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Clinical Applications of Chemiluminescence of Granulocytes Russell W. Steele
From the Division of Infectious Diseases/Immunology, Department of Pediatrics, Louisiana State University Medical Center and Children's Hospital, New Orleans, Louisiana
It has been common medical practice for decades to determine a complete blood cell count in the evaluation of a patient with fever or other evidence of systemic infection. The absolute white-cell concentration and granulocyte and bandform counts have all been used in making decisions about patient management, and this approach has been supported by a number of clinical studies [1-3]. Correlations between these parameters and disease imply that granulocytes can reflect in vivo inflammatory processes, but simple measurement of their numbers or maturity is evidently inadequate. Nearly a century ago Bodkin [4] suggested that a detailed cytologic examination of white blood cells in peripheral blood smears would offer useful information in evaluating infective states, and almost 50 years ago Ponder et a1. [5] better quantitated correlations between clinical conditions of patients and morphologicchangesof polymorphonuclearleukocytes (PMNs), such as toxic granulation, ameboid cell outline, vacuolation, and pyknotic nuclei. However, routine application was obviously impractical, since such analysis is extremely timeconsuming and requires a high degree of training and experience. Recent technologic advances in the measurement of oxygenation of leukocytes have reawakened interest in detailed examination of granulocytes as a method for early detection of inflammatory diseases. Changes in these cells appear to precede other acute-phase reactants and to more closely parallel severity of infection. The most promising lab-
Received 17 July 1990; revised 24 October 1990. Reprints and correspondence: Dr. Russell W. Steele, Children's Hospital, 200 Henry Clay Avenue, New Orleans, Louisiana 70118. Reviews of Infectious Diseases 1991;13:918-25 © 1991 by The University of Chicago. All rights reserved. 0162-0886/91/1305-0042$02.00
oratory tool for these analyses is measurement of chemiluminescence, a fairly simple technique. The methods have been reviewed in previous publications [6], but equipment, reagents, newly developed whole-blood techniques, and approaches to the analysis of data continue to be refined. The present communication reviews progress in the clinical application of chemiluminescence as a laboratory assay. Methodology of Chemiluminescence Chemiluminescence was originally developed as a means of examining the intracellular respiratory burst, production of oxygenating radicals, and generation of other agents that function to eliminate microbial pathogens. An early observation was that chemiluminescence was absent following stimulation of PMNs from patients with chronic granulomatous disease [7, 8], thus further confirming a specific defect in the redox metabolism of leukocytes in patients with this inherited disorder. Subsequent investigations focused on chemiluminescence as an indicator of microbicidal activity. Assays have shown that the chemiluminescence response in neutrophils is correlated with bacterial killing in vitro as measured by more standard assays such as bacterial colony counts and superoxide production [9-11]. Additional studies have directly compared chemiluminescence to other measurements of neutrophil function in patients with infection. For example, one report obtained concomitant measurements of chemotaxis, phagocytosis (using uptake of radiolabeled bacteria), and cellular ratios of cyclic guanosine monophosphate to cyclic AMP [12]. Increases in phagocytosis and chemotactic responsiveness during active bacterial infection closely correlated with changes in the chemiluminescence of leukocytes.
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The oxidative metabolic activity of granulocytes can be directly examined by chemiluminescence, a laboratory technique that measures photon emission during well-defined inflammatory or microbicidal events. Numerous studies have utilized chemiluminescence to examine early changes during infectious diseases and other pathologic processes. Studies have suggested that receptors on cell surfaces and oxygenation of granulocytes can reflect the severity of disease as well as provide early diagnostic information. Diseases within virtually every subspecialty of medicine have been studied in this respect, but most investigations have focused on infectious and autoimmune conditions. The present review summarizes current progress in laboratory methods and evaluates the potential application of recently published clinical data. It is apparent that during disease myeloperoxidase- and oxidase-dependent oxygenation activities reflect separate host responses, and independent measurements of these activities will offer a more meaningful understanding of host defense. Immune complexes and other factors in serum may also interact with granulocytes to alter the receptors on cell surfaces and subsequent metabolic activity. In some circumstances, enhanced function of granulocytes may be detrimental to the host.
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Clinical Applications of Chemiluminescence
General Assessment of Activity of PMN In Vivo Circulating granulocytes from healthy donors and granulocytes stimulated with various mediators of inflammation show well-recognized differences [6, 13-16]. For example, fMet-Leu-Phe and C5a desArg increase the total number of C3b receptors by 8- to 10-fold and activate potential metabolic activity of normal circulating PMN s [17], while the simple warming of cells, analogous to development of clinical fever, will increase the number of C3b and fMet-Leu-Phe receptors [18]. Abundant indirect evidence indicates that enhanced oxygenation as well as other cellular changes occur in vivo during infectious diseases and inflammatory conditions [12, 19-25]. One explanation for increased potential activity in circulating neutrophils from infected individuals is the recently observed influence of numerous cytokines. These include colony-stimulating factors, interleukin-l-o and interIeukin-l-d, tumor necrosis factors, and interferons, which most specifically increase phagocytic activity and oxidative metabolism. Data related to such activation of neutrophils by recom-
binant cytokines, both direct and in synergy with other stimulants, were reviewed in this journal recently [26]. In separate recent publications, Ueno [20] and Zgliczynski et al. [21]used luminol-enhanced chemiluminescence of whole blood to evaluate the functional states of phagocytic cells in patients with various diseases. Both studies demonstrated increased activity of granulocytes obtained from those individuals with systemic bacterial infections. With increased severity of disease, some patients exhibited markedly decreased responses, leading Zgliczynski et al. [21] to suggest a profile of four functional states: resting, as seen in normal healthy individuals; standby, as exhibited during early or mild disease; activated, as in individuals with moderate infection; and exhausted, as with severe clinical manifestations. The four functional states could be reproduced by preincubating cells with bacterial components, fMet-Leu-Phe, or the products of activated complement. These studies confirmed earlier reports of increased or "primed" chemotactic, phagocytic, metabolic, and bactericidal responses of granulocytes in infected patients as assessed with various assays. Increased oxidative metabolism, as indicated by direct measurement of the hexose-monophosphate shunt [27] and increased reduction of nitroblue tetrazolium dye [27-29], is well documented. Random motility [12,28-30], response to chemoattractants [12, 28-30], release and activation of lysosomal enzymes [27], carrier-mediated membrane transport of 2-deoxyglucose [31], and neutrophil adherence [32] are likewise increased. However, observed differences were often highly variable, and values were not greatly different from control values. It may be that during disease a mixed population of granulocytes in the peripheral circulation accounts for the relatively high variability. To examine this possibility, Bass et al. [33] employed a quantitative flow cytometry assay of the formation of HzOz-dependent oxidative products and the intracellular oxidation of 2',7'-dichlorofluorescein after stimulation of cells with PMA. Flow cytometry demonstrated that bacteremic patients possess two populations of peripheral blood granulocytes, one with normal oxidative metabolism and another with up to five times more oxidative product than in normal cells. Among infected cases this "primed" population of phagocytes averaged 40 %, with a range of 0-80 %. There were no distinct differences between patients with gram-negative and gram-positive bacteremia. Sera as well as other body fluids from patients with inflammatory diseases may generate chemiluminescence in normal peripheral blood leukocytes. This observation was confirmed for active autoimmune diseases [34] as well as for a wide variety of infectious processes. In a recent report from our laboratories, peak chemiluminescence generated by sera from individuals with bacterial infection was significantly higher than that produced by sera obtained during viral infections or by control samples [35]. These and other studies suggest that there is a direct correlation between severity of infection and chemiluminescence [35, 36]. When sera were examined
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The chemiluminescence response in granulocytes can be magnified with secondary substrates, i.e., chemiluminigenic probes such as luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (10,10'-dimethyl-9,9'-biacridinium dinitrate). These two probes can be used to measure different metabolic events within PMNs. Luminol-enhanced chemiluminescence reflects primarily myeloperoxidase activity, while lucigenin-dependent chemiluminescence is myeloperoxidase independent and measures oxidase-associated oxygenation. Proof for this differentiation is provided by appropriate blocking experiments; azide, which inhibits myeloperoxidase but not oxidases, substantially reduces luminol-enhanced chemiluminescence, while superoxide dismutase alters the superoxide anion and thereby lucigenin-dependent activity. Various PMN stimulants can be selected to separate receptor-related events from those not requiring receptors on the cell surface. Immunoglobulin and complement opsonized particles are phagocytized following contact with Fe and C3b receptors, respectively. Purified components of complement have also been employed. Activity is obviously dependent on the quantity or potential quantity of expressed receptors. In contrast, phorbol myristate acetate (PMA), purified from croton oil, induces redox metabolism, degranulation of PMNs, and measurable chemiluminescence without using receptors or provoking events on the cell surface associated with phagocytosis. Expression of receptors may also be examined indirectly by using extremely low concentrations of stimulants and thereby obtaining rate-limiting kinetics. If such low concentrations of stimulants still produce maximum stimulation, then the total number of receptors or, more correctly, the expression of receptors, is probably low.
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Susceptibility to Infection Chronic granulomatous disease is a metabolic disorder of granulocytes that is characterized by a heterogeneous group of deficiencies that prevent hexose-monophosphate shunt activity and the generation of superoxide and hydroxyl microbicidal radicals. This defect involves neutrophils, macrophages, and eosinophils. In two-thirds of affected individuals, the genetic inheritance pattern is X-linked recessive, while in onethird the pattern is autosomal recessive; occasional autosomal dominant transmission has.. been reported [8]., The bestdescribed specific defect is absence of or decreased levels of cytochrome b558, the heme protein closely linked to the nicotinamide-adenine dinucleotide phosphate oxidase complex, but a number of genetic defects involving constituents of this complex have now been identified and characterized [40]. All forms of this disease can be diagnosed by measuring the respiratory burst, and chemiluminescence provides a quan-
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titative assessment of this activity [7, 8]. Therefore, partial defects and a carrier state can be detected. Opsonized microorganisms are classically used to document disease, but all types ofchronic granulomatous disease may also be tested with stimuli such as PMA that do not require membrane-associated phagocytic events. Patients with AIDS develop a number of defects in host defense, including defects in aspects of the metabolic activity of their PMNs. Compared with healthy controls, AIDS patients in one report showed a 50% reduction of chemiluminescence induced by opsonized zymosan [41]. Some patients with AIDS-related complex as characterized by lymphadenopathy also demonstrated decreased activity of granulocytes. Stohr et al. suggest that measurement of the chemiluminescence of granulocytes is an important parameter in determining both susceptibility to infection and ultimate prognosis in patients with AIDS. In a similar fashion, chemiluminescence has been shown to be useful as a predictive factor in patients with cancer [42]; opsonophagocytosis was assessed by using zymosan opsonized by serum obtained from healthy control subjects or from patients with leukemia and multiple myeloma. The PMN responses of patients were reduced, particularly when autologous sera were used. In addition, the chemiluminescence of control PMNs was significantly inhibited by patient sera. Normal sera completely restored the chemiluminescence of patients with multiple myeloma but only partially corrected the defect in patients with leukemia. Significant correlations were observed between the chemiluminescence of PMNs in patients with cancer and four clinical parameters: disease stage, presence of cancer-related symptoms, subsequent bacterial infections, and long-term outcome. As concluded for patients with AIDS, evaluation of chemiluminescence appeared to be useful in predicting the prognosis for infection and disease. Defects in the myeloperoxidase activity of granulocytes can be evaluated with techniques using chemiluminescence. Patients most frequently present with recurrent superficial staphy lococcal abscesses that often require surgical drainage. In one report, impaired luminol-dependent chemiluminescence was associated with slow release of myeloperoxidase, an unusual congenital abnormality of the degranulation process [43]. Chemiluminescence in monocytes from patients with Shwachman syndrome is also decreased, but in these patients PMN activity is increased [44]. Reasons for differences between these populations of phagocytes have not been delineated, but the increased oxidation with PMNs may account for the well-recognized impaired chemotaxis of purified cells. The susceptibility of newborns to severe infection is well known and likely attributable to an immature immune system that does not function well, particularly during the stress of disease. Although many studies have suggested that fullterm neonates have normal phagocytic and bactericidal activity, those who become infected usually exhibit depressed
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for immune complexes by using a Clq-binding assay, those sera that generated the highest level of chemiluminescence had detectable complexes. Similar correlations were observed in sera from patients with systemic lupus erythematosus (SLE) , thereby leading investigators to conclude that circulating immune complexes cause neutrophil activation [34]. It is also possible that complexes indirectly affect PMNs by activating the complement system and thereby generating biologically active split products such as C5a. Kapp et al. [37] have quantitated the activity of C5a by measuring a specific peak of lucigenin-enhanced chemiluminescence seen within 2 minutes of the addition of test serum. A standard radioimmunoassay of the C5a peptide confirmed the specificity of the chemiluminescence technique. Overnight effluent from patients undergoing chronic ambulatory peritoneal dialysis was examined for opsonic capacity by using luminol-dependent chemiluminescence [38]. Low levels of response were associated with an increased incidence of peritonitis, while no patients with high response levels developed peritonitis during 12 months ofcontinuous monitoring. Opsonic activity was both immunoglobulin and complement dependent. Synovial fluid from patients with rheumatoid arthritis has been shown to have an enhancing influence in assays of chemiluminescence. Preincubation of normal granulocytes with 10% synovial fluid increased spontaneous as well as PMAand zymosan-induced chemiluminescence [39]. Additional studies suggested that the primary mechanism is increased release of myeloperoxidase within granulocytes caused by a factor present in the fluid. This factor may be immune complexes. The ultimate result is detrimental to the host, since oxygen radicals are implicated as mediators of tissue destruction within joints. If the factor is released from the breakdown of tissue, an obvious cycle of continued inflammation would be perpetuated.
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Clinical Applications of Chemiluminescence
Serious Infections Most investigators agree that the chemiluminescence ofhuman PMNs increases during early serious bacterial infection and that such activity returns to normal during the course of successful medical management [12, 19-25]. However, some investigators have also observed that critically ill patients or those whose diagnosis was delayed exhibit impaired neutrophil activity [20, 25, 49]. Studies designed to use chemiluminescence to examine the bactericidal function of neutrophils during serious bacterial infections were first undertaken just 10 years ago. In one of the earliest studies [12], Barbour et al. used opsonized zymosan to stimulate the metabolic activity ofPMNs. Patients had a variety of infections; most common were pneumonia, abscesses, and endocarditis. Mortality in this study group was
30 %. Data clearly showed that chemiluminescence was increased in patients compared with uninfected control subjects (P < .01). This activated state remained with persistent infection but returned to control levels with appropriate therapy. Patients who eventually died were not noted in this report to differ from others, but whether these patients were followed sequentially or analyzed as a separate group is not clear. A number of studies have examined patients with surgical infections, and results have suggested that early activation of PMN function, followed by depression of responses to stimulants, occurs in patients with more-severe disease. Salo et al. [25] quantitated the luminol-enhanced chemiluminescence of isolated granulocytes from patients with postoperative infection, primarily minor wound infections but some cases of sepsis or intraabdominal abscesses. Stimulants of PMN activity included the bacterium isolated from each patient, a standard strain of Escherichia coli, and opsonized zymosan. Responses to all stimulants were significantly reduced in those patients who had a major infection, while responses in those with minor infections were similar to the responses of noninfected control subjects. Stimulation with the autologous bacterium did not yield data more sensitive in delineating severity of infection. Four patients with major infections died, but the chemiluminescence in their cells was no more suppressed than that of eight others who survived equally serious postoperative complications. Two similar reports confirmed these findings for severe disease but observed increased luminoldependent chemiluminescence in patients with mild to moderate infection [20, 49]. Furthermore, one of these publications documented an inverse correlation between the concentration of bacteria in the infective site and the chemiluminescence of peripheral blood neutrophils [49]. In all patients in the study, response to antimicrobial therapy was associated with normalization of chemiluminescence. A possible explanation for immunologic changes following surgical procedures is the effect of anesthesia. Using chemiluminescence, Busoni et al. [50] demonstrated a transient depression in the production of singlet oxygen in PMNs of children undergoing herniorrhaphy that was dependent on the duration of halothane anesthesia. If the entire surgical procedure was accomplished under general anesthesia, changes in chemiluminescence persisted for as long as 24 hours. In a similar fashion the trauma of an operation itself may alter immune function, including chemiluminescence responses to opsonized zymosan and bacteria, for approximately 24 hours [51, 52]; this depression of activity may be prolonged for 3-4 days with more-extensive procedures such as cardiac surgery [53]. However, in the studies of surgical infections discussed above [20, 25, 49], effects of anesthesia and operative trauma were controlled by enrolling only those patients who had undergone operations >14 days prior to the study. In a recent study peripheral blood was obtained from pediatric patients with bacterial and viral meningitis and was examined by a whole-blood microassay system using chemilu-
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activity [45]. Differences are even more apparent in premature and low-birth-weight neonates, but in contrast to responses in full-term newborns, peak chemiluminescence responses of PMN to PMA and zymosan in one study were consistently diminished, even when pretenn infants were not ill [46]. A more-recent publication, however, suggested that depressed function wasnot a universal phenomenon in premature neonates but rather a defect present in a segment of the population; when chemiluminescence was used to assess microbicidal activity, granulocytes from infected and stressed pretenn infants but from only a small minority of stable low-birth-weight neonates demonstrated defective PMN oxidative metabolic responses [47]. These abnormalities were often present prior to recognition of any clinical illness, and in most patients the chemiluminescence of PMNs remained depressed following episodes of serious infection. One conclusion by Driscoll et al. was that pretenn neonates have deficient intracellular activity of myeloperoxidase in all or a portion of their circulating PMNs. Patients without specifically defined immune deficiency syndromes but with a predisposition to infection have also been studied for possible abnormalities in the oxygenation of PMN. Results in some reports have suggested that subtle defects in phagocytosis and oxidative metabolism of PMNs may be major determinants of susceptibility to infection. Bondestam et al. evaluated multiple functional properties of PMNs in a group of Swedish children who had experienced severe multifocal infections [48]. Parameters included random migration, chemotaxis, chemoattractant activity of serum, phagocytosis, and production of chemiluminescence. Most of these patients had at least one defect in PMN function that appeared to be inherited and was a primary cause of increased susceptibility to infection. It is quite possible, however, that defects were seen because children were infected at the time of examination. Because the design of the study did not provide longitudinal analysis of these pediatric patients, the latter interpretation cannot be excluded.
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Minor Infections Animal studies have best documented the effect of mild to moderate infectious diseases on endogenous chemiluminescence activity in host granulocytes [56]. Twelve hours after a live vaccine strain of Francisella tularensis was injected into rodent models, enhanced chemiluminescence in PMNs occurred concomitantly with the onset of fever. It is interesting that immune animals challenged with microorganisms also exhibited a level of response higher than those of control subjects but significantly lower than those of nonimmune animals. Viral infection in animal models [57] as well as in humans does not enhance responses and often depresses the oxidation of PMNs. A number of published clinical studies have examined the chemiluminescence of PMNs from patients with various nonlife-threatening infections. Essentially all reports noted that responses were enhanced compared with those of control subjects [12,24,25,58-60]. Diagnoses have included some moreunusual infectious diseases whose pathogenesis is poorly un-
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derstood, such as yersinia arthritis [58], periodontitis [59], and familial Mediterranean fever [60]. An additional study of patients with lymphocytic meningoencephalitis found no alteration in the oxygenation of PMNs but rather a fourfold enhancement of the chemiluminescence of peripheral blood monocytes [61]. This enhancement persisted in some patients for as long as 3 weeks. The magnitude of change correlated with the cell count and level of protein in CSF but not with the IgG content. The association of the clinical courses of individual patients with enhanced chemiluminescence of peripheral blood monocytes was highly significant, as higher levels of chemiluminescence predicted rapid recovery. The increased oxidative metabolism seen in peripheral blood neutrophils probably occurs to a greater extent in cells present at the site of infection. Here, activated granulocytes produce oxidant damage and other tissue-destructive events during their attempt to eradicate invading pathogens [62]. In a rat model of pyelonephritis, Meylan et al. [63] used lucigeninenhanced chemiluminescence of activated PMNs to show that dapsone could inhibit myeloperoxidase-mediated reactions even though it had no direct effect on killing of bacteria. This finding correlated with a 65 % reduction in renal scars, although there was no alteration in bacterial counts, inflammatory swelling, or infiltration of PMNs during acute pyelonephritis. Chemiluminescence could thus provide a tool for evaluating the therapeutic management of inflammatory events during this common infection.
Autoimmune Disease It is well documented that autoimmune diseases are risk factors for recurrent and severe infection, even when patients with these diseases are not receiving immunosuppressive chemotherapy. This predisposition may be secondary to alteration in immune function attributable to the disease process itself. Early studies suggested that granulocyte phagocytic and killing functions were most affected by these rheumatic diseases [64]. However, subsequent investigations yielded various results for alterations in these aspects of host defense, and differences have been in large part dependent on the methods employed for assessment, particularly inclusion of autologous sera. It is now clear that immune complexes can either augment or inhibit phagocytosis, and the concentration of immune complexes may vary with the activity of the disease. According to measurements of luminol-enhanced chemiluminescence of normal PMNs, sera of patients with active SLE stimulated the oxygenation of granulocytes, and the chemiluminescence responses directly correlated with disease activity [34]. In contrast, sera from patients with SLE in remission yielded results similar to those of control subjects. Stimulatory capacity was concentration dependent and was augmented by the addition of normal serum complement. These observations suggest activation of PMNs by immune complexes. It
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minescence [54]. White blood cells were stimulated with complement-opsonized zymosan, C5a, tMet-Leu-Phe, and PMA. The chemiluminigenic probes luminol and lucigenin were used to measure myeloperoxidase- and oxidase-associated oxygenation, respectively. Integral and peak.chemiluminescence response adjusted for the total number of granulocytes in the sample were calculated. Integral responses for bacterial versus viral meningitis were significantly higher when granulocytes were stimulated with complement-opsonized zymosan or the phorbol ester. Control values were similar to those for aseptic meningitis. When opsonized zymosan was used as a standard stimulus and a combination of opsonized zymosan and C5a was used to elicit a maximum granulocyte response, a percent reserve could be calculated. Compared with patients with viral meningitis, patients with bacterial infection had a markedly decreased functional reserve of granulocytes, and there was no overlap between these two groups of patients. Children who ultimately died demonstrated rapidly decreasing responses. Determination of the reserve of granulocytes in this study was a very sensitive method for differentiating bacterial from aseptic meningitis and determining the prognosis for patients with clinically more-severe bacterial infection. Phagocytic oxygenation activity has been carefully examined in bum patients and monitored during their prolonged hospital courses [55]. Alterations in chemiluminescence consistently anticipated changes in clinical condition in that sepsis was associated with a marked decrease in chemiluminescence. Similar to studies of postoperative surgical infections [20, 25, 49], these studies demonstrate the clinical utility of determining the chemiluminescence of granulocytes as a method for monitoring patient populations who are at risk of developing sepsis during a defined period.
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Clinical Applications of Chemiluminescence
is therefore likely that in vivo exposure of granulocytes to immune complexes during relapse of disease decreases subsequent functional capacity, as measured in other reports [65]. Chemiluminescence of peripheral blood neutrophils is usually increased in patients with active rheumatic autoimmune disorders, including rheumatoid arthritis [66], SLE [67], and progressive systemic sclerosis [67, 68], but not always in patients with ankylosing spondylitis or psoriatic arthritis [68]. Changes are not related simply to the presence or absence of the B27 antigen, as arthritic patients all show equal changes during relapse regardless of this histocompatibility marker [69, 70].
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ness of the split products ofIgG and suggested wide variations of specific antibody among these products. In summary, the measurement of chemiluminescence has potential application for clinical medicine. Luminometers for measuring chemiluminescence have now been vastly improved, and optimal reagent materials are readily available. What remains is for assays to be optimized and standardized so that additional clinical studies can better define parameters that are sensitive and specific enough to direct decisions regarding diagnosis and patient management. Such investigations are currently in progress. References
Chemiluminigenic probes have also been employed in highly sensitive laboratory assays for detecting microbial antigens and antibodies to potential pathogens [71, 72]. In these assays chemiluminescence is induced following the interaction of luminol and hydrogen peroxide with an enzyme such as horseradish peroxidase conjugated to antigen or antibodies. This produces a chemiluminescence-enhanced ELISA. The resulting peroxidase-catalyzed oxidation of luminol can be detected by luminometers, which directly measure light emission, or can be recorded on photographic film. This method has been used to measure antibody to cytomegalovirus [73], rubella virus [74], and herpes simplex virus [74] and to detect the presence of viral antigens for herpes simplex virus [75], respiratory syncytial virus [76], and rotavirus [76]. Detection limits have been as low as 0.01 ng in these assays, which contrasts with 1.0 ng in other ELISAs for determining microbial antigen protein. In similar investigations for which immunoassays of chemiluminescence were used to detect hormones in plasma or body secretions, protein concentrations as low as 1-2 pg could be measured. An interesting diagnostic application of chemiluminescence is detection of neutrophil antibodies [77]. In this assay, the chemiluminescence of peripheral blood monocytes is induced with antibody-coated granulocytes, a technique more rapid and more sensitive than any currently available that use indirect immunofluorescence. With chemiluminescence, IgG antibodies can be differentiated from IgM autoimmune mechanisms by including simple blocking experiments. These data may be relevant for determining which patients might benefit from intravenous immunoglobulin therapy, since those with IgG antibodies are more likely to respond. Chemiluminescence is ideal for determining the functional capacity of antibody, since results have been shown to correlate with clinical protection. We recently published the results of studies that used chemiluminescence to compare the opsonizing activity of five commercially available intravenous immunoglobulin preparations for important microbial pathogens [78]. The data did not support potential clinical useful-
1. McGowan JE Jr, Bratton L, Klein JO, Finland M. Bacteremia in febrile children seen in a "walk-in" pediatric clinic. N Engl J Med 1973; 288:1309-12 2. Teele DW, Pelton SI, Grant MJA, Herskowitz J, Rosen DJ, Allen CE, Wimmer RS, Klein JO. Bacteremia in febrile children under 2 years of age: results of cultures of blood of 600 consecutive febrile children seen in a "walk-in" clinic. J Pediatr 1975;87:227-30 3. Waskerwitz S, Berkelhamer JE. Outpatient bacteremia: clinical findings in children under two years with initial temperatures of 39.5°C or higher. J Pediatr 1981;99:231-3 4. Bodkin E. Uber die LoslichKeit der weissen Blutkorperchen in Peptonlosungen. Virchows Arch [A] 1892;137:476-85 5. Ponder E, Ponder RV, Mineola LI. The cytology of the polymorphonuclear leucocyte in toxic conditions. J Lab Clin Med 1942;28: 316-22 6. Allen RC. Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. Methods Enzymol 1986; 133:449-93 7. Allen RC, Stjernholm RL, Reed MA, Harper TB III, Gupta S, Steele RH, Waring WW. Correlation of metabolic and chemiluminescent responses of granulocytes from three female siblings with chronic granulomatous disease. J Infect Dis 1977;136:510-8 8. Capsoni F, Minonzio F, Venegoni E, Lazzarin A, Galli M, Silvani C, Ongari AM, Zanussi C. Chronic granulomatous disease in a 23-yearold female. J Clin Lab Immunol 1986;19:149-54 9. Horan TD, English D, McPherson TA. Association of neutrophil chemiluminescence with microbicidal activity. Clin Immunol 00munopathol 1982;22:259-69 10. Welch WD. Correlation between measurements of the luminol-dependent chemiluminescence response and bacterial susceptibility to phagocytosis. Infect Immun 1980;30:370-4 11. Grebner JV, Mills EL, Gray BH, Quie PG. Comparison of phagocytic and chemiluminescence response of human polymorphonuclear neutrophils. J Lab Clin Med 1977;89:153-9 12. Barbour AG, Allred CD, Solberg CO, Hill HR. Chemiluminescence by polymorphonuclear leukocytes from patients with active bacterial infection. J Infect Dis 1980;141:14-26 13. Gallin 11, Seligmann BE. Mobilization and adaptation of human neutrophil chemoattractant fMet-Leu-Phe receptors. Fed Proc 1984;43: 2732-6 14. Hotfstein ST, Friedman RS, Weissmann G. Degranulation, membrane addition, and shape change during chemotactic factor-induced aggregation of human neutrophils. J Cell Biol 1982;95:234-41 15. Gresham HD, McGarr JA, Shackelford PG, Brown EJ. Studies on the molecular mechanisms of human Fe receptor-mediated phagocytosis. J Clin Invest 1988;82:1192-1201 16. Lehrer RI, Ganz T, Selsted ME, Babior BM, Curutte JT. Neutrophils and host defense. Ann Intern Med 1988;109:127-42
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39. Bender JG, Van Epps DE, Searles R, Williams RC Jr. Altered function of synovial fluid granulocytes in patients with acute inflammatory arthritis; evidence for activation of neutrophils and its mediation by a factor present in synovial fluid. Inflammation 1986;10:443-53 40. Clark RA. The human neutrophil respiratory burst oxidase. J Infect Dis 1990;161:1140-7 41. Stohr L, Altmeyer P, Sessler MJ, Scharrer I, Helm EB, Holzmann H. Granulocytic chemiluminescence in AIDS, lymphadenopathy, and hemophilia. Z Hautkr 1985;60:1214-23 42. Nagler A, Obendeanu N, Tatarsky I, Hocherman I, Merzbach D, Carter A. Chemiluminescence response of polymorphonuclear leucocytes to serum-opsonized zymosan. A predictive parameter for infections and prognosis in multiple myeloma patients. Clin Lab Haematol 1988;10: 365-75 43. Edwards SW, Hart CA, Davies JM, Pattison J, Hughes V, Si11s JA. Impaired neutrophil killing in a patient with defective degranulation of myeloperoxidase. J Clin Lab Immunol 1988;25:201-6 44. Repo H, Savilahti E, Leirisalo-Repo M. Aberrant phagocyte function in Shwachman syndrome. Clin Exp Immunol 1987;69:204-12 45. Shigeoka AO, Santos Jl, Hill HR. Functional analysis of neutrophil granulocytes from healthy, infected, and stressed neonates. J Pediatr 1979;95 :454-60 46. Peden DB, VanDyke K, Ardekani A, Mullett MD, Myerberg DZ, VanDyke C. Diminished chemiluminescent responses of polymorphonuclear leukocytes in severely and moderately preterm neonates. J Pediatr 1987;111:904-6 47. Driscoll MS, Thomas VL, Ramamurthy RS, Casto DT. Longitudinal evaluation of polymorphonuclear leukocyte chemiluminescence in premature infants. J Pediatr 1990;116:429-34 48. Bondestam M, Hakansson L, Foucard T, Venge P. Defects in polymorphonuclear neutrophil function and susceptibility to infection in children. Scand J Clin Lab Invest 1986;46:685-94 49. Belotskii SM, Snastina TI, Filiukova OB, Zviagin AA. The microbial factor in the chemiluminescence of the peripheral blood neutrophils of patients with suppurative surgical infection. Zh Mikrobiol Epidemiol Immunobiol 1988;8:87-90 50. Busoni P, Sarti A, DeMartino M, Graziani E, Santoro S. The effect of general and regional anesthesia on oxygen-dependent microbicidal mechanisms of polymorphonuclear leukocytes in children. Anesth Analg 1988;67:453-6 51. Burrows FA, Steele RW, Marmer DJ, Van Devanter SH, Westerman GR. Influence of operations with cardiopulmonary bypass on polymorphonuclear leukocyte function in infants. J Thorac Cardiovasc Surg 1987;93 :253-60 52. Solomkin JS, Bauman MP, Nelson RD, Simmons RL. Neutrophils dysfunction during the course of intra-abdominal infection. Ann Surg 1981;194:9-17 53. Perttila J, Lehtonen O-P, Salo M, Tertti R. Effects of coronary bypass surgery under high-dose fentanyl anaesthesia on granulocyte chemiluminescence. Br J Anaesth 1986;58:1027-30 54. Steele RW, Allen RC. Granulocyte oxygenation activity and functional reserve during bacterial meningitis [abstract]. Pediatr Res 1990;27:184 55. Allen RC, Pruitt BA Jr. Humoral-phagocyte axis of immune defense in burn patients. Chemoluminigenic probing. Arch Surg 1982;117: 133-140 56. McCarthy JP, Bodroghy RS, Jahrling PB, Sobocinski PZ. Differential alterations in host peripheral polymorphonuclear leukocyte chemiluminescence during the course of bacterial and viral infections. Infect Immun 1980;30:824-31 57. Bellanti JA, Krasner RI, Bartelloni PJ, Yang MC, Beisel WR. Sandfly fever: sequential changes in neutrophil biochemical and bactericidal functions. J Immunol 1972;108:142-51 58. Koivuranta-Vaara P, Leirisalo-Repo M, Repo H. Polymorphonuclear leucocyte function and previous yersinia arthritis: enhanced chemoki-
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17. Fearon DT, Collins LA. Increased expression of C3b receptors on polymorphonuclear leukocytes induced by chemotactic factors and by purification procedures. J Immunol 1983;130:370-5 18. Gallin Jl, Seligmann BE, Fletcher M. Dynamics of human neutrophil receptors for the chemoattractant fMet-Leu-Phe. Agents Actions Suppl 1983;12:290-308 19. Malech HL, Gallin JI. Neutrophils in human diseases. N Engl J Med 1987;317:687-94 20. Ueno N. Chemiluminescence of whole blood. Analysis of the kinetics and application of clinical examination of phagocytic functions of whole blood from various types of disease. Hokkaido Igaku Zasshi 1986; 61:947-60 21. Zgliczynski JM, Kwasnowska E, Stelmaszynska T, Olszowska E, Olszowski S, Knapik JM. Functional states of neutrophils as suggested by whole blood chemiluminescence. Acta Biochim Pol 1988;35:331-42 22. Van Dyke K, Van Dyke C. Cellular chemiluminescence associated with disease states. Methods Enzymol 1986;133:493-507 23. Campbell AK, Hallett MB, Weeks I. Chemiluminescence as an analytical tool in cell biology and medicine. Methods Biochem Anal 1985;31:317-416 24. Miyata H, Moriguchi N, Kinoshita T. The chemiluminescence response of polymorphonuclear leukocytes from febrile patients. Clin Chim Acta 1988;173:337-42 25. Salo M, Perttila J, Lehtonen O-P. Granulocyte chemiluminescence in patients with postoperative infections. Arch Surg 1988;123:17-22 26. Steinbeck MI, Roth JA. Neutrophil activation by recombinant cytokines. Rev Infect Dis 1989;11:549-68 27. McCall CE, DeChatelet LR, Cooper MR, Shannon C. Human toxic neutrophils. m. Metabolic characteristics. J Infect Dis 1973;127:26-33 28. McCall CE, Bass DA, DeChatelet LR, Link AS Jr, Mann M. In vitro responses of human neutrophils to N-formyl-methionyl-leucylphenylalanine: correlation with effects of acute bacterial infection. J Infect Dis 1979;140:277-86 . 29. Hill HR, Kaplan EL, Dajani AS, Wannamaker LW,Ollie PG. Leukotactic activity and reduction of nitroblue tetrazolium by neutrophil granulocytes from patients with streptococcal skin infection. J Infect Dis 1974;129:322-6 30. Hill HR, Gerrard JM, Hogan NA, Quie PG. Hyperactivity of neutrophil leukotactic responses during active bacterial infection. J Clin Invest 1974;53:996-1002 31. Bibi SS, DeChatelet LR, McCall CEo Human toxic neutrophils. IV. Incorporation of amino acids and uptake of 2-deoxy-n-glucose. J Infect Dis 1977;135:949-51 32. Lentnek AL, Schreiber AD, MacGregor RR. The induction of augmented granulocyte adherence by inflammation. Mediation by a plasmid factor. J Clin Invest 1976;57:1098-1103 33. Bass DA, Olbrantz P, Szejda P, Seeds MC, McCall CEo Subpopulations of neutrophils with increased oxidative product formation in blood of patients with infection. J Immunol 1986;136:860-6 34. Via CS, Allen RC, Welton RC. Direct stimulation of neutrophil oxygenation activity by serum from patients with systemic lupus erythematosus: a relationship to disease activity. J RheumatoI1984;11:745-53 35. Steele RW, Steele RW, Truax DL, Allen RC. Stimulation of neutrophil chemiluminescence activity by serum from pediatric patients with bacterial and viral infections. Clin Res 1989;37:60A 36. Kharazmi A, Eriksen HO, Willumsen L, Ronne-Rasmussen JO, Permin H, Faber V. Opsonic activity of serum in the acute phase of meningitis. Acta Pathol Microbiol Immunol Scand [C] 1986;94:167-70 37. Kapp A, Maly FE, Schopf E. Detection of complement activation in human serum using C5a-induced chemiluminescence of human granulocytes. Arch Dermatol Res 1985;278:41-3 38. Coles GA, Alobaidi HMM, Topley N, Davies M. Opsonic activity of dialysis effluent predicts those at risk of Staphylococcus epidermidis peritonitis. Nephrol Dial Transplant 1987;2:359-65
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netic migration and oxygen radical production correlate with the severity of the acute disease. Ann Rheum Dis 1987;46:307-13 Whyte GJ, Seymour GJ, Cheung K, Robinson MF. Chemiluminescence of peripheral polymorphonuclear leukocytes from adult periodontitis patients. J Clin Periodontol 1989;16:69-74 Anton PA, Targan SR, Vigna SR, Durham M, Schwabe AD, Shanahan F. Enhanced neutrophil chemiluminescence in familial Mediterranean fever. J Clin Immunol 1988;8:148-56 Hammann KP, Corradini C, Dillmann D, Fischer A, Kleider A, Hopf He. Correlation of the chemiluminescence-activity of peripheral blood monocytes with CSF parameters of inflammation and the clinical course of patients with lymphocytic meningoencephalitis. Acta Neurol Scand 1985;72:198-202 Weiss S1. Tissue destruction by neutrophils. N Engl J Med 1989; 320:365-76 Meylan PR, Markert M, Bille J, Glauser MP. Relationship between neutrophil-mediated oxidative injury during acute experimental pyelonephritis and chronic renal scarring. Infect Immun 1989;57:2196-202 Landry M. Phagocyte function and cell-mediated immunity in systemic lupus erythematosus. Arch Dermatol 1977;113:147-54 Doll NJ, Wilson MR, Salvaggio IE: Inhibition of polymorphonuclear leukocyte chemiluminescence for detection of immune complexes in human sera. J Clin Invest 1980;66:457-464 Magaro M, Altomonte L, Zoli A, Mirone L, DeSole P, DiMario G, Lippa S, Oradei A. Influence of diet with different lipid composition on neutrophil chemiluminescence and disease activity in patients with rheumatoid arthritis. Ann Rheum Dis 1988;47:793-6 Czirjak L, Danko K, Sipka S, Zeher M, Szegedi G. Polymorphonuclear neutrophil function in systemic sclerosis. Ann Rheum Dis 1987; 46:302-6 Kovacs ill, Thomas RHM, Mackay AR, Rustin MHA, Kirby JDT. Increased chemiluminescence of polymorphonuclear leucocytes from
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patients with progressive systemic sclerosis. Clin Sci 1986;70:257-61 69. Bohm D, Meretey K, Sesztak M, Hodinka L, Koo E, Zahumenszky Z. Defekte opsonisationsaktivitat des serums von patienten mit chronischer Arthritis. Z Rheumatol 1988;47:161-5 70. Tertti R. Polymorphonuclear leukocyte chemiluminescence response in HLA-B27 positive and negative arthritis patients. Rheumatol Int 1989;8:279-82 71. Barnard GJR, Kim JB, Williams JL. Chemiluminescence immunoassays and immunochemiluminometric assays. In: Collins WP, ed. Alternative immunoassays. New York: John Wiley & Sons, 1985:123-52 72. Dudley RF. Chemiluminescence immunoassay: an alternative to RIA. Laboratory Medicine 1990;21:216-22 73. Nickless GG, Thorpe GHG, Kricka U, Whitehead TP, Wells U, Ala FA. A rapid chemiluminescent enzyme-linked immunosorbent assay for cytomegalovirus immunoglobulin G antibodies using instant photographic film. J Virol Methods 1985;12:313-21 74. Thorpe GHG, Haggart R, Kricka U, Whitehead TP. Enhanced luminescent enzyme immunoassays for rubella antibody, immunoglobulin E and digoxin. Biochem Biophys Res Commun 1984;119:481-7 75. Pronovost AD, Baumgarten A, Hsiung GD. Sensitive chemiluminescent enzyme-linked immunosorbent assay for quantification of human immunoglobulin G and detection of herpes simplex virus. J Clin Microbiol 1981;13:97-101 76. Hornsleth A, Aaen K, Gundestrup M. Detection of respiratory syncytial virus and rotavirus by enhanced chemiluminescence enzyme-linked immunosorbent assay. J Clin Microbiol 1988;26:630-5 77. Hadley AG, Holburn AM, Bunch C, Chapel H. Anti-granulocyte opsonic activity and autoimmune neutropenia. Br J Haematol 1986; 63:581-9 78. Steele RW, Steele RW. Functional capacity of immunoglobulin G preparations and the F (ab')zsplit product. J Clin MicrobiolI989;27:640-3
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64.
Clinical Applications of Chemiluminescence
Clinical Applications of Chemiluminescence of Granulocytes Russell W. Steele
From the Division of Infectious Diseases/Immunology, Department of Pediatrics, Louisiana State University Medical Center and Children's Hospital, New Orleans, Louisiana
It has been common medical practice for decades to determine a complete blood cell count in the evaluation of a patient with fever or other evidence of systemic infection. The absolute white-cell concentration and granulocyte and bandform counts have all been used in making decisions about patient management, and this approach has been supported by a number of clinical studies [1-3]. Correlations between these parameters and disease imply that granulocytes can reflect in vivo inflammatory processes, but simple measurement of their numbers or maturity is evidently inadequate. Nearly a century ago Bodkin [4] suggested that a detailed cytologic examination of white blood cells in peripheral blood smears would offer useful information in evaluating infective states, and almost 50 years ago Ponder et a1. [5] better quantitated correlations between clinical conditions of patients and morphologicchangesof polymorphonuclearleukocytes (PMNs), such as toxic granulation, ameboid cell outline, vacuolation, and pyknotic nuclei. However, routine application was obviously impractical, since such analysis is extremely timeconsuming and requires a high degree of training and experience. Recent technologic advances in the measurement of oxygenation of leukocytes have reawakened interest in detailed examination of granulocytes as a method for early detection of inflammatory diseases. Changes in these cells appear to precede other acute-phase reactants and to more closely parallel severity of infection. The most promising lab-
Received 17 July 1990; revised 24 October 1990. Reprints and correspondence: Dr. Russell W. Steele, Children's Hospital, 200 Henry Clay Avenue, New Orleans, Louisiana 70118. Reviews of Infectious Diseases 1991;13:918-25 © 1991 by The University of Chicago. All rights reserved. 0162-0886/91/1305-0042$02.00
oratory tool for these analyses is measurement of chemiluminescence, a fairly simple technique. The methods have been reviewed in previous publications [6], but equipment, reagents, newly developed whole-blood techniques, and approaches to the analysis of data continue to be refined. The present communication reviews progress in the clinical application of chemiluminescence as a laboratory assay. Methodology of Chemiluminescence Chemiluminescence was originally developed as a means of examining the intracellular respiratory burst, production of oxygenating radicals, and generation of other agents that function to eliminate microbial pathogens. An early observation was that chemiluminescence was absent following stimulation of PMNs from patients with chronic granulomatous disease [7, 8], thus further confirming a specific defect in the redox metabolism of leukocytes in patients with this inherited disorder. Subsequent investigations focused on chemiluminescence as an indicator of microbicidal activity. Assays have shown that the chemiluminescence response in neutrophils is correlated with bacterial killing in vitro as measured by more standard assays such as bacterial colony counts and superoxide production [9-11]. Additional studies have directly compared chemiluminescence to other measurements of neutrophil function in patients with infection. For example, one report obtained concomitant measurements of chemotaxis, phagocytosis (using uptake of radiolabeled bacteria), and cellular ratios of cyclic guanosine monophosphate to cyclic AMP [12]. Increases in phagocytosis and chemotactic responsiveness during active bacterial infection closely correlated with changes in the chemiluminescence of leukocytes.
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The oxidative metabolic activity of granulocytes can be directly examined by chemiluminescence, a laboratory technique that measures photon emission during well-defined inflammatory or microbicidal events. Numerous studies have utilized chemiluminescence to examine early changes during infectious diseases and other pathologic processes. Studies have suggested that receptors on cell surfaces and oxygenation of granulocytes can reflect the severity of disease as well as provide early diagnostic information. Diseases within virtually every subspecialty of medicine have been studied in this respect, but most investigations have focused on infectious and autoimmune conditions. The present review summarizes current progress in laboratory methods and evaluates the potential application of recently published clinical data. It is apparent that during disease myeloperoxidase- and oxidase-dependent oxygenation activities reflect separate host responses, and independent measurements of these activities will offer a more meaningful understanding of host defense. Immune complexes and other factors in serum may also interact with granulocytes to alter the receptors on cell surfaces and subsequent metabolic activity. In some circumstances, enhanced function of granulocytes may be detrimental to the host.
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Clinical Applications of Chemiluminescence
General Assessment of Activity of PMN In Vivo Circulating granulocytes from healthy donors and granulocytes stimulated with various mediators of inflammation show well-recognized differences [6, 13-16]. For example, fMet-Leu-Phe and C5a desArg increase the total number of C3b receptors by 8- to 10-fold and activate potential metabolic activity of normal circulating PMN s [17], while the simple warming of cells, analogous to development of clinical fever, will increase the number of C3b and fMet-Leu-Phe receptors [18]. Abundant indirect evidence indicates that enhanced oxygenation as well as other cellular changes occur in vivo during infectious diseases and inflammatory conditions [12, 19-25]. One explanation for increased potential activity in circulating neutrophils from infected individuals is the recently observed influence of numerous cytokines. These include colony-stimulating factors, interleukin-l-o and interIeukin-l-d, tumor necrosis factors, and interferons, which most specifically increase phagocytic activity and oxidative metabolism. Data related to such activation of neutrophils by recom-
binant cytokines, both direct and in synergy with other stimulants, were reviewed in this journal recently [26]. In separate recent publications, Ueno [20] and Zgliczynski et al. [21]used luminol-enhanced chemiluminescence of whole blood to evaluate the functional states of phagocytic cells in patients with various diseases. Both studies demonstrated increased activity of granulocytes obtained from those individuals with systemic bacterial infections. With increased severity of disease, some patients exhibited markedly decreased responses, leading Zgliczynski et al. [21] to suggest a profile of four functional states: resting, as seen in normal healthy individuals; standby, as exhibited during early or mild disease; activated, as in individuals with moderate infection; and exhausted, as with severe clinical manifestations. The four functional states could be reproduced by preincubating cells with bacterial components, fMet-Leu-Phe, or the products of activated complement. These studies confirmed earlier reports of increased or "primed" chemotactic, phagocytic, metabolic, and bactericidal responses of granulocytes in infected patients as assessed with various assays. Increased oxidative metabolism, as indicated by direct measurement of the hexose-monophosphate shunt [27] and increased reduction of nitroblue tetrazolium dye [27-29], is well documented. Random motility [12,28-30], response to chemoattractants [12, 28-30], release and activation of lysosomal enzymes [27], carrier-mediated membrane transport of 2-deoxyglucose [31], and neutrophil adherence [32] are likewise increased. However, observed differences were often highly variable, and values were not greatly different from control values. It may be that during disease a mixed population of granulocytes in the peripheral circulation accounts for the relatively high variability. To examine this possibility, Bass et al. [33] employed a quantitative flow cytometry assay of the formation of HzOz-dependent oxidative products and the intracellular oxidation of 2',7'-dichlorofluorescein after stimulation of cells with PMA. Flow cytometry demonstrated that bacteremic patients possess two populations of peripheral blood granulocytes, one with normal oxidative metabolism and another with up to five times more oxidative product than in normal cells. Among infected cases this "primed" population of phagocytes averaged 40 %, with a range of 0-80 %. There were no distinct differences between patients with gram-negative and gram-positive bacteremia. Sera as well as other body fluids from patients with inflammatory diseases may generate chemiluminescence in normal peripheral blood leukocytes. This observation was confirmed for active autoimmune diseases [34] as well as for a wide variety of infectious processes. In a recent report from our laboratories, peak chemiluminescence generated by sera from individuals with bacterial infection was significantly higher than that produced by sera obtained during viral infections or by control samples [35]. These and other studies suggest that there is a direct correlation between severity of infection and chemiluminescence [35, 36]. When sera were examined
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The chemiluminescence response in granulocytes can be magnified with secondary substrates, i.e., chemiluminigenic probes such as luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (10,10'-dimethyl-9,9'-biacridinium dinitrate). These two probes can be used to measure different metabolic events within PMNs. Luminol-enhanced chemiluminescence reflects primarily myeloperoxidase activity, while lucigenin-dependent chemiluminescence is myeloperoxidase independent and measures oxidase-associated oxygenation. Proof for this differentiation is provided by appropriate blocking experiments; azide, which inhibits myeloperoxidase but not oxidases, substantially reduces luminol-enhanced chemiluminescence, while superoxide dismutase alters the superoxide anion and thereby lucigenin-dependent activity. Various PMN stimulants can be selected to separate receptor-related events from those not requiring receptors on the cell surface. Immunoglobulin and complement opsonized particles are phagocytized following contact with Fe and C3b receptors, respectively. Purified components of complement have also been employed. Activity is obviously dependent on the quantity or potential quantity of expressed receptors. In contrast, phorbol myristate acetate (PMA), purified from croton oil, induces redox metabolism, degranulation of PMNs, and measurable chemiluminescence without using receptors or provoking events on the cell surface associated with phagocytosis. Expression of receptors may also be examined indirectly by using extremely low concentrations of stimulants and thereby obtaining rate-limiting kinetics. If such low concentrations of stimulants still produce maximum stimulation, then the total number of receptors or, more correctly, the expression of receptors, is probably low.
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Susceptibility to Infection Chronic granulomatous disease is a metabolic disorder of granulocytes that is characterized by a heterogeneous group of deficiencies that prevent hexose-monophosphate shunt activity and the generation of superoxide and hydroxyl microbicidal radicals. This defect involves neutrophils, macrophages, and eosinophils. In two-thirds of affected individuals, the genetic inheritance pattern is X-linked recessive, while in onethird the pattern is autosomal recessive; occasional autosomal dominant transmission has.. been reported [8]., The bestdescribed specific defect is absence of or decreased levels of cytochrome b558, the heme protein closely linked to the nicotinamide-adenine dinucleotide phosphate oxidase complex, but a number of genetic defects involving constituents of this complex have now been identified and characterized [40]. All forms of this disease can be diagnosed by measuring the respiratory burst, and chemiluminescence provides a quan-
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titative assessment of this activity [7, 8]. Therefore, partial defects and a carrier state can be detected. Opsonized microorganisms are classically used to document disease, but all types ofchronic granulomatous disease may also be tested with stimuli such as PMA that do not require membrane-associated phagocytic events. Patients with AIDS develop a number of defects in host defense, including defects in aspects of the metabolic activity of their PMNs. Compared with healthy controls, AIDS patients in one report showed a 50% reduction of chemiluminescence induced by opsonized zymosan [41]. Some patients with AIDS-related complex as characterized by lymphadenopathy also demonstrated decreased activity of granulocytes. Stohr et al. suggest that measurement of the chemiluminescence of granulocytes is an important parameter in determining both susceptibility to infection and ultimate prognosis in patients with AIDS. In a similar fashion, chemiluminescence has been shown to be useful as a predictive factor in patients with cancer [42]; opsonophagocytosis was assessed by using zymosan opsonized by serum obtained from healthy control subjects or from patients with leukemia and multiple myeloma. The PMN responses of patients were reduced, particularly when autologous sera were used. In addition, the chemiluminescence of control PMNs was significantly inhibited by patient sera. Normal sera completely restored the chemiluminescence of patients with multiple myeloma but only partially corrected the defect in patients with leukemia. Significant correlations were observed between the chemiluminescence of PMNs in patients with cancer and four clinical parameters: disease stage, presence of cancer-related symptoms, subsequent bacterial infections, and long-term outcome. As concluded for patients with AIDS, evaluation of chemiluminescence appeared to be useful in predicting the prognosis for infection and disease. Defects in the myeloperoxidase activity of granulocytes can be evaluated with techniques using chemiluminescence. Patients most frequently present with recurrent superficial staphy lococcal abscesses that often require surgical drainage. In one report, impaired luminol-dependent chemiluminescence was associated with slow release of myeloperoxidase, an unusual congenital abnormality of the degranulation process [43]. Chemiluminescence in monocytes from patients with Shwachman syndrome is also decreased, but in these patients PMN activity is increased [44]. Reasons for differences between these populations of phagocytes have not been delineated, but the increased oxidation with PMNs may account for the well-recognized impaired chemotaxis of purified cells. The susceptibility of newborns to severe infection is well known and likely attributable to an immature immune system that does not function well, particularly during the stress of disease. Although many studies have suggested that fullterm neonates have normal phagocytic and bactericidal activity, those who become infected usually exhibit depressed
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for immune complexes by using a Clq-binding assay, those sera that generated the highest level of chemiluminescence had detectable complexes. Similar correlations were observed in sera from patients with systemic lupus erythematosus (SLE) , thereby leading investigators to conclude that circulating immune complexes cause neutrophil activation [34]. It is also possible that complexes indirectly affect PMNs by activating the complement system and thereby generating biologically active split products such as C5a. Kapp et al. [37] have quantitated the activity of C5a by measuring a specific peak of lucigenin-enhanced chemiluminescence seen within 2 minutes of the addition of test serum. A standard radioimmunoassay of the C5a peptide confirmed the specificity of the chemiluminescence technique. Overnight effluent from patients undergoing chronic ambulatory peritoneal dialysis was examined for opsonic capacity by using luminol-dependent chemiluminescence [38]. Low levels of response were associated with an increased incidence of peritonitis, while no patients with high response levels developed peritonitis during 12 months ofcontinuous monitoring. Opsonic activity was both immunoglobulin and complement dependent. Synovial fluid from patients with rheumatoid arthritis has been shown to have an enhancing influence in assays of chemiluminescence. Preincubation of normal granulocytes with 10% synovial fluid increased spontaneous as well as PMAand zymosan-induced chemiluminescence [39]. Additional studies suggested that the primary mechanism is increased release of myeloperoxidase within granulocytes caused by a factor present in the fluid. This factor may be immune complexes. The ultimate result is detrimental to the host, since oxygen radicals are implicated as mediators of tissue destruction within joints. If the factor is released from the breakdown of tissue, an obvious cycle of continued inflammation would be perpetuated.
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Clinical Applications of Chemiluminescence
Serious Infections Most investigators agree that the chemiluminescence ofhuman PMNs increases during early serious bacterial infection and that such activity returns to normal during the course of successful medical management [12, 19-25]. However, some investigators have also observed that critically ill patients or those whose diagnosis was delayed exhibit impaired neutrophil activity [20, 25, 49]. Studies designed to use chemiluminescence to examine the bactericidal function of neutrophils during serious bacterial infections were first undertaken just 10 years ago. In one of the earliest studies [12], Barbour et al. used opsonized zymosan to stimulate the metabolic activity ofPMNs. Patients had a variety of infections; most common were pneumonia, abscesses, and endocarditis. Mortality in this study group was
30 %. Data clearly showed that chemiluminescence was increased in patients compared with uninfected control subjects (P < .01). This activated state remained with persistent infection but returned to control levels with appropriate therapy. Patients who eventually died were not noted in this report to differ from others, but whether these patients were followed sequentially or analyzed as a separate group is not clear. A number of studies have examined patients with surgical infections, and results have suggested that early activation of PMN function, followed by depression of responses to stimulants, occurs in patients with more-severe disease. Salo et al. [25] quantitated the luminol-enhanced chemiluminescence of isolated granulocytes from patients with postoperative infection, primarily minor wound infections but some cases of sepsis or intraabdominal abscesses. Stimulants of PMN activity included the bacterium isolated from each patient, a standard strain of Escherichia coli, and opsonized zymosan. Responses to all stimulants were significantly reduced in those patients who had a major infection, while responses in those with minor infections were similar to the responses of noninfected control subjects. Stimulation with the autologous bacterium did not yield data more sensitive in delineating severity of infection. Four patients with major infections died, but the chemiluminescence in their cells was no more suppressed than that of eight others who survived equally serious postoperative complications. Two similar reports confirmed these findings for severe disease but observed increased luminoldependent chemiluminescence in patients with mild to moderate infection [20, 49]. Furthermore, one of these publications documented an inverse correlation between the concentration of bacteria in the infective site and the chemiluminescence of peripheral blood neutrophils [49]. In all patients in the study, response to antimicrobial therapy was associated with normalization of chemiluminescence. A possible explanation for immunologic changes following surgical procedures is the effect of anesthesia. Using chemiluminescence, Busoni et al. [50] demonstrated a transient depression in the production of singlet oxygen in PMNs of children undergoing herniorrhaphy that was dependent on the duration of halothane anesthesia. If the entire surgical procedure was accomplished under general anesthesia, changes in chemiluminescence persisted for as long as 24 hours. In a similar fashion the trauma of an operation itself may alter immune function, including chemiluminescence responses to opsonized zymosan and bacteria, for approximately 24 hours [51, 52]; this depression of activity may be prolonged for 3-4 days with more-extensive procedures such as cardiac surgery [53]. However, in the studies of surgical infections discussed above [20, 25, 49], effects of anesthesia and operative trauma were controlled by enrolling only those patients who had undergone operations >14 days prior to the study. In a recent study peripheral blood was obtained from pediatric patients with bacterial and viral meningitis and was examined by a whole-blood microassay system using chemilu-
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activity [45]. Differences are even more apparent in premature and low-birth-weight neonates, but in contrast to responses in full-term newborns, peak chemiluminescence responses of PMN to PMA and zymosan in one study were consistently diminished, even when pretenn infants were not ill [46]. A more-recent publication, however, suggested that depressed function wasnot a universal phenomenon in premature neonates but rather a defect present in a segment of the population; when chemiluminescence was used to assess microbicidal activity, granulocytes from infected and stressed pretenn infants but from only a small minority of stable low-birth-weight neonates demonstrated defective PMN oxidative metabolic responses [47]. These abnormalities were often present prior to recognition of any clinical illness, and in most patients the chemiluminescence of PMNs remained depressed following episodes of serious infection. One conclusion by Driscoll et al. was that pretenn neonates have deficient intracellular activity of myeloperoxidase in all or a portion of their circulating PMNs. Patients without specifically defined immune deficiency syndromes but with a predisposition to infection have also been studied for possible abnormalities in the oxygenation of PMN. Results in some reports have suggested that subtle defects in phagocytosis and oxidative metabolism of PMNs may be major determinants of susceptibility to infection. Bondestam et al. evaluated multiple functional properties of PMNs in a group of Swedish children who had experienced severe multifocal infections [48]. Parameters included random migration, chemotaxis, chemoattractant activity of serum, phagocytosis, and production of chemiluminescence. Most of these patients had at least one defect in PMN function that appeared to be inherited and was a primary cause of increased susceptibility to infection. It is quite possible, however, that defects were seen because children were infected at the time of examination. Because the design of the study did not provide longitudinal analysis of these pediatric patients, the latter interpretation cannot be excluded.
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Minor Infections Animal studies have best documented the effect of mild to moderate infectious diseases on endogenous chemiluminescence activity in host granulocytes [56]. Twelve hours after a live vaccine strain of Francisella tularensis was injected into rodent models, enhanced chemiluminescence in PMNs occurred concomitantly with the onset of fever. It is interesting that immune animals challenged with microorganisms also exhibited a level of response higher than those of control subjects but significantly lower than those of nonimmune animals. Viral infection in animal models [57] as well as in humans does not enhance responses and often depresses the oxidation of PMNs. A number of published clinical studies have examined the chemiluminescence of PMNs from patients with various nonlife-threatening infections. Essentially all reports noted that responses were enhanced compared with those of control subjects [12,24,25,58-60]. Diagnoses have included some moreunusual infectious diseases whose pathogenesis is poorly un-
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derstood, such as yersinia arthritis [58], periodontitis [59], and familial Mediterranean fever [60]. An additional study of patients with lymphocytic meningoencephalitis found no alteration in the oxygenation of PMNs but rather a fourfold enhancement of the chemiluminescence of peripheral blood monocytes [61]. This enhancement persisted in some patients for as long as 3 weeks. The magnitude of change correlated with the cell count and level of protein in CSF but not with the IgG content. The association of the clinical courses of individual patients with enhanced chemiluminescence of peripheral blood monocytes was highly significant, as higher levels of chemiluminescence predicted rapid recovery. The increased oxidative metabolism seen in peripheral blood neutrophils probably occurs to a greater extent in cells present at the site of infection. Here, activated granulocytes produce oxidant damage and other tissue-destructive events during their attempt to eradicate invading pathogens [62]. In a rat model of pyelonephritis, Meylan et al. [63] used lucigeninenhanced chemiluminescence of activated PMNs to show that dapsone could inhibit myeloperoxidase-mediated reactions even though it had no direct effect on killing of bacteria. This finding correlated with a 65 % reduction in renal scars, although there was no alteration in bacterial counts, inflammatory swelling, or infiltration of PMNs during acute pyelonephritis. Chemiluminescence could thus provide a tool for evaluating the therapeutic management of inflammatory events during this common infection.
Autoimmune Disease It is well documented that autoimmune diseases are risk factors for recurrent and severe infection, even when patients with these diseases are not receiving immunosuppressive chemotherapy. This predisposition may be secondary to alteration in immune function attributable to the disease process itself. Early studies suggested that granulocyte phagocytic and killing functions were most affected by these rheumatic diseases [64]. However, subsequent investigations yielded various results for alterations in these aspects of host defense, and differences have been in large part dependent on the methods employed for assessment, particularly inclusion of autologous sera. It is now clear that immune complexes can either augment or inhibit phagocytosis, and the concentration of immune complexes may vary with the activity of the disease. According to measurements of luminol-enhanced chemiluminescence of normal PMNs, sera of patients with active SLE stimulated the oxygenation of granulocytes, and the chemiluminescence responses directly correlated with disease activity [34]. In contrast, sera from patients with SLE in remission yielded results similar to those of control subjects. Stimulatory capacity was concentration dependent and was augmented by the addition of normal serum complement. These observations suggest activation of PMNs by immune complexes. It
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minescence [54]. White blood cells were stimulated with complement-opsonized zymosan, C5a, tMet-Leu-Phe, and PMA. The chemiluminigenic probes luminol and lucigenin were used to measure myeloperoxidase- and oxidase-associated oxygenation, respectively. Integral and peak.chemiluminescence response adjusted for the total number of granulocytes in the sample were calculated. Integral responses for bacterial versus viral meningitis were significantly higher when granulocytes were stimulated with complement-opsonized zymosan or the phorbol ester. Control values were similar to those for aseptic meningitis. When opsonized zymosan was used as a standard stimulus and a combination of opsonized zymosan and C5a was used to elicit a maximum granulocyte response, a percent reserve could be calculated. Compared with patients with viral meningitis, patients with bacterial infection had a markedly decreased functional reserve of granulocytes, and there was no overlap between these two groups of patients. Children who ultimately died demonstrated rapidly decreasing responses. Determination of the reserve of granulocytes in this study was a very sensitive method for differentiating bacterial from aseptic meningitis and determining the prognosis for patients with clinically more-severe bacterial infection. Phagocytic oxygenation activity has been carefully examined in bum patients and monitored during their prolonged hospital courses [55]. Alterations in chemiluminescence consistently anticipated changes in clinical condition in that sepsis was associated with a marked decrease in chemiluminescence. Similar to studies of postoperative surgical infections [20, 25, 49], these studies demonstrate the clinical utility of determining the chemiluminescence of granulocytes as a method for monitoring patient populations who are at risk of developing sepsis during a defined period.
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Clinical Applications of Chemiluminescence
is therefore likely that in vivo exposure of granulocytes to immune complexes during relapse of disease decreases subsequent functional capacity, as measured in other reports [65]. Chemiluminescence of peripheral blood neutrophils is usually increased in patients with active rheumatic autoimmune disorders, including rheumatoid arthritis [66], SLE [67], and progressive systemic sclerosis [67, 68], but not always in patients with ankylosing spondylitis or psoriatic arthritis [68]. Changes are not related simply to the presence or absence of the B27 antigen, as arthritic patients all show equal changes during relapse regardless of this histocompatibility marker [69, 70].
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ness of the split products ofIgG and suggested wide variations of specific antibody among these products. In summary, the measurement of chemiluminescence has potential application for clinical medicine. Luminometers for measuring chemiluminescence have now been vastly improved, and optimal reagent materials are readily available. What remains is for assays to be optimized and standardized so that additional clinical studies can better define parameters that are sensitive and specific enough to direct decisions regarding diagnosis and patient management. Such investigations are currently in progress. References
Chemiluminigenic probes have also been employed in highly sensitive laboratory assays for detecting microbial antigens and antibodies to potential pathogens [71, 72]. In these assays chemiluminescence is induced following the interaction of luminol and hydrogen peroxide with an enzyme such as horseradish peroxidase conjugated to antigen or antibodies. This produces a chemiluminescence-enhanced ELISA. The resulting peroxidase-catalyzed oxidation of luminol can be detected by luminometers, which directly measure light emission, or can be recorded on photographic film. This method has been used to measure antibody to cytomegalovirus [73], rubella virus [74], and herpes simplex virus [74] and to detect the presence of viral antigens for herpes simplex virus [75], respiratory syncytial virus [76], and rotavirus [76]. Detection limits have been as low as 0.01 ng in these assays, which contrasts with 1.0 ng in other ELISAs for determining microbial antigen protein. In similar investigations for which immunoassays of chemiluminescence were used to detect hormones in plasma or body secretions, protein concentrations as low as 1-2 pg could be measured. An interesting diagnostic application of chemiluminescence is detection of neutrophil antibodies [77]. In this assay, the chemiluminescence of peripheral blood monocytes is induced with antibody-coated granulocytes, a technique more rapid and more sensitive than any currently available that use indirect immunofluorescence. With chemiluminescence, IgG antibodies can be differentiated from IgM autoimmune mechanisms by including simple blocking experiments. These data may be relevant for determining which patients might benefit from intravenous immunoglobulin therapy, since those with IgG antibodies are more likely to respond. Chemiluminescence is ideal for determining the functional capacity of antibody, since results have been shown to correlate with clinical protection. We recently published the results of studies that used chemiluminescence to compare the opsonizing activity of five commercially available intravenous immunoglobulin preparations for important microbial pathogens [78]. The data did not support potential clinical useful-
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Chemiluminescence as a Laboratory Method for Detecting Antigen and Antibody
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64.
Clinical Applications of Chemiluminescence
