Studies on the efficient application by STKS and 8200]

We examined the usefulness of differential leukocyte counts provided by the automated STKS (Coulter) using the VCS method, the cell analyzer 8200 (Hitachi) using the image analysing as compared with that of the traditional manual method. In the STKS, about 8000 leukocytes can be counted within one minute and 5 part differential counts (neutrophils, eosinophils, monocytes, lymphocytes) are provided. The STKS may provided adequate identification of patients who have significant leukocyte abnormalities that require further study. Potential deficiencies of this system included an inability to detect abnormal lymphocytes such as ATL cells. In addition, low numbers of erythroblasts were missed, although these cells could be warned by a flagging system. In the cell analyzer 8200, the differential leukocyte count is done by the microscopic examination of stained blood films which is prepared by automatic device. Results from counts performed with this system correlated well with those obtained by the manual method. This system also missed low numbers of erythroblasts and ATL cells. Another potential deficiencies of the system is that number of cells observed within one minute were limited to about 100. When a prescribed set of cut-off limits, carefully chosen qualitatively, is introduced into these systems, automated leukocyte differential counts, with combination of STKS, 8200 and the manual method, clearly improve turn around time for laboratory blood assessments and this help to reduce unnecessary differential leukocyte counts by the manual method.     

Hematologic parameters and leukocyte histogram patterns in infectious mononucleosis

Blood samples from 60 patients with serologically positive infectious mononucleosis were analyzed on the Coulter S-Plus IV. Hemoglobins and platelet counts were essentially normal, and leukocyte counts were elevated in less than half the cases. Reactive lymphocytes were present on blood smears from all patients. The S-Plus IV automated three-part leukocyte differentials showed significantly lower numbers of lymphocytes and more mononuclears and granulocytes than manual microscopic differentials. On the leukocyte histograms, lymphocyte peaks were consistently wider than normal and asymmetrical. The instrument generated R2 flags on 43 of 60 (72%) of the samples. Using a combination of "R" flags, mononuclear backlighting, and "H" flags, the Coulter S-Plus IV identified 59 of 60 infectious mononucleosis samples as requiring further review.     

Accuracy and utility of differential white blood cell count in the neonatal intensive care unit.

The clinical utility of the complete blood cell count (including the differential white blood cell count) as a means to follow the course of infants in a neonatal intensive care unit was assessed. Utility was judged for three purposes: (1) predicting the onset of clinically unrecognized disease, (2) assessing the severity of current disease, and (3) following a trend during treatment. Neither conventional nor automated differential counts were useful for surveillance (predicting the onset of clinically unrecognized disease). The white blood cell count, the platelet count, and the absolute immature neutrophil count and immature/total neutrophil ratio were useful to assess the severity of current clinical events. The white blood cell count was superior to the differential count for following trends in patients' conditions. Information regarding nuclear immaturity derived from automated counts and the cost and slowness of the manual differential count are good indications for decreased use of conventional counts, increased use of certain features of the automated differential, or both, in neonatal intensive care units.     

Manual differential cell counts help predict bacterial infection. A multivariate analysis

We developed logistic regression models that combine information from the automated CBC and manual 100-cell differential counts to predict bacterial infection. The logistic models were fitted from a case group of 116 patients with proven bacterial infection and a control group of 930 presumably uninfected outpatients. A 4-variable, 15-parameter model, which includes automated absolute neutrophil, manual band, and manual immature granulocyte counts, performed best with a receiver operating characteristic (ROC) curve area of 89%. A more practical 2-variable model including automated absolute neutrophil and manual band counts performed almost as well with an ROC curve area of 86%. The automated neutrophil count-only model is less informative with an ROC curve area of 78%. The combined information from automated and manual differential cell counts more accurately predicts bacterial infection than automated counting alone. Despite these modest improvements, the high cost of manual differential cell counts dictates careful patient selection. The supplemental information gained from manual differential counts is most useful for patients with low to normal neutrophil counts (8,000/microL [8.0 x 10(9)/L] or less). Further studies are indicated to determine the characteristic patient populations deriving maximal benefit from this information.     

Performance of an automated immature granulocyte count as a predictor of neonatal sepsis

Neonatologists use immature granulocytes (IG) in manual differential counts as an indicator of sepsis. This study was designed to compare the predictive ability of automated vs manual IG counts for neonatal sepsis. Infants undergoing sepsis evaluation were identified prospectively for study if a CBC count was obtained in temporal proximity to the blood culture. Automated IG counts were obtained from the research software of the Sysmex XE-2100 (Sysmex, Kobe, Japan). Manual average IG counts were obtained from two 100-cell manual differential counts independently performed by a technologist and a hematopathology resident. A comparative analysis of manual and automated IG counts showed considerable overlap of ranges. The highest positive blood culture rate occurred in the nonneutropenic preterm subset of infant older than 7 days (21/55 [38%]). For these infants, elevated IG counts by manual and automated methods were associated significantly with positive blood culture results (odds ratio, manual, 3.74; odds ratio, automated, 3.63), albeit with low sensitivity.     

An evaluation of the Sysmex NE-8000 hematology analyzer

The Sysmex NE-8000 is a new, fully automated hematology analyzer capable of providing a five-part white blood cell differential count and identifying abnormal specimens. This instrument was evaluated on 5,000 consecutive blood specimens and compared to the Coulter S Plus-IV analyzer and manual differential cell counts to determine the efficacy of its five-cell differential and screening capabilities. There was a high correlation between the commercial counters for the standard parameters, white blood cell count, red blood cell count, hemoglobin level, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, and platelet count (r greater than 0.95), except for the mean corpuscular hemoglobin concentration (r = 0.51), for which the NE-8000 was considered the more accurate measurement. Precision and linearity studies were excellent. The white blood cell count, red blood cell count, hemoglobin level, and platelet count were reproducible on specimens stored at 4 degrees C or room temperature for 72 hours and the differential counts were reproducible for 12 hours. The correlations between automated and manual counts for neutrophils, eosinophils, basophils, and lymphocytes were excellent: r = 0.912, 0.945, 0.332, and 0.964, respectively. The monocyte correlation improved with software modification from 0.306 to 0.801. The NE-8000 gave accurate and reproducible differential counts for neutrophils, lymphocytes, and monocytes on specimens with white blood cell counts as low as 0.6 x 10(9)/1. The ability of the instrument to 'flag' abnormal specimens was excellent. The false-positive rate on normal samples was 1.8%, and the false-negative rate on known abnormal samples was 0.3%, due only to nonrecognition of a mild left shift. The identification of specific abnormalities was less precise. The NE-8000 is a powerful hematology analyzer that can perform a five-part white blood cell differential count accurately for a wide range of WBCs and reliably indicate abnormal specimens. It is an excellent screening tool for distinguishing between normal and abnormal specimens and identifying those that require microscopy. Its reliability significantly reduces the need for manual film examination.     

Elimination of instrument-driven reflex manual differential leukocyte counts. Optimization of manual blood smear review criteria in a high-volume automated hematology laboratory

Automated peripheral blood leukocyte differential counts (LDCs) are widely accepted in routine practice. However, many laboratories still reflexively perform manual LDCs based solely on abnormal automated results or instrument "flags," before any manual triage step. We describe our transition to a procedure that uses manual methods to validate, rather than to replace, automated LDCs (an approach recommended early in the development of automated methods, but still not used in many clinical laboratories). Manual microscopic scans were performed in lieu of manual LDCs. Each scan that revealed cell types not quantifiable by the instrument triggered a manual LDC. However, if the manual scan simply confirmed the cell types seen on automated LDC, then the automated result was released, even if clinically significant quantitative abnormalities were present. This policy reduced manual LDCs by more than 70% and was validated by a manual retrospective audit. Patient care and laboratory operations can be optimized by using manual microscopic examination as a validation procedure rather than as a reflexive substitute for automated methods. There is no clinical rationale for reflex performance of manual LDCs based solely on instrument warnings.     

用流式细胞仪检测HIV感染者和AIDS患者的T细胞亚群

目的用流式细胞仪（ｆｌｏｗｃｙｔｏｍｅｔｅｒ,ＦＣＭ）检测周围血中ＣＤ４＋、ＣＤ８＋淋巴细胞,结合临床情况对ＨＩＶ感染者和ＡＩＤＳ患者的免疫状况进行评价。方法将抗凝全血进行白细胞分类计数,用双色荧光标记的单克隆抗体染色,经溶血和固定后,用ＦＣＭ计数,从而得出ＣＤ４＋、ＣＤ８＋淋巴细胞数。结果ＨＩＶ感染者和ＡＩＤＳ患者的ＣＤ４＋淋巴细胞数都比正常人低,特别是ＡＩＤＳ患者,他们的ＣＤ４＋淋巴细胞数都低于２００个／ｍｍ３,临床表现也很差。结论ＦＣＭ检测结果与临床评价高度一致,而且ＦＣＭ比一般人工计数法更准确、方便、迅速,同时也证明ＦＣＭ是监测ＨＩＶ感染者和ＡＩＤＳ患者的免疫状况的最佳方法。     

Manual and automated leukocyte differentiation in bronchoalveolar lavage fluids from rodent models of pulmonary inflammation

Manual total and differential leukocyte counting in bronchoalveolar lavage fluids (BALF) by visual microscopy is a standard means of evaluating airway inflammation and the anti-inflammatory properties of therapeutics in various animal models of lung disease. The manual cell counting method derives total leukocyte counts from BALF with a hemocytometer, and cell differentials (mononuclear, neutrophil, eosinophil) are calculated from the percentage of each cell type taken from a count of at least 200 cells on a stained cytocentrifuge preparation of the BALF cells. These manual methods are time-consuming and have inherent error–variability. The ADVIA 120 Hematology System is an automated analyzer designed to perform total and differential leukocyte analysis of blood. With the light scattering, cell lysis resistance, and cytochemical staining data from a BALF sample processed by the ADIVA, a BALF total leukocyte count and differential analysis is provided in approximately 30 s. In order to correlate automated BALF leukocyte counting by the ADVIA 120 Hematology System with manual counting, we developed a manual red blood cell lysing and white blood cell staining technique for BALF cells similar to the process used by the ADVIA. Significant correlations for BALF white blood cells were obtained for the manual (microscopic analysis) and the automated (ADVIA) methods. Comparison of manual and automated cell counts also generates the same conclusions about anti-inflammatory drug efficacy. Both manual and automated cell counting methods agree that 3 mg/kg orally administered dexamethasone inhibited cigarette-smoke-induced total BALF cell counts by ∼65% in mice and 42 μg/kg fluticasone propionate delivered by nose-only inhalation inhibited allergen-induced total BALF cells by 77% in rats. The use of the ADVIA to perform total and differential leukocyte counts in BALF will save time spent manually counting cells and this instrument will standardize the analysis of white blood cells across the laboratories currently using various manual counting preparations and procedures.     

Assessment of automated DNA extraction coupled with real-time PCR for measuring Epstein-Barr virus load in whole blood, peripheral mononuclear cells and plasma.

BACKGROUND: Epstein-Barr virus (EBV) DNA load monitoring in blood has been shown to be essential for the diagnosis of EBV-associated diseases. However, the methods currently used to assess EBV DNA load are often time-consuming and require prior blood separation. OBJECTIVES: The aim of this study was to evaluate the relative diagnostic value of EBV DNA load monitoring in whole blood, peripheral blood mononuclear cells (PBMCs) and plasma after automated DNA extraction using the MagNA Pure extractor followed by LightCycler real-time quantitative PCR (LC-PCR). STUDY DESIGN: First, EBV DNA load was assessed retrospectively after automated or manual extraction on 104 PBMC specimens. Second, EBV DNA load was determined prospectively with the automated extraction procedure in the whole blood, PBMCs and plasma of 100 samples from patients with EBV-related diseases (group 1, n = 20), HIV-seropositive individuals (group 2, n = 66), and healthy EBV carriers (group 3, n = 14). RESULTS: A good correlation was observed between automated and manual extraction on 104 PBMC specimens (r = 0.956; P < 0.0001). In the prospective study, 67 samples were positive in both whole blood and PBMCs, with a good correlation between EBV DNA loads in whole blood and PBMCs (r = 0.936; P < 0.0001). Only 18/100 samples were positive in plasma. Higher viral loads were regularly observed in the three blood compartments from group 1 than from groups 2 and 3. CONCLUSION: This study demonstrated that an automated extraction of EBV DNA is easier to perform in whole blood or plasma than in PBMCs and facilitates the standardisation of EBV DNA measurement by real-time quantitative PCR. The quantitative detection of EBV DNA load in whole blood appeared more sensitive than in plasma for infectious mononucleosis in immunocompetent patients, probably because of a rapid loss of plasmatic EBV DNA. In transplant patients, EBV DNA load monitoring in whole blood and in plasma turned out to be equivalent in terms of feasibility and accuracy for the early diagnosis of post-transplant lymphoproliferative diseases (PTLDs).     

Evaluation of the Coulter S-Plus IV three-part differential in an acute care hospital

An evaluation of the three-part white cell differential count on the Coulter S-Plus IV was carried out in a 500-bed acute care hospital. Automated counts were compared with manual differential counts over a seven-month period. The S-Plus IV categorizes cells by volume after treatment with diluent and lysing solution. The three categories are labeled lymphocyte, mononuclear, and granulocyte, and, in normal samples, these correspond with lymphocytes, monocytes, and neutrophils. Eosinophils are counted either as mononuclear cells or granulocytes. Automated counts agree well with manual counts for the lymphocyte and granulocyte categories. Agreement is poor for the mononuclear category; this is likely due to the imprecision of the manual differential count. The S-Plus IV accurately classifies most samples as normal or abnormal. When all parameters of the hematology panel were evaluated, the false negative rate was less than 1%. Sixteen percent of results from the S-Plus IV were flagged, and 79% of these indicated an abnormality. False positive flags are usually due to a failure to adequately segregate mononuclear cells from granulocytes. Most commonly encountered abnormalities and hematologic disorders have abnormal three-part differential counts. The authors think the three-part differential cannot totally replace the manual differential but can decrease the number of repeat manual differentials on inpatients. It also offers quality control features useful to the hematology laboratory.     

The "diff-if". Use of microcomputer analysis to triage blood specimens for microscopic examination

Increasingly, all automated blood counts are not accompanied by a microscopic white blood cell differential. A popular strategy is to obtain a manual differential if any part of the automated blood count and differential is outside specified limits (the "diff-if" strategy). The authors compared two sets of criteria to triage blood counts for manual differentials: previously recommended numeric values, and the analysis of a microcomputer program. In a population of subjects with a high percentage of hematologic disorders, the microcomputer program and the numeric criteria were equally specific (excluding normal blood smears); the program was more sensitive for bands, immature granulocytes, monocytes, nucleated red blood cells, reticulocytosis, teardrops, red blood cell fragments, and hypersegmented neutrophils. The numeric criteria were more sensitive for eosinophilia (less than 1.0 X 10(9)/L) and mandated fewer manual differentials. In a population of predominantly normal subjects, the program was more sensitive for increased bands and equally sensitive for eosinophilia, the only abnormalities observed on the smear. In a population of subjects with predominantly abnormal blood counts, but excluding most primary hematologic disorders, there were few blood smears with abnormalities beyond eosinophilia or increased bands. In both of these groups, the computer program mandated more manual differentials than did the numeric criteria. The authors conclude that microcomputer analysis by the program tested was more sensitive than numeric criteria to identify specimens with abnormal blood smears. Specificity depended on the patient population. The choice of a triage strategy should be based on the individual laboratory's patient population.     

Laboratory evaluation of the Coulter "three-part electronic differential"

Comparisons were made between "electronic" white blood cell (WBC) differential counts based on nuclear volume, differential counts performed by an automated image analyzer, and conventional manual counts performed by experienced technologists in a routine laboratory setting. The correlation between methods was excellent for lymphocytes (r = 0.9) and granulocytes (r = 0.9), but none of the three methods that were tested produced good results in enumeration of mononuclear cells (r = 0.6). Approximately 85% of the samples on which differentials were requested for an inpatient population could be processed by the electronic counter. Although the electronic counter failed to flag all abnormal samples (there were 6% false negatives), the performance of technologists doing 200 cell differentials was similar. Rapidly generated "electronic differentials" might be a useful and cost-effective adjunct to inpatient hospital practice if certain suggestions were implemented.     

The new hematology analyzer Sysmex XE-2100: performance evaluation of a novel white blood cell differential technology

CONTEXT: The new hematology analyzer Sysmex XE-2100 (TOA Medical Electronics, Kobe, Japan) has a novel, combined, white blood cell differential technology and a special reagent system to enumerate nucleated red blood cells. DESIGN: Performance evaluation of both technologies of the Sysmex XE-2100 according to the H20-A protocol of the National Committee for Clinical and Laboratory Standards and comparison of the results with those for the hematology analyzer Sysmex NE-8000 (TOA Medical Electronics). SPECIMENS: Five hundred forty-four blood samples randomly chosen from various inpatient and outpatient departments of the Vienna University hospital. RESULTS: Five-part white blood cell differential counts on the XE-2100 revealed excellent correlation with the manual reference method for neutrophils, lymphocytes, and eosinophils (r =.925,.922, and.877, respectively) and good correlation for monocytes and basophils (r =.756 and.763, respectively). The efficiency rates of flagging for the presence of >/=1% abnormal white blood cells were 83% (XE-2100) and 66% (NE-8000). The correlation of automated and microscopic nucleated red blood cell counts was excellent (r =.97). CONCLUSIONS: From the present evaluation and our former experience with other types of Sysmex analyzers, we conclude that the new white blood cell differential technology of the XE-2100 represents a further development toward more efficient flagging of abnormal white blood cells.     

The white blood cell differential. Evaluation of rapid impression scanning versus the routine manual count

A quick, one and a half-minute qualitative microscopic scan was investigated as an alternative approach to the more labor-intensive 100-cell differential white blood cell count. The scanning results of 400 randomly selected hospital cases were compared with the on-line results of the 100-cell counts. Additionally, 50 cases selected to have a high percentage of abnormal results were each scanned and manually counted by four different readers. The results indicate that the scanning differential is equivalent to the 100-cell manual count in the detection of the presence of abnormal cell types such as immature granulocytes and blasts. Its ability to properly estimate the relative proportions of normal cells, especially lymphocytes, however, does not appear as reliable as the manual count. Most importantly, the analysis demonstrates that the scanning differential count exhibits a set of advantages and disadvantages that is complementary to those of the "three-part differential" technic provided by the newer generation automated hematologic analyzers. The authors therefore propose that these two procedures used in combination offer a suitable alternative to the manual 100-cell differential count.     

An intralaboratory evaluation of the Coulter S+II lymphocyte percent (CLy%) as indicative of actual lymphocyte numbers in blood

A field trial of the medical significance and possible error rate associated with the Coulter S+II lymphocyte percent was performed by comparing 1,500 sequential differentials performed on Geometric Data Hematrak instruments to the results of Coulter volume displacement enumeration of lymphocytes on three S+II instruments. The results of this study indicate that a statistically significant but medically insignificant bias is present between lymphocyte counting technics, that the statistical imprecision of the determination of lymphocytes by the Coulter lymph percent (CLy%) falls within the expected range for statistical inaccuracy of determination of lymphocytes by manual differential technics and that in virtually all cases no medically significant errors would result from accepting the CLy% as indicating the actual numbers of lymphocytes present, as all blood specimens with medically significant abnormalities would have had manual differential and slide review triggered by other abnormalities in the blood count results. The Coulter lymph percent is a useful addition to the other complete blood count parameters.     

A study of comparison between Technicon THMS H-2 and Hematrak 590]

Using 655 peripheral blood samples from normal individuals and patients, we evaluated the relationship between the measurements of red blood cell (RBC) and white blood cell (WBC) through the Hematrak 590 (Hematrak) and the generated flag systems in the Technicon H-2 (H-2). 1) Among the 12 VAR (Variation) flag positive samples in H-2, 10 samples were obtained from the patients receiving blood transfusions. The samples with high values of HDW (Hemoglobin distribution width) in H-2 seemed to have hypochromatic and polychromatic changes in RBC. 2) Hematrak is likely to be more sensitive than H-2 in discovering atypical lymphocytes (AL). When AL counts in Hematrak were over 5 or 6% of WBC, these results were coincident with ATYP flag positive in H-2. 3) In our examinations, when %Blast in H-2 was over 4%, BLASTS flag always generated positively. We think that H-2 is a tool with high reproducibility for the detection of the blast-like abnormal cells. 4) We evaluated the results of WBC differential count by the three methods of H-2, Hematrak and manual procedures in the peripheral blood samples from normal individuals. We recognized close correlations in neutrophils, lymphocytes and eosinophils (coefficients of correlation = 0.826-0.911), and relatively close ones in monocytes (r = 0.280-0.562), but no correlations in basophils (r = 0.106-0.281). To comprehend the pathophysiological states of the various diseases hematologically, it is necessary to understand the characteristics of the different measurement principles between the two instruments (H-2 and Hematrak).     

A comparison of differential white blood cell counts using manual technic and the Coulter S-Plus IV

The results of 100-cell and 500-cell manual differential white blood cell counts were compared with those obtained using a Coulter S-Plus IV electronic particle counter. Significant (P less than 0.001) correlations were observed between the manual and instrument-derived data for percentages of granulocytes and lymphocytes.     

Fragmented red cells are the major cause of spuriously high platelet counts in patients with fragmented red cells when counts are obtained by automated blood cell counter

Platelet counts measured by automated blood cell counter often show spuriously high values when measuring samples contain particles of equal size to platelets. The major cause of spuriously high platelet counts in samples with fragmented red cells (FRC) is thought to be the FRC themselves. We studied the correlation between FRC and spuriously high platelet counts in 40 patients demonstrating FRC on blood smears. FRC were measured by manual hemocytometry and by flow cytometry using a monoclonal antibody against glycophorin A (GPA method). There was a significant correlation between spuriously high platelet counts and FRC by manual hemocytometry (r=0.60, p<0.001) or FRC by the GPA method (r=0.45, p<0.005). These data suggest that FRC are the major cause of spuriously high platelet counts in samples with FRC.     

Total hematological analysis system as a screening test for hematological malignancy

Measurement of complete blood cell count and white blood cell differentiation is an essential laboratory test and the most important screening test for hematological malignancy. Recently, several automated blood cell analyzers have been developed to improve accuracy and precision. When flag messages generated in the presence of morphological abnormalities of the samples are displayed, manual revision is necessary. In our laboratory, the manual revision rate has been 35-40%. Therefore blood cell analyzers are useful in screening for abnormalities as well as greatly reducing expensive and time-consuming manual differential procedures. In addition, automated blood cell analyzers can provide several types of useful information including the leukocyte distribution scattergram. However, most such information is not utilized in the clinical field. In the future, a total hematological analysis system will be constructed so that all information provided by automated blood cell analyzer and by manual methods are available.