fully in Chapter 28):

    1. Glucosuria without hyperglycemia
    a. Glucosuria of pregnancy (lactosuria may occur as well as glucosuria)
    b. Renal glucosuria
    c. Certain inborn errors of metabolism (Fanconi’s syndrome)
    d. After certain nephrotoxic chemicals (carbon monoxide, lead, mercuric chloride)
    2. Glucosuria with hyperglycemia
    a. Diabetes mellitus
    b. Alimentary glucosuria (hyperglycemia is very transient)
    c. Increased intracranial pressure (tumors, intracerebral hemorrhage, skull fracture)
    d. Certain endocrine diseases or hormone-producing tumors (Cushing’s syndrome, pheochromocytoma)
    e. Hyperthyroidism (occasionally)
    f. Occasionally, transiently, after myocardial infarction
    g. After certain types of anesthesia, such as ether

    The most commonly used tests for urine glucose are glucose oxidase enzyme paper dipsticks such as Clinistix, Tes-Tape, and the multiple-test strips, all of which are specific for glucose. Another widely used test is Clinitest, which, like the old Benedict’s method (rarely used today), is based on copper sulfate reduction by reducing substances and is therefore not specific for glucose or even sugar. In several reported comparisons of these tests, Tes-Tape proved most sensitive but also gave occasional false positives. Clinistix gave few false positives but sometimes gave equivocal results or missed a few positive results that Tes-Tape detected. The copper reduction tests included about 10% positive results from reducing substances other than glucose. Clinitest is a copper reduction tablet test that seemed to have median sensitivity, whereas Benedict’s method, which is a standard type of chemical procedure, was least sensitive, missing 10%-15% of tests positive by glucose oxidase, but with very few false positives apart from other reducing substances.

    Dipstick problems. Although theoretically specific and relatively sensitive, the enzyme papers are not infallible. False positive results have been reported due to hydrogen peroxide and to hypochlorites (found in certain cleaning compounds). These substances give negative copper reduction test results. Large amounts of vitamin C may produce false negative results with the glucose oxidase methods but false positive results with reducing substance methods. False negative results using Clinistix (but not Tes-Tape) have been reported from homogentisic acid (alkaptonuria), levodopa, and large doses of aspirin. Also, the enzyme papers may unaccountably miss an occasional positive result that is detected by copper sulfate reduction techniques. As with urine protein dipsticks, studies have demonstrated considerable variation in test reports by different persons on the same specimens, especially at glucose levels that are borderline between two test strip color change levels.

    Clinitest (copper reduction) false positive results are produced by sugars other than glucose (galactose, lactose) and by hemogentisic acid (alkaptonuria). Other potentially troublesome substances are p-aminosalicylic acid (PAS), methyldopa (Aldomet), heavy salicylate therapy (salicyluric acid excreted), and heavy concentrations of urates. Various beta-lactam antibiotics in large doses (especially the cephalosporins, but also the penicillins, monobactams, and carbapenems) may interfere with Clinitest interpretation by forming various colors. An important technical consideration is to keep Clinitest tablets and the enzyme papers free from moisture before use.

    Change in Clinitest methodology. The original methodology for Clinitest used five drops of urine. When urine contains large amounts of sugar, the color change typical of a high concentration may be unstable and quickly change to another color, which could be misinterpreted as an end point produced by smaller amounts of sugar (“pass-through” phenomenon). Many laboratories now use a two-drop specimen, which avoids the pass-through effect. However, the color changes that represent different concentrations of reducing substances are not the same with five drops as with two drops. Knowledge of the method used is essential for correct interpretation by the patient or the physician.

    Acetone and diacetic acid (ketone bodies)

    Ketone bodies include b-hydroxybutyric acid (BHBA), diacetic (acetoacetic) acid (DAA), and acetone. Under normal conditions, anaerobic metabolism of glucose proceeds to eventual formation of a compound called acetyl–coenzyme A, or acetyl-CoA. It can also be produced by metabolism of fatty acids. Most acetyl-CoA is used in the tricarboxylic (citric acid) metabolic cycle. However, acetyl-CoA can also enter the pathway to ketone body formation in the liver. Diacetic acid is formed initially, with the great majority being metabolized to BHBA and a small amount spontaneously decarboxylated to acetone and carbon dioxide. The major (but not the only) impetus toward increased ketone formation is either carbohydrate restriction or impaired carbohydrate metabolism. Skeletal and cardiac muscle can utilize a limited amount of DAA and BHBA as an energy source when normal glucose-derived energy is insufficient; but after a certain point, metabolic capacity is exceeded and excess ketone bodies are excreted by the kidney. However, renal excretion rate is also limited, and when ketone formation exceeds muscle utilization and renal excretory capacity, ketone bodies accumulate in the plasma, a condition known as ketosis. Normal plasma ketone composition is about 78% BHBA, 20% DAA, and 2% acetone.

    Technical methods and their drawbacks. Most chemical tests for ketones employ a nitroprusside reagent. Nitroprusside detects both DAA and acetone but not BHAA. The most commonly used product of this type is a tablet reagent formulation called Acetest. Acetest will detect concentrations of 5 mg/100 ml of DAA as well as react with acetone and can be used with serum, plasma, or urine. In addition, there is a dipstick method called Ketostix that is specific for DAA and detects 10 mg/100 ml (980 µmol/L). This method is also incorporated into certain multiple-test urine dipsticks from several manufacturers. Dipstick false positive results have been reported with levodopa, mesna, Captopril, and N-acetylcysteine. Since the concentration of DAA is several times that of acetone, and also because Acetest is only about one fifth as sensitive to acetone as it is to DAA, the dipstick tests that detect only DAA give equivalent results in urine to those of Acetest. However, the majority of literature references prefer Acetest when testing plasma or serum. The majority also recommend crushing the Acetest tablet for serum but not for urine.

    Some case reports describe instances in which the dipstick test for ketones failed to detect ketones in the plasma of certain patients with diabetic ketoacidosis. Acetest tablets were able to detect the ketones. The manufacturer states that the ketone portion of multiple-test strip dipsticks is more sensitive to effects of moisture than the other test areas and that the dipstick container lid must be replaced and fastened immediately after a test strip is removed (which is an unrealistic expectation in many laboratories). Free sulfhydride compounds (such as N-acetylcysteine used to treat acetaminophen overdose) will produce false positive reaction for ketones in tests based on nitroprusside. False positive reactions due to sulfhydride can be detected by adding glacial acetic acid to the reagent area after the reaction and seeing the reaction fade completely by 30 seconds or less.

    Interpretation of test results. Standard chemical tests for ketone bodies do not detect the small amounts in normal plasma or urine. A positive test result in both urine and plasma is uncommon and usually indicates severe excess of ketones. This is most often due to severe metabolic acidosis, of which the classic example is diabetic acidosis. Detectable urine ketones (ketonuria) with nondetectable plasma (or serum) ketones is fairly common and indicates mild or moderate over-production of ketone bodies. In one study, it was reported in 14%-28% of all hospital admission urines. Ketonuria is a classic finding in diabetic acidosis. However, ketonuria may also be found in starvation, in alcoholism, in many normal pregnancies at time of delivery, and especially in severe dehydration from many causes (vomiting, diarrhea, severe infections). In children, ketone bodies are prone to appear on what, by adult standards, would be relatively small provocation. A weakly positive urine test result for ketones is usually of little significance.

    It must be emphasized that diabetes is practically the only disease in which ketonuria has real diagnostic importance. Slight to moderate degrees of ketonuria are fairly common in certain other conditions, as mentioned earlier, but are only incidental. Serum ketone results are usually negative. In my experience, trace, 1+, and 2+ (using a scale of 0-4+) urine ketone results are rarely accompanied by positive serum ketone results, and the same was true even for some patients with 3+ ketonuria. In diabetes the presence of large amounts of urine ketones raises the question of a possible dangerous degree of diabetic ketoacidosis. In well-established diabetic ketoacidosis the serum or plasma acetone test result will usually be positive (exceptions are the rare cases of hyperosmolar coma or lactic acidosis). In fact, the quantity of plasma ketones, estimated by testing dilutions of plasma, provides some (although not an exact) indication of the severity of acidosis and the amount of insulin needed. In the urine, nitroprusside test results usually will be strongly positive. In most cases, there will be glucosuria as well as marked ketonuria. The major exception is renal insufficiency with oliguria due to severe renal disease, shock, or exceptionally severe acidosis with secondary renal shutdown, since some of these patients may be unable to filter the increased serum ketone bodies into the urine. The blood urea nitrogen (BUN) level is frequently elevated in diabetic acidosis; even so, there usually will be ketonuria. If there is any question, a plasma or serum ketone test should be done.

    In summary, the urine ketone result in severe diabetic acidosis usually is very strongly positive and the plasma ketone result is positive. However, if symptoms of diabetic acidosis are present, lack of strongly positive urine ketone results does not rule out the diagnosis if renal failure is present.

    Serum ketone quantitative measurement. Biochemical methods (usually enzymatic) are available to measure BHBA, which actually is the dominant ketone form in ketosis and provides a better estimation of severity in diabetic acidosis than DAA measurement. In early ketoacidosis, the BHBA level becomes elevated, whereas the DAA level is still normal. However, BHBA has not replaced DAA because of the ease, convenience, speed, and low cost of Acetest and the dipsticks.

    Hemoglobin (blood)

    Characteristics of test methods. Several biochemical procedures for detecting hemoglobin in urine are on the market. Some have a sensitivity of approximately 1:100,000, about that of the old benzidine method. A representative of this type is an orthotolidine reagent tablet method called Occultest. This is reported to detect fewer than five RBCs per high-power field in centrifuged urine sediment. Hematest is another tablet test with the same basic reagents but is adjusted to approximately 1:20,000 sensitivity, slightly more than the guaiac method. Experimentally, it will not reliably detect fewer than 200 RBCs per high-power field unless some of the cells have hemolyzed. It is much more sensitive to free hemoglobin, where it can detect amounts produced by hemolysis of only 25-30 RBCs per high-power field. It has been used for feces as well as urine, although at this sensitivity there is an appreciable number of false positives. A dipstick called Hemastix and the occult blood section of different multitest dipsticks are likewise based on orthotolidine but are more sensitive than the reagent tablets, detecting 0.04 mg/100 ml of hemoglobin, or, in my experience, more than two to three RBCs per high-power field of centrifuged urine sediment. There is a small but probably significant variation in sensitivity among the different dipsticks. The usefulness of these chemical tests is enhanced by their ability to detect hemoglobin even after some or all of the RBCs have disintegrated and are no longer visible microscopically. Red blood cell lysis is particularly likely to occur in alkaline or dilute urine. Failure to mix a specimen before testing could result in testing a supernate without RBCs and could yield a false negative dipstick reaction. Large doses of vitamin C interfere with the test chemical reaction and may produce a false negative result. One report indicates that providone-iodine (Betadine) can produce a false positive reaction. Contamination from vaginal secretions in the female may introduce RBCs into the specimen as an artifact. Myoglobin reacts as well as hemoglobin.

    Biochemical methods versus microscopic examination. There is disagreement in the literature as to whether the dipstick tests for hemoglobin could be used in place of microscopic examination (for RBCs) of the urine sediment. In my experience with one company’s multitest dipstick, almost all patients with microscopic findings of two or more RBCs per high-power field were detected by the dipstick. However, testing with dipstick only would not reveal abnormal numbers of squamous epithelial cells, which would raise the question of possible contamination artifact. Also, detection of urine hemoglobin or hematuria would necessitate a microscopic sediment examination for RBC casts. Etiologies of hematuria are discussed later in the section on microscopic sediment examination.

    Leukocyte esterase (white blood cells)

    A dipstick called Chemstrip-L that will detect WBCs in urine by means of hydrolysis of the reagent by esterase enzyme contained in the cytoplasm of neutrophils has been introduced. The same reagent has been incorporated into some of the multitest dipsticks. Reports indicate that the leukocyte esterase test will detect about 85%- 95% (range, 73%-99%) of patients with abnormal numbers of WBCs in centrifuged urinary sediment. The test reportedly will also detect abnormal numbers of WBCs that have lysed due to alkaline urine or other factors. The leukocyte esterase test is about 80%-90% sensitive in detecting urinary tract infection (defined as 100,000 colony-forming units/ml) compared with urine culture. When performed with the nitrite test (discussed later), sensitivity versus that of quantitative urine culture improves to about 85%-90% (range, 78%-100%). Several reports suggest false positive results due to Trichomonas organisms, but others suggest relatively little interference. In addition, leukocyte esterase will not differentiate urinary tract WBCs from WBCs added through contamination of the specimen (especially by vaginal secretions) during specimen collection.

    Nitrite

    Many bacteria produce an enzyme called reductase that can reduce urinary nitrates to nitrites. Some of the multitest dipsticks contain a reagent area that reacts with nitrite to produce a color, thus indirectly suggesting the presence of bacteria in certain quantity. Sensitivity of the nitrite test versus that of quantitative urine culture is only about 50% (range, 35%-69%). However, it can enhance the sensitivity of the leukocyte esterase test to detect urinary tract infection.

    Bile (conjugated bilirubin)

    Conjugated bilirubin is not normally detected in urine but can appear secondary to biliary tract obstruction, either extrahepatic (common duct obstruction) or intrahepatic (cholestatic liver cell injury due to such conditions as active cirrhosis or hepatitis virus hepatitis). Typically, in hepatitis virus hepatitis the urine appears dark 2 or 3 days before the patient becomes icteric and clears swiftly once skin jaundice develops.

    In pulmonary infarction, some elevation in the serum unconjugated bilirubin level may occur, but unconjugated bilirubin does not appear in urine.

    Tests for conjugated bilirubin in urine include Fouchet’s reagent, Ictotest tablets, or dipstick tests. The simple foam test (yellow foam after shaking) may be all that is necessary (false positive result may be produced by pyridium). Ictotest and the dipstick tests are based on the same chemical reaction and are fairly sensitive, detecting as little as 0.1 mg/100 ml (7.1 µmol/L). According to one report, the dipsticks detect about 70% of patients with serum conjugated bilirubin between 1.0-2.0 mg/100ml (17.1-34.2 µmol/L) and nearly all above 2.0 mg/100 ml (34.2 µmol/L). These tests are reasonably specific, but chlorpromazine may produce false positive results. If urine is allowed to stand for some time before testing, conjugated bilirubin decomposes and a false negative result may occur. Ictotest is somewhat less sensitive to this change than the dipsticks and therefore is a little less likely to become falsely negative.

    Most requests for urine bile could be questioned, since the serum bilirubin level would provide more useful information (Chapter 20).

    Urobilinogen

    Urobilinogen is produced from conjugated bilirubin by metabolic activity of bacteria in the intestine, followed by reabsorption into the bloodstream (Chapter 20). Urine urobilinogen becomes increased from either a marked increase in production secondary to increase in serum unconjugated bilirubin (usually from hemolytic processes) or because of inability of damaged liver parenchymal cells to metabolize normal amounts of urobilinogen absorbed from the intestine (usually due to cirrhosis or severe hepatitis). The metabolic pathways involved are discussed in the chapter on liver function tests.

    Tests for the presence of urobilinogen include both a standard chemistry method and a dipstick procedure, both based on Ehrlich’s reagent. Either a random urine specimen or a 24-hour specimen can be used. The 24-hour specimen must contain a preservative.

    For routine specimens, it is of great importance that the test be done within 30 minutes after voiding. Urobilinogen rapidly oxidizes in air to nondetectable urobilin, and without special preservative it will decrease significantly after 30 minutes. This unrecognized fact probably accounts for the test’s poor reputation. If the procedure cannot be done relatively soon after voiding, it is better to get a 24-hour specimen with preservative.

    Urine urobilinogen determination is rarely needed, since liver function or other tests provide more useful information.

    Urine urobilinogen methods based on Ehrlich’s reagent may also detect porphobilinogen; methods using other reagents will not.

    Microscopic examination of urinary sediment

    Urine microscopic examination is routinely done on centrifuged urinary sediment. There is no widely accepted standardized procedure for this, and the varying degrees of sediment concentration that are produced make difficult any unquestioning interpretation of quantitative reports. However, it is fairly safe to say that normally so few cellular elements are excreted that most results of examination in normal uncontaminated specimens fall within normal clinical values no matter how much the specimen is centrifuged. The majority of sources (including my experience) use a reference range of 0-2 RBCs or 0-5 WBCs per high-power field and only an occasional cast. However, there is disagreement in the literature regarding the reference range for both WBCs and RBCs. Reference ranges for WBC can be found that vary from 1 WBC per high-power field to as many as 20 WBCs per high-power field. RBC ranges are discussed in the section on hematuria investigation in Chapter 13. Some commercial systems are available for preparation of the urine sediment that can improve reproducibility.

    The main pathologic elements of urinary sediment include RBCs, WBCs, and casts. Less important elements are yeasts, crystals, or epithelial cells.

    Red blood cells. Gross urinary bleeding is usually associated with stones, acute glomerulonephritis, and tumor, although these conditions may (less often) be manifested only by microscopic hematuria. Tuberculosis traditionally has been listed as a fourth cause but is rare today. In several series, benign prostate hyperplasia and bladder or urethral infection were also frequent etiologies. There are conditions that frequently are associated with significant microscopic hematuria and occasionally approach gross bleeding. Some of the more important include bleeding and clotting disorders (e.g., purpura or anticoagulants), blood dyscrasias (including sickle cell anemia or leukemia), renal infarction, malignant hypertension, subacute bacterial endocarditis, collagen diseases (especially lupus and polyarteritis nodosa), Weil’s disease, and certain lower urinary tract conditions such as cystitis, urethritis, and prostatitis. In the female, vaginal blood or leukocytes may contaminate ordinary voided specimens; finding significant numbers of squamous epithelial cells in the urinary sediment suggests such contamination. Yeast may simulate RBCs (discussed later).

    Red blood cell casts are the most reliable way to localize the source of bleeding to the kidney. Some reports indicate that the presence of “dysmorphic RBCs” (distorted or shrunken RBCs) using ordinary microscopy, phase contrast, Wright’s stain on a sediment smear, or a Papanicolaou cytology technique is also helpful in suggesting renal origin.

    White blood cells. WBCs may originate anywhere in the urinary tract. Hematogenous spread of infection to the kidney usually first localizes in the renal cortex. Isolated cortical lesions may be relatively silent. Retrograde invasion from the bladder tends to involve calyces and medulla initially. Urinary tract obstruction is often a factor in retrograde pyelonephritis. Generally speaking, pyuria of renal origin is usually accompanied by significant proteinuria. Pyuria originating in the lower urinary tract may be associated with proteinuria, but it tends to be relatively slight.

    Urinary tract infections tend to be accompanied by bacteriuria. Tuberculosis of the kidney, besides producing hematuria, characteristically is associated with pyuria without bacteriuria. An ordinary urine culture would not reveal tuberculosis organisms. WBC casts are definite evidence that urinary WBCs originate in the kidney (discussed later); WBCs in clumps are suggestive of renal origin but are not diagnostic.

    Correlation of pyuria with urine culture. Although many physicians regard pyuria as a good screening test for urinary tract infection, false normal results are reported in approximately 25%-30% of patients (literature range 0%-87%) who have a positive quantitative urine culture result. WBC counting of uncentrifuged urine in a WBC counting chamber is reported to be more accurate than urine sediment, but very few laboratories use this technique. Abnormal numbers of WBCs are a reasonably good indicator of urinary tract infection, although reports indicate 2%-13% false positive results compared to urine culture. In females, one should take precautions to avoid artifactual increases in urine WBCs from contamination by vaginal or labial secretions. Several studies (including my own experience) have suggested female urine contamination rates as high as 30%. In any specimen it is important to perform the microscopic examination promptly (i.e., within 1 hour after voiding) or else use some method of preservation. WBCs lyse in hypotonic or alkaline urine. Some studies reported that as many as 30%-50% of specimens containing abnormal numbers of WBCs were normal after 2-3 hours of standing at room temperature.

    Casts. Casts are protein conglomerates that outline the shape of the renal tubules in which they were formed. Factors involved in cast formation include the following:

    1. pH: Protein casts tend to dissolve in alkaline medium.
    2. Concentration: Casts tend to dissolve in considerably dilute medium. Concentration also has an important role in the formation of casts; a concentrated urine favors precipitation of protein.
    3. Proteinuria: Protein is necessary for cast formation, and significant cylindruria (cast excretion) is most often accompanied by proteinuria. Proteinuria may be of varied etiology. The postrenal type is added beyond the kidneys and obviously cannot be involved in cast formation.
    4. Stasis: Stasis is usually secondary to intratubular obstruction and thus allows time for protein precipitation within tubules.

    Mechanisms of cast formation. Mechanisms of cast formation are better understood if one considers the normal physiology of urine formation. The substance filtered at the glomerulus is essentially an ultrafiltrate of plasma. In the proximal tubules, up to 85% of filtered sodium and chloride is reabsorbed, with water passively accompanying these ions. In the thick ascending loop of Henle, sodium is actively reabsorbed; however, since the membrane here is impermeable to water, an excess of water (free water) remains. As water passes along the distal tubules, some may be reabsorbed with sodium ions, and in the collecting tubules, up to 5% of the glomerular filtrate is osmotically reabsorbed due to the relatively high osmolality of the renal interstitial cells. Water reabsorption in distal and collecting tubules is under the control of antidiuretic hormone. At this point, the urine reaches its maximum concentration and proceeds to the bladder relatively unchanged. Thus, cast formation takes place ordinarily in the distal and collecting tubules, where acidification takes place and concentration reaches its height.

    Cast types. There are two main types of casts, cellular and hyaline, depending on their main constituents. Other types include fatty casts, broad casts, and hemoglobin casts.

    CELLULAR CASTS. RBCs, WBCs, desquamated renal epithelial cells, or any combination of these may be trapped inside renal tubules in a protein matrix. The cast is named for the cells inside it. This proves renal orgin of the RBCs and WBCs. (Fig. 12-1).

    Formation of casts

    Fig. 12-1 Formation of casts.

    As the cellular cast moves slowly down the nephron, the cells begin to disintegrate. Eventually, all that is left of the cells are relatively large fragments, chunks, or granules. The cellular cast has now become a coarsely granular cast. It is composed entirely of large irregular or coarse solid granules.

    If disintegration is allowed to continue, the coarse granules break down to small granules, and a relatively homogeneous finely granular cast is formed.

    The end stage of this process is the production of a homogeneous refractile material still in the shape of the tubule, known as a waxy cast. The waxy cast is translucent, without granules, and reflects light, which gives it a somewhat shiny, semisolid appearance. What stage of cast finally reaches the bladder depends on how long the cast takes to traverse the nephron and thus how long it remains in the kidney, where the forces of disintegration may work. A waxy cast thus indicates fairly severe stasis in the renal tubules.

    HYALINE CASTS. Hyaline casts are composed almost exclusively of protein alone, and they pass almost unchanged down the urinary tract. They are dull, nearly transparent, and reflect light poorly compared with waxy casts. Thus, hyaline casts are often hard to see, and the microscope condenser usually must be turned down to give better contrast.

    Sometimes cellular elements may be trapped within hyaline casts. If hyaline material still predominates, the result is considered a hyaline cast with cellular inclusions. Degenerative changes can take place similar to those of regular cellular casts, with the production of hyaline coarsely granular and hyaline finely granular casts.

    FATTY CASTS. Fatty casts are a special type of cellular cast. In certain types of renal tubular damage, fatty degeneration of the tubular epithelial cells takes place. These cells desquamate and are incorporated into casts. The epithelial cells contain fatty droplets. As the cells degenerate, the fatty droplets remain and may even coalesce somewhat. The final result is a cast composed mainly of fatty droplets and protein. Sometimes either renal epithelial cells with fatty degeneration or the remnants thereof containing fat droplets are found floating free in the urine and are called oval fat bodies.

    When oval fat bodies or fatty casts are present in significant number, the patient usually has a disease that is associated with the nephrotic syndrome, such as primary lipoid nephrosis or nephrosis secondary to Kimmelstiel-Wilson syndrome, systemic lupus, amyloid, subacute glomerulonephritis, the nephrotic stage of chronic glomerulonephritis, certain tubule poisons such as mercury, and rare hypersensitivity reactions such as those from insect bites. Oval fat bodies have essentially the same significance as fatty casts. Fat droplets have a Maltese cross appearance in polarized light but are recognized without polarized light. They can also be identified using fat stains such as Sudan IV.

    BROAD CASTS. When very severe stasis occurs regardless of the type of cast involved, cast formation may take place in the larger more distal collecting tubules, where large ducts drain several smaller collecting tubules. If this occurs, a broad cast is formed, several times wider than ordinary-sized casts. This is always indicative of severe localized tubule damage. These broad casts may be of any cast type, and due to their peculiar mode of formation they are sometimes spoken of as “renal failure casts.” This is not entirely accurate, because the kidney may often recover from that particular episode and the problem sometimes involves only part of a kidney.

    HEMOGLOBIN CASTS. Hemoglobin casts are derived from RBC casts that degenerate into granular (rarely, even waxy) material but still have the peculiar orange-red color of hemoglobin. Not all casts derived from RBCs retain this color. Strongly acid urine changes the characteristic color of hemoglobin to the nonspecific gray-brown color of acid hematin. Also, one must differentiate the brown or yellow-brown coloring derived from bile or other urine pigments from the typical color of hemoglobin.

    Significance. The significance of casts in the urine varies according to the type of cast. Fatty casts, RBC casts, and WBC casts are always significant. The only general statement one can make about ordinary hyaline or granular casts is that their appearance in significant numbers has some correlation with the factors involved in cast formation—proteinuria, concentration, and stasis. Of these, the most general correlation is with stasis, although one cannot tell whether the stasis is generalized or merely localized. It is common for showers of casts to clear dramatically once the underlying cause of their formation is corrected. On the other hand, significant numbers of casts that persist for some time despite therapeutic efforts may suggest serious intrarenal derangement. In this respect, granular casts in the late stages of development will be seen. Generally speaking, hyaline casts alone are of little practical importance and are usually only an acute, mild, and temporary phenomenon. Thus, to have any meaning, the appearance of casts must be correlated with other findings and with the clinical situation. A few hyaline or granular casts in themselves have no importance.

    Crystals. Crystals are often overemphasized in importance. They may, however, be a clue to calculus formation and certain metabolic diseases. Crystals tend to be pH dependent:

    Acid urine: uric acid, cystine, calcium oxalate.
    Alkaline urine: phosphates, as triple phosphate (magnesium ammonium phosphate), which comprises many staghorn calculi. Note: alkaline urine is most often produced by (Proteus) infection.
    Amorphous (loose finely granular) crystals: If the pH is acid, the material is urate; if the pH is alkaline, it is phosphate. Rarely one might find sulfa crystals in acid urine, since sulfadiazine is still sometimes used. Most sulfa crystals resemble needle-like structures in bunches, but this appearance can be mimicked by certain nonpathologic crystals.

    Epithelial cells. Squamous epithelial cells are useful as an index of possible contamination by vaginal secretions in females or by foreskin in uncircumcised males. I have found (unpublished study) that more than 10 squamous epithelial cells per low-power (10 Ч objective) field provide an adequate guidepost for suspicion of contamination. However, contamination (especially bacteriologic contamination) might occur without abnormal numbers of squamous cells, and the presence of abnormal numbers of squamous cells does not mean that RBCs and WBCs are present only because of contamination. The squamous cells only alert one to a definite possibility of contamination and, if abnormalities are present, suggest the desirability of a carefully collected midstream specimen. Unfortunately, however, even specimens that supposedly are collected by careful midstream voiding technique may be contaminated, especially if the patient collects the specimen without expert assistance.

    Miscellaneous microscopic sediment findings.

    Trichomonas. Female vaginal infection by the protozoan parasite Trichomonas is fairly common; it is often associated with mild proteinuria, WBCs, and epithelial cells in the urine sediment. The organism is frequently found in urine specimens, where diagnosis is greatly assisted by the organism’s motility (so a fresh specimen is essential). When nonmotile, it resembles a large WBC or small tubular epithelial cell. Fluorescent antibody methods are now available for diagnosis and are more sensitive than ordinary urine sediment examination but are relatively expensive. Trichomonas is found occasionally in the male.

    Spermatozoa. Spermatozoa may appear in the urine of males and, occasionally, in that of females.

    Yeast. Yeast is a relatively common sediment finding; the most common is Candida albicans (Monilia). Yeast cells are often misdiagnosed as RBCs. Differential points are budding (not always noticed without careful search), ovoid shape (not always pronounced), an appearance slightly more opaque and homogeneous than that of the RBC, and insolubility in both acid and alkali. If the cells are numerous and their identity is still in doubt, a chemical test for blood is recommended.

    Pitfalls in microscopic examination of urine sediment. The most common and most important mistake is failure to mix the specimen sufficiently before a portion of it is poured into a centrifuge tube for concentration of the sediment. Certain other points deserve reemphasis. If the specimen remains at room temperature for a long time, there may be disintegration of cellular elements, bacterial growth, and change toward an alkaline pH. If RBC or WBC casts are not reported, this does not mean that they are not present, since it usually takes careful and somewhat prolonged search to find them (especially RBC casts).

    Other urine tests

    Calcium. Urine can be tested for calcium with Sulkowitch reagent; it is a semiquantitative method in which results are reported as 0-4+ (normal is 1+) or as negative, moderately positive, and strongly positive. Standard clinical chemistry quantitative methods have almost entirely replaced this test. Interpretation of urine calcium is discussed in Chapter 25. Excessively concentrated or diluted urine may produce unreliable results, just as it does for protein.

    Porphobilinogen. Porphobilinogen is diagnostic of acute porphyria (episodes of abdominal crampy pain with vomiting and leukocytosis, Chapter 34). Porphobilinogen is colorless and is easily identified by rapid screening tests, such as the Watson-Schwartz procedure.

    Urinary coproporphyrins. Coproporphyrin type III excretion is increased in lead poisoning (Chapter 35) and has been used as a relatively simple screening test for that condition. However, increased coproporphyrin III is not specific for lead poisoning.

    Phenylpyruvic acid. Phenylpyruvic acid is excreted in phenylketonuria (Chapter 34). A ferric chloride test is the classic means of detection. There is a dipstick method (Phenistix) for the diagnosis of phenylketonuria that depends on the reaction between ferric ions and phenylpyruvic acid, as does the classic ferric chloride test. It is more sensitive than ferric chloride, detecting as little as 8 mg/100 ml or trace amounts of phenylpyruvic acid in urine. False positive results occur if large amounts of ketone bodies are present. Reactions will take place with salicylates, PAS, and phenothiazine metabolites, but the color is said to be different from that given by phenylpyruvic acid. The urine screening tests have become less important since neonatal blood test screening has become mandatory in many areas. It takes approximately 3-6 weeks after birth before phenylpyruvic acid becomes detectable in urine, and by that time treatment is much less effective.

    Points to remember in interpretation of urinalysis results

    1. Laboratory reports usually err in omission rather than commission (except occasionally in RBCs vs. yeast). If technicians cannot identify something, they usually do not mention it at all.
    2. Certain diagnostic findings, such as RBC casts, may not appear in every high-power (or even every low-power) field. If hematuria is present, one should look for RBC casts and, similarly, for WBC casts and other such structures in the appropriate sediment settings.
    3. If laboratory personnel know what the physician is trying to find, they usually will give more than a routine glance. Otherwise, the pressure of routine work may result in examination that is not sufficient to detect important abnormalities.
    4. In many laboratories, reports will include only items specifically requested, even when other abnormalities are grossly visible (e.g., bile).
    5. Contamination of urine by vaginal secretions in the female may introduce RBCs, WBCs, or protein into the specimen as an artifact. The presence of more than 10 squamous epithelial cells per low-power field is a warning of possible contamination. Nevertheless, abnormalities should not be dismissed on the basis of possible contamination without careful and repeated attempts to obtain a noncontaminated specimen, because contamination may be superimposed on true abnormality. In some cases this problem may necessitate expert help in specimen collection.

    One fact that should be stressed when commercial tests of any type are used is to follow all the directions exactly. Any dipstick or table method requires a certain length of time in contact with the specimen. When this is true, a quick dip-and-read technique, or, on the other hand, overly prolonged contact with the specimen, may lead to false results. Moisture may affect some of the test reagents, so the tops on reagent bottles should be replaced and tightened immediately after the dipsticks or tablets are taken out.

    Laboratory evaluation of urinary tract calculi. Laboratory evaluation consists of (1) stone mineral analysis, and (2) investigation of stone-formation etiology. Stone mineral analysis is performed by x-ray diffraction, infrared spectroscopy, optical crystallography (polarization microscopy), or some combination of these techniques. Standard chemical analysis of crushed stone material is no longer recommended, due to fragment inaccuracies, interferences, lack of sensitivity, and problems with sufficient specimen if the stone is small. There is no universally accepted standard workup protocol to investigate pathogenesis. However, the majority of investigators require both a 24-hour urine specimen and a fasting serum specimen obtained on the same day, on three separate occasions. The urine specimen should be kept on ice or in the refrigerator during collection. In the laboratory, the specimen is preserved in the refrigerator if necessary tests are performed in house; if the specimen is sent to an outside laboratory, appropriate preservatives should be added. The patient is usually on his or her regular diet (at least initially). The substances measured vary somewhat among investigators, but measurements most often include calcium, phosphate, uric acid, oxalate, urine pH, and urine volumes. Citrate and cystine are also frequently assayed.

    Representative reference ranges (24-hour urine) are as follows: calcium, <250 mg (6.24 mmol)/day on normal diet and <150 mg/(6.24 mmol)/day on low-calcium diet; uric acid, 250-800 mg (1.49-4.76 mmol)/day on normal diet and <600 mg (3.57 mmol)/day on low-purine diet; phosphate, 400-1,300 mg (12.9-42 mmol)/day; and oxalate, 10-40 mg (0.11-0.44 mmol)/day.

  • Urinalysis

    Urinalysis is an indispensable part of clinical pathology. It may uncover disease anywhere in the urinary tract. Also, it may afford a semiquantitative estimate of renal function and furnish clues to the etiology of dysfunction. Certain systemic diseases may produce quantitative or qualitative alterations of urine constituents or excretion of abnormal substances, quite apart from direct effects on the kidneys. Conversely, urinary tract disease may produce striking systemic symptoms.

    The standard urinalysis test panel includes specimen appearance, pH, specific gravity, protein semiquantitation, presence or absence of glucose and ketones, and microscopic examination of the centrifuged urinary sediment for white blood cells (WBCs), red blood cells (RBCs), and other abnormalities. (Table 12-1). These tests (not including microscopic examination) can be performed separately or together in various combinations on dipsticks available from several manufacturers. Some manufacturers have added one or more of the following tests to their dipsticks: hemoglobin, bile (conjugated bilirubin), urobilinogen, nitrite, leukocyte esterase, and specificgravity. Instruments that process and read the dipsticks are now available, thereby improving accuracy by eliminating some of the subjective element that is inherent in color changes read by human eye.

    Most important conditions screened for in basic urinalysis

    Table 12-1 Most important conditions screened for in basic urinalysis

  • Hemolytic Reactions

    The presence of unexpected alloantibodies (antibodies against red cell antigens) in patient serum found in pretransfusion screening of recipients is 0.7%-1.6%. This averages 9% (range, 6%-36%) in multitransfused patients. Infants less than 4 months old usually do not form alloantibodies against transfused red cell antigens that they lack. After that, age per se does not appear to affect red blood cell (RBC) antigen sensitization. Immunosuppressive therapy can diminish this response. In the case of Rh system D (Rho) antigen, chance of sensitization has some correlation with antigen dose, but this is not exact or linear. Sensitization of D-negative recipients of D-positive cells has ranged from 8% to 70%. Although antibodies to bacterial or many other antigens usually appear in 7-21 days, alloantibodies usually take 3-4 months after transfusion with a minimum (in one study) of 1 month. Once formed, the antibodies remain detectable for variable periods of time, depending to some extent (but not entirely) on the particular antibody. Anti-D is particularly likely to be detectible for many years; anti-C and anti-Kidd are more likely to become nondetectable (50% loss in 5 years in one study). However, nondetectable antibodies can be reactivated by anamnesthic antigen exposure.

    Hemolytic reactions may be caused by either complete or incomplete antibodies. In reactions caused by complete antibodies, such as occur in the ABO blood group system, there is usually intravascular hemolysis. The amount of hemolysis depends on several factors, such as quantity of incompatible blood, antibody titer, and the nature of the antibody involved. However, there is an element of individual susceptibility; some patients die after transfusion of less than 100 ml of incompatible blood, whereas others survive transfusion of several times this amount. The direct Coombs’ test result is often positive, but this depends on whether all the RBCs attacked by the complete antibody have been lysed, whether more antibody is produced, and, to some extent, on how soon the test is obtained. If the sample is drawn more than 1 hour after the ABO transfusion reaction is completed, the chance of a positive direct Coombs’ test result is much less. A Coombs’ reagent acting against both gamma globulin and complement (broad spectrum) is needed; most laboratories now use this type routinely. Free hemoglobin is released into the plasma from lysed RBCs and is carried to the kidneys, where it is excreted in the urine. Some of the intravascular hemoglobin is converted to bilirubin in the reticuloendothelial system so that the serum nonconjugated (indirect) bilirubin level usually begins to increase. In reactions caused by incomplete antibodies, such as the Rh system, there is sequestration of antibody-coated cells in organs such as the spleen, with subsequent breakdown by the reticuloendothelial system. With incomplete antibody reactions, RBC breakdown is extravascular rather than intravascular; in small degrees of reaction, plasma free hemoglobin levels may not rise, although indirect bilirubin levels may eventually increase. In more extensive reactions, the plasma hemoglobin level is often elevated, although the elevation is sometimes delayed in onset. The direct Coombs’ test should be positive in hemolytic reactions due to incomplete antibodies (unless all affected RBCs have been destroyed).

    Undesirable effects of blood or blood product transfusion

    RECIPIENT REACTION TO DONOR ANTIGENS ON DONOR CELLS
    Clinical types of reactions
    Hemolytic reaction
    Nonhemolytic febrile reactions
    Allergic reactions
    Cytopenic reactions
    Tissue-organimmunologic reactions
    Anaphylactic reactions

    ANTIGEN GROUPS INVOLVED
    ABO system
    Rh and minor blood groups
    Histocompatibility leukocyte antigen (HLA) system
    Platelet antigens

    INFECTIONS
    Hepatitis viruses
    Human immunodeficiency virus-1 and 2 (HIV-1 and 2)
    Human T-cell lymphotropic virus-1 and 2 (HTLV-1 and II)
    Cytomegalovirus (CMV)
    Epstein-Barr virus
    Syphilis
    Malaria
    Other

    OTHER TRANSFUSION PROBLEMS
    Citrate overload
    Hyperkalemia
    Depletion of coagulation factors
    Depletion of platelets
    Transfused blood temperature
    Donor medications

    Hemolytic transfusion reactions are usually caused by incompatible blood but are occasionally caused by partial hemolysis of the RBCs before administration, either before leaving the blood bank or just before transfusion if the blood is warmed improperly. Analysis of fatal cases of hemolytic transfusion reaction reported to the Food and Drug Administration in 1976-1983 shows that almost 65% were due to problems of mistaken identity and about 9% were due to clerical error. Only about 18% were due to error while performing blood bank tests. Frequent mistakes included transfusion into one patient of blood meant for another patient, obtaining a recipient crossmatch specimen from the wrong patient, mixup of patient crossmatch specimens in the blood bank, and transcription of data belonging to one patient onto the report of another.

    The great majority of hemolytic transfusion reactions occur during transfusion. Symptoms and laboratory evidence of hemolysis usually are present by the time transfusion of the incompatible unit is completed, although clinical symptoms or laboratory abnormalities may sometimes be delayed for a few minutes or even a few hours. Occasionally, hemolytic reactions take place after completion of transfusion (delayed reaction). In one kind of delayed reaction, the reaction occurs 4-5 days (range, 1-7 days) following transfusion and is due to anamnestic stimulation of antibodies that were already present but in very low titer. The Kidd (Jk) system is frequently associated with this group. The intensity of the reaction may be mild or may be clinically evident, but most are not severe. In a second kind of delayed reaction, the reaction occurs several weeks after transfusion and is due to new immunization by the transfused RBC antigens with new antibody production. This type of reaction is usually mild and subclinical. In both types of reaction, and occasionally even in an immediate acute reaction, a hemolytic reaction may not be suspected and the problem is discovered accidentally by a drop in hemoglobin level or by crossmatching for another transfusion.

    Symptoms of hemolytic transfusion reaction include chills, fever, and pain in the low back or legs. Jaundice may appear later. Severe reactions lead to shock. Renal failure (acute tubular necrosis) is common due either to shock or to precipitation of free hemoglobin in the renal tubules. Therefore, oliguria develops, frequently accompanied by hemoglobinuria, which is typically manifest by red urine or nearly black “coffee ground” acid hematin urine color.

    Tests for hemolytic transfusion reaction

    These tests include immediate rechecking and comparison of patient identification and the unit or units of transfused donor blood. Tubes of anticoagulated and nonanticoagulated blood should be drawn. The anticoagulated tube should be centrifuged immediately and the plasma examined visually for the pink or red color produced by hemolysis. A direct Coombs’ test and crossmatch recheck studies should be performed as soon as possible. A culture and Gram stain should be done on the remaining blood in the donor bag.

    Plasma free hemoglobin. The presence of free hemoglobin in plasma is one of the most valuable tests for diagnosis of acute hemolytic transfusion reaction. Most cases of severe intravascular hemolysis due to bivalent antibodies such as the ABO group produce enough hemolysis to be grossly visible. (Artifactual hemolysis from improperly drawn specimens must be ruled out.) If chemical tests are to be done, plasma hemoglobin is preferred to serum hemoglobin because less artifactual hemo-lysis takes place before the specimen reaches the laboratory. Although hemolytic reactions due to incomplete antibodies (such as Rh) have clinical symptoms similar to those of ABO, direct signs of hemolysis (e.g., free plasma hemoglobin) are more variable or may be delayed for a few hours, although they become abnormal if the reaction is severe. Another drawback in the interpretation of plasma hemoglobin values is the effect of the transfusion itself. The older erythrocytes stored in banked blood die during storage, adding free hemoglobin to the plasma. Therefore, although reference values are usually considered to be in the range of 1-5 mg/100 ml, values between 5 and 50 mg/100 ml are equivocal if stored blood is given. Hemolysis is barely visible when plasma hemoglobin reaches the 25-50 mg/100 ml range. Frozen blood has a greater content of free hemoglobin than stored whole blood or packed cells and may reach 300 mg/100 ml.

    Additional tests. If visual inspection of patient plasma does not suggest hemolysis and the direct Coombs’ test result is negative, additional laboratory tests may be desirable to detect or confirm a hemolytic transfusion reaction. Procedures that may be useful include serum haptoglobin measurement, both immediately and 6 hours later; serum nonconjugated (“indirect”) bilirubin measurement at 6 and 12 hours; and urinalysis.

    A pretransfusion blood specimen, if available, should be included in a transfusion reaction investigation to provide baseline data when the various tests for hemolysis are performed.

    Urinalysis. Urine can be examined for hemoglobin casts, RBCs, and protein and tested for free hemoglobin. RBCs and RBC casts may be found in hemolytic reactions since the kidney is frequently injured. If no hematuria or free hemoglobin is found, this is some evidence against a major acute hemolytic transfusion reaction. Abnormal findings are more difficult to interpret unless a pretransfusion urinalysis result is available for comparison, since the abnormality might have been present before transfusion. In addition, abnormal urine findings could result from hypoxic renal damage due to an episode of hypotension during surgery rather than a transfusion reaction.

    Hemoglobin passes through the glomerular filter into the urine when the plasma hemoglobin level is above 125 mg/100 ml. Therefore, since hemolysis should already be visible, the major need for urine examination is to verify that intravascular hemolysis occurred rather than artifactual hemolysis from venipuncture or faulty specimen processing. Conversely, unless the plasma free hemoglobin level is elevated, urine hemoglobin may represent lysed RBCs from hematuria unrelated to a hemolytic reaction. However, if sufficient time passes, the serum may be cleared of free hemoglobin while hemoglobin casts are still present in urine. Hemosiderin may be deposited in renal tubule cells and continue to appear in the urine for several days.

    Serum haptoglobin. Haptoglobin is an alpha globulin that binds free hemoglobin. Two types of measurements are available: haptoglobin-binding capacity and total haptoglobin. Binding capacity can be measured by electrophoresis or chemical methods. Total haptoglobin is usually estimated by immunologic (antibody) techniques; a 2-minute slide test is now commercially available. The haptoglobin-binding capacity decreases almost immediately when a sufficient quantity of free hemoglobin is liberated and remains low for 2-4 days. Stored banked blood, fortunately, does not contain enough free hemoglobin to produce significant changes in haptoglobin-binding capacity. The total haptoglobin level decreases more slowly than the binding capacity after onset of a hemolytic reaction and might not reach its lowest value until 6-8 hours later. Haptoglobin assay is much less helpful when frozen RBCs are transfused because of the normally increased free hemoglobin in most frozen RBC preparations. Bilirubin determinations are not needed if haptoglobin assay is done the same day.

    In common with other laboratory tests, serum haptoglobin levels may be influenced by various factors other than the one for which the test is ordered. Haptoglobin levels may be decreased by absorption of hemoglobin from an extravascular hematoma and also may be decreased in severe liver disease, hemolytic or megaloblastic anemia, estrogen therapy, and pump-assisted open-heart cardiac surgery. Haptoglobin is one of the body’s “acute-phase reactant” proteins that are increased by infection, tissue injury or destruction, widespread malignancy, and adrenocorticosteroid therapy. Therefore, a mild decrease in haptoglobin level could be masked by one of these conditions. In order to aid interpretation, it is helpful to perform the haptoglobin assay on a pretransfusion serum specimen as well as the posttransfusion specimen.

    Sensitivity of tests in hemolytic reaction. Data regarding frequency of test abnormality in transfusion reaction are difficult to obtain. In one series, comprising predominantly hemolytic reactions not due to ABO antibodies, free hemoglobin was detected in plasma or urine in 88% of cases involving immediate reactions and in 52% of cases involving incomplete antibodies or delayed reactions. Serum haptoglobin levels were decreased in 92% of the patients in whom it was assayed. Various factors influence this type of data, such as the amount of incompatible blood, the antibody involved, the time relationship of specimen to onset of reaction, and the test method used.

    Nursing station action in possible hemolytic reaction

    Transfusion should be stopped at the first sign of possible reaction and complete studies done to recheck compatibility of the donor and recipient blood. If these are still satisfactory, and if results of the direct Coombs’ test and the studies for intravascular hemolysis are negative, a different unit can be started on the assumption that the symptoms were pyrogenic rather than hemolytic. Whatever unused blood remains in the donor bottle, the donor bottle pilot tube, and a new specimen drawn from the patient must all be sent to the blood bank for recheck studies. Especially dangerous situations exist in transfusion during surgery, where anesthesia may mask the early signs and symptoms of a reaction. Development during surgery of a marked bleeding or oozing tendency at the operative site is an important danger signal. A hemolytic transfusion reaction requires immediate mannitol or equivalent therapy to protect the kidneys.

    Hemolytic disease of the newborn (HDN)

    The other major area where blood banks meet blood group hemolytic problems is in hemolytic disease of the newborn (HDN). It may be due to ABO, Rh, or (rarely) minor group incompatibility between fetal and maternal RBCs. ABO and the Rh antigen D (Rho) are by far the most common causes. The Rh antigen c and the blood group Kell antigen are next most important. Hemolytic disease of the newborn results from fetal RBC antigens that the maternal RBCs lack. These fetal RBC antigens provoke maternal antibody formation of the IgG type when fetal RBCs are introduced into the maternal circulation after escaping from the placenta. The maternal antibodies eventually cross the placenta to the fetal circulation and attack the fetal RBCs.

    Hemolytic disease of the newborn due to Rh. Hemolytic disease of the newborn due to Rh incompatibility varies in severity from subclinical status, through mild jaundice with anemia, to the dangerous and often fatal condition of erythroblastosis fetalis. The main clinical findings are anemia and rapidly developing jaundice. Reticulocytosis over 6% accompanies the anemia, and the jaundice is mainly due to unconjugated (indirect) bilirubin released from the reticuloendothelial sequestration and destruction of RBCs. The direct Coombs’ test result is positive. In severe cases there are usually many nucleated RBCs in the peripheral blood. Jaundice is typically not present at birth except in very severe cases (since the mother excretes bilirubin produced by the fetus) but develops several hours later or even after 24 hours in mild cases. Diseases that cause jaundice in the newborn, often accompanied by anemia and sometimes a few peripheral blood nucleated RBCs, include septicemia, cytomegalic inclusion disease, toxoplasmosis, and syphilis. Physiologic jaundice of the newborn is a frequent benign condition that may be confused with hemolytic disease or vice versa. There is, however, no significant anemia. A normal newborn has a (average) hemoglobin value of 18 gm/100 ml, and a value less than 15 gm/100 ml (150 g/L) indicates anemia. Anemia may be masked if heelstick (capillary) blood is used, since neonatal capillary hemoglobin values are higher than venous blood values.

    Hemolytic disease due to Rh incompatibility occurs in an Rh-negative mother whose fetus is Rh positive. Usually, the mother and fetus are ABO compatible. The mother develops antibodies against the RBC Rh antigen after being exposed to Rh-positive RBCs. This may occur due to pregnancy, abortion, ectopic pregnancy, amniocentesis (or other placental trauma), blood transfusion with Rh-positive RBCs, or transfusion of certain RBC-contaminated blood products such as platelets. The most common maternal contact with Rh-positive RBCs occurs during pregnancy when fetal RBCs escape through the placenta into the maternal circulation. This may happen at any time after the 16th week of pregnancy, and both the quantity of cells and the frequency of exposure increase until delivery. The largest single dose of fetal RBCs occurs during delivery. Not all mothers have detectable fetal RBCs in their circulation, and of those who do, not all become sensitized during any one pregnancy. There is approximately a 10%-13% risk of sensitization in previously nonsensitized Rh-incompatible pregnancies. When fetal-maternal ABO incompatibility (with the mother being group O) is present, the usual risk for Rh sensitization is decreased, presumably because sufficient fetal cells are destroyed that the stimulus for sensitization is reduced below the necessary level.

    The first child is usually not affected by Rh hemolytic disease if the mother has not been exposed to Rh-positive RBCs before pregnancy, and full sensitization usually does not develop until after delivery in those mothers who become sensitized. However, occasional firstborn infants are affected (5%-10% of HDN infants) either because of previous maternal exposure (e.g., a previous aborted pregnancy) or because of unusually great maternal susceptibility to Rh stimulus during normal pregnancy. Once maternal sensitization takes place, future exposure to Rh antigen, as during another pregnancy with an Rh-positive fetus, results in maternal antibody production against the Rh antigen, which can affect the fetus.

    Current recommendations of the American College of Obstetricians and Gynecologists (ACOG) are that every pregnant woman should have ABO and Rh typing and a serum antibody screen as early as possible during each pregnancy. If results of the antibody screen are negative and the mother is Rh positive, the antibody screen would not have to be repeated before delivery. Theoretically, if the mother is Rh positive, or if the mother is Rh negative and the father is also Rh negative, there should be no risk of Rh-induced fetal disease. However, the antibody screen is still necessary to detect appearance of non-Rh antibodies. If results of the antibody screen are negative and the mother is Rh negative, the father should be typed for Rh and the antibody screen should be repeated at 28 weeks’ gestation. If the antibody screen results are still negative, a prophylactic dose of Rh immune globulin is recommended (discussed later). If the antibody screen detects an antibody, subsequent testing or action depends on whether the antibody is Rh or some other blood group and whether or not this is the first pregnancy that the antibody was detected. The father should be tested to see if he has the antigen corresponding to the antibody. If the antibody is one of the Rh group, antibody titers should be performed every 2 weeks. Titers less than 1:16 suggest less risk of fetal hazard. Titer equal to or greater than 1:16 is usually followed by amniocentesis (as early as 24 weeks’ gestation) for spectrophotometric examination of amniotic fluid bilirubin pigment density. The greater the pigment density the greater the degree of fetal RBC destruction. Antibodies that are not Rh are managed by amniotic fluid examination without antibody titers, since there is inadequate correlation of titers to fetal outcome.

    Rh immune globulin. Studies have now proved that nearly all nonsensitized mothers with potential or actual Rh incompatibility problems can be protected against sensitization from the fetus by means of a “vaccine” composed of gamma globulin with a high titer of anti-Rh antibody (RhIg). This exogenous antibody seems to prevent sensitization by destroying fetal RBCs within the mother’s circulation and also by suppressing maternal antibody production. Abortion (spontaneous or induced), ectopic pregnancy, amniocentesis, and platelet or granulocyte transfusions (which may be contaminated with Rh-positive RBCs) may also induce sensitization and should be treated with RhIg. Current ACOG recommendations are to administer one unit (300 µg) of RhIg to all Rh-negative women at 28 weeks’ gestation and a second unit within 72 hours after delivery. A minidose of 50 µg is often used rather than the standard dose when abortion occurs before 12 weeks’ gestation. Although 72 hours after delivery is the recommended time limit for RhIg administration, there is some evidence that some effect may be obtained up to 7 days after delivery. Untreated Rh-negative women have about a 10% (range, 7%-13%) incidence of Rh antibody production (sensitization). When RhIg is administered postpartum it reduces incidence to about 1%-2%, and antepartum use combined with postpartum use reduces the incidence to about 0.1%-0.2%. RhIg will produce a positive Rh antibody screening test result if present in sufficient titer, so that antibody screening should be performed before administration rather than after. The half-life of exogenous gamma globulin (which applies to RhIg) is about 25 days (range 23-28 days). Injected RhIg may be detected in maternal blood after intramuscular injection within 24-48 hours with a peak at about 5 days, and is often still detectable for 3 months (sometimes as long as 6 months). Detection after 6 months postpartum suggests patient sensitization (failure of the RhIg). There have been a few reports that some Rh-positive infants whose mother received antepartum RhIg had a weakly positive direct Coombs’ test result at birth due to the RhIg. The blood bank should always be informed if RhIg has been given antepartum to properly interpret laboratory test results. The standard dose of 300 µg of RhIg neutralizes approximately 15 ml of Rh-positive RBCs (equivalent to 30 ml of fetal whole blood). In some patients the RBCs received from the fetus may total more than 15 ml.

    There are several methods used to quantitate fetal RBCs in the maternal circulation, none of which are considered ideal. The Kleihauer acid elution test is a peripheral smear technique that stains the hemoglobin F (Hb F) of fetal RBCs. Other tests that have been used are the weak D (Du) test read microscopically (to detect the mixed field agglutination of the relatively small number of fetal RBCs involved) and the RhIg crossmatch technique. None of these three procedures has proved adequately sensitive. For example, a proficiency survey in 1980 found that about 10% of the laboratories using acid elution or microscopic Du techniques failed to detect the equivalent of 30 ml of fetal RBCs (twice the significant level). When a normal blood sample was tested, about 10% of those using microscopic Du obtained false positive results, and those using acid elution had 40% or more false positive results (especially with one commercial adaptation of the acid elution technique called Fetaldex). Also, false positive acid elution test results can be produced if increased maternal Hb F is present, as seen in beta thalassemia minor and in hereditary persistence of fetal hemoglobin. Newer procedures, such as the erythrocyte rosette test, have proved much more sensitive and somewhat more reproducible. However, the rosette test is not quantitative, so that screening is done with the rosette test (or equivalent) and a positive result is followed by quantitation with the acid elution procedure. If the quantitative test for fetal RBCs indicates that more than 15 ml is present, additional RhIg should be administered. If this is not done, failure rates for RhIg of 10%-15% have been reported; if it is done, the failure rate is approximately 2%. The current American Association of Blood Banks (AABB) recommendation is to administer twice the dose of RhIg indicated by formulas, depending on the percent of fetal RBCs detected by acid elution methods. This is done because of variation above and below the correct result found on proficiency surveys.

    ABO-induced hemolytic disease of the newborn

    Fifty percent or more of HDN is due to ABO incompatibility between mother and fetus. There usually is no history of previous transfusion. Most cases are found in mothers of blood group O with group A or B infants. Infant anti-A and anti-B production begins between 3 and 6 months after birth. Until then, ABO antibodies in the infant’s serum originate from the mother. If the mother possesses antibodies to the A or B RBC antigens of the fetus, hemolytic disease of the fetus or newborn may result, just as if the fetus or newborn had received a transfusion of serum with antibodies against his or her RBCs. Nevertheless, although 20%-25% of all pregnancies display maternal-fetal ABO incompatibility, only about 40% of these infants develop any evidence of hemolytic disease. In those who do, the condition is usually clinically milder than its counterpart caused by Rh incompatibility with only 4%-11% displaying clinical evidence of disease. There usually are no symptoms at birth. Some infants, however, will suffer cerebral damage or even die if treatment is not given. Therefore, the diagnosis of ABO disease and its differential diagnosis from the other causes of jaundice and anemia in the newborn are of great practical importance.

    There are two types of ABO antibodies: the naturally occurring complete saline-reacting type discussed earlier and an immune univalent (incomplete) type produced in some unknown way to fetal A or B antigen stimulation. In most cases of clinical ABO disease the mother is group O and the infant is group A or B. The immune anti-A or anti-B antibody, if produced by the mother, may cause ABO disease because it can pass the placenta. Maternal titers of 1:16 or more are considered dangerous. The saline antibodies do not cross the placenta and are not significant in HDN.

    Results of the direct Coombs’ test done on the infant with ABO hemolytic disease are probably more often negative than positive, and even when positive the test tends to be only weakly reactive. Spherocytes are often present but the number is variable. Good evidence for ABO disease is detection of immune anti-A or anti-B antibodies in the cord blood of a newborn whose RBCs belong to the same blood group as the antibody. Detection of these antibodies only in the serum of the mother does not conclusively prove that ABO disease exists in the newborn.

    Laboratory tests in hemolytic disease of newborns (HDN)

    When an infant is affected by Rh-induced HDN, the result of the direct Coombs’ test on cord blood is nearly always positive. In ABO-induced HDN the direct Coombs’ test result on cord blood is frequently (but not always) positive. The direct Coombs’ test result on infant peripheral blood is usually positive in Rh-induced disease but is frequently negative in ABO-induced disease, especially when done more than 24 hours after delivery. The direct Coombs’ test should be performed in all cases of possible HDN, because incomplete antibodies occasionally coat the surface of fetal RBCs to such an extent as to interfere with proper Rh typing. Cord blood bilirubin is usually increased and cord blood hemoglobin is decreased in severe HDN. There is disagreement whether cord blood hemoglobin level has a better correlation with disease severity than the cord blood bilirubin or infant venous or capillary hemoglobin level. Infant hemoglobin levels tend to be higher than cord hemoglobin levels if blood from the cord and placenta is allowed to reach the infant after delivery.

    Laboratory criteria for hemolytic disease of the newborn

    Infants with HDN can frequently be saved by exchange transfusion. Commonly accepted indications for this procedure are the following:

    1. Infant serum indirect bilirubin level more than 20 mg/100 ml (342 µmol/L) or, in considerably premature or severely ill infants, 15 mg/100 ml (257 µmol/L).
    2. Cord blood indirect bilirubin level more than 3 mg/100 ml (51 µmol/L) (some require 4 mg/100 ml).
    3. Cord blood hemoglobin level less than 13 gm/dl (130 g/L)(literature range, 8-14 gm/100 ml).
    4. Maternal Rh antibody titer of 1:64 or greater, although this is not an absolute indication if the bilirubin does not rise very high.

    Bilirubin levels in hemolytic disease of the newborn. Most infants with HDN can be treated effectively enough that exchange transfusion is not needed. The level of infant bilirubin is generally used as the major guideline for decision and to monitor results of other treatment such as phototherapy. An infant total bilirubin level of 12-15 mg/100 ml (205 µmol/L) depending to some extent on the clinical situation (degree of prematurity, presence of anemia or infection, severity of symptoms, etc.) is the most commonly accepted area at which therapy is begun. However, there is surprising variation in the levels quoted in various medical centers. Rarely, kernicterus has been reported in seriously ill infants at bilirubin levels near 10 mg/100 ml (and in one case even as low as 6 mg/100 ml). The rapidity with which the bilirubin level rises is also a factor, as noted previously. To further complicate matters, autopsy studies have shown yellow staining of brain tissue typical of kernicterus in some infants who did not have clinical symptoms of kernicterus.

    Bilirubin levels and kernicterus. The most feared complication of Rh-induced hemolytic disease of the newborn is kernicterus, defined as bilirubin staining of the central nervous system (CNS) basal ganglia with death or permanent neuro-logic or mental abnormalities. When this syndrome was first studied in the 1950s, Rh-induced hemolytic disease was the usual etiology, and the nonconjugated bilirubin level of 20 mg/100 ml in term infants (15 mg/dl in premature infants) was established as the level at which the kernicterus syndrome was most likely to develop and thus the level at which exchange transfusion was required. It was also reported that various other factors, such as acidosis, respiratory distress, infection, and very low birth weight could be associated with the kernicterus syndrome at bilirubin levels less than 15 mg/100 ml (several case reports included a few infants whose total bilirubin level was as low as 9-10 mg/100 ml). Eventually the nonconjugated bilirubin level rather than infant symptoms became the center of attention (however, since neonatal bilirubin except in rare cases is almost all nonconjugated, total bilirubin level is routinely assayed instead of nonconjugated bilirubin). As time went on, the advent of RhIg therapy markedly reduced Rh hemolytic disease, and neonatal jaundice became over 90% nonhemolytic. More recent studies have questioned the relationship between total bilirubin level and the kernicterus syndrome. Although phototherapy can reduce total bilirubin levels, there is some question whether in fact this can prevent kernicterus. Therefore, in the early 1990s there is very low incidence of the kernicterus syndrome, the mechanisms and pathologic basis for this syndrome is uncertain, the relationship and interpretation of nonconjugated or total bilirubin levels is being questioned, and the classic guidelines for therapy are being disputed. Nevertheless, while the situation is unclear, the majority of investigators appear to be using 15 mg/100 ml as the level to begin phototherapy (less if the infant is premature and severely ill) and 20 mg/100 ml as the level to consider intensive therapy (possibly, but not necessarily including exchange transfusion). Exchange transfusion appears to be reserved more for infants with severe hemolytic disease; for example, Rh or Kell incompatibility, neonatal glucose 6-phosphate dehydrogenase hemolysis, and sepsis.

    In addition, there are certain technical problems involving bilirubin assay. Phototherapy breaks down nonconjugated bilirubin into nontoxic bilirubin isomers, which, however, are measured and included with unconjugated bilirubin in standard bilirubin assays. Finally, it must be mentioned that bilirubin is one of the least accurate of frequently or routinely performed chemistry assays, with proficiency surveys consistently showing between-laboratory coefficients of variation of 10% or more. To this is added variances due to specimens obtained under different conditions (venous vs. capillary or heelstick), state of infant hydration, and differences between laboratories because of different methodologies. At present, the bilirubin measurement within the capability of most laboratories that is considered to best correlate with clinical kernicterus is unconjugated (indirect) bilirubin. In HDN, most of the bilirubin will be unconjugated. Therefore, it usually is sufficient to obtain total bilirubin levels in order to follow the patient’s progress. If there is a question about the diagnosis, one request for conjugated/unconjugated fractionation is sufficient. A definite consideration is the additional blood needed to assay both the conjugated and unconjugated fractions since large specimens may be difficult to obtain from an infant heel puncture.

    Albumin-bound bilirubin. Unconjugated bilirubin is presumed to be the cause of kernicterus. However, unconjugated or total bilirubin levels do not always correlate well with development of kernicterus. In the 1980s there was interest in measurement of free nonconjugated bilirubin and bilirubin-binding capacity. Most of the unconjugated bilirubin in serum is tightly bound to serum albumin, with a small portion being loosely bound (“free”). Since the free portion rather than the tightly bound portion theoretically should be the most important element in kernicterus, various methods have been devised to assay free unconjugated bilirubin rather than total bilirubin. Until approximately 1980, direct measurement was difficult, and most attention was given to indirect methods, chiefly estimation of the bilirubin-binding capacity of albumin. In general, when more bilirubin binds to albumin, the residual binding capacity becomes smaller and less binding of serum free bilirubin takes place. Therefore, free bilirubin levels are more likely to increase. Several methods have been proposed to measure albumin-binding capacity; the most popular involved a Sephadex resin column. Sephadex resin competes with albumin for loosely bound bilirubin. When unconjugated bilirubin levels exceed the bilirubin-binding capacity of albumin, the excess binds to the sephadex column. There are conflicting reports in the literature on the value of the albumin-binding capacity assay. Some reports indicated that it was very helpful; others found that results from individual patients were either too often borderline or did not correlate sufficiently well with the clinical picture. More recently, an instrument called the hematofluorometer that measures total bilirubin-binding capacity (TBBC) has become available. Total bilirubin-binding capacity as measured by the hematofluorometer is reported to correlate well with unbound bilirubin. Nevertheless, there does not appear to be convincing evidence that albumin-binding capacity or free bilirubin measurements have shown clear-cut superiority over traditional guidelines. In summary, although a total bilirubin level of 20 mg/100 ml is a reasonably good cutoff point for substantial risk of kernicterus in full-term infants, no adequate cutoff point has been found for sick premature infants; and consideration of risk factors such as sepsis, acidosis, pulmonary distress, hypothermia, hypoalbuminemia, or bilirubin-binding capacity has not produced totally reliable criteria for determining whether to use therapy or not.

    Role of amniocentesis in hemolytic disease of the newborn. Amniocentesis has been advocated in selected patients as a means of estimating risk of severe HDN while the fetus is still in utero. A long needle is introduced into the amniotic fluid cavity by a suprapubic puncture approach in the mother. The amniotic fluid is subjected to spectro photometric estimation of bilirubin pigments. Markedly increased bilirubin pigment levels strongly indicate significant degrees of hemolytic disease in the fetus. If necessary, delivery can be induced prematurely once the 32nd week of gestation has arrived. Before this, or if the fetus is severely diseased, intrauterine exchange transfusion has been attempted using a transabdominal approach. The indications for amniocentesis are development of significant titer of Rh antibody in the mother or a history of previous erythroblastosis. Significant maternal antibody Rh titers do not always mean serious fetal Rh disease, but absence of significant titer nearly always indicates a benign prognosis. Also, if initial amniocentesis at 32 weeks does not suggest an immediately dangerous situation, even though mild or moderate abnormalities are present, a fetus can be allowed to mature as long as possible (being monitored by repeated studies) to avoid the danger of premature birth.