Validation of Sysmex XT-2000iV generated quantitative bone marrow differential counts in untreated Wistar rats.

BACKGROUND: Preclinical drug trials frequently require assessment of bone marrow toxicity in animals to evaluate hematopoietic safety. Since the gold standard, cytologic evaluation, is time consuming and requires highly trained individuals, automated methods remain intriguing. OBJECTIVE: The Sysmex XT-2000iV hematology analyzer allows user-developed customizable gating. This study was conducted to validate the gating of bone marrow cell populations in Sysmex cytograms from untreated rats. METHODS: B- and T-lymphocytes and myeloid cells were experimentally depleted from Charles River Wistar Han IGS (CRL: WI [Han]) rat whole bone marrow suspension using a magnetic cell sorting (MACS) method. The positively and negatively selected populations were used to verify select gates within the Sysmex cytogram. Intra- and inter-animal precision, comparability between right and left femur, as well as agreement with microscopic myelograms based on 500 counted cells, were determined. RESULTS: Intra-sample precision and right-to-left femur comparability confirmed that gating was reproducible and stable. In 50 tested rats, myeloid to erythroid ratios (M:E) were 1.32 ± 0.33 in males and 1.38 ± 0.29 in females by Sysmex compared to 1.36 ± 0.32 in males and 1.42 ± 0.32 in females by microscopic evaluations. Bland-Altman differences between methods was ≤ ± 0.35 units for M:E, ≤ 5.4% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, ≤ 6.0% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, and ≤ 4.1% for lymphocytes. CONCLUSIONS: In untreated control Charles River Wistar Han IGS (CRL: WI [Han]) rats, the Sysmex XT-2000iV produced an automated M:E and 5-part differential count equivalent to microscopic differential counts.

The application of Beckman Coulter VCS technology at a major cancer center, with emphasis on the detection of circulating immature plasma cells in plasma cell leukemia.

The St. Vincent's Comprehensive Cancer Center (SVCCC) has a large multiple myeloma program in downtown New York City. The laboratory at SVCCC is an integral part of the diagnosing and monitoring of its myeloma patients. Circulating plasma cells are not a common finding in multiple myeloma. Being able to detect plasma cells in peripheral blood is important because they are a prognostic indicator that correlates with disease progression. Furthermore, the peripheral blood plasma cell population can demonstrate morphologic variability. Immature plasma cells, both plasmablasts and proplasmacytes are associated with more aggressive disease and shortened survival. We encountered 3 multiple myeloma patients with circulating immature plasma cells that appeared as distinct populations on our hematology analyzer's automated white blood cell (WBC) differential. The immature plasma cells, given their unique cellular characteristics, appeared in a common place within the WBC differential scatterplot in each patient. In our laboratory, we have utilized this common graphic pattern to screen for immature plasma cells. This pattern has proven to be a useful tool in our large population of multiple myeloma patients. We have also used examination of the scatterplots in other hematologic malignancies such as chronic lymphocytic leukemia. Using this review policy, the laboratory has been able to achieve a smear review of 25% in our highly abnormal patient population.

Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoietic cells.

Two important parameters for determination of deleterious effects of cellular processing on hematopoietic progenitor cells are cell viability and concentration. The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Hospital evaluated the Beckman Coulter Vi-Cell automated instrument for the measurement of these two parameters. Using 33 thawed hematopoietic progenitor cell samples, automated Vi-Cell viability results were compared to those obtained using the standard trypan blue manual method. In addition, cell concentrations from these samples were compared with results from the Model Z2 Coulter Counter. Chinese Hamster Ovary cells were used for the evaluation of Vi-Cell linearity at the Beckman Coulter Cellular Analysis Development Center. Significant correlation was obtained when the two methods were compared for both cell concentration and percentage viability (P < .0001). The results of the linearity study indicated that the Vi-Cell is linear from approximately 5 x 10(4) to greater than 1 x 10(7) cells/mL. The Vi-Cell uses sample volumes as low as 0.5 mL; cell diameters may be 2 to 70 microns. The Vi-Cell automated instrument offers many significant advantages for cell analyses in today's busy laboratory environment.

Direct analysis of pesticide residues in olive oil by on-line reversed phase liquid chromatography-gas chromatography using an automated through oven transfer adsorption desorption (TOTAD) interface.

A fully automated on-line reversed phase liquid chromatography-gas chromatography system is described. The system uses a prototype of the automated through oven transfer adsorption desorption interface. The system is demonstrated by presenting a new rapid method for the determination of pesticide residue in olive oil, which is injected directly with no sample pretreatment step other than filtration. Methanol:water is used as the eluent in the LC preseparation step, while the LC fraction containing the pesticide is automatically transferred to the gas chromatograph. Detection limits of pesticides varied from 0.18 to 0.44 mg/L when a flame ionization detector was used. As an example, relative standard deviation and linear calibration are presented for terbutryne.

Reversible shear-mediated platelet dysfunction during cardiac surgery as assessed by the PFA-100 platelet function analyzer.

We undertook this investigation to assess alterations in shear-mediated platelet function during cardiac surgery and to determine the potential for the PFA-100 to predict post-operative bleeding. Platelet aggregation and PFA-100 closure times were determined in 18 adult patients at five intervals during cardiac surgery. Associations between post-operative bleeding and closure times were examined in an additional 58 patients. Statistical analysis consisted of Student's t, Wilcoxon signed rank, and Spearman correlation tests. All results are reported as mean +/- SEM. Collagen/epinephrine closure times were prolonged prior to and throughout surgery. Collagen/adenosine-5'-diphosphate (ADP) closure times were significantly prolonged by heparin administration, 141 +/- 15 s versus 115 +/- 10 s (P = 0.01), and subsequent initiation of cardiopulmonary bypass (CPB), 203 +/- 12 s (P= 0.0001); however, 15 min after protamine administration, closure times returned to near pre-operative values, 138 +/- 12 s (P = not significant). In contrast, platelet aggregation in response to ADP remained impaired in 17 of 19 patients after CPB. Neither ex vivo correction of sample hematocrits nor supplementation with Humate P affected closure times. Positive and negative predictive values for post-CPB collagen/ADP closure times to predict bleeding were 18 and 96%, respectively. These results suggest that factors both intrinsic and extrinsic to the platelet contribute to reversible shear-mediated platelet dysfunction during CPB, and that the PFA-100 may prove useful after CPB to identify patients unlikely to benefit from platelet transfusions.

Selection and implementation for coagulation instruments/reagents in a multiple hospital/clinic network.

Selection, standardization, and implementation of instrumentation and reagents throughout a health care facility network can often be a difficult process. However, in today's ever-changing health care setting, it is often mandated. The Veteran's Integrated Systems Network 16 (VISN 16) was faced with such a task early in 1999, with the targeted area being its coagulation laboratories. The plan outlined in this paper was drafted to help facilitate the selection, standardization and implementation of coagulation systems for 17 health care facilities that make up the VISN 16 network. The VISN, encompassing 170,000 square miles, has 10 tertiary care hospitals, six of which have close relationships with affiliate universities. There are 299,733 patients enrolled in this health delivery system. The facilities range from large institutions performing both tertiary and outpatient care to small outpatient clinics. Because of the plan's detailed, comprehensive content, which included analyses of a large number of performance parameters as well as cost-efficiency, the selection process was carried out using a checklist that could be helpful to other organizations selecting equipment and reagents for coagulation studies. An implementation process was devised, resulting in coagulation standardization across the Integrated Health Network.

A barbiturate screening assay for the Abbott AxSYM analyzer.

A fluorescence polarization immunoassay for barbiturates on the Abbott AxSYM analyzer is described. The assay displayed dilution linearity up to 1200 ng/mL; coefficients of variation varied from 5.96 to 8.61%; recovery varied from 94.9 to 105.3%; and sensitivity was less than 40 ng/mL. Good correlation between the standard six- and factory two-point calibration methods was observed. The Immunoassay demonstrated good cross-reactivity to several commonly prescribed barbiturates; low cross-reactivity with structurally similar compounds; low interference from endogenous substances, dyes, preservatives, and several commonly available adulterants; and good correlation with the TDx Barbiturate Urine assay.

[Experimental analytical electron microscopic studies on the quantitative analysis of elemental concentrations in biological thin specimens and its application to dental science].

The aim of this study is to employ an energy dispersive X-ray spectrometer (EDS) in developing a computer software system for the quantitative analysis of elemental concentrations in biological specimens. The methods and the software were applied to the examination of the coronal dentin of human deciduous and permanent teeth. Results 1. Examination methods. Chemical compounds known for their calcium (Ca) and phosphorus (P) contents were used to determine optimum conditions for analysis. The following were the best analytical conditions: 100 kV accelerating voltage, 2 x 10(-10) A probe current, 10 ekV energy scale, 100 sec. counting time, and 100-150 nm section thickness. Under these conditions, it is possible to obtain statistically sufficient integral spectra values. By calculating with the computer software(t-factor) developed in this study, it was possible to arrive at analytical calcium and phosphorus concentration values that are very close to theoretical values. 2. Application to human dentin. Deciduous intertubular dentin contained 24.9% (w/w) Ca and 12.1% (w/w) P; peritubular dentin in the same teeth contained 30.7% (w/w) Ca and 15.3% (w/w) P. Permanent intertubular dentin contained 25.5% (w/w) Ca and 12.5% (w/w) P; peritubular dentin in the same teeth contained 34.5% (w/w) Ca and 16.9% (w/w) P. These results show that, in both permanent and deciduous teeth, concentrations of Ca and P are higher in peritubular than in intertubular dentin. Concentrations Ca and P in both peritubular and intertubular dentin are lower in deciduous than in permanent teeth. The computer software developed for this study differs from chemical analysis and may prove very useful in microanalysis of mineralized tissues on the basis of their ultrastructures.

Primary hyperparathyroidism: its clinical pattern and results of surgical treatment in Hong Kong Chinese.

Primary hyperparathyroidism has been increasingly diagnosed among whites since the advent of the biochemical autoanalyzer. However, the condition remains uncommon among Orientals. Our experience with 31 patients at the Queen Mary Hospital, University of Hong Kong, in the periods before and after we began to use the biochemical autoanalyzer was reviewed. The prevalence of primary hyperparathyroidism rose slightly from 3.1 to 3.7 per 100,000 hospital population. In both periods skeletal manifestation was the major clinical presentation, followed by hypercalcemic symptoms and urologic disease. Asymptomatic hypercalcemia occurred in three of 31 patients despite the use of the biochemical autoanalyzer. Preoperative localization was carried out in 27 patients and was helpful in nine (33.3%) of them. The surgeon explored all four parathyroids, removed the diseased gland(s), and examined a biopsy specimen of one normal-appearing gland. There were 27 adenomas, two carcinomas, one four-gland hyperplasia, and one sarcoidosis. Twenty-eight patients had transient hypocalcemia after parathyroidectomy and required calcium supplements for variable periods. Before and after we began to use the biochemical autoanalyzer, there was minimal change in the prevalence and clinical pattern of primary hyperparathyroidism seen in our hospital. In our experience, successful parathyroidectomy depends more on the surgeon's operative technique than on preoperative localization.

A rule-based process control method with feedback.

This paper describes a method for developing a rule-based control algorithm for process control that includes feedback and modification of the rule base from samples of the process output. The rules are used to guide the process toward the desired goal, or goals, and as the process operates new data samples allow the inference of new rules so that the process is automatically optimized and the rules for controlling the process are automatically generated.

A risk of inaccuracy in biochemical analysis of urinary stone-forming salts.

We investigated the occasional inaccuracy in the biochemical measurement of urine by comparing the values from untreated samples to those obtained following our experimental pre-treatment regimen, which prevents precipitation and dissolves visible crystals. Discrepancies were observed between some of the paired samples, especially with regard to calcium and uric acid (urate) concentrations. Moreover, considerable differences were recognized in fresh as well as stored urine specimens. Our findings demonstrate the need to develop accurate methods of processing urine for biochemical analysis with multichannel autoanalyzers.

Automated sample cleanup for pesticide multiresidue analysis. Part II--Design and evaluation of column chromatography module.

An automated, continuous flow system is described for Florisil column chromatography of pesticide residues from food extracts. Evaluation of the system using 5 common organochlorine and organophosphorus pesticides in 2 crop matrices demonstrates essentially no difference in recovery or precision between automated and currently used manual analyses. The automated procedure uses only 20% of the solvents and adsorbents used in the manual procedure and is 3 times faster.

Simultaneous determination of protein (nitrogen), phosphorus, and calcium in animal feedstuffs by multichannel flow-injection analysis.

A 3-channel flow-injection procedure was developed, which enables the simultaneous determination of protein, phosphorus, and calcium in a wide range of animal feeds from a single digestion. Samples are digested with a block digestor, diluted, and analyzed at a rate of 82 samples/h. Protein (nitrogen) as ammonia is determined colorimetrically by the indophenol method. Phosphorus and calcium are determined by measuring the absorbances of the molybdenum blue and calcium-cresolphthalein complexes at 660 and 580 nm, respectively. Protein is determined in the range from 0 to 75%, phosphorus in the range from 0 to 6%, and calcium in the range from 0 to 6%. The results obtained do not differ significantly from those obtained by proven manual methods, and considerable time, space, and reagents are saved.

[Automated screening and identification of irregular anti-erythrocyte antibodies]. / Dépistage et identification automatisés des anticorps irréguliers anti-érythrocytaires.

Screening of irregular antibodies performed on a Groupamatic MG50, a two or three channels auto-analyser, and with manual techniques, revealed 4,445 positive sera which were than identified on this auto-analyser system. It was concluded that the "saline-PVP-4 degrees C" channel recovers 17% of the screened sera, which appeared negative with the bromelin and polybrene channels. Saline -4 degrees C detected sera include all of the -M and -P1 specificities and about half of the -K, -Lea and Leb. Identification of cold and namely anti-I antibodies on this channel also leads to an improved analysis of polyspecific sera. The high sensitivity of this 3 channels auto-analyser seems to be optimal for screening and identification of irregular antibodies.

Low results for inorganic phosphorus with the SMAC continuous-flow analyzer.

Serum inorganic phosphorus concentrations as measured with the SMAC are lower than those found with other methods. To resolve this problem we analyzed patients' specimens and performed analytical recovery studies with four different systems (SMAC, AutoAnalyzer II, aca, and the Fiske--SubbaRow method). With the SMAC, results for patients' specimens are significantly lower (p less than 0.0001) than with any of the other three methods. The SMAC recovered only about 87% of the added inorganic phosphorus. The value assigned to SMAC Reference I for inorganic phosphorus was 0.876 of the value obtained when the material was analyzed by the reference method. Thus there is a significant systematic error in the SMAC method for inorganic phosphorus determination, attributable to an erroneous inorganic phosphorus concentration assigned the SMAC calibration material by the supplier (Technicon).

Automated gel permeation chromatographic cleanup of animal and plant extracts for pesticide residue determination.

Applications of an improved gel-solvent system for cleanup of pesticide residues by gel permeation chromatography (GPC) were investigated. Elution characteristics using Bio-Beads SX-3 gel and a toluene-ethyl acetate (1+3) elution solvent were determined for 16 nonionic chlorinated pesticides, 3 polychlorinated biphenyls (PCBs), 14 chlorophenoxy herbicide esters, and 7 organophosphate insecticides. Elution patterns for vegetable and animal lipids were also studied. Quantitative recoveries of the pesticides were achieved. No liquid-liquid partitioning cleanup steps were required with any type of nonionic chlorinated pesticide or sample matrix. Only GPC cleanup was required for the nonionic chlorinated pesticides, PCBs, and organophosphate pesticide residues in chicken and turkey fat samples. Electron capture and flame photometric detectors were used in the gas chromatographic method for the respective pesticides. Samples containing up to 0.5 g lipid each were processed at the rate of one every 30-40 min with the automated system. Results were in excellent agreement with those from accepted manual partitioning methods and were achieved with significant savings of both labor and chemicals.

A proposed blind routine quality control system for multichannel analyzers.

A blind quality control system using a special type of commercial reference serum is described. As packaged, the product contains six pools of analyzed reference sera with vials identified by coded numbers. The test constituents in the pools cover the range of multichannel analyzers in a random manner, i.e., each pool contains constituents in the low, normal and elevated ranges. As specific pools are not identified, and the analyst is unaware of the expected values for each constituent, results collected for quality control purposes are unbiased, i.e., free of conscious or unconscious editing. Data analysis in the proposed system is by least-squares technics. During an eight-week period, performance was studied using the standard error of the estimate to evaluate precision and the slope of the regression line to evaluate accuracy on the SMA and Mark X instruments. As an empirical comparison, the data are expressed as relative errors and precision and accuracy of analyzer performance evaluated as mean and standard deviation. The proposed regression analysis-based technic has the advantage of simultaneously evaluating linearity, as well as checking accuracy and precision over the total dynamic range of the instrument by use of a series of related serum pools.