College of American Pathologists
Printable Version

  Clinical Abstracts





cap today



August 2006

Michael Bissell, MD, PhD, MPH

Latex immunoturbidimetric assay for fibrin monomer
Role of antineutrophil cytoplasmic antibodies
Rapid urease tests for Helicobacter pylori infection
International variations in anemia management
Genetics of the Ko phenotype
Evaluation of the Pentra 80 hematology analyzer
Vitamin B12 and folate intake and cognitive decline in elderly patients
Chlamydia pneumoniae in coronary artery disease

bullet Latex immunoturbidimetric assay for fibrin monomer

Fibrin monomer and its derivatives in blood, produced by the cleavage of one or both A peptides and both A and B peptides from fibrinogen by thrombin, are found in early stage thrombosis. When produced in blood, they form complexes with fibrinogen and exist as soluble complexes called soluble fibrin complex (SFC). Because increased SFC concentrations in plasma indicate that thrombin has converted fibrinogen to fibrin, increased SFC is considered to be a molecular marker of imminent thrombotic events. Other hemostatic molecular markers, such as D-dimer and thrombin-antithrombin (TAT) complexes, have been developed for diagnosing disseminated intravascular coagulation (DIC). These markers are very sensitive but not specific for the diagnosis of DIC. D-dimer reflects the combined effect of coagulation and fibrinolysis. Similarly, TAT complexes may reflect thrombin generation but not thrombin activity in vivo. Several methods have been described for the specific detection of SFC using monoclonal antibodies (MoAbs) to fibrin monomer or SFC. Sheefers-Borchel, et al, first reported a MoAb to the newly exposed NH2 terminus of the fibrin a-chain, obtained by using α-chain N-terminal peptides as the immunogen, and established an enzyme-linked immunosorbent assay method using that MoAb. Later, Dempfle, et al, also established a MoAb to fibrin monomer in the same manner and developed an ELISA method for detecting fibrin monomer in human plasma, which involved pretreating the samples with sodium thiocyanate (NaSCN). However, NaSCN pretreatment, used to expose the SFC sites recognized by the antibody, also exposed sites of fibrin degradation products, such as cross-linked fibrin degradation products. The authors of this study obtained a monoclonal antibody that specifically reacts with SFC, with desAA-fibrin as the immunogen, and developed a rapid and sensitive latex immunoturbidimetric assay using latex-immobilized anti-SFC monoclonal antibody. The assay system was based on the increase in turbidity induced by the reaction of the latex-immobilized anti-SFC monoclonal antibody with SFC in plasma, and the assay procedure was fully automated on a Hitachi 911 analyzer. The authors found that the method had an analytical range of 3 to 300 mg/L. Intra- and interassay precision studies indicated that this system provided reproducible data (coefficients of variation, less than three percent and less than two percent, respectively). The assay detection limit was less than 0.5 mg/L. There was no interference from bilirubin (up to 440 mg/L), hemoglobin (up to 9.6 g/L), Intralipid (up to 10 percent), D-dimer (up to 200 mg/L), and rheumatoid factor (up to 470,000 IU/L). SFC concentrations in plasma from patients with thrombotic diseases (mean [SD], 48.9 [57.6] mg/L; n=160) were significantly higher than those in plasma from healthy individuals (1.8 [2.1] mg/L; P<0.001; n=304). The authors concluded that in terms of linearity, precision, and sensitivity, the latex immunoturbidimetric assay, performed on a Hitachi 911 automated analyzer, may be useful for measuring SFC in plasma.

Hamano A, Umeda M, Ueno Y, et al. Latex immunoturbidimetric assay for soluble fibrin complex. Clin Chem. 2005;51:183–188.

Reprints: Mamoru Umeda, Central Research Laboratory of Nissui Pharmaceutical Co. Ltd., 1075-2, Hokunanmoro, Yuuki-shi, Ibaraki, 307-0036, Japan;

bullet Role of antineutrophil cytoplasmic antibodies

Antineutrophil cytoplasmic antibodies are autoantibodies that are directed against different neutrophil antigens. When detected by immunofluorescence on ethanol-fixed human neutrophils, antineutrophil cytoplasmic antibodies (ANCA) usually display three major patterns: cytoplasmic (cANCA), perinuclear (pANCA), and atypical (xANCA). The main targets of ANCA are either myeloperoxydase (MPO) or proteinase 3 (PR3), but ANCA may also be directed against lactoferrin, elastase, cathepsin, lysozyme, bactericidal permeability increasing protein (BPI), and azurocidin. ANCA are observed in a large spectrum of diseases. Their specificity can make them a helpful tool in diagnosing primary systemic vasculitides. Anti-PR3 antibodies (Abs) are strongly associated with Wegener’s disease and, to a lesser extent, with microscopic polyarteritis and necrotizing crescentic glomerulonephritis. Anti-MPO antibodies are often associated with systemic vasculitis but can also be found in various autoimmune disorders and connective tissue diseases without evidence of vasculitis. Other specificity is not considered to be a helpful serological marker of any diseases. The authors studied nine patients with chronic unexplained neutropenia and ANCA. They extensively analyzed clinical charts, and all patients underwent hematological and immunological investigations. All patients (six women and three men) were Caucasian and were a mean age of 49 years (range, 16–67 years). All presented with a neutropenia below 1.5 X 109/L for more than six months. The neutropenia was less than 0.5 X 109/L in six cases and moderate in three. There was no evidence of toxic- or drug-related neutropenia or of a hematological malignancy. Autoimmune anemia or thrombocytopenia, or both, were present in five patients. ANCA, with various specificities, were present in all patients. ANCA were associated with various other autoantibodies in eight patients, including antisurface-neutrophil antibodies in three cases. Four of the six patients with severe neutropenia experienced infections. Five patients were treated with hematopoietic growth factors, steroids, intravenous immunoglobulins, splenectomy, methotrexate, or cyclophosphamide, allowing the neutrophil count to be restored transiently or permanently. The authors concluded that a subset of patients with neutropenia of possible autoimmune origin may develop ANCA. Their detection would provide strong evidence of an autoimmune mechanism. Neutropenia should be added to the list of ANCA-associated diseases.

Coppo P, Ghez D, Fuentes V, et al. Antineutrophil cytoplasmic antibody-associated neutropenia. Eur J Intern Med. 2004;15:451–459.

Reprints: K. Lassoued, Service d’Immunologie, Faculté de Medecine, 3 rue des Louvels, F-80036 Amiens Cedex 1, France; kaiss.las

bullet Rapid urease tests for Helicobacter pylori infection

Many invasive and noninvasive diagnostic methods have been developed to detect Helicobacter pylori infection. Current concepts for managing H. pylori infection recommend a test-and-treat approach. Consequently, rapid and accurate diagnostic methods are necessary if patients are to receive appropriate therapy immediately after endoscopy. Endoscopy is an important tool for primary diagnosis in dyspeptic patients, and biopsy-based tests are appropriate for detecting H. pylori. However, in biopsy-based tests, the accuracy of histological tests depends to a large degree on the expertise of the pathologist, while the accuracy of cultures depends on the conditions in which the specimens are transported and processed. Obtaining the results of culture and histology is time consuming. In contrast, rapid urease testing (RUT) is a simple, inexpensive, rapid, and reliable method for detecting urease activity in gastric specimens and is considered by clinicians to be the initial test of choice for diagnosing H. pylori. The CLO agar gel test (Delta West Bentley, WA, Australia) is the most widely used and best studied RUT, but the reading time of up to six hours does not satisfy the claim that RUTs are fast. The authors conducted a study to evaluate the accuracy and positive reaction times of two new RUTs (ProntoDry and HpOne) in comparison with the CLO test. They included in the study 51 patients (26 men, 25 women; mean age, 52.4 years), and all underwent esophagogastroduodenoscopy. None of the patients had received H. pylori eradication therapy. H. pylori infection status was evaluated by histology, culture, 13C-UBT, and RUT. H. pylori infection was considered to be positive if the culture was positive or if two of the other three tests were positive. If culture were negative and only one of the other three tests were positive, or if all four tests were negative, the result was interpreted as negative. Of these 51 patients, two were excluded and 29 (59.1 percent) were infected with H. pylori. The authors concluded that the sensitivities, specificities, positive predictive values, and negative predictive values of the three RUTs were not significantly different. The mean positive reaction times of the three RUTs—CLO test, ProntoDry, and HpOne—were 67.8±12.0, 16.5±2.2, and 17.8±2.1 minutes, respectively. ProntoDry and HpOne had significantly faster reaction times than the CLO test, but there was no significant difference between ProntoDry and HpOne. The authors concluded that different media of RUTs may influence the rapidity of a positive reaction time. ProntoDry and HpOne were superior to the CLO test in terms of accuracy, reaction time, and cost effectiveness.

Tseng C-A, Wang W-M, Wu D-C. Comparison of the clinical feasibility of three rapid urease tests in the diagnosis of Helicobacter pylori infection. Dig Dis Sci. 2005;50:449–452.

Reprints: Dr. Deng-Chyang Wu, Division of Gastroenterology, Dept. of Internal Medicine, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Rd., Kaohsiung 807, Taiwan;

bullet International variations in anemia management

The authors of this study collected data from nationally representative samples of hemodialysis patients (n=11,041) in 2002 and 2003 and used the data to describe current anemia management for long-term hemodialysis patients at 309 dialysis units in 12 countries. Analyses of associations and outcomes were adjusted for demographics, 15 comorbid classes, laboratory values, country, and facility clustering. The authors found that for patients on dialysis therapy for longer than 180 days, 23 percent to 77 percent had a hemoglobin (Hgb) concentration of less than 11 g/dL (<110 g/L), depending on country; 83 percent to 94 percent were administered erythropoietin (EPO). Mean Hgb levels were 12 g/dL (120 g/L) in Sweden; 11.6 to 11.7 g/dL (116 to 117 g/L) in the United States, Spain, Belgium, and Canada; 11.1 to 11.5 g/dL (111 to 115 g/L) in Australia/New Zealand, Germany, Italy, United Kingdom, and France; and 10.1 g/dL (101 g/L) in Japan. Hgb levels were substantially lower for new patients with end-stage renal disease, and EPO use before ESRD ranged from 27 percent (United States) to 65 percent (Sweden). By patient, EPO use significantly declined with greater Hgb concentration (adjusted odds ratio, 0.61 per 1 g/dL [10 g/L] greater Hgb level; P<0.0001), as did EPO dosage. Case-mix-adjusted mortality and hospitalization risk declined by five percent and six percent per 1 g/dL greater patient baseline Hgb level (P=0.003 each), respectively. Furthermore, patient mortality and hospitalization risks were 10 percent to 12 percent lower for every 1 g/dL greater facility mean Hgb level. Patients were significantly more likely to have Hgb levels of 11 g/dL or greater (110 g/L) if they were older; were men; had polycystic kidney disease; had greater albumin, transferrin saturation, or calcium levels; were not receiving dialysis with a catheter; or had lower ferritin levels. Facilities with greater intravenous iron use showed significantly greater facility mean Hgb concentrations. Mean EPO dose varied from 5,297 (Japan) to 17,360 U/week (United States). Greater country mean EPO doses were significantly associated with greater country mean Hgb concentrations. Several patient characteristics were associated with greater EPO doses. Even in some countries with high intravenous iron use, 35 percent to 40 percent of patients had a transferrin saturation of less than 20 percent, which was below guidelines. The authors concluded that these findings indicate large international variations in anemia management, with significant improvements during the last five years, although many patients remain below current anemia guidelines, suggesting large and specific opportunities for improvement.

Pisoni RL, Bragg-Gresham JL, Young EW, et al. Anemia management and outcomes from 12 countries in the Dialysis Outcomes and Practice Patterns Study (DOPPS). Am J Kidney Dis. 2004;44:94–111.

Reprints: Dr. Ronald L. Pisoni, URREA, 315 W. Huron, Ste. 260, Ann Arbor, MI 48103;

bullet Genetics of the Ko phenotype

The rare Ko phenotype is defined by total absence of Kell antigens on red blood cells and occurs by homozygosity or compound heterozygosity for silent alleles at the KEL locus. The absence of Kell glycoprotein has no known disease association: Ko RBCs have normal discoid shape, but a decreased amount of XK protein is expressed even though Kx antigen expression appears elevated. In contrast, the absence of the XK protein leads to the McLeod phenotype, which is associated with RBC acanthocytosis and late-onset neurologic defects and with the depressed expression of Kell protein. Three reports have been published in which a total of 10 alleles with different point mutations giving rise to the Ko phenotype have been described in several populations. In this study, three Ko samples were identified by blood banks in Uppsala, Umea, and Linköping, Sweden, during a 20-year period. The respective blood banks performed Kell antigen typing with standard serologic techniques, and Ko status was confirmed by the International Blood Group Reference Laboratory in Bristol, England. Polymerase chain reaction and DNA sequencing of the KEL coding region (exons 1–19) were performed on genomic DNA. The authors found that the Uppsala Ko was homozygous for a 1540C>T substitution in exon 13, leading to an immediate stop codon. The Umea Ko was homozygous for 1023delG in exon 8 that results in a frameshift and a premature stop codon in exon 9. In the Linköping Ko, a previously reported mutation g>a at +1 of intron 3 was found. The authors concluded that two novel and one previously reported null alleles at the KEL locus are described. The identified nonsense mutations abolish expression of the Kell glycoprotein and are thus responsible for the Ko phenotype in these Swedish families.

Wester ES, Storry JR, Schneider K, et al. Genetic basis of the Ko phenotype in the Swedish population. Transfusion. 2005;45: 545–549.

Reprints: Dr. Jill R. Storry, Blood Center, University Hospital, SE221 85, Lund, Sweden;

bullet Evaluation of the Pentra 80 hematology analyzer

The Pentra 80 automated cell counter is a new 80-sample-per-hour hematology analyzer manufactured by ABX Diagnostics (Montpellier, France). According to the specifications of the manufacturer, it provides complete blood cell counts, including a five-part white blood cell differential count, at the same time that it provides enumeration of large immature cells and atypical lymphocytes. Measurements are based on principles of impedance, cytochemistry, and the measurement of light absorbance using the so-called double-hydrodynamic sequential system, or DHSS, technology associated with staining samples with a cytochemical reagent (Eosinophix, ABX Diagnostics) before analyzing cell volume and light absorbance. Because it is newly available, no studies have been reported in which the Pentra 80 is evaluated in detail in a clinical diagnostic hematology laboratory for its performance in calculating the five-part WBC differential and identifying the related quantitative and/or morphologic abnormalities to be flagged. The authors evaluated the performance of the Pentra 80 in enumerating the most frequent subsets of WBCs in peripheral blood, atypical lymphocytes, and large immature cells by comparing results with those obtained by manual microscopic counts, another hematology analyzer, and flow cytometric immunophenotyping. Identification and enumeration of neutrophils and lymphocytes with the Pentra 80 showed high correlation with all three reference methods (R2=0.92 and R2=0.88, respectively); quantification of eosinophils showed good correlation with the other analyzer and flow cytometric immunophenotyping (R2=0.70); lower correlation coefficients were found for comparisons with conventional microscopy (R2=0.50). For monocytes, lower but acceptable correlation and agreement were found; marginal correlation was found for basophils. The Pentra 80 showed good performance in detecting large immature cells but was less effective for identifying atypical lymphocytes in relatively low frequencies in abnormal peripheral blood samples. The Pentra 80 also showed good performance for five-part leuKocyte differential analyses, especially for enumerating neutrophils, lymphocytes, eosinophils, and large immature cells.

Arroyo ME, Tabernero MD, Garcia-Marcos MA, et al. Analytic performance of the PENTRA 80 automated blood cell analyzer for the evaluation of normal and pathologic WBCs. Am J Clin Pathol. 2005;123:206–214.

Reprints: Dr. Alberto Orfao, Servicio General de Citometria, Laboratorio de Hematologia, Hospital Universitario Salamanca, Paseo San Vincente, 58-182, 37007 Salamanca, Spain

bullet Vitamin B12 and folate intake and cognitive decline in elderly patients

In addition to showing the well-established importance of vitamin B12 deficiency to neurologic disease, a number of cross-sectional studies have observed lower cognition among people with insufficient serum or plasma levels of vitamin B12 and folate. Furthermore, inadequate levels of either vitamin can lead to elevated plasma homocysteine levels, which may be independently associated with Alzheimer’s disease, thus raising interest in these vitamins as a means of preventing the disease. As of January 1998, the FDA required the fortification of grain products with folic acid to prevent neural tube defects. The long-term effects of the program on other health outcomes or in special populations are not yet known. The authors examined the associations of dietary folate and vitamin B12 with six-year cognitive change in a prospective study performed from 1993 to 2002 in a geographically defined biracial community in Chicago. A total of 3,718 residents, 65 years and older, who completed two to three cognitive assessments and a food frequency questionnaire participated in the study. The main outcome measure was change in cognitive function measured at baseline and three-year and six-year followup using the average z score of four tests: East Boston tests of immediate and delayed recall, Mini-Mental State Examination, and Symbol Digit Modalities test. The authors found that high folate intake was associated with a faster rate of cognitive decline in mixed models adjusted for multiple risk factors. The rate of cognitive decline among persons in the top fifth of total folate intake (median, 742 µg/d) was more than twice that of those in the lowest fifth of intake (median, 186 µg/d), a statistically significant difference of 0.02 standardized unit per year (P=.002). A faster rate of cognitive decline was also associated with high folate intake from food (P for trend=.04) and with folate vitamin supplementation of more than 400 µg/d compared with nonusers (β= –.03; P<.001). High total vitamin B12 intake was associated with slower cognitive decline only among the oldest study participants. The authors concluded that high intake of folate may be associated with cognitive decline in older persons. These unexpected findings call for further study of the cognitive implications of high levels of dietary folate in older populations.

Morris MC, Evans DA, Bienias JL, et al. Dietary folate and vitamin B12 intake and cognitive decline among community-dwelling older persons. Arch Neurol. 2005;62:641–645.

Reprints: Martha Clare Morris, Rush Institute for Healthy Aging, 1645 W. Jackson, Suite 675, Chicago, IL 60612;

bullet Chlamydia pneumoniae in coronary artery disease

Infectious pathogens, particularly Chlamydia pneumoniae, recently have been deemed potential risk factors for atherosclerosis. It has been proposed that during respiratory tract infection, C. pneumoniae reaches vascular tissue via infected leuKocytes. Some seroepidemiologic studies have found an association between C. pneumoniae and coronary artery disease (CAD). It has not been demonstrated whether antibiotic treatment eradicates C. pneumoniae from vascular tissue. The authors conducted a study to assess the effect of clarithromycin on C. pneumoniae in the vascular tissue of patients with CAD. Patients who had CAD and who were waiting for coronary artery bypass graft surgery were enrolled in a randomized, double-blind, placebo-controlled trial. Patients were treated with clarithromycin at 500 mg or placebo once daily from the day they started the study until surgery. Several vascular tissue specimens were obtained during surgery. The presence of C. pneumoniae in vascular tissue specimens was examined by immunohistochemical staining and two polymerase chain-reaction assays. Chlamydia immunoglobulin G (IgG) titers were determined by an enzyme-linked immunosorbent assay at the time subjects enrolled in the study and eight weeks after surgery. A total of 76 patients were included, and 180 vascular tissue specimens were obtained—80 specimens from the group treated with clarithromycin and 100 specimens from the group treated with placebo. Thirty-five patients received clarithromycin (mean duration, 27 days; standard deviation [SD], 12.2 days), and 41 patients received placebo (mean duration, 27 days; SD, 13.9 days). Immunohistochemical staining detected the C. pneumoniae major outer membrane protein antigen in 73.8 percent of the specimens from the group treated with clarithromycin and 77 percent of the specimens from the group treated with placebo (P was not significant). Chlamydia lipopolysaccharide antigen was found in only one specimen from the group that received placebo. C. pneumoniae DNA was not detected in any specimen. Baseline Chlamydia-specific IgG titers were equally distributed in both groups and were not significantly different after treatment. There was no indication of an active C. pneumoniae infection in vascular tissue. Chlamydia-specific IgG titers remained unchanged throughout the study in the antibiotic- and placebo-treated patients. The authors concluded that it is unlikely that antibiotic treatment will have any effect in patients with advanced atherosclerosis.

Berg HF, Maraha B, Van der Zee A, et al. Effect of clarithromycin treatment on Chlamydia pneumoniae in vascular tissue of patients with coronary artery disease: a randomized, double-blind, placebo-controlled trial. J Clin Microbiol. 2005;43:1325–1329.

Reprints: Hans F. Berg, Dept. of Medical Microbiology, St. Elisabeth Hospital, St. Ignatiusstraat 91-C, 4817 KC Breda, Netherlands;

Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus.