Reticulocyte count and extended reticulocyte parameters by Mindray BC-6800: Reference intervals and comparison with Sysmex XE-5000.

INTRODUCTION: In this study, analytic performance (imprecision, carryover, time stability) and diagnostic efficiency of Mindray BC-6800 analyzer to quantify reticulocytes and extended reticulocyte parameters was evaluated. Moreover, reference intervals on adult population were determined. Results were compared with those obtained by Sysmex XE-5000 analyzer. METHODS: One hundred and eighty-four healthy adults of both sexes, and 368 subjects affected by various pathologic conditions (nutritional anemias before and after treatment, hemolytic and posthemorragic anemias, acute and chronic inflammations, malignancy under therapy, and beta thalassemia trait) were selected. RESULTS: Reference intervals were as follows: reticulocytes (×109 /L): 23.2-93.2; immature reticulocyte fraction: 0.015-0.14; mean reticulocyte hemoglobin equivalent (RHE) (pg): 30.9-35.7; mean reticulocyte volume (fL): 97.8-118. Imprecision on reticulocyte count at all concentrations was close to analytic goal based on within-subject biological variation. Carryover (2.3%) was negligible, and time-stability was excellent up to 8 hours. CONCLUSION: When compared with XE-5000, BC-6800 shows a good overall correlation on counting despite evidence of difference in the upper limit of reference intervals (93.2 vs 101.3). Comparison of diagnostic efficiency of extended parameters shows a good total agreement of RHE (91.6%).

Role of visceral proteins in detecting malnutrition in the elderly.

OBJECTIVE: In the clinical practice, visceral proteins are used as indirect markers of protein energy malnutrition (PEM), but their reliability could be reduced with advancing age. The aim of this work is to investigate the reliability of albumin, prealbumin, retinol-binding protein (RBP) and transferrin in evaluating nutritional status in old patients and their relationship with fat-free mass (FFM). DESIGN: Cross-sectional study. SETTING: Padua, Italy. SUBJECTS: In 44 underweight (body mass index < 20 kg/m(2)) (66-97 years) and 69 normal weight or overweight elderly subjects (62-98 years), albumin, prealbumin, transferrin and RBP were determined in the plasma. Body composition and particularly FFM was obtained by dual X-ray absorptiometry. FFM was also expressed as FFM index (FFMI) calculated as FFM divided by height squared. Subjects affected by acute illnesses and inflammatory states were excluded. RESULTS: Albumin, prealbumin and RBP mean values were significantly lower in underweight subjects. No differences between two groups were found for transferrin. Albumin prealbumin and RBP resulted under the normal range in 55, 25 and 54% of underweight subjects, respectively. Transferrin's values were low in about 40% of underweight and normal weight subjects, respectively. In all subjects, FFMI shows a significant correlation with albumin (r: 0.52), prealbumin (r: 0.64) and RBP (r: 0.57). No correlation between FFMI and transferrin was found. CONCLUSIONS: Visceral proteins, except for transferrin, seem to be useful indexes in detecting malnutrition in the elderly; low values still in the normal range should also be carefully evaluated because they could suggest a poor nutritional status.

Laboratory diagnosis of anemia: are the old and new red cell parameters useful in classification and treatment, how?

INTRODUCTION: Anemia is a global problem affecting the population in both developing and developed countries, and there is a debate on which hemoglobin level limit should be used to define anemia in general population and particularly in the elderly. We present herein a laboratory approach to diagnosing the possible causes of anemia based on traditional and new erythroid parameters. In this article, we provide practical diagnostic algorithms that address to differential diagnosis of anemia. Based on both morphological and kinetic classifications, three patterns were considered: microcytic, normocytic, and macrocytic. METHODS: Main interest is on the clinical usefulness of old and new parameters such as mean cell volume (MCV), red blood cell distribution width (RDW), hypochromic and microcytic erythrocytes, immature reticulocyte fraction (IRF), and some reticulocyte indices such as reticulocyte hemoglobin content and mean reticulocyte volume. The pathophysiologic basis is reviewed in terms of bone marrow erythropoiesis, evaluated by reticulocyte count (increased or normal/decreased) and IRF. The utility of reticulocyte indices in the diagnosis of iron-deficient erythropoiesis (absolute or functional) and in monitoring of response to treatment in nutritional anemia (iron and cobalamin) was also investigated. RESULTS: For each parameter, the availability, the possible clinical applications, and the limitations were evaluated. A discussion on intraindividual biological variation and its implication on the usefulness of conventional reference intervals and in longitudinal monitoring of the patients was also reported. CONCLUSION: Red cell parameters and reticulocyte indices play an essential role in differential diagnosis of anemia and in its treatment. More efforts are needed in harmonizing parameters whose results are still too different when produced by different analyzers.

Time-course of interleukin-2 receptor expression in interferon beta-treated multiple sclerosis patients.

The time-course of CD25 (the 55-kD/alpha subunit of the interleukin-2 (IL-2) receptor) expression on CD4+ T lymphocytes, and serum levels of soluble IL-2 receptors (sIL-2R) and IL-2 were evaluated in relapsing-remitting multiple sclerosis (RRMS) patients treated with interferon beta-1b (IFNbeta1b). Peripheral blood samples were collected before therapy (T0), and 1 (T1), 2 (T2), 3 (T3), 6 (T4), and 12 (T5) months after therapy initiation. While at T1 and T2, half the patients showed an increased number of circulating CD4+ CD25+ lymphocytes and an up-regulation of CD25 expression, at T3 this T-cell subset was significantly reduced in all the patients. From T4 to T5, however, the progressive return to pretreatment values was observed. Serum sIL-2R levels were not significantly affected by IFNbeta1b at any time point. IL-2 was detected in only a few patients at T0, and never at T1 to T5. The transient up-regulation of CD25+ expression that occurred in about 50% of the patients may explain the unchanged relapse rate observed during the first 2 to 3 months after starting IFNbeta1b therapy. Our study demonstrates that IFNbeta1b down-regulates CD25 expression in vivo. This effect, however, was observed only after 3 months of therapy, and was followed by the return to pretreatment values after 6 to 12 months. Taken all together, our findings suggest that IFNbeta1b only transiently affects CD25 expression in vivo, and that this effect cannot account for the reported long-lasting beneficial action of IFNbeta1b on RRMS.

Effect of IFNbeta and anti-IFNbeta antibodies on NK cells in multiple sclerosis patients.

We analysed longitudinally the numbers of CD3-CD16+ (natural killer cells, NK) and CD3-CD57+ cells (a subset of NK) in 15 IFNbeta1b- and 12 IFNbeta1a-treated relapsing-remitting multiple sclerosis (RRMS) patients. IFNbeta1b (Betaferon)-treated RRMS patients showed a rapid and marked reduction in the number of both NK subsets which started 1 month after therapy initiation, and reached highest significance after 3 months (P=0.000); however, figures reverted to pre-treatment values following the appearance of anti-IFNbeta antibodies. In IFNbeta1a (Avonex)-treated RRMS patients, the decrease in both CD3-CD16+ and CD3-CD57+ cell number was slower but more persistent; anti-IFNbeta antibodies were only rarely detected in these patients, and at lower titers than in IFNbeta1b-treated ones. Our findings suggest that NK cells might be one of the major immunological targets of IFNbeta-based treatments.

Sysmex SE-9000 hematology analyzer: performance evaluation on leukocyte differential counts using an NCCLS H20-A protocol. National Committee for Clinical Laboratory Standards.

We evaluated the performance (ie, imprecision, inaccuracy, and analytic sensitivity) of the Sysmex SE-9000 commercial hematology analyzer (TOA Medical Electronics, Kobe, Japan) on differential leukocyte counts according to the National Committee for Clinical Laboratory Standards H20-A protocol. The results obtained were compared with those from the Bayer H6000 and H3 (Bayer Diagnostic Division, Tarrytown, NY), the Coulter MAXM (Miami, Fla), and the microscopic method. Altogether, samples from 462 subjects were analyzed. The results show a substantial superimposition of reference intervals between the methods. The imprecision of the SE-9000 is low for all the leukocyte subpopulations, with the exception of basophils (coefficient of variation: neutrophils, 3.35%; lymphocytes, 4.25%; monocytes, 7.9%; eosinophils, 9.5%; and basophils, 44.2%) and is consistently lower than that of manual counts. The correlation with other methods is high, with the exception of basophils (r2: neutrophils, 0.94-0.95; lymphocytes, 0.93-0.97; monocytes, 0.76-0.85; eosinophils, 0.96-0.99; and basophils, 0.02-0.56). When compared with the microscopic method, an overestimation of neutrophils is seen mostly at low concentrations (mean difference, 2.63), and an underestimation of lymphocytes is seen at high concentrations (mean difference, -3.1). The clinical sensitivity was good, with an agreement of 75.7% on morphologic and 89.6% on distributional abnormalities. With a new analytical channel for immature cells (IMI), the analyzer shows high sensitivity in detecting immature cells of the granulocytic lineage (from 94.4% for immature granulocytes to 96% for myeloblasts).

Flow cytometric reticulocyte counting. Parallel evaluation of five fully automated analyzers: an NCCLS-ICSH approach.

We performed a parallel evaluation of 5 automated reticulocyte analyzers. The guidelines were those proposed by the National Committee for Clinical Laboratory Standards and the International Council for Standardisation in Haematology. Duplicate analyses were performed for 225 healthy subjects and 115 patients affected by various diseases. The reference intervals were different for each method (ADVIA 120, 27-125 x 10(3)/microL [27-125 x 10(9)/L]; CELL DYN 4000, 25-108 x 10(3)/microL [25-108 x 10(9)/L]; GEN-S, 20-85 x 10(3)/microL [20-85 x 10(9)/L]; SE 9500 RET, 23-95 x 10(3)/microL [23-95 x 10(9)/L]; and VEGA RETIC, 30-130 x 10(3)/microL [30-130 x 10(9)/L]). The comparisons of percentage counts with the microscopic reference method were satisfactory for all automated methods. However, a tendency to overestimate at low counts was noted. This progressively increased from the SE 9500 RET to the VEGA RETIC. The imprecision was excellent for all the methods at normal and high concentrations. This was higher at low concentrations. When compared with the microscopic reference, the analyzers showed satisfactory sensitivity at low counts and excellent sensitivity at high counts. The overall agreement varied from 74.8% for the GEN-S to 86.1% for the SE 9500 RET.

Evaluation of four automated hematology analyzers. A comparative study of differential counts (imprecision and inaccuracy).

The authors evaluated the performance of four modern, commercially available hematology analyzers for imprecision and inaccuracy in determining the leukocyte differential count. The evaluation was performed according to International Committee for Standardization in Haematology protocols and the National Committee for Clinical Laboratory Standards H20-T standard, using the same group of patients simultaneously. Imprecision was very low among all the analyzers for neutrophils and lymphocytes (coefficient of variation maximum = 4.12%), whereas for the other leukocyte populations it tended to increase as their presence percentage decreased. The imprecision of the analyzers was still lower than that of the microscopic method. The correlation with the manual 800 cell count (inaccuracy) was good for neutrophils, lymphocytes, and eosinophils (r = 0.974 to 0.888), less so for monocytes (r = 0.757 to 0.490), whereas it was poor for basophils (r = 0.532 to 0.078).

Nutritional parameters, body composition, and progression of disability in older disabled residents living in nursing homes.

BACKGROUND: The evaluation of nutritional status is one of the primary components of multidimensional geriatric assessment. We investigated the relationship between some markers of malnutrition and the modifications in functional status in a sample of older disabled residents living in nursing homes. METHODS: Ninety-eight subjects who were independent in at least two activities of daily living (ADLs) were enrolled in a 2-year longitudinal study. Anthropometric, nutritional, and metabolic parameters, as well as body composition, were measured at baseline and after 2 years. RESULTS: Deteriorating functional status (> or =2 additional lost ADLs) was associated with baseline albumin levels (Tertile 3 vs Tertile 1; odds ratio [OR] 0.16, 95% confidence interval [CI] 0.04-0.67) and subscapular skinfold thickness (Tertile 3 vs. Tertile 1; OR 0.06, 95% CI 0.006-0.50). After multivariate adjustment, the OR for increasing disability was >4 in subjects with decreasing body cell mass (BCM), compared with subjects with a stable BCM. The degree of BCM reduction was strongly related to the number of additional ADLs lost at follow-up (test for trend, p = .003). CONCLUSIONS: In a sample of older disabled nursing home residents, signs of malnutrition seem to predict further worsening in functional status. Furthermore, BCM declines proportionally to the loss in ADLs, suggesting the existence of a strong relationship between BCM loss and the progressive deterioration of functional status.

Laboratory evaluation of the Miles H.3 automated reticulocyte counter. A comparative study with manual reference method and Sysmex R-1000.

OBJECTIVE: To evaluate the performance of the new commercial Miles H.3 RTX analyzer in counting reticulocytes. METHODS AND PATIENTS: The results from the counter were compared to those obtained from microscopic methods, following the National Committee for Clinical Laboratory Standards H44-P guidelines, and to the results from the Sysmex R-1000 counter. In total, 279 samples were analyzed in duplicate with each of the three methods. One hundred thirty-three samples were from healthy subjects, while 146 were from patients with various pathologies, 10 of whom presented with posttherapeutic aplasia of the bone marrow and 9 with iron-deficiency anemia. RESULTS: The reference intervals for the normal controls are different for each of the three methods (manual: 0.35-2.35%, 16 to 116 x 10(9)/L; Miles H.3: 0.65-2.30%, 35.1 to 112.0 x 10(9)/L; Sysmex R-1000: 0.6-1.85%, 28.0 to 85.0 x 10(9)/L). The overall imprecision was lower for the instruments than for the microscopic method (Miles H.3: coefficient of variation, 11.6%; R-1000: coefficient of variation, 4.2%; microscopic method: coefficient of variation, 24.2%). The Miles H.3 shows a good correlation with the other methods, yet it overestimated the low values with respect to both the microscopic method (intercept, 0.55; slope, 0.70) and the R-1000 (intercept, 0.44; slope, 0.78). This became particularly pronounced in patients with marrow aplasia. CONCLUSIONS: Miles H.3 can produce results with an acceptable degree of accuracy. The agreement with the dedicated fluorescence-based flow cytometer R-1000 at normal and high concentrations is also good. The possibility of providing reticulocyte indices as well as erythrocyte indices (mean volume, mean hemoglobin content, mean hemoglobin concentration) and the relative dispersion indices could be useful in understanding red cell pathophysiology in normal and iron deficient patients.

Diagnostic performance: a comparative study of the leukocyte differential count on four automated haematology analysers.

The diagnostic performance of the haematology analysers, Technicon H1, Sysmex NE 8000, Coulter STKS and Sequoia Cell Dyn 3000 was determined by comparing their results for automated differential counting with the result obtained for 400 cells by the manual microscopic method. These comparative studies were performed on a group of adults, containing both normal individuals (n = 150) and those affected by various pathologies (n = 113). The number of morphologically false negatives is low for all the systems (from 1.8% for the Cell Dyn 3000 to 6.2% for the NE 8000), while the number of distributionally false negatives was slightly higher (from 4.4% for the Cell Dyn 3000 to 7% for the H1 and NE 8000). The morphological flags, though useful for improving the diagnostic performance of the instruments, show a rather modest sensitivity when taken individually: immature granulocytes/bands from 71% for the STKS to 43% for the NE 8000; blasts from 66.6% for the H1 to 53.3% for the STKS, atypical lymphocytes from 59% for the H1 to 13.6% for the NE 8000; erythroblasts from 42.8% for the STKS to 7% for the NE 8000.

Reticulocyte quantification by Coulter MAXM VCS (volume, conductivity, light scatter) technology.

In this study the ability of the Coulter MAXM analyzer to quantify reticulocytes was evaluated. The results were compared with those obtained by a microscopic method according to NCCLS H44-P recommendations and with the results from the automated analyzer Sysmex R-1000. Duplicate samples from 330 patients were analyzed. The reference intervals obtained with the three methods were: MAXM 0.37-1.80%, median 0.83%; manual 0.40-2.30%, median 1.00%; R-1000 0.60-1.95%, median 1.06%. The imprecision (CV) at all concentrations is lower than the microscopic method (low 16.1% vs 67%; normal 16.9% vs 28.9%; high 9.5% vs 13.0%). The MAXM shows a good overall correlation with the microscopic method (intercept 0.01, slope 0.89, r2 = 0.87) despite evidence of a significant overestimation at low concentrations [difference (D) 0.30] and a moderate underestimation at normal (D -0.15) and high (D -1.04) concentrations; the same behavior is shown in comparison with results from the R-1000, with an overall underestimation by MAXM (D -0.26). When compared with the microscopic method, MAXM shows a modest sensitivity at low reticulocyte counts (54.8%) and satisfactory sensitivity for high counts (87.3%), with an overall agreement of 81.3%.

Time-course analysis of CD25 and HLA-DR expression on lymphocytes in interferon-beta 1b-treated multiple sclerosis patients.

To identify immunological markers that could be used to monitor relapsing-remitting multiple sclerosis (RRMS) course/activity during interferon beta 1b (IFN beta 1b) therapy, we longitudinally studied HLA-DR and CD25 expression by T lymphocytes in 15 IFN beta 1b-treated RRMS patients. Peripheral blood T cell subsets were analysed before therapy (T0), and after 1 (T1), 2 (T2), 3 (T3), 6 (T4) and 12 (T5) months after therapy initiation. HLA-DR expression and the CD3+HLA-DR+ T cell number showed a peculiar trend in almost all (14/15) the patients: a significant decrease at T1 and T2 followed by a return to pre-treatment levels from T3 to T5. At T1 and T2, eight patients showed an up-regulation of CD25 on CD4, as well as an increase in the CD4+CD25+ cell number. However, a marked, significant reduction of this T cell subset was observed in all the patients at T3, followed by the progressive return to pre-treatment values from T4 to T5. All the patients developed anti-IFN beta 1b 'binding' antibodies within the first three months of therapy. Our findings demonstrate that: (1) the expression of HLA-DR and CD25 on T cells, as well as the number of circulating CD3+HLA-DR+ and CD4+CD25+ cells, are only transiently reduced in vivo in IFN beta 1b-treated RRMS patients, (2) the expression of HLA-DR and CD25 on T lymphocytes cannot be used to monitor MS course/activity during IFN beta 1b therapy, (3) the long-lasting beneficial effect of IFN beta 1b on RRMS reported in the literature cannot be explained by the down-regulation of MHC class II antigens and/or interleukin-2 receptor expression induced by this cytokine.

International Council for Standardization in Haematology (ICSH) recommendations for "surrogate reference" method for the packed cell volume.

The spun packed cell volume (PCV, hematocrit) is a key measurement on which are based hematology instrument calibration, reference range determination, and assignment of values to calibrators/controls. In 2001, the International Council for Standardization in Haematology (ICSH) recommended a Reference PCV method, which is fully traceable to the ICSH reference hemoglobin method. Because of its complexity, however, this method is impractical for occasional use in routine laboratories and is therefore intended primarily for use by manufacturers of capillary microhematocrit tubes, liquid calibrators, and multichannel analyzers. In response to the need for a simpler method--accessible to all routine laboratories--the ICSH offers this "Surrogate Reference" PCV procedure. It is traceable to the original ICSH Reference PCV method and is based on spun PCVs obtained using borosilicate capillary tubes with an already-known relationship to this reference procedure. This ICSH "Surrogate Reference" PCV method is substantially simpler, thus putting it within the reach of most routine hematology laboratories.