Articles on Medical Diseases and Conditions

Tag: Laboratory findings

  • Rheumatoid Arthritis (RA)

    Rheumatoid arthritis (RA) is a chronic systemic disease whose most prominent symptom is inflammation of joints. The small joints of the hands and feet, especially the proximal interphalangeal joints, are most frequently affected; involvement of larger joints of the extremities is somewhat less frequent, and occasionally nonextremity joints may be affected. Polyarticular involvement is much more common than monoarticular disease. Articular disease activity may or may not be preceded or accompanied by systemic symptoms such as low-grade fever, myalgias, malaise, and fatigue. Rheumatoid arthritis tends to be a slow, intermittently active, migratory process that is frequently symmetric. Onset is gradual in 75%-80% of affected adults and more severe and abrupt in 20%-25%. Subcutaneous nodules with distinctive microscopic appearance occur in 15%-20% of patients, most frequently distal to (but not far from) the elbows. Inflammatory involvement of nonarticular organs or tissues such as the heart or lungs may sometimes occur. Patients with RA have increased frequency of the antigen HLA-DR4.

    Laboratory findings. In active adult-onset RA, anemia is present in about 40% of men and 60% of women. The anemia usually appears within 2 months after onset of clinical disease, usually does not become more severe, and is usually of mild or moderate degree, with a hemoglobin value less than 10 gm/100 ml (100 g/L) in fewer than 10% of cases. There is said to be some correlation between the degree of anemia and the initial severity of illness. The anemia of RA is usually included with the anemia of chronic disease, which typically is normocytic and normochromic. However, anemia in RA is more likely to be hypochromic (reported in 50%-100% of cases), although microcytosis is found in less than 10% of cases.

    White blood cell (WBC) counts are most often normal or only minimally elevated. About 25% of RA patients are said to have leukocytosis, usually not exceeding 15,000/mm3 (15 Ч 109/L), which is more apt to be present when onset of disease is severe and abrupt. Leukopenia is found in about 3% of cases, usually as part of Felty’s syndrome (RA plus splenomegaly and leukopenia).

    Anemia and leukocytosis are more common in juvenile-onset RA than adult-onset RA.

    In active RA, nonspecific indicators of acute inflammation, such as the erythrocyte sedimentation rate (ESR) and C-reactive protein level, are elevated in most (but not all) patients. The serum uric acid level is normal in most patients. The serum iron level is generally low-normal or decreased, and iron-binding capacity is also low-normal or decreased.

    Rheumatoid factor. RA and related diseases are associated with production of a group of immunoglobulins called rheumatoid factors (RFs) that include IgG, IgM, and IgA varieties. These immunoglobulins (antibodies) have specificity for IgG that has been altered in certain ways. It is still not certain whether the altered IgG is the cause of the inflammatory abnormalities in RA or is a body response against the inflammatory process. From the laboratory standpoint, the most important of the is the one that is an IgM macroglobulin. RF combines with its altered IgG antigen in vivo, accompanied by complement fixation. IgM RF, like other antibodies, is produced by lymphocytes and plasma cells of B-cell origin. In some persons, especially in infants, IgM antibody production against some infectious organism not associated with rheumatoid disease may result in concurrent production of varying amounts of IgM RF. Outside the body, IgM RF can combine with normal gamma globulin without complement fixation (in fact, some patient serum contains excess C1q component of complement, which may cause a nonspecific RF test reaction that can be avoided by heat inactivation of complement before the test).

    Serologic tests. Serologic tests are the usual method of laboratory diagnosis in adult-onset RA. Various types of serologic tests may be set up utilizing reaction of IgM RF with IgG gamma globulin, differing mainly in the type of indicator system used to visually demonstrate results. The original method was known as the “Rose-Waaler test,” or “sheep cell agglutination test.” Anti-sheep red blood cell (RBC) antibodies were reacted with tannic acid-treated sheep RBCs, then the RF in the patient’s serum was allowed to combine with the antibody gamma globulin coating the sheep cells. Clumping of RBCs indicated a positive test result. It was found subsequently that synthetic particles such as latex could be coated with gamma globulin and the coated particles could be clumped by RF, thus giving a flocculation test. Just as happened with the serologic test for syphilis, many combinations of ingredients have been tried, with resulting variations in sensitivity and specificity. These tests are too numerous to discuss individually, but a distinction must be made between tube tests and rapid slide tests. The slide tests in general have a slightly greater sensitivity than tube tests but also produce more false positive results. Therefore, slide tests should be used mainly for screening purposes. As noted previously, some patient serum contains a nonspecific C1q agglutinator that can be eliminated by inactivating patient serum by heating at 56°C for 30 minutes.

    The latex fixation tube test for RA, known also as the “Plotz-Singer latex test,” currently is considered the standard diagnostic method. The average sensitivity in well-established clinical cases of adult RA is about 76% (range, 50%-95%). Clinically normal controls have about 1%-2% positive results (range, 0.2%-4%). Latex slide tests offer an average sensitivity of approximately 85% (literature range, 78%-98%), with positive results seen in approximately 5%-8% of normal control persons (range, 0.2%-15%). It may take several weeks or months after onset of clinical symptoms, even as long as 6 months, before RA serologic test results become abnormal.

    False positive results. Certain diseases, especially those associated with increased gamma globulin (“hyperglobulinemia”), produce a significantly high number of positive reactions analogous to the “biologic false positive” reactions of syphilis serology. These include collagen diseases, sarcoidosis, syphilis, viral hepatitis and cirrhosis, bacterial infections (especially subacute bacterial endocarditis[SBE]), and even old age (as many as 10%-25% positive over age 70). The incidence of reactive RA tests is higher with the slide than the tube tests. The percentage of positive reactions in the diseases listed ranges from 5%-40%. Sjцgren’s syndrome (75%-96%) and SBE (50%) are most likely to produce false positive results.

    Differential diagnosis. RA is usually part of the differential diagnosis of joint pain. However, other causes must be considered, especially if symptoms, location of joint involvement, laboratory test results, or other features are atypical. Even a positive test result is not conclusive evidence for RA. Other diseases that frequently enter the differential diagnosis are the so-called seronegative spondyloarthropathies, septic (infectious) arthritis, systemic lupus erythematosus (SLE) and other collagen-vascular diseases, crystal-deposition arthritis, and acute rheumatic fever (ARF). These conditions will be discussed later in this chapter.

  • Human Immunodeficiency Virus 1 (HIV-1)

    The HIVs are retroviruses; their genetic information (genome) is composed of RNA rather than the usual DNA. To reproduce, the virus uses an enzyme known as reverse transcriptase to produce a DNA copy of its genetic RNA and incorporates this material into the host cell genetic material. Some of the copied viral genome also exists in the host cell without being incorporated into host chromosomes. Thus, the host cell nucleus reproduces the virus as well as itself. The HIVs have an unusual property, similar to the herpesvirus group, that active viral reproduction and infection can coexist with presence of antibodies against the virus. In most other virus infections, appearance of specific antibody marks the end of the infection and confers partial or complete protection against subsequent infection by that virus. The HIVs attack a subgroup of T-lymphocytes known as helper (inducer) T-cells (CD4 cells). Helper T-cells are important in cell-mediated immunity (delayed hypersensitivity), which is the immunologic mechanism classically defending against chronic lower virulence infections such as tuberculosis, fungus, and parasites. Monocytes, macrophages, and possibly Langerhans cells also become infected.

    The first HIV to be discovered was isolated from patients with the acquired immunodeficiency syndrome (AIDS) by three different groups of investigators who each gave the virus a different name (human T-cell lymphotropic virus type III, or HTLV-III; lymphadenopathy-associated virus, or LAV; and AIDS-associated retrovirus, or ARV). The current terminology for this virus is human immunodeficiency virus type 1 (HIV-1). This virus is present endemically in Central Africa. A related virus that produces a syndrome similar to AIDS is found in West Africa and has been named HIV-2 (originally called HTLV-IV). The HIV viruses are related to a similar virus found in African green monkeys. They are also related, but less closely, to certain animal viruses called lenteviruses (“slow viruses”), of which the most well known is the visna virus of sheep. Besides the HIV virus group that injures or destroys helper T-cells, there is another group of viruses that affects T-cells but that causes excessive T-cell proliferation rather than destruction. This group has retained the name of HTLV and includes HTLV-I (which causes human T-cell leukemia) and HTLV-II (which may be associated with hairy cell leukemia). Similar to the HIV viruses, the HTLV virus group is related to a monkey T-cell leukemia virus and more distantly to a T-cell leukemia virus of cattle.

    Clinical findings

    HIV-1 can be isolated from many body fluids (including blood or blood products, semen, cervical secretions, saliva, tears, cerebrospinal fluid, breast milk, urine, and various tissues including the cornea). However, urine and saliva appear to have relatively little infectious capacity. HIV-1 is predominantly transmitted in three ways: by sexual intercourse (heterosexual or male homosexual), by transfusion or inoculation of infected blood or blood products, and by mother to fetus through the placenta. After exposure, there is an incubation period that typically lasts 2-6 weeks (range, 6 days-8 weeks, but sometimes lasting several months or years). In about 50% of patients (range, 4%-70%) this is followed by an acute viral type of illness (sometimes called the “acute seroconversion” or “acute HIV syndrome”) resembling infectious mononucleosis or CMV infection that usually lasts 2-3 weeks (range, 3 days-several weeks). Symptoms usually include fever, sore throat, and lymphadenopathy; often include skin rash, myalgias, diarrhea, vomiting, and aseptic meningitis; and sometimes thrombocytopenia. Some patients never develop the initial acute febrile illness or any clinical infection; they may recover completely from the initial exposure (although this is probably uncommon) or may become an asymptomatic carrier. Those who develop the initial acute illness exhibit a wide spectrum of possible outcomes. After recovery they may become asymptomatic carriers; may have defective immunologic responses without clinical disease; may develop persistent generalized lymphadenopathy (PGL); may develop a variety of non-life-threatening fungal, bacterial, or viral infections (e.g., oral Candida) as part of the so-called AIDS-related complex (ARC); or may develop the AIDS syndrome.

    AIDS is the most severe manifestation of HIV-1 infection, defined briefly as serologic evidence of HIV antigen or antibody plus certain opportunistic infections or Kaposi’s sarcoma (a malignant tumor of fibroblasts and capillary-sized blood vessels) in a patient who is immunocompromised without known cause. The most frequent opportunistic organism producing active infection in AIDS is pneumocystis carinii (about 60% of cases; range, 35%-80%); other common agents include Cryptococcus neoformans (4%-13% of cases), Candida albicans esophagitis (14%-25%), “atypical” mycobacteria of the Mycobacterium avium-intracellulare complex (22%-30%), and protozoans such as Toxoplasma(3%-12%) and Cryptosporidium (4%-13%). Other organisms with evidence of frequent past or recent infection include CMV (66%94%) and HSV (4%-98%, the lower figures being active infection). Incidence of Kaposi’s sarcoma varies according to risk group; in male homosexuals with AIDS the incidence is about 35% (range, 25%-50%) in clinical studies and 30%-75% in autopsy studies; but in drug abusers and hemophiliacs it is found in less than 5%. Some 50%-80% of patients with AIDS develop various types of neurologic disorders with or without dementia, which may precede other evidence of AIDS in 25% of patients. In one series, 66% of these patients had elevated CSF protein levels (42-189 mg/100 ml; 0.42-1.89g/L); 20% had a small degree of mononuclear cell count elevation (4-51 WBCs); and three of seven patients tested had oligoclonal bands detected in their CSF. Cerebral abnormalities were found in two thirds of the patients with AIDS who were autopsied. There is an increased incidence of B-cell lymphoma, especially primary CNS lymphoma (2%-6% of patients).

    In the United States as of 1992, about 58% of AIDS patients were male homosexuals, usually those who had multiple sex partners. About 23% were intravenous drug abusers; about 6% were persons infected heterosexually; and about 4% were of undetermined etiology. However, incidence of heterosexual infection (as opposed to current incidence of AIDS, a late-stage development of HIV-1 infection) is becoming more frequent. Infection has been reported after a single heterosexual encounter, although more commonly it takes more than one episode. After an infected person develops detectable antibody, the current approximate calculated progression to AIDS per year is about 2.5% for asymptomatic patients, 3.5% for PGL patients, and 8.5% for ARC patients. Progression to AIDS is highest among infected male homosexuals (4%-10%/year) and low among transfusion-infected hemophiliacs. About 20%-40% (range, 10%-73%) of infected mothers transmit the virus to the fetus during pregnancy. A few infants appear to become infected during delivery and some during breast feeding.

    Laboratory findings

    In the few small studies in AIDS patients that contain hematologic data, anemia was present in about 80% (range, 45%-95%), leukopenia in about 65% (range, 40%-76%), thrombocytopenia in about 25%-30% (range, 3%-59%; about 5%-10%, range 3%-15% in HIV-infected non-AIDS patients) and pancytopenia in 17%-41%. Lymphocytopenia was reported in about 70%-80% (range, 30%-83%).

    Diagnosis of HIV-1 infection

    Culture. HIV-1 can be isolated from concentrated peripheral blood lymphocytes and less frequently from body fluids. Isolation rates in already seropositive patients average about 50%-60% (range, 8%-100%; more likely positive just before or during the first half of the acute HIV syndrome). Culture is difficult, is expensive, takes several days, is available only at a relatively few laboratories, and is positive more often in early stages of infection than in later stages. Culture may be the only method that can confirm infection in the first 2-3 weeks after exposure. Culture detects only about 50% of neonates infected in utero in the newborn period up to the first month of life (range, 30%-50%) but a greater percentage at 3 and 6 months. Culture is positive in CSF from about 30% (range, 20%-65%) of seropositive adult patients whether or not CNS symptoms are present, but about 20% more often in more advanced states of disease.

    Antigen detection. Viral antigen may become detectable as soon as 2 weeks after infection (in one report it was detected 4 days after a transplant operation). Antigenemia (viremia) lasts roughly 3 months (range, 1 week-5 months). In several reports, antigen could be detected from a few days to as many as 6-9 months before first-generation ELISA antibody test results became positive. Several methods have been used, including RIA, fluorescent antibody, and ELISA. Until about 1990, sensitivity was usually less than 50% and varied considerably between kits of different manufacturers. It was discovered that varying amounts of the circulating p24 antigen were bound to immune complexes. Methods are now available that break up (dissociate) the bound complexes before testing. In one study this increased test sensitivity to 60%-65% in patients without symptoms and 80%-90% in patients with symptoms.

    Nucleic acid probe kits with PCR amplification (NA-PCR) have become available. These detect HIV antigen within infected peripheral blood lymphocytes. Sensitivity appears to be about 40%-60% in the first 1-2 weeks of life and up to 98% by age 3 months. NA-PCR detects about 96%-100% of seropositive pediatric patients over age 6 months or adult patients with CD4 counts over 800/mm3 and about 85%-97% of those with CD4 counts below 200/mm3. NA-PCR is more sensitive than culture in HIV-infected but seronegative patients and can detect HIV in CSF from about 60% of seropositive patients. As with all laboratory tests, all manufacturer’s NA-PCR probes are not identical in sensitivity.

    Antibody detection. Seroconversion occurs on the average about 6-10 weeks after infective exposure (range, 12 days-5 years), which corresponds to the last part of the acute HIV syndrome stage or up to several weeks afterward (in some degree depending on the sensitivity of the test). Two types of antibodies are produced, IgM and IgG. IgM antibodies are detectable first, and several studies report IgM antibody present in some patients 1-10 weeks before IgG antibody (using first-generation IgG ELISA methods). In general, IgM antibody becomes detectable about 1-2 weeks after onset of the “acute HIV syndrome” (about 5-6 weeks after infection), peaks about 2-3 weeks after first detection, and becomes nondetectable 2-4 months after first detection. IgG antibody becomes detectable 1-2 weeks after IgM antibody, peaks several weeks later, and persists for life (there is controversy over whether a few patients lose antibody shortly before death from AIDS). However, one recent study using a second-generation IgG ELISA found little difference. Commercial ELISA IgM, second-generation IgG, and rapid slide LA methods are now available. Many of these use antibody against one (sometimes more) selected protein components of HIV-1 obtained through recombinant DNA techniques (see Confirmatory Methods section). Test results in HIV-1 infection are shown in Fig. 17-10.

    Tests in HIV-1 infection

    Fig. 17-10 Tests in HIV-1 infection.

    The bulk of published test kit evaluations involve first-generation ELISA methods, which are based on crude extracts of the whole virus. There were a considerable number of such methods commercially available, but even the first was introduced in only mid-1985. These tests detect antibody in 94%-99.5% of patients with confirmed AIDS, depending on the particular assay and the investigator. Positive tests in random blood donors have averaged about 0.9% (range, 0.2%-2.56%). However, in some (not all) of these first-generation kits only about 25%-35% (range, 17%-44%) of initially positive ELISA test results on random blood donors remain reactive when retested with the same kit. Of those whose results were repeatedly positive, only about 20%-35% (range, 15%-62%) were positive on confirmatory tests. This means that only about 10%-15% (range, 3%-22%) of the initial positive results on random blood donors from these particular kits eventually were confirmed positive. Some manufacturer’s kits were shown to be more sensitive than others, and some produced more false positive results than others. Some of this discrepancy is explained on the basis of different appearance times or quantity present of different viral antigens being detected by the different kit antibodies being used. There is also controversy whether reactivity against only a single antigen or a certain type (e.g., the core proteins [“group-specific antigen” or gag] p24 and p55 or the envelope glycoproteins gp 120/160 and gp 41) is sufficient to consider the test truly reactive and thus indicative of HIV-1 infection in the absence of reactivity against any other important structural protein. When this happens, it is often considered a false positive or an “indeterminant” reaction, although its significance has not yet been definitely established. In addition to these controversies, definite false negative results and false positive results may occur. Previously mentioned have been false negative results due to variable time periods before antibody is produced and variation in sensitivity of different methods and different manufacturers kits. Also, in the late or terminal stages of AIDS, antibody may disappear from patient serum in about 2%-4% (range, 0%-7%) of those who previously had antibody.

    When HIV infection is acquired in utero, IgM (and IgA) antibody is slow to rise until 3-6 months after birth. In several reports, IgA was detectable in 6%-17% at one month of age, 57%-67% at 3 months, and 77%-94% at 6 months. IgG antibody was not helpful for the first 6 months of life (range, 3-18 months) because it may be acquired from the mother through the placenta. False negative and positive results can be due to technical and clerical errors. False positive results in some kits may be due to patient antibodies against certain HLA antigens (most often, DR4) in antigenic material from infected H9 cells used in the kits to capture the patient antibodies. Antigenic material from different cell lines or synthetic (recombinant) antigen does not have this problem. Some kits, but not others, have an increased incidence of false positive results in active alcoholic cirrhosis, renal failure, and autoimmune diseases. Gamma globulin used for HBV prophylaxis may contain antibody against HIV-1, although the gamma globulin is noninfectious due to certain steps in its manufacture. This passively transferred antibody may be detectable for as long as 3 months. Antibody detection methods for urine have been developed with sensitivity reported to be comparable to serum tests.

    Confirmatory antibody detection methods. Until 1988 these tests consisted of Western blot and immunofluorescent methods. Western blot is an immunochromatographic technique in which virus material from cell culture is separated into its major component proteins by electrophoresis, transferred (“blotted”) onto a chromatography support medium, and exposed to patient serum. Antibody in patient serum, if present, attaches to whatever virus component proteins it recognizes. Then the preparation is stained to display visually what protein areas had reacted. Not all the virus proteins are considered specific for HIV-1. There is some controversy as to what specific proteins must be present for the results to be considered definitely positive. This affects sensitivity of the test. The two most specific proteins are the virus envelope glycoprotein gp41 and the group-specific antigen (gag) core protein p24 (the numbers refer to molecular weight). However, other proteins, particularly a precursor envelope protein called gp160 (from which gp41 is derived), often appears before either of the more specific proteins. Western blot in general has been shown to detect antibody earlier than most of the first-generation ELISA tests but not as early as IgM or antigen-detection methods (or “second-generation” IgG tests). Unfortunately, Western blot is time consuming, takes 2 days to complete, and reliable results are considerably technique dependent. False negative and false positive results have been reported, although the exact incidence is currently unknown, due to lack of quality control surveys. The test is currently available only in large medical centers and reference laboratories. Immunofluorescence procedures are also available and are claimed to produce results equivalent to Western blot. Immunofluorescence is easier to perform and produces same-day results. However, a minority of investigators found Western blot to be more reliable. Both of these techniques are generally considered suitable to confirm screening test results. The Western blot, however, is currently considered the gold standard. There is also a radioimmunoprecipitation (RIPA) technique that has also been used as a confirmatory procedure. This method is slightly more sensitive than Western blot. However, it is technically difficult and currently is used only in research laboratories.

    Recently, tests have become available based on genetically engineered HIV proteins, most often gp160, gp120, gp41, and p24. One or more of these are used in “second-generation” ELISA or LA tests. In general, these tests are somewhat more sensitive and specific than the “first-generation” tests. One kit (HIVAGEN) consists of separate ELISA tests for antibody against several of these antigens, thus becoming, in effect, a sort of ELISA version of the Western blot.

    Tests for immunologic status. As noted previously, HIV-1 selectively infects T-lymphocyte CD4 cells (also called helper/inducer, Leu3, or OKT4 cells; CD means cluster designation), which eventually leads to defective immune function. CD8 T-cells (suppressor/cytotoxic or OKT8 cells) are normal or become increased. The 1993 CDC revised classification system for HIV infection considers 500 CD4 T-cells/mm3 or more to be normal; 200-499, moderately decreased; and less than 200, severely decreased. CD4 absolute or relative counts are considered to be the best index of HIV disease severity. Eighty percent to 95% of AIDS patients have a decreased absolute number of helper T-cells (<400/mm3) and a reversed (inverted) helper T-cell/suppressor T-cell ratio, with a T4/T8 ratio less than 1.0. One possible cause of false T4 decrease is the recent report that about 20% of African Americans have helper T-cells that fail to react with the OKT4 antibody but do react with the Leu3 and certain other helper T-cell antibodies. A lesser but substantial number of AIDS patients display more nonspecific immune system abnormalities, such as lymphocytopenia (<1,500/mm3) and nonreactivity to skin test challenge by standard delayed sensitivity antigens such as Candida, mumps, or Trichophyton. Tests of immune function usually do not become abnormal until relatively late stages of HIV-1 infection. These tests are not tests for HIV infection, nor are they diagnostic of AIDS. CD4 cell levels are currently considered the best overall indicator of HIV-1 disease severity and prognosis.

    Beta-2 microglobulin. Beta-2 microglobulin (B2M) is a small polypeptide that forms the light chain of the class I histocompatibility complex antigen (HLA) molecules present on the surface of many nucleated cells, including lymphocytes. It is released into serum by cell destruction or membrane turnover and is filtered by the renal glomerulus, after which it is more than 99% reabsorbed and metabolized by the proximal renal tubules. About 50% of serum B2M is derived from lymphocytes. Therefore, B2M levels have been used as a nonspecific marker for lymphocyte proliferation or turnover, as seen in immunologic stimulation or lymphoproliferative disorders. Since it is excreted by the kidney, it has been used to estimate renal function. Since CD4 (T-helper) lymphocytes are affected in HIV infection, B2M is reported to be elevated in about 85%-90% (range, 68%-100%) of patients with AIDS or ARC, 45% of patients with PGL, in smaller numbers of other persons with HIV infection but few or no symptoms, and in varying numbers of clinically healthy male homosexuals (20%-44% in two studies). Some investigators have used B2M as a marker for progression to AIDS, because in general the degree of B2M elevation corresponds roughly with degree of HIV illness severity and inversely with CD4 count. B2M can be assayed by RIA, EIA, or immunodiffusion.

    B2M may also become elevated in persons with poor renal function; in various lymphomas and leukemias, especially (but not exclusively) those of B-cell origin, in myeloma, in nonlymphoid malignancies, in sarcoidosis, in infectious mononucleosis and certain other viral infections, in various chronic inflammatory diseases including active liver disease, and in autoimmune disorders.

    Neopterin. Neopterin is an intermediate substance in the biopterin synthesis pathway. It is produced by macrophages when stimulated by gamma interferon released by activated T-cells (lymphocytes also produce neopterin but to a minor degree). Therefore, neopterin is an indirect indicator of increased T-cell activity. Neopterin is excreted by the kidney. Plasma or urine neopterin levels can be elevated in acute or active chronic infections or noninfectious inflammatory conditions, similar to B2M or C-reactive protein (CRP). The neopterin level is likely to be elevated in both viral and bacterial infection, whereas the CRP level is more likely to become elevated in bacterial than in viral infection. Also, the neopterin level is more likely than the CRP level to be elevated in immunologic graft rejection, whereas both become elevated in graft infection. In general, like B2M, the neopterin level becomes elevated in HIV infection; and the incidence and degree of elevation have a rough correlation to degree of HIV severity and inverse correlation to CD4 count. One reference states that the neopterin level is elevated in about 90% of seropositive but asymptomatic HIV-infected persons. Another found B2M elevated in 75% of these asymptomatic HIV patients and the neopterin level elevated in 60%; both were elevated in all ARC patients. B2M thus far seems to have aroused more interest than neopterin as a marker for severity in HIV infection.

    Summary of human immunodeficiency virus 1 tests. In summary, ELISA tests for HIV-1 antibody are used as a general screen to detect persons infected with HIV-1. Western blot (or equivalent) tests help establish presence of infection. Culture, tests for HIV-1 antigen, and possibly ELISA-IgM antibody tests, may detect infection earlier than the ELISA screening tests. Tests for decreased immunologic function (especially CD4 lymphocyte absolute counts) are useful to help confirm a clinical impression of advanced-stage HIV-1 and AIDS.

  • Epstein-Barr Virus (EBV)

    The Epstein-Barr virus is a member of the herpesvirus group and is reported to infect 80% or more of the U.S. population. It is thought to be spread from person to person, most likely through saliva, with the majority of infections occurring in childhood, adolescents, and young adults. The EBV infects B-lymphocytes. In common with the other herpesviruses, once infection (with or without symptoms) takes place the virus eventually becomes dormant but can be reactivated later into clinical disease. Reactivation is said to occur in 15%-20% of healthy persons and in up to 85% in some groups of immunosuppressed patients. Epstein-Barr virus infection in young children is usually asymptomatic. Primary infection by EBV in older children, adolescents, or young adults produces the infectious mononucleosis syndrome in up to 50% of cases. The EBV is also strongly associated with Burkitt’s lymphoma in Africa and nasopharyngeal carcinoma in southern China.

    Infectious mononucleosis (infectious mono; IM)

    Infectious mononucleosis (IM) patients are most often adolescents and young adults, but a significant number are older children and middle-aged or even older adults. When IM is part of a primary infection, the incubation period is 3-7 weeks (range, 2-8 weeks). The acute phase of illness in those patients who are symptomatic lasts about 2-3 weeks (range, 0-7 weeks). Convalescence takes about 4-8 weeks. The most common features of the acute illness are fever, pharyngitis, and adenopathy, with lymph node enlargement occurring in 80%-90% of patients. The posterior cervical nodes are the ones most commonly enlarged. Soft palate petechiae are found in 10%-30% of cases. Jaundice, usually mild, is found in about 5%-10% (range, 4%-45%) of patients in large series. The spleen is mildly enlarged in about 50% of patients (range, 40%-75%) and hepatomegaly is present in about 10% (range, 6%-25%).

    Laboratory findings. Patients usually have normal hemoglobin values. Mild thrombocytopenia is reported in 25%-50% of patients (range, 15%-50%). Leukocytosis between 10,000 and 20,000/ mm3 (10 Ч 109-20 Ч 109/L) occurs in 50%-60% of patients (range, 40%-70%) by the second week of illness. About 10% (range, 5%-15%) of patients develop a leukocytosis over 25,000/mm3 (25 Ч 109/L). However, during the first week there may be leucopenia. About 85%-90% (range, 80%-100%) of patients with IM have laboratory evidence of hepatic involvement (Table 17-2). Peak values are reported to occur 5-14 days after onset of illness for aspartate aminotransferase (AST), bilirubin, and alkaline phosphatase (ALP); and between 7 and 21 days for gamma-glutamyltransferase (GGT). The AST and ALP levels return to normal in nearly all patients by 90 days, but occasionally there may be some degree of GGT elevation persisting between 3-12 months. Total LDH is elevated in about 95% of patients. LDH isoenzyme fractionation by electrophoresis can show three patterns: elevation of all five fractions; elevation of LDH 3, 4, and 5; or elevation of LDH-5 only.

    Liver function tests in EBV-induced infectious mononucleosis

    Table 17-2 Liver function tests in EBV-induced infectious mononucleosis

    Peripheral blood smear. The first of three classic findings is a lymphocytosis, with lymphocytes making up more than 50% of the total white blood cells (WBCs). Lymphocytosis is said to be present in 80%-90% of patients (range, 62%-100%), peaks during the second or third week, and lasts for an additional 2-6 weeks. The second classic criterion is the presence of a “significant number” of atypical lymphocytes on Wright-stained peripheral blood smear. There is disagreement as to whether greater than 10% or greater than 20% must be atypical. These atypical lymphocytes are of three main types (Downey types). Type I has vacuolated or foamy blue cytoplasm and a rounded nucleus. Type II has an elongated flattened nucleus and large amounts of pale cytoplasm with sharply defined borders and often some “washed-out” blue cytoplasm coloring at the outer edge of the cytoplasm. Type III has an irregularly shaped nucleus or one that may be immature and even may have a nucleolus and resemble a blast. All three types are larger than normal mature lymphocytes, and their nuclei are somewhat less dense. Most of the atypical lymphocytes are activated T-lymphocytes of the CD-8 cytotoxic-suppressor type. Some of the Downey III lymphocytes may be EBV-transformed B lymphocytes, but this is controversial. These atypical lymphocytes are not specific for IM, and may be found in small to moderate numbers in a variety of diseases, especially cytomegalovirus and hepatitis virus acute infections. In addition, an appearance similar to that of the type II variety may be created artificially by crushing and flattening normal lymphocytes near the thin edge of the blood smear. IM cells are sometimes confused with those of acute leukemia or disseminated lymphoma, although in the majority of cases there is no problem.

    Although most reports state or imply that nearly all patients with IM satisfy the criteria for lymphocytosis and percent atypical lymphocytes, one study found only 55% of patients had a lymphocytosis and only 45% had more than 10% atypical lymphocytes on peripheral smear when the patients were first seen. Two studies found that only about 40% of patients with IM satisfied both criteria.

    Serologic tests. The third criterion is a positive serologic test for IM either based on heterophil antibodies or specific anti-EBV antibodies. The classic procedure is the heterophil agglutination tube test (Paul-Bunnell test). Rapid heterophil antibody slide agglutination tests have also been devised. Slide tests now are the usual procedure done in most laboratories. However, since the basic principles, interpretation, and drawbacks of the slide tests are the same as those of the older Paul-Bunnell tube test, there are some advantages in discussing the Paul-Bunnell procedure in detail.

    Serologic tests based on heterophil antibodies.

    Paul-Bunnell antibody is an IgM-type antibody of uncertain origin that is not specific for EBV infection but is seldom found in other disorders (there are other heterophil antibodies that are not associated with EBV infection). Paul-Bunnell antibodies begin to appear in the first week of clinical illness (about 50% of patients detectable; range, 38%-70%), reaching a peak in the second week (60%-78% of patients positive) or third (sometimes the fourth) week (85%-90% positive; range, 75%-100%), then begin to decline in titer during the fourth or fifth week, most often becoming undetectable 8-12 weeks after beginning of clinical illness. However in some cases some elevation is present as long as 1-2 years (up to 20% of patients). In children less than 2 years old, only 10%-30% develop heterophil antibodies; about 50%-75% of those 2-4 years old develop heterophil antibodies. One report states that these antibodies are rarely elevated in Japanese patients of any age. Once elevated and returned to undetectable level, heterophil antibodies usually will not reelevate in reactivated IM, although there are some reports of mild heterophil responses to other viruses.

    The original Paul-Bunnell test was based on the discovery that the heterophil antibody produced in IM would agglutinate sheep red blood cells (RBCs). In normal persons the sheep cell agglutination titer is less than 1:112 and most often is almost or completely negative. The Paul-Bunnell test is also known as the “presumptive test” because later it was found that certain antibodies different from those of IM would also attack sheep RBCs. Examples are the antibodies produced to the Forssman antigen found naturally in humans and certain other animals and the antibody produced in “serum sickness” due to certain drug reactions. To solve this problem the differential absorption test (Davidsohn differential test) was developed. Guinea pig kidney is a good source of Forssman antigen. Therefore, if serum containing Forssman antibody is allowed to come in contact with guinea pig kidney material, the Forssman antibody will react with the kidney antigen and be removed from the serum when the serum is taken off. The serum will then show either a very low or a negative titer, whereas before it was strongly positive. The IM heterophil antibody is not significantly absorbed by guinea pig kidney but is nearly completely absorbed by bovine (beef) RBCs, which do not significantly affect the Forssman antibody. The antibody produced in serum sickness will absorb both with beef RBCs and guinea pig kidney.

    The level of Paul-Bunnell titer does not correlate well with the clinical course of IM. Titer is useful only in making a diagnosis and should not be relied on to follow the clinical course of the disease or to assess results of therapy.

    In suspected IM, the presumptive test is performed first; if necessary, it can be followed by a differential absorption procedure.

    “Spot” tests were eventually devised in which the Paul-Bunnell and differential absorption tests are converted to a rapid slide agglutination procedure without titration. Most of the slide tests use either horse RBCs, which are more sensitive than sheep RBCs, or bovine RBCs, which have sensitivity intermediate between sheep and horse RBC but which are specific for IM heterophil antibody and therefore do not need differential absorption. Slide test horse cells can also be treated with formalin or other preservatives that extend the shelf life of the RBC but diminish test sensitivity by a small to moderate degree.

    Heterophil-negative infectious mononucleosis.

    This term refers to conditions that resemble IM clinically and show a similar Wright-stained peripheral blood smear picture, but without demonstrable elevation of Paul-Bunnell heterophil antibody (see the box) About 65% (range, 33%-79%) are CMV infection, about 25% (15%-63%) are heterophil-negative EBV infections, about 1%-2% are toxoplasmosis, and the remainder are other conditions or of unknown etiology.

    Diagnosis of infectious mononucleosis. When all three criteria for IM are satisfied, there is no problem in differential diagnosis. When the results of Paul-Bunnell test or differential absorption test are positive, most authors believe that the diagnosis can be made, although there are reports that viral infections occurring after IM can cause anamnestic false positive heterophil reelevations. When the clinical picture is suggestive of IM but results of the Paul-Bunnell test, differential absorption procedure, or the spot test are negative, at least one follow-up specimen should be obtained in 14 days, since about 20%-30% of IM patients have negative heterophil test results when first seen versus 10%-15% negative at 3 weeks after onset of clinical symptoms (although the usual time of antibody appearance is 7-10 days, it may take as long as 21 days and, uncommonly, up to 30 days). About 10% (range, 2%-20%) of patients over age 5 years and 25%-50% or more under age 5 years never produce detectable heterophil antibody. Another potential problem is that several evaluations of different heterophil kits found substantial variation in sensitivity between some of the kits. If the clinical picture is typical and the blood picture is very characteristic (regarding both number and type of lymphocytes), many believe that the diagnosis of IM can be considered probable but not established. This may be influenced by the expense and time lapse needed for specific EBV serologic tests or investigation of CMV and the various other possible infectious etiologies.

    Some “Heterophil-Negative” Mononucleosis Syndrome Etiologies

    Viruses

    EBV heterophil-negative infections
    Cytomegalovirus
    Hepatitis viruses
    HIV-1 seroconversion syndrome
    Other (rubella, herpes simplex, herpesvirus 6, mumps, adenovirus)

    Bacteria

    Listeria, tularemia, brucellosis, cat scratch disease, Lyme disease, syphilis, rickettsial diseases

    Parasites

    Toxoplasmosis, malaria

    Medications

    Dilantin, azulfidine, dapsone, “serum sickness” drug reactions

    Other

    Collagen diseases (especially SLE, primary or drug-induced)
    Lymphoma
    Postvaccination syndrome
    Subacute bacterial endocarditis (SBE)

    In summary, the three classic criteria for the diagnosis of IM are the following:

    1. Lymphocytes comprising more than 50% of total WBC count.
    2. Atypical lymphocytes comprising more than 10% (liberal) or 20% (conservative) of the total lymphocytes.
    3. Significantly elevated Paul-Bunnell test and/or differential absorption test result. A positive slide agglutination test result satisfies this criterion.

    Serologic tests based on specific antibodies against EBV. The other type of serologic test for IM detects patient antibodies against various components of the EBV (Table 17-3). Tests are available to detect either viral capsid antigen-IgM or IgG (VCA-IgM or IgG) antibodies. VCA-IgM antibody is usually detectable less than one week after onset of clinical illness and becomes nondetectable in the late convalescent stage. Therefore, when present it suggests acute or convalescent EBV infection. Rheumatoid factor (RF) may produce false positive results, but most current kits incorporate some method to prevent RF interference. VCA-IgG is usually detectable very soon after VCA-IgM, but remains elevated for life after some initial decline from peak titer. Therefore, when present it could mean either acute, convalescent, or old infection. Tests are available for Epstein-Barr nuclear antigen (EBNA) IgM or IgG antibody, located in nuclei of infected lymphocytes. Most kits currently available test for IgG antibody (EBNA-IgG or simply EBNA). EBNA-IgM has a time sequence similar to that of VCA-IgM. The more commonly used EBNA-IgG test begins to rise in late acute stage (10%-34% positive) but most often after 2-3 weeks of the convalescent stage. It rises to a peak after the end of the convalescent stage (90% or more positive), then persists for life. Elevated EBNA/EBNA-IgG is suggestive of nonacute infection when positive at lower titers and older or remote infection at high or moderately high titer. A third type of EBV test is detection of EBV early antigen (EA), located in cytoplasm of infected cells. There are two subtypes; in one the antigen is spread throughout the cytoplasm (“diffuse”; EA-D) and in the other, the antigen is present only in one area (“restricted”; EA-R). The EA-D antibody begins to rise in the first week of clinical illness, a short time after the heterophil antibody, then peaks and disappears in the late convalescent stage about the same time as the heterophil antibody. About 85% (range, 80%-90%) of patients with IM produce detectable EA-D antibody, which usually means EBV acute or convalescent stage infection, similar to VCA-IgM or heterophil antibody. However, EA-D may rise again to some extent in reactivated EBV disease, whereas VCA-IgM does not (whether heterophil antibody ever rises is controversial, especially since it may persist for up to a year or even more in some patients). EA-D is typically elevated in EBV-associated nasopharyngeal carcinoma. EA-R is found in about 5%-15% of patients with clinical IM. It is more frequent (10%-20%) in children less than 2 years old with acute EBV infection and is typically elevated in patients with EBV-related Burkitt’s lymphoma. Expected results from the various serologic tests in different stages of EBV infection are summarized in Table 17-3 and Fig. 17-9.

    Antibody tests in EBV infection

    Table 17-3 Antibody tests in EBV infection

    Tests in EBV infection

    Fig. 17-9 Tests in EBV infection.

    Specific serologic tests for EBV are relatively expensive compared to heterophil antibody tests and are available predominantly in university centers and large reference laboratories. Such tests are not needed to diagnose IM in the great majority of cases. The EBV tests are useful in heterophil-negative patients, in problem cases, in patients with atypical clinical symptoms when serologic confirmation of heterophil results is desirable, and for epidemiologic investigations. If the initial heterophil test is nonreactive or equivocal, it is desirable to freeze the remainder of the serum in case specific EBV tests are needed later.

    Summary of Epstein-Barr Antibody Test Interpretation
    Never infected (susceptible) = VCA-IgM and IgG both negative.
    Presumptive primary infection = Clinical symptoms, heterophil positive.
    Primary infection: VCA-IgM positive (EBNA-IgG negative; heterophil positive or negative)
    Reactivated infection: VCA-IgG positive; EBNA-IgG positive; EA-D positive (heterophil negative, VCA-IgM negative)
    Old previous infection: VCA-IgG positive; EBNA-IgG positive; EA-D negative (VCA-IgM negative, heterophil negative).

  • Hemolytic Anemias Due to Extracorpuscular Agents

    Anemias due to isoagglutinins (isoantibodies)

    These anemias are hemolytic reactions caused by antibodies within the various blood group systems. The classification, symptomatology, and diagnostic procedures necessary for detection of such reactions and identification of the etiology are discussed in Chapter 9 and Chapter 11.

    Anemias due to autoagglutinins (autoantibodies)

    Autoagglutinins are antibodies produced by an individual against certain of his or her own body cells. This discussion concerns autoantibodies produced against his or her own RBCs. The anemia associated with this condition has been called autoimmune hemolytic anemia or acquired hemolytic anemia.
    Autoantibodies of the autoimmune hemolytic anemias form two general categories: those that react best in vitro above room temperature (37°C, warm autoantibodies) and those that react best in vitro at cold temperatures (cold autoantibodies or cold agglutinins). For each type there are two general etiologies, idiopathic and secondary to some known disease.

    Warm autoantibodies are IgG antibodies usually directed against Rh antigens on the RBC membrane. They comprise about 50%-70% of Coombs’-positive autoantibodies. The presence of the autoantibody and the RBC antigen against which it reacts can often (not always) be proven by detaching (eluting) the antibody from affected RBC. Clinical disease from warm autoantibodies is more frequent than clinical abnormality from cold autoantibodies, and the idiopathic variety is twice as frequent as that secondary to known disease. Clinically, anemia due to warm-reacting autoantibodies appears at any age and may be either chronic or acute. When chronic, it is often low grade. When acute, it is often severe and fatal. The laboratory signs are those of any hemolytic anemia and depend on the degree of anemia. Thus, there are varying degrees of reticulocyte elevation. The direct Coombs’ test result is usually, although not always, positive. Most patients have spherocytes in peripheral blood, especially if the anemia is acute; and splenomegaly is frequent.

    Cold agglutinins are IgM antibodies usually directed against the I antigen on RBC membranes. These comprise about 15%-30% of Coombs’-positive autoantibodies. Complement can often be detected on affected RBC but no antibody usually can be eluted. Clinical disease from cold-reacting agglutinins is seen much less frequently than hemolytic disease from warm-reacting autoantibodies. Cold agglutinin disease is seen predominantly in adults, particularly in the elderly. The most common cause of symptomatic hemolytic anemia induced by cold agglutinins is mycoplasma infection. After mycoplasma-induced disease, the idiopathic and the secondary forms occur in nearly equal incidences. Clinically, the disease is often worse in cold weather. Raynaud’s phenomenon is common. Splenomegaly is not common. Laboratory abnormalities are not as marked as in the warm autoantibody type, except for a usually positive direct Coombs’ test result, and the anemia tends to be less severe. The reticulocyte count is usually increased but often only slightly. Spherocytes are more often absent than present. WBCs and platelets are usually normal unless altered by underlying disease. However, exceptions to these statements may occur, with severe hemolytic anemia present in all its manifestations. As noted in the discussion of mycoplasma pneumonia (Chapter 14), cold agglutinins may occur in many normal persons but only in titers up to 1:32. In symptomatic anemia due to cold agglutinins the cold agglutinin titer is almost always more than 1:1,000.

    Paroxysmal cold hemoglobinuria (PCH)

    Paroxysmal cold hemoglobinuria is a rare syndrome in which an antibody (Donath-Landsteiner antibody) of the IgG class binds to and sensitizes RBCs at cold temperatures and then produces complement-activated RBC lysis at warmertemperatures. PCH comprises about 2%-5% of Coombs’-positive autoantibodies, much more common in children than in adults. Paroxysmal cold hemoglobinuria was originally associated with syphilis, but more cases occur idiopathically or following viral infection than from syphilis. Hemoglobinuria is produced after patient exposure to cold temperatures and may be accompanied by back or leg pain, chills, and cramps, similar to symptoms of hemolytic transfusion reaction. The IgG-specific Coombs’ reagent produces positive direct Coombs’ test results at cold temperatures and Coombs’ reagents containing non-gamma non-IgG-specific antibody (sometimes called broad-spectrum Coombs’ reagent) produce positive direct Coombs’ test results at the usual Coombs’ test temperature of 37°C. The major diagnostic procedure for paroxysmal cold hemoglobinuria is the Donath-Landsteiner test, in which the development of hemolysis in patient and normal blood is compared at cold temperature.

    Secondary acquired autoimmune hemolytic anemia

    The causes of acquired hemolytic anemia of the secondary type, either warm or cold variety, can be divided into three main groups. The first group in order of frequency is leukemia and lymphoma; most often chronic lymphocytic leukemia, to a lesser extent lymphocytic lymphoma, and occasionally Hodgkin’s disease. The second group in order of frequency is collagen disease, notably lupus erythematosus. The third group is a miscellaneous collection of systemic diseases in which overtly hemolytic anemia rarely develops but may do so from time to time. These diseases include viral infections, severe liver disease, ovarian tumors, and carcinomatosis. It should be emphasized that in all three disease groups, anemia is a common or even frequent finding, but the anemia is usually not hemolytic, at least not of the overt or symptomatic type.

    Drug-induced hemolytic anemia

    Drug-induced hemolytic anemia is sometimes included with the autoimmune hemolytic anemias. However, in most cases antibodies are formed primarily against the drug, and action against the RBC is secondary to presence of the drug on the RBC surface. These cause about 10%-20% of Coombs’-positive autoantibodies. There are four basic mechanisms proposed, as follows:

    1.
    Combination of the drug with antidrug antibody to form an immune complex that is adsorbed onto RBCs, often activating complement. Quinidine is the best-known drug of this type. The antiquinidine antibody is of the IgM class.
    2.
    Binding of the drug to the RBC membrane and acting as a hapten. Penicillin (in very large doses, і10 million units/day for 7 days or more) is the major drug of this type, although abnormality develops in fewer than 3% of these cases.
    3.
    Nonspecific coating of RBC by drug with absorption of various proteins. The antibiotic cephalothin has been shown to act by this mechanism. A positive direct Coombs’ test result is produced by antibodies against proteins absorbed onto the cell or onto cephalothin. There is no hemolysis, however. Cephalothin may occasionally act as a hapten and in these cases may be associated with hemolytic anemia.
    4.
    Unknown mechanism. a-Methyldopa is the predominant drug of this type and may be the most common agent associated with drug-induced hemolytic anemia. The antimethyldopa antibody is of the IgG class and usually has Rh group specificity. Besides coating of RBCs, a-methyldopa–treated patients may have circulating autoantibodies demonstrated by an indirect Coombs’ test, which is unusual for other drugs. Patients taking a-methyldopa may also develop a syndrome resembling systemic lupus erythematosus, with antinuclear antibodies and lupus erythematosus cells (Chapter 23). Up to 25% of patients (literature range 10%-36%) develop a positive direct Coombs’ test, and about 1% (literature range 0%-5%) develop hemolytic anemia. The direct Coombs’ test result remains positive 1-24 months after the end of therapy.

    Laboratory investigation of possible drug-induced hemolytic anemia is usually difficult for the ordinary laboratory. The procedure usually involves washing off (eluting) the antibody from the RBC, if possible, and trying to determine whether the antibody has specificity against drug-coated RBCs rather than normal RBCs.

    Traumatic (microangiopathic) hemolytic anemia

    This category includes several diseases that produce hemolytic anemia with many schistocytes, the schistocytes being formed through some kind of trauma. Representative conditions are disseminated intravascular coagulation and thrombotic thrombocytopenic purpura (in which RBCs strike fibrin clots in small vessels), the hemolytic-uremic syndrome (thrombi in renal glomerular capillaries and small vessels), the cardiac prosthesis syndrome (in which RBCs are damaged while passing through the artificial heart valve), and hemolytic anemia associated with vascular grafts and some long-term indwelling catheters. The same type of hemolytic anemia may be found in a few patients with malignancy (most commonly gastric carcinoma), in Zieve’s syndrome associated with cirrhosis, in the first few hours after extensive severe burns, and in Clostridium welchii septicemia. As noted in Chapter 2, schistocytes can be found in smaller numbers in other conditions. Microangiopathic hemolytic anemia is discussed in greater length in Chapter 8.

    Paroxysmal nocturnal hemoglobinuria (PNH)

    Patients with paroxysmal nocturnal hemoglobinuria (PNH) develop an acquired blood cell membrane defect in which RBCs, WBCs, and platelets demonstrate abnormal sensitivity to the effect of activated serum complement. This is manifest by hemolytic anemia, granulocytopenia, and thrombocytopenia. Not all patient RBCs have the same degree of abnormality, and resistance to lysis varies from relatively normal to markedly abnormal. It is often associated with aplastic anemia and is said to develop in 5%-10% of these patients without regard to the cause of the marrow depression (with the exception that PNH is not associated with radiation marrow damage). It may appear either at the beginning of aplasia, during the aplastic period, or during recovery. About 50% of cases develop without prior evidence of aplastic marrow. It may also develop in some patients with erythroleukemia, myelofibrosis, or refractory anemia.

    RBCs that are abnormally sensitive to complement have markedly decreased acetylcholinesterase levels, but this is not thought to be the cause of the defect in PNH.

    Paroxysmal nocturnal hemoglobinuria most often affects young or middle-aged adults, with the usual age range being 10-60 years. The disease presents as hypoplastic anemia in about 25% of cases, as an episode of abdominal pain in about 10%, and with hemoglobinuria in about 50%. Clinically, there is a chronic hemolytic anemia, with crisis episodes of hemoglobinuria occurring most often at night. However, hemoglobinuria is present at disease onset only in about 50% of cases. Another 20% develop it within 1 year, and eventually it occurs in more than 90% of patients. Anemia is usually of moderate degree except during crisis, when it may be severe. A crisis is reflected by all the usual laboratory parameters of severe hemolysis, including elevated plasma hemoglobin levels. No spherocytosis or demonstrable antibodies are present. The disease gets its name because hemoglobinuric episodes turn urine collected during or just after sleep to red or brown due to large amounts of hemoglobin. Urine formed during the day is clear. Stimuli known to precipitate attacks in some patients include infections, surgery, and blood transfusion.

    Laboratory findings. In addition to anemia, leukopenia (granulocytopenia) is present in about 50% of patients, and some degree of thrombocytopenia is present in about 70%. This is in contrast to most other hemolytic anemias, in which hemolysis usually provokes leukocytosis. The MCV is elevated in about 83%, normal in about 13%, and decreased in about 5%. The reticulocyte count is elevated in about 90%. Loss of iron in the urine (in the form of hemoglobin and hemosiderin) leads to chronic iron deficiency in some patients. For some reason the kidney in PNH is not damaged by the hemoglobin or by renal tubular cell deposition of hemosiderin.

    Venous thrombosis is frequent in PNH, and patients have a considerably increased tendency toward infection (predominantly lung and urinary tract). There may be episodes of abdominal pain related to venous thrombosis.

    Tests for paroxysmal nocturnal hemoglobinuria. A good screening test is a urine hemosiderin examination. However, a positive urine hemosiderin value may be obtained in many patients with chronic hemolytic anemia of various types and also may be produced by frequent blood transfusions, especially if these are given over periods of weeks or months. A much more specific test is the acid hemolysis (Ham) test. The RBCs of PNH are more susceptible to hemolysis in acid pH. Therefore, serum is acidified to a certain point that does not affect normal RBCs but will hemolyze the RBCs of PNH. Another widely used procedure is the sugar-water (sucrose hemolysis) test, which is easier to perform than the Ham test and may be more sensitive. It is based on evidence that RBCs in PNH are more susceptible to hemolysis in low ionic strength media than normal RBCs. Many laboratories screen with the sugar-water test and confirm a positive result with the Ham test. The sugar-water test is apt to produce more weak positive reactions in patients who do not have verifiable PND than does the Ham test. In my experience (also reported by others) there occasionally is discrepancy between results of the sugar-water test and the Ham test in the same patient, resulting in diagnostic problems.

    Hemolytic anemia due to toxins

    Chemical. Lead poisoning is the most frequent cause in this group. Ingestion of paint containing lead used to be frequent in children and still happens occasionally. Auto battery lead, gasoline fumes, and homemade whiskey distilled in lead-containing apparatus are the most common causes in adults. It takes several weeks of chronic exposure to develop symptoms unless a large dose is ingested. The anemia produced is most often mild to moderate, and the usual reason for seeking medical treatment is development of other systemic symptoms, such as convulsions from lead encephalopathy, abdominal pain, or paresthesias of hands and feet. The anemia is more often hypochromic but may be normochromic; it is usually normocytic. Basophilic stippling of RBCs is often very pronounced and is a classic diagnostic clue to this condition. Basophilic stippling may occur in any severe anemia, especially the hemolytic anemias, but when present to an unusual degree should suggest lead poisoning unless the cause is already obvious. The stippled cells are reticulocytes, which, for some unknown reason, appear in this form in these patients. However, in some patients, basophilic stippling is minimal or absent. Tests useful in lead poisoning for screening purposes or for diagnosis are discussed in Chapter 35.

    Other chemicals were mentioned in the discussion of G-6-PD deficiency anemia. Benzene toxicity was discussed in the section on hypoplastic bone marrow anemias. Other chemicals that often produce a hemolytic anemia if taken in sufficient dose include naphthalene, toluene, phenacetin, and distilled water given intravenously. Severe extensive burns often produce acute hemolysis to varying degrees.

    Bacterial. Clostridium welchii septicemia often produces a severe hemolytic anemia with spherocytes. Hemolytic anemia is rarely seen with tuberculosis. The anemia of infection is usually not overtly hemolytic, although there may be a minor hemolytic component (not demonstrable by the usual laboratory tests).

    Hemolytic anemia due to parasites

    Among hemolytic anemias due to parasites, malaria is by far the most frequent. It must be considered in persons who have visited endemic areas and who have suggestive symptoms or no other cause for their anemia. The diagnosis is made from peripheral blood, best obtained morning and afternoon for 3 days. Organisms within parasitized RBCs may be few and often are missed unless the laboratory is notified that malaria is suspected. A thick-drop special preparation is the method of choice for diagnosis. With heavy infection, the parasites may be identified on an ordinary (thin) peripheral blood smear. A hemolytic anemia is produced with the usual reticulocytosis and other laboratory abnormalities of hemolysis. Most patients have splenomegaly. Bartonella infection occurs in South America, most often in Peru. This is actually a bacterium rather than a parasite, but in many textbooks it is discussed in the parasite category. The organisms infect RBCs and cause hemolytic anemia clinically similar to malaria. Babesiosis is an uncommon protozoan infection of RBC similar in some respects to malaria. This condition is discussed in Chapter 18.

    Hypersplenism

    Hypersplenism is a poorly understood entity whose main feature is an enlarged spleen associated with a deficiency in one or more blood cell elements. The most common abnormality is thrombocytopenia, but there may be a pancytopenia or any combination of anemia, leukopenia, and thrombocytopenia. Hypersplenism may be primary or, more commonly, secondary to any disease that causes splenic enlargement. However, splenic enlargement in many cases does not produce hypersplenism effects. Portal hypertension with secondary splenic congestion is the most common etiology; the usual cause is cirrhosis. If anemia is produced in hypersplenism, it is normocytic and normochromic without reticulocytosis. Bone marrow examination in hypersplenism shows either mild hyperplasia of the deficient peripheral blood element precursors or normal marrow.

    Several mechanisms have been proposed to explain the various effects of hypersplenism. To date, the weight of evidence favors sequestration in the spleen. In some cases, the spleen may destroy blood cells already damaged by immunologic or congenital agents. In some cases, the action of the spleen cannot be completely explained.