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April 2005

Editors:
Michael Bissell, MD, PhD, MPH, Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus
Ronald Domen, MD, Professor of Pathology, Medicine, and Humanities, Penn State University College of Medicine, Hershey, Pennsylvania

Assessing immune function in renal transplant patients
Platelet integrity in volume-reduced platelet concentrates
Predictive risk of malignancy in iron deficiency anemia
Use of immunoblot to detect Streptococcus pneumoniae
NT-proBNP reference ranges
Tests for diagnosing variegate porphyria
Automated and manual schistocyte counting
Cytomegalovirus and graft-versus-host disease

Assessing immune function in renal transplant patients

Despite advances in renal allograft survival with current immunosuppressive therapies, including cyclosporine A and mycophenolate mofetil, the risks of major immunosuppressive complications, such as malignancy and infection, persist. Longer graft survival involves greater exposure to immunosuppression and a potential increase in these risks. A reproducible laboratory assessment of the extent of immunosuppression that predicted the risks would improve prevention and minimize immunosuppressive therapy. The effect of immunosuppressive drugs on transplant patients has been shown to have inhibitory effects on the humoral and cellular arms of the immune system. Correlations have also been noted between the immune defect measured and the incidence of immunosuppressive complications. However, it has been difficult to quantify the level of immunosuppression on an individual basis, perhaps because in most studies, only one parameter of immune function was assessed. The current study attempted to quantify the extent of immunosuppression by the simultaneous evaluation of several laboratory measures of immune function. These included assessment of neutrophil phagocytosis and reactive oxygen species generation, enumeration of lymphocyte subsets, T-cell mitogen responses, and levels of circulating immunoglobulins. A derived score of immune competence was developed from these tests. Patients with the worst score had increased incidence and severity of infection. The authors assessed the immune status of 152 transplant recipients (138 renal and 14 pancreas/renal) by measuring lymphocyte subsets, mitogen-induced T-cell proliferative responses, neutrophil phagocytic capacity, and reactive oxygen species generation. They devised a scoring system based on the average number of these parameters below the 10th percentile of normal. They found that the most common abnormality was B-cell lymphopenia (85 percent), followed by reduced neutrophil reactive oxygen species production (63 percent), NK cell lymphopenia (50 percent), lymphocyte mitogen response (49 percent), and CD4 number (23 percent). The abnormalities were unrelated to the duration of immunosuppression (up to 15 years) and variable combinations of cyclosporine A, azathioprine, prednisolone, and mycophenolate mofetil (MMF), except for a consistent reduction in lymphocyte mitogen response in MMF-treated patients. Retrospective comparison of infective episodes showed a significantly greater index of infections in patients with the worst score compared with a normal score. The authors concluded that this quantification of immune function may allow assessment of the level of host immune defense reflecting the level of drug-induced immunosuppression and thus risks of immunosuppressive complications.

Hutchinson P, Chadban SJ, Atkins RC, et al. Laboratory assessment of immune function in renal transplant patients. Nephrol Dial Transplant. 2003;18:983-989.

Reprints: Paul Hutchinson, Dept. of Clinical Immunology, Monash Medical Centre, 246 Clayton Rd., Clayton, VIC 3168, Australia; paul.hutchinson@med.monash.edu.au

Platelet integrity in volume-reduced platelet concentrates

The volume of plasma in platelet concentrates may need to be reduced prior to transfusion, particularly in pediatric patients. This is particularly true for low-birth-weight infants and infants undergoing open-heart surgery with extracorporeal circulation. Few studies have examined the effect of plasma reduction on platelet function. The authors of this study examined platelet activation in apheresis platelets. They studied 20 apheresis platelets from healthy donors. The donors did not ingest antiplatelet drugs for 10 days prior to donation. The volume of the collected platelets was 220 to 250 mL, and all concentrates were leukocyte-reduced by filtration prior to storage at 22° with constant agitation. Platelets were volume-reduced by centrifugation (four minutes at 3,500 g and 22°). Plasma was removed to achieve a residual volume of 90 mL. In vitro studies were performed before and after plasma depletion. No difference in mean platelet volume was noted before and after plasma depletion. There was a significant loss in adenosine diphosphate (ADP)-induced aggregability in the volume-reduced platelets (P=0.03). Collagen-induced aggregation was not significantly altered. As evaluated by flow cytometry, there was a significant decrease in ADP-induced spontaneous activation in the volume-reduced platelets (PN=0.001). Collagen induction was not significantly different. The authors concluded that plasma depletion is an activator of platelet function but that little is known about the efficacy of transfused, volume-reduced platelets.

Schoenfeld H, Muhm M, Doepfmer UR, et al. The functional integrity of platelets in volume-reduced platelet concentrates. Anesth Analg. 2005;100:78-81.

Correspondence: Dr. Helge Schoenfeld, Dept. of Anesthesiology and Intensive Care Medicine, Charite, University of Medicine Berlin, Campus Charite Mitte, Humboldt-University Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany; helge.schoenfeld@charite.de

Predictive risk of malignancy in iron deficiency anemia

Iron deficiency is the most common cause of anemia, and chronic blood loss is a common cause of iron deficiency anemia. Occult bleeding from gastrointestinal malignancy is a cause of iron deficiency anemia (IDA) in approximately 10 percent to 17 percent of cases. In clinical practice, however, the cause of IDA is often under-investigated. The authors of this study prospectively studied the cause of IDA, the prevalence of gastrointestinal malignancy in this group of patients, and the predictive risk factors for malignancy in IDA patients. Patients with IDA and profound menstruation were excluded from the study. A total of 148 patients were studied (88 male, 60 female) who were a mean age of 66.2 years (range, 14 to 96 years) and had a mean hemoglobin at enrollment of 83 g/L. Four groups of causes for IDA were identified: Malignant tumors were found in 18 patients (12.2 percent), who were labeled as group A; benign tumors were found in 10 patients (6.8 percent), who were labeled as group B; benign causes such as peptic ulcer, gastritis, polyps, and hemorrhoids were found in 96 patients (64.8 percent), who were labeled as group C; and no detectable cause was found in 24 patients (16.2 percent), who were labeled as group D. Only hemoglobin and lactate dehydrogenase (LDH) levels were found to differ significantly between the four groups. Hemoglobin was significantly lower and LDH significantly higher in the malignant group than in the nonmalignant groups. Univariate analysis found that IDA patients ran a higher risk of malignancy if they were older than 60 years and had serum ferritin of 10 µg/L or less, hemoglobin of 80 g/L or less, or LDH greater than 250 U/L. By multivariate logistic regression, a hemoglobin of 80 g/L or less lost its significance for malignancy. The combination of a serum ferritin of 10 µg/L or less and an LDH greater than 250 U/L was associated with a 74.33 times greater chance for malignancy. Clinical symptoms and the presence of fecal occult blood did not appear to be significant variables in predicting malignancy. The authors concluded that an elderly patient with a high LDH level and a low serum ferritin level is at high risk for a gastrointestinal malignancy and should be evaluated fully.

Ho CH, Chau WK, Hsu HC, et al. Predictive risk factors and prevalence of malignancy in patients with iron deficiency anemia in Taiwan. Am J Hematol. 2005;78:108-112.

Correspondence: Dr. Chao-Hung Ho, Division of Hematology, Taipei Veterans General Hospital, Taipei, Taiwan, ROC; chho@vghtpe.gov.tw

Use of immunoblot to detect Streptococcus pneumoniae

Pneumococcal infections involve colonization early in childhood, often by two months of age, and may lead to bacteremia, pneumonia, meningitis, and repeated otitis media episodes. A new pneumococcal heptavalent protein conjugate vaccine (Prevnar, Wyeth-Lederle Vaccines), licensed in the United States in February 2000 for use in children up to nine years of age, has been shown to be efficacious against invasive pneumococcal disease, pneumonia, otitis media, and nasopharyngeal (NP) colonization. Children are serially and simultaneously colonized in the nasopharynx by various S. pneumoniae serotypes. Although a single serotype usually predominates at any given time, there is ample evidence for the carriage of multiple serotypes. The authors sought to develop a method that could detect the carriage of multiple serotypes and that would be highly sensitive, less labor-intensive than the stereotyping of multiple colonies, and reproducible. The intent of developing such a method was specifically for use in a study of the effect of a conjugate pneumococcal vaccine on NP carriage. The authors developed an immunoblot method with monoclonal antibodies (MAbs) to detect S. pneumoniae and to identify serotypes. NP specimens stored in skim milk-tryptone-glucose-glycerol medium were assessed by the immunoblot method and the reference culture method. In the latter method, four optochin-sensitive alpha-hemolytic colonies resembling pneumococci were typed by the Quellung reaction. In the immunoblot method, a nitrocellulose membrane blot of surface growth was reacted with a pneumococcal surface adhesion (PsaA) Mab and visualized. Of 47 NP specimens, 32 (68 percent) were found to be positive and 13 (28 percent) were found to be negative for pneumococci by both methods; each method alone yielded one positive result. The sensitivity and specificity of the immunoblot method for detecting pneumococci were 97 percent and 93 percent, respectively. To identify serotypes, blots were tested with serotype-specific MAbs (4, 6A, 6B, 9V, 14, 18C, 19F, and 23F). To detect the remaining serotypes, positive serotype-specific replicate blots were compared visually to an original anti-PsaA-positive blot; four unidentified colonies were subcultured and serotyped by the Quellung reaction. Fifty-eight S. pneumoniae-positive NP specimens containing 69 pneumococcal strains (23 serotypes) were tested; 68 (98.6 percent) of the strains were detected by the immunoblot method and 66 (95.6 percent) were detected by the reference culture method. For 11 specimens found to contain two serotypes, both methods detected both serotypes in seven (63.6 percent); the immunoblot method alone detected the two serotypes in three (27.3 percent); and the reference culture method alone detected both serotypes in one (nine percent). The immunoblot method identified multiple clones and minor populations of pneumococci in NP secretions. The authors concluded that this method is useful for detecting specific serotypes and carriage of multiple serotypes in epidemiologic surveillance and carriage studies.

Bronsdon MA, O'Brien KL, Facklam RR, et al. Immunoblot method to detect Streptococcus pneumoniae and identify multiple serotypes from nasopharyngeal secretions. J Clin Microbiol. 2004;42:1596-1600.

Reprints: Katherine L. O'Brien, Center for American Indian Health, Johns Hopkins Bloomberg School of Public Health, 621 N. Washington St., Baltimore, MD 21205; klobrien@jhsph.edu

NT-proBNP reference ranges

Natriuretic peptides are increasingly gaining recognition as diagnostic markers in heart failure. Brain natriuretic peptide (BNP) and its amino terminal portion, N-terminal pro brain natriuretic peptide (NT-proBNP), which may have advantages because of its greater stability, have proved especially promising. Concentrations of BNP and NT-proBNP are related to left ventricular filling pressure and wall stress. All previous studies of the diagnostic value of BNP in heart failure, whether focusing solely on left ventricular systolic dysfunction or on a clinical diagnosis of heart failure, have consistently reported a very high negative predictive value for BNP (0.98-1.00), while the positive predictive value has been lower (0.16-0.42). The high negative predictive value makes BNP appear well suited for population screening; however, the low positive predictive value may pose a problem in any population screening or diagnostic setting because of the large number of false-positive results that are likely to be found. The authors of this population-based study attempted to identify potentially confounding variables for interpreting the plasma concentration of NT-proBNP, which might improve the diagnostic performance of the marker. Randomly selected subjects completed a heart failure questionnaire and underwent pulse and blood pressure measurements, electrocardiogram, echocardiography, and blood sampling. Subjects were recruited from four Copenhagen general practices located in the same urban area and were examined in a Copenhagen University hospital. They included 382 women and 290 men in four age groups: 50 to 59 years (n=174); 60 to 69 years (n=204); 70 to 79 years (n=174); and 80 years and older (n=120). The main outcome measures were associations between the plasma concentration of NT-proBNP and a range of clinical variables. In the undivided study sample, female gender (P<0.0001), greater age (P<0.0001), increasing dyspnoea (P=0.0001), diabetes mellitus (P=0.01), valvar heart disease (P=0.002), low heart rate (P<0.0001), left ventricular ejection fraction of less than or equal to 45 percent (P<0.0001), abnormal ECG (P<0.0001), high log10 [plasma creatinine] (P=0.0009), low log10 [plasma glycosylated hemoglobin A1c] (P=0.0004), and high log10 [urine albumin] (P<0.0001) were independently associated with a high plasma log10 [plasma NT-proBNP] by multiple linear regression analysis. The authors concluded that a single reference interval for the normal value of NT-proBNP is unlikely to suffice. There are several confounders for interpreting a given NT-proBNP concentration and, at the very least, adjustment should be made for the independent effects of age and gender.

Raymond I, Groenning BA, Hildebrandt PR, et al. The influence of age, sex and other variables on the plasma level of N-terminal pro brain natriuretic peptide in a large sample of the general population. Heart. 2003;89:745-751.

Reprints: Dr. Bjoern A. Groenning, Dept. of Cardiology and Endocrinology, Copenhagen University Hospital Frederiksberg, 57 Nordre Fasanvej, DK-2000 Frederiksberg, Denmark; bjoerng@dadlnet.dk

Tests for diagnosing variegate porphyria

Variegate porphyria is an autosomal dominant disorder associated with deficiency of the enzyme protoporphyrinogen oxidase, the penultimate enzyme of the hemesynthetic pathway. Protoporphyrinogenoxidase (PPOX) activity is decreased by about half in patients with variegate porphyria (VP), how ever, penetrance is low. Clinically, patients with VP may manifest a photodermatitis characterized by skin fragility, erosions, blisters, milia, and chronic pigmentary changes in sun-exposed areas, particularly the backs of the hands. These individuals are also at risk for acute attacks of porphyria characterized by episodes of severe abdominal pain, autonomic disturbance, and a motor neuropathy that may progress to flaccid quadriparesis and death. Until recently, a gold standard for diagnosis based on the underlying genetic abnormality has been lacking, so the sensitivity and specificity of the stool porphyrin analysis in use have not been known precisely. Plasma fluorescence scanning has been suggested as a more robust test than fecal porphyrin analysis for the diagnosis of VP. Identification of the cDNA for PPOX and recognition that the founder R59W mutation accounts for 95 percent of all South African patients with VP has enabled the assignment of an accurate genotypic diagnosis in patients and has, in turn, allowed the accurate determination of sensitivity and specificity of biochemical population testing. The authors evaluated all patients in their laboratory for whom the genotype and a plasma scan or fecal porphyrin result were available. Mutations were detected by restriction digest analysis. Plasma fluorescence scanning was conducted according to published methods. Fecal porphyrins were identified and quantified by thin-layer chromatography. Plasma fluorescence scanning was assessed in 679 patients (205 with VP who were carriers of a PPOX mutation, either with disease symptoms or asymptomatic) and fecal analysis in 473 (190 with VP). The sensitivity and specificity of both tests were higher in adults than in children and higher for adults with disease symptoms than for asymptomatic carriers. In a direct comparison in 168 adults (73 with VP), plasma scanning was significantly more sensitive than fecal porphyrin analysis [sensitivity, 0.96 (95 percent confidence interval, 0.89-0.99) versus 0.77 (0.66-0.85)]. Fecal coproporphyrin [area under the curve, 0.87 (0.83-0.90)] was a better predictor of VP than protoporphyrin [0.80 (0.76-0.84)]. The authors concluded that plasma scanning is a more sensitive and specific test for VP than fecal porphyrin analysis. Neither test is sensitive in children, and both are less sensitive in asymptomatic carriers than in symptomatic cases. DNA analysis, therefore, remains the preferred method for identifying carriers, particularly among children.

Hift RJ, Davidson BP, van der Hooft C, et al. Plasma fluorescence scanning and fecal porphyrin analysis for the diagnosis of variegate porphyria: precise determination of sensitivity and specificity with detection of protoporphyrinogen oxidase mutations as a reference standard. Clin Chem. 2004;400:915-923

Reprints: Richard J. Hift, Dept. of Medicine, Medical School, Anzio Rd., Observatory, South Africa 7925; rjh@liver.uct.ac.za

Automated and manual schistocyte counting

Schistocytes are circulating red blood cell fragments. Their presence in the blood is considered abnormal and indicates the possibility of a thrombotic microangiopathy (TMA), in particular, one of the two major syndromes of thrombocytopenic purpura (TTP) or hemolytic uremic syndrome (HUS), with an unfavorable prognosis if untreated. The detection and quantification of schistocytes is, consequently, an urgent test with great diagnostic and therapeutic importance. Conversely, the counting of schistocytes often needs to be done on an iterative basis in the followup for medullary transplants. (TMA is a vital risk if it occurs during a reaction of a transplant against the host.) Finally, the presence of schistocytes in heart valve recipients is evidence of valve dysfunction. Yet the identification of schistocytes remains difficult because the shapes to which they correspond are still under discussion. A consensus report was prepared by the Groupe Français d'Hématologie Cellulaire, or French Group of Cellular Hematology. Automated hematology systems permit one to obtain precise cellular counts on an urgent basis. The Advia 120 analyzer (Bayer Health Care, Tarrytown, NY) is the first to offer the possibility of direct measurement of abnormal RBCs and RBC fragments. The authors compared schistocyte counts performed by different biologists and technicians with the automated counts by the Advia 120. Their objective was to compare the results obtained by the analyzer (RBC fragments) with those obtained by the usual microscopic count using stained blood smears. The authors then compared automated schistocyte detection with the presence or absence of thrombotic disease. Because a link has been found between the severity of thrombocytopenia and TMA prognosis, the authors also studied the relationship between the percentage of schistocytes and the number of platelets. Their study validated the automated count provided by the analyzer. The agreement between the Advia 120 and the average of the observers gave a correlation coefficient of 0.72741 (95 percent confidence interval, 0.6285-0.8019). The Advia 120 had a tendency to overestimate the count (average, +0.445 percent). No false-negative case was recorded. The maximum sensitivity (detection of 100 percent of samples with schistocytes) of the analyzer was determined at a threshold value of 0.25 percent, but the specificity was 20 percent. Therefore, a blood smear evaluation remains necessary to confirm the presence of schistocytes. However, the clinical features correlated particularly with negative automated RBC fragments, and the high negative predictive value of RBC fragments ruled out thrombotic events (macroangiopathies or microangiopathies).

Lesesve J-F, Salignac S, Alla F, et al. Comparative evaluation of schistocyte counting by an automated method and by microscopic determination. Am J Clin Pathol. 2004;121:739-745.

Reprints: Dr. Jean-François Lesesve, Service d’Hématologie Biologique, CHU Nancy-Brabois, 54 511-Vandoeuvre, France

Cytomegalovirus and graft-versus-host disease

Cytomegalovirus is a major cause of morbidity and mortality after allogeneic stem-cell transplantation, but its role as a risk factor for chronic graft-versus-host disease has been controversial. Cytomegalovirus (CMV) seropositivity in the donor and seropositivity in the donor and recipient have been correlated with chronic graft-versus-host disease (GvHD), but other studies have not been able to confirm that association. One possible explanation for the differing results is that varying transplant procedures could be more important as risk factors for chronic GvHD than CMV. In this single-center study, the authors chose a study group that was as homogenous as possible. They evaluated factors that could be associated with the development of extensive chronic GvHD in patients receiving transplants with allogeneic hematopoietic stem cells from human leukocyte antigen-identical sibling donors at their hospital between 1984 and 2000. The aim of the study was to investigate the effects of polymerase chain reaction-based monitoring for CMV-DNA with preemptive antiviral therapy on demand on the development of extensive chronic GvHD. The authors retrospectively analyzed 262 consecutive patients undergoing conventional allogeneic stem-cell transplantation with human leukocyte antigen-identical sibling donors and who were given cyclosporine A and methotrexate as GvHD prophylaxis and survived more than three months after stem-cell transplantation. Most patients received transplants because of a hematologic malignancy (n=226), but 36 patients with nonmalignant disorders were included in the analysis. Ninety-nine patients were monitored for CMV infection with rapid virus isolation and 163 patients by either a pp65 antigenemia test (n=5) or a qualitative polymerase chain reaction assay for CMV-DNA (n=158). A total of 130 (50 percent) patients developed chronic GvHD, of whom 17 (6.5 percent) developed extensive chronic GvHD. Risk factors for developing extensive chronic GvHD were determined by multivariate logistic regression. The strategy of polymerase chain reaction-based monitoring for CMV-DNA, giving preemptive antiviral therapy on demand, significantly decreased the risk for developing extensive chronic GvHD (odds ratio=0.32, P=0.03). No other factors tested, including recipient and donor age and gender, source of graft, cell dose, and acute GvHD, had a significant effect on the development of extensive chronic GvHD. The authors concluded that the risk for extensive chronic GvHD in this homogenous group of patients was reduced by the use of polymerase chain reaction-based preemptive therapy.

Larsson K, Aschan J, Remberger M, et al. Reduced risk for extensive chronic graft-versus-host disease in patients receiving transplants with human leukocyte antigen-identical sibling donors given polymerase chain reaction-based preemptive therapy against cytomegalovirus. Transplantation. 2004;77: 526-531.

Reprints: Kajsa Larsson, Dept. of Hematology, Huddinge University Hospital, SE-14186 Stockholm, Sweden; kajsa.larsson@medhs.ki.se