Prevalence of iron deficiency across different tumors and its association with poor performance status, disease status and anemia.

BACKGROUND: Only limited data on the prevalence of iron deficiency (ID) and its correlation with clinical parameters are available in cancer. ID frequently contributes to the pathogenesis of anemia in patients with cancer and may lead to several symptoms such as impaired physical function, weakness and fatigue. PATIENTS AND METHODS: Parameters of iron status and clinical parameters were evaluated in 1528 patients with cancer who presented consecutively within a four-month period at our center. One thousand fifty-three patients had solid tumors and 475 hematological malignancies. RESULTS: ID [transferrin saturation (TSAT) < 20%] was noted in 645 (42.6%) of the 1513 patients with TSAT tests available and 500 (33.0%) were anemic. ID rates were highest in pancreatic (63.2%), colorectal (51.9%) and lung cancers (50.7%). Of the 409 iron-deficient patients in whom serum ferritin levels were available additionally to TSAT, 335 (81.9%) presented with functional ID (FID) (TSAT < 20%, serum ferritin ≥30 ng/ml) and 74 (18.1%) with absolute ID. In patients with solid tumors, prevalence of ID correlated with cancer stage at diagnosis (P = 0.001), disease status (P = 0.001) and ECOG performance status (P = 0.005). CONCLUSIONS: ID was frequently noted in cancer and was associated with advanced disease, close proximity to cancer therapy, and poor performance status in patients with solid tumors.

Immunoglobulin heavy/light chain ratios improve paraprotein detection and monitoring, identify residual disease and correlate with survival in multiple myeloma patients.

The novel heavy/light chain (HLC) assay was used for the detection and measurement of monoclonal immunoglobulins, response evaluation and prognostication. This test allows identification and quantification of the different light chain types of each immunoglobulin class (for example, IgGκ and IgGλ) and enables calculation of ratios of monoclonal/polyclonal immunoglobulin (HLC ratio). Sequential sera of 156 patients with IgG or IgA myeloma started on first-line therapy and followed for a median of 46.1 months were analyzed. Results were compared with those obtained with conventional techniques (serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE), nephelometry (NEPH), and the free light chain test (FLC)). Our data show that the HLC assay allowed quantification of monoclonal proteins not accurately measurable by SPEP or NEPH. When both HLC and FLC testing were applied for response assessment, clonal excess was noted in 14/31 patients with complete response (CR). HLC ratio indicated presence of disease in 8/31 patients who achieved CR and, in sequential studies indicated evolving relapse in three patients before IFE became positive. Highly abnormal HLC ratios at presentation were significantly associated with shorter overall survival (40.5 months vs median not reached, P=0.016). Multivariate analysis revealed HLC ratio (P=0.03) and ß(2)-microglobulin (P<0.01) as independent risk factors for survival.

Comparison of enoxaparin and unfractionated heparin in endovascular interventions for the treatment of peripheral arterial occlusive disease: a randomized controlled trial.

BACKGROUND: Although unfractionated heparin (UFH) is an effective antithrombotic agent in endovascular interventions for the treatment of peripheral occlusive arterial disease (PAOD), it produces a highly variable anticoagulant response. Intravenous (i.v.) enoxaparin might be an effective and safe alternative. PATIENTS AND METHODS: In a prospective, open-label, randomized, single-center trial, 210 patients with PAOD (Fontaine stage IIb to IV) were randomly assigned in a 1 (UFH): 2 (enoxaparin) fashion to receive an i.v. bolus of 60 units UFH per kg body weight or 0.5 mg enoxaparin per kg body weight, respectively, before endovascular intervention. The primary composite endpoint assessed the clinical performance of enoxaparin by comparing the peri-interventional rate of thromboembolia/occlusion (efficacy) of endovascularly reconstructed areas, of bleeding according to the Global Utilization of Streptokinase and t-PA for Occluded Coronary Arteries (GUSTO) criteria (safety) and of any necessary re-intervention for any percutaneous transluminal angioplasty (PTA)-related bleeding. The secondary endpoint evaluated anti-factor (F)Xa levels during intervention. RESULTS: The primary composite endpoint showed a better performance of enoxaparin (10.5% vs. 2.5% absolute difference - 8.0%; P < 0.05). The concomitant use of acetylsalicylic acid (ASA) significantly (P < 0.05) increased the risk of a complication in the UFH group, but not in the enoxaparin group. Within 15 min, anti-Xa levels were reached by 63.7% of patients treated with enoxaparin and only by 39.1% with UFH. CONCLUSION: Enoxaparin has a better performance than UFH in endovascular interventions for the treatment of PAOD. In patients with concomitant use of ASA, the risk of complications with UFH increases significantly compared with enoxaparin.

Traditional versus endoscopic saphenous vein stripping: a prospective randomized pilot trial.

INTRODUCTION: The aim of this pilot study was to compare two methods of removing the great saphenous vein (GSV) from the groin to the limit of distal venous incompetence. Our aim was to compare endoscopically assisted GSV stripping to conventional stripping. DESIGN: Randomised pilot study. PATIENTS AND METHODS: 60 patients presenting with primary GSV incompetence and symptomatic varicose veins were randomly assigned to sapheno-ligation and either conventional GSV stripping or endoscopically assisted GSV stripping. The primary endpoint was the number of adverse events including haematoma in the thigh, ecchymosis, seroma, wound healing complications and wound infections. The SF-36 health survey was completed before treatment and one and four weeks postoperatively. The study was approved by the local ethics committee (EK 07-041-VK). RESULTS: 60 patients were enrolled in the study and randomized to endoscopic (n=30) and to traditional (n=30) stripping. The patients age ranged from 30 to 75 years (mean 53 years), 18 patients were male, 42 female. The combined rate of postoperative morbidity at week 1 was 32 events (53%), 13 (42%) events in the endoscopic and 19 (63%) in the conventional group (not significant). The SF-36 assessment one week postoperatively showed that patients in the endoscopic group reported significantly less pain (P=0.03, Mann-Whitney). At four weeks, patients in the endoscopic group had significantly less pain (P<0.005) and better physical function (P<0.005) and physical role (P=0.01). For all other parameters no significant difference noted. CONCLUSION: The results of this study suggest that endoscopic GSV excision showed no difference in adverse events between treatments, although our pilot study may have been under-powered to demonstrate this. The SF-36 assessment suggests more rapid return to normal activities post-operatively in the endoscopic group.

Detection of ZAP-70 in patients with chronic lymphocytic leukemia.

PURPOSE: To determine the presence and prognostic significance of ZAP-70 in patients with B chronic lymphocytic leukemia (B-CLL). Expression of ZAP-70 in B-CLL is a continuum ranging from absent to high, affecting the prognostic reliability of this marker. PATIENTS AND METHODS: 32 patients with B-CLL treated from March 2006 to February 2007 were prospectively studied. Patients were stratified in two groups: those diagnosed with B-CLL within 18 months and a retrospective group diagnosed before 18 months since the beginning of this study. Patients were predominantly males (78.1%), over 60 years old (81.25%). ZAP-70 was detected on EPICX-XL flow cytometer with System II software. Directly conjugated antibodies CD19 PE, ZAP-70 FITC (clone 1E7.2) and CD2 APC were used. Leuoperm was used for permeabilization. All samples with over 20% ZAP-70/CD19 double positive B lymphocytes were considered positive. RESULTS: ZAP-70 was absent in a group of 22 patients with B-CLL diagnosed over 18 months from the beginning of this study, while 2 out of 10 (20%) patients from the group of newly diagnosed patients were ZAP-70(+). During follow up both ZAP-70(+) cases showed worsening of their clinical status resulting to death in one patient. In borderline cases with ZAP-70(+) ranging from 10.9-19.9% there was no correlation of the level of ZAP-70 expression with disease activity or clinical scoring systems. CONCLUSION: Very high percentage of ZAP-70(+) cells is a sign of poor prognosis in B-CLL. Borderline expression of ZAP-70, although more frequent, could not be successfully assigned into a risk group.

Homocysteine and early re-stenosis after carotid eversion endarterectomy.

BACKGROUND: Homocysteine (Hcy) appears to be involved in the development of intimal hyperplasia and arterial thrombosis. The purpose of this study was to evaluate the association of plasma Hcy with early re-stenosis following carotid eversion endarterectomy. PATIENTS AND METHODS: Of 398 consecutive patients, 363 were included in this study. 62% of patients had symptomatic internal carotid artery (ICA) stenosis. Patients had preoperative assessment of Hcy and other well established atherosclerosis risk factors. Intraoperatively, completion angiography was performed in 2 planes. Patients had clinical, Hcy and duplex follow up at 1, 3, 18 and 36 months postoperatively. RESULTS: Complete follow up data were available for 312 patients. Five patients suffered from strokes and 2 patients died during the peri-operative period (combined stroke and death rate of 2%). Mean follow up was 26+/-5 months (range 17 to 36 months). Seventeen and six patients (5.5%) developed a 50-69% and >70% re-stenosis, respectively. Serum creatinine was significantly higher in patients with early re-stenosis, occlusion or stroke after CEA (P=0.043). High grade re-stenosis, occlusion and stroke ipsilateral to the operated side (17 patients) was associated with HbA1C and creatinine (P=0.043 and 0.046, respectively) but not Hcy. CONCLUSION: While Hcy is a recognized independent risk factor for atherothrombosis, our study suggests that there is no association of Hcy with early re-stenosis after eversion endarterectomy.

Plasma D-lactate as a potential early marker for colon ischaemia after open aortic reconstruction.

BACKGROUND AND AIM: The breakdown of mucosal barrier function due to intestinal hypo-perfusion is the earliest dysfunction of ischaemic colitis. Severe colon ischaemia after aortic reconstruction is associated with mortality rates up to 90%. Therefore, early detection and treatment of patients with extensive ischaemic colitis is of crucial importance. In experimental studies, both D-lactate and bacterial endotoxin have been reported as markers of intestinal mucosal barrier impairment. However, evidence of their value in clinical practice is lacking. The aim of this pilot prospective cohort study was to assess the association between ischaemia of the colon (assessed histologically) and plasma levels of D-lactate and endotoxin in patients undergoing open aortic reconstruction. PATIENTS AND METHODS: Twelve consecutive patients underwent surgery between February and April 2003. Six patients underwent emergency surgery and six patients elective aortic surgery. D-Lactate and endotoxin levels were measured in blood samples collected according to a standardised protocol. For histological examination biopsies were obtained by sigmoidoscopy on days 4-6 after surgery, or earlier if indicated clinically. RESULTS: As early as 2 h postoperatively, elevated plasma levels of d-lactate were measured in patients with histologically proven ischaemic colitis. The peak of D-lactate elevation was on postoperative days 1 and 2. Concentration of plasma endotoxin was not significantly different in patients with or without ischaemic colitis. CONCLUSION: Our data suggest that plasma D-lactate levels are a useful marker for early detection of ischaemic colitis secondary to aortic surgery.

Carotid endarterectomy under local anaesthesia does not increase plasma homocysteine concentration.

INTRODUCTION: The purpose of this cohort study was to evaluate the effect of carotid endarterectomy under local anaesthesia on homocysteine (Hcy) concentrations. PATIENTS AND METHODS: Of 100 patients with internal carotid artery (ICA) stenosis >70%, the complete data set was available for 91 patients (39 asymptomatic and 52 symptomatic). All patients underwent eversion endarterectomy of the ICA under regional anaesthesia. RESULTS: Thirty-two percent of the examined patients had a total Hcy above 15 micromol/l. The mean Hcy levels preoperatively were 13.9+/-4.8 micromol/l. The Hcy levels on day 5 were 13.1+/-5.0 micromol/l and after 6 months 14.0+/-5.8 micromol/l. There was no significant change during follow-up. No intraoperative strokes and deaths were observed and during the 6 months follow-up no recurrent strokes, TIAs or deaths occurred. CONCLUSION: Patients undergoing carotid endarterectomy under regional anaesthesia do not have an increase in total Hcy postoperatively. This finding is in contrast to results from cardiac surgery and carotid endarterectomy in a recently published animal study, both performing surgery under general anaesthesia.

Topical PDGF-BB results in limited healing in a patient with Werner's syndrome and chronic leg ulcers.

Impaired wound healing is a feature of Werner's syndrome. Treatment of one such patient with painful chronic leg ulcers included topical application of PDGF-BB. Granulation increased slightly, enabling full-thickness skin grafting to take place.

Immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) in autoimmune disease diagnostics--technique, benefits, limitations and applications.

Autoimmune diseases are relatively frequent disease complexes, affecting approximately five to seven percent of the population. After cardio-vascular and malignant diseases they come third in mortality. As the clinical diagnosis of rheumatic autoimmune diseases is difficult, laboratory tests are helpful in differential diagnosis and for verification of the clinical diagnosis. The most commonly used assay is the determination of ANAs (anti nuclear antibodies) by indirect immunofluorescence (IFA). However, this method lacks reliable standardisation and is very dependable on the qualification of the observer. Enzyme Immunoassays (EIA) and Immunoblotting techniques, on the contrary, attain good standardisation and comparability. However, the latter methods are limited to the presentation of defined autoantibodies only. There is a need to select a suitable strategy for the use of laboratory parameters in order to support the clinical diagnosis more efficiently. A possible strategy is to replace IFA as a first line screening-step by second-generation ANA-EIA kits.

A simple method for measurement of CD14weak CD16strong monocytes in peripheral blood.

Mounting evidence for the clinical significance of the CD 14weak CD16strong monocyte subpopulation in peripheral blood induced the demand for an efficient method for its determination. We propose a simple, fast, no-wash flow cytometric method using fluorescence-labelled anti-CD14, anti-CD16, and anti-HLA-DR antibodies and ammonium chloride-based erythrocyte lysis. This type of analysis can be performed on a standard three-colour flow cytometer. The method avoids interference by NK-cells and neutrophil granulocytes without defining monocytes by stringent light scatter criteria that might lead to a loss of CD14weak CD16strong monocytes. It, therefore, offers high reliability and accuracy. Its performance recommends the method to be used for routine clinical measurements of CD14weak CD16strong monocytes.

External evaluation of LIAISON tumour marker assays on the fully automated chemiluminescent LIAISON immunoassay analyser.

The LIAISON immunoassay analyser was tested in a multicentre evaluation performed by 8 laboratories. The analytes evaluated were CA 15-3, CA 19-9, CA 125II, AFP, CEA, NSE and PSA. Excellent results were obtained for within-run and between-run precision with most assays showing within-run CVs < 5% and between-run CVs between 4 and 8%. The linearity of all assays was acceptable, however, for PSA, NSE and CA 19-9 a recovery > 110% was obtained for some of the samples tested. None of the assays revealed a high-dose hook effect. Method comparisons were performed by using the routine method of the respective study centre. Results generally showed an acceptable agreement between the LIAISON system and the different methods of comparison. The reference ranges for all assays were found to be in accordance with data known from the literature. All assays showed similar results for serum, heparinised plasma and EDTA plasma. Additionally, two experiments were performed with only one of the analytes tested: the sample-to-sample carry-over, using the CA 19-9 assay (3.3 x 10(-6)-2.3 x 10(-5)) and the functional sensitivity for the PSA assay (0.2 ng/ml).

Evaluation of the LIAISON thyroid chemiluminescence immunoassays.

The LIAISON thyroid hormone assays TSH, FT4, FT3, T4 and T3 were evaluated by determining the imprecision, the reference ranges, the functional sensitivity (TSH), the dilution characteristics (accuracy) (FT4, FT3), and the recovery after spiking (TSH, T4, T3). Furthermore, inter-method comparisons were performed with following methods: Elecsys (Roche Diagnostics; TSH), AxSYM (Abbott Diagnostics; TSH, FT4, FT3, T4), ACS:180 (Bayer Diagnostics; all analytes), Amerlex-M (Johnson & Johnson; T4) and LISO-Phase (Techno Genetics; FT4). The fully automated LIAISON random access analyser is based on microparticle immunoassays and chemiluminescence. The coefficients of variation (CV) of intra-assay imprecision were between 0.2-6.0%, except for the control sample with extremely low TSH concentrations and low T3 concentrations. Inter-assay imprecision was performed by measuring controls covering the measuring range over a period of 9 to 20 days, with CVs ranging from 2.3-16.0%. The suitability of the sample material was determined by analysing serum and samples treated with EDTA, citrate or heparin in parallel. The results showed good correlations of the thyroid hormone concentrations between serum and plasma samples except for LIAISON FT3, for which lower results were observed with EDTA-plasma. The regression analysis of correlation studies gave slopes from 0.849 to 0.957 for TSH, from 1.023 to 1.375 for FT4, from 0.670 to 0.911 for FT3, from 0.917 to 1.166 for T4 and 1.00 for T3 depending on the concentration range and the method of comparison. The LIAISON FT4 assay showed a trend towards higher values in the high concentration range when compared with the ACS:180. The ranges of thyroid hormone concentrations determined in serum taken from apparently healthy subjects were found to be in accordance with published data. The clinical sample study confirmed that the LIAISON thyroid hormone assays are sensitive methods for the differentiation of euthyroid subjects and patients with hyper- and hypothyroidism. In conclusion, the automated thyroid hormone immunoassays on the random-access LIAISON immunoassay analyser proved to be very satisfactory, both from the analytical and the clinical point of view.

Multicenter evaluation of the Elecsys CA 125 II assay.

The technical performance and clinical usefulness of the newly developed Elecsys CA 125 II assay (Boehringer Mannheim) was evaluated in a multicenter study. Imprecision studies were carried out using control sera and human pool sera with CA 125 concentrations from 11 to 1026 U/ml. Within-run CVs between 0.7 to 4.8% (median 1.7%) and between-day CVs between 2.4 to 10.9% (median 5.7%) were found. Method comparison studies with Enzymun-Test CA 125 II carried out in four laboratories yielded slopes between 0.94 to 1.07 and intercepts < 3 U/ml. A good comparability of the Elecsys CA 125 II assay was also found with one MEIA and the Centocor" IRMA. For a second MEIA and a second IRMA the slopes were 1.23 and 1.42, and the corresponding correlation coefficients were 0.987 and 0.977, respectively. The Elecys CA 125 II concentrations are clearly related to the tumor stage of ovarian carcinoma patients. The maximum of diagnostic efficiency of ovarian carcinoma patients compared with patients of benign gynecological diseases is reached at 150 U/ml with a specificity of 93% and a sensitivity of 69%. Follow-up studies of ovarian carcinoma patients reflect the status of the disease and the effect of various therapeutic applications. The technical and clinical evaluation of the Elecsys CA 125 II assay show a superior analytical performance with a broad measuring range up to 5000 U/ml and a short measuring time of 18 minutes.

Differential expression of tumor necrosis factor receptor subtypes on leukocytes in systemic inflammatory response syndrome.

OBJECTIVE: To determine the expression of tumor necrosis factor (TNF) receptor in patients with systemic inflammatory response syndrome (SIRS). DESIGN: Prospective study. SETTING: Intensive care unit and central laboratory. PATIENTS: Blood specimens from 18 healthy volunteers (controls) and 16 patients with SIRS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Using monoclonal antibodies, fluorescence labeling, and high sensitivity flow cytometry, we measured the expression of membrane TNF receptor subtypes TNF-R55 and TNF-R75 on peripheral blood leukocytes. Receptor expression is expressed as mean fluorescence intensity +/- SD (units: detection channel number). In controls, TNF-R55 was only weakly expressed (monocytes: 2.5+/-1.8; neutrophils: 0.7+/-0.8), whereas expression of TNF-R75 was higher (monocytes: 28.6+/-9.0; neutrophils: 4.8+/-1.0) and was also found on lymphocytes (on CD8+ lymphocytes: 5.7+/-1.8; CD16+: 5.5+/-1.2; CD4+: 9.7+/-3.7). In SIRS, we observed increased expression of TNF-R55 on monocytes (6.9+/-3.4, p<.001) and neutrophils (2.2+/-1.9, p<.01), as well as decreased expression of TNF-R75 on monocytes (17.3+/-13.2; p<.001). The extent of TNF-R55 up-regulation did not correlate with that of TNF-R75 down-regulation. TNF-R55 on monocytes and neutrophils strongly correlated with body temperature but not with survival, whereas monocyte TNF-R75 was considerably lower in nonsurvivors, albeit not significantly (12.3+/-7.1 vs. 23.9+/-16.7; p = .07). CONCLUSIONS: These data indicate that leukocyte TNF-R55 and TNF-R75 react differentially and probably serve different functions in SIRS, which prompts the investigation of receptor subtype-specific therapeutic approaches.

Analytical and clinical evaluation of an electrochemiluminescence immunoassay for the determination of CA 125.

The CA 125 II assay on the Elecsys(R) 2010 analyzer was evaluated in an international multicenter trial. Imprecision studies yielded within-run CVs of 0.8-3.3% and between-day CVs of 2.4-10.9%; CVs for total imprecision in the manufacturer's laboratory were 2.4-7.8%. The linear range of the assay extended to at least 4500 kilounits/L (three decades). Interference from triglycerides (10.3 mmol/L), bilirubin (850 micromol/L), hemoglobin (1.1 mmol/L), anticoagulants (plasma), and several widely used drugs was undetectable. Method comparisons with five other CA 125 II assays showed good correlation but differences in standardization. A 95th percentile cutoff value of 35 kilounits/L was calculated from values measured in 593 apparently healthy (pre- and postmenopausal) women. In 95% of patients with benign gynecological diseases CA 125 was 190 kilounits/L. A comparison of CA 125 values obtained with the Elecsys test and with other common CA 125 tests in monitored patients being treated for ovarian cancer showed identical patterns. In conclusion, the Elecsys CA 125 II assay is linear over a broad range, yields precise and accurate results, is free from interferences, and compares well with other assays.

Measurement of absolute concentration and viability of CD34+ cells in cord blood and cord blood products using fluorescent beads and cyanine nucleic acid dyes.

Conventional flow cytometric methods for CD34+ cell counting may be affected by the high number of nucleated red blood cells or nonviable cells in cord blood and its products. We developed a simple flow cytometric no-wash procedure that avoids these shortcomings because it provides absolute CD34+ cell counts and assesses cell viability. Samples were incubated with phycoerythrin (PE)-labeled anti-CD34 (Becton Dickinson Immunocytometry Systems [BD], San Jose, CA) and peridinin chlorophyll protein (PerCP)-labeled anti-CD45 (BD) in bead-containing TRUCOUNT tubes (BD). After red cell lysis with a fixative-free reagent, the impermeant nucleic acid dye YO-PRO-1 (Molecular Probes, Eugene, OR) was added and samples were analyzed on a single-laser FACSCalibur (BD). A comparison with the ProCOUNT progenitor cell assay (BD) in 57 samples revealed excellent correlation of results (r = 0.98, intercept -0.2 cells/microl, slope 1.01). Precision studies conveyed coefficients of variation of 6.4 and 8.9% at concentrations of 35 and 16 CD34+ cells/microl, respectively. In untreated and leukocyte-enriched cord blood 4.5+/-3.8% of CD34+ cells were stained by YO-PRO-1, representing apoptotic or necrotic cells. In post-thawing cryopreserved samples this number increased to 10.4+/-5.5%. Isotype controls showed very low blank values of viable cells (0.1+/-0.4 cells/microl, maximum 2.4) and seemed unnecessary. We found no washing-related alteration of results in 35 samples, indicating that the method may also be performed with cell washing. Replacing YO-PRO-1 with TO-PRO-3 facilitated four-color analysis of subpopulations of viable CD34+ cells on a FACSCalibur equipped with a second (diode) laser. We found the proposed method to be a rapid, efficient, and flexible procedure that improved validity of CD34+ cell counts.

FMC7 antigen expression on normal and malignant B-cells can be predicted by expression of CD20.

Most antibody panels proposed for flow cytometric immunophenotyping of non-Hodgkin's lymphomas and chronic lymphoid leukemias include anti-CD20 and FMC7 antibodies. As in our experience, reactivity of B-cells with these antibodies seemed to be correlated, we evaluated whether the simultaneous use of anti-CD20 and FMC7 antibodies is justified. Using flow cytometry, we measured the binding of these 2 antibodies to the B-cells of 67 bone marrow aspirates, 31 lymph node biopsies, 18 peripheral blood specimens, and 12 tissue samples from other locations. The diagnoses included 50 cases without overt abnormalities, 5 reactive lymphadenopathies, 56 lymphomas and chronic lymphoid neoplasias, and 17 cases with other malignancies. Although CD20 expression was consistently higher, we observed a significant and strong correlation between CD20 and FMC7 antigen expression on B-lymphocytes, irrespective of the nature of the sample or disease (r=0.910; P < 0.001). Moreover, FMC7 antigen expression on B-cells could be predicted by CD20 expression with a sensitivity of 96%, a specificity of 94% and an efficiency of 96%. Our results show that although differing in intensity, expression of CD20 on B-cells closely parallels that of FMC7 antigen. We, therefore, conclude that little additional information is revealed by using FMC7 in immunophenotyping of non-Hodgkin's lymphomas or chronic lymphoid leukemias if intensity of CD20 expression is taken into consideration.

Toward a new reference method for the leukocyte five-part differential.

A flow cytometric method performing a five-part leukocyte differential based on three-color staining with anti-CD45-fluorescein isothiocyanate (FITC), anti-CD-14-phycoerythrin (PE)/Cy5, and a cocktail of PE-labeled anti-CD2, anti-CD16, and anti-HLA-DR antibodies was evaluated. Results obtained by using three different sample preparation procedures and two different flow cytometers were compared with those of a 1,000-cell manual differential for evaluation of accuracy. We observed excellent correlations with the manual differential for all leukocyte subclasses and even higher correlations between the different flow cytometric methods. Flow cytometric basophil results were identical to the manual counts, regardless of which sample preparation technique or flow cytometer was used. Therefore, we propose our flow cytometric method as the first acceptable automated reference method for basophil counting. The flow cytometric results for the other leukocyte subclasses were apparently influenced by the sample preparation, which could not be explained by cell loss during washing steps. Moreover, a small influence of the flow cytometer was also observed. Assessing the influence of sample storage, we found only minimal changes within 24 h. In establishing reference values, high precision of flow cytometric results facilitated detection of a significantly higher monocyte count for males (relative count: 7.08 +/- 1.73% vs. 6.44 +/- 1.33%, P < 0.05; absolute count: 0.536 +/- 0.181 x 10(9)/liter vs. 0.456 +/- 139 x 10(9)/liter, P < 0.01). Our data indicate that monoclonal antibody-based flow cytometry is a highly suitable reference method for the five-part differential: It also shows, however, that studies will have to put more emphasis on methodological issues to define a method that shows a high interlaboratory reproducibility.