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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 × 109/liter vs. 0.456 ± 139 × 109/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. Cytometry 30:72–84, 1997. © 1997 Wiley‐Liss, Inc.

Changes in key enzymes of oxidative metabolism at the mitochondrial level are known to be associated with the aging process, apoptosis, and many diseases. Considering the risk of acquiring a myelodysplastic syndrome (MDS) with age, the aim of this study was to quantify mRNA synthesis of the carnitine palmitoyltransferases (CPT1 and CPT2), carnitine acetyltransferase (CRAT), human specific microsomal CPT, and OCTN2 (organic cation transporter) in mononuclear cells of healthy humans of different age groups and MDS patients. Using quantitative reverse transcriptase real-time PCR we compared mRNA synthesis of the above mentioned enzymes in mononuclear cells from peripheral blood of 23 healthy persons (mean age 45 years), 9 blood and 22 bone marrow samples of 31 MDS patients with varying proportions of apoptotic cells (mean age 78 years), and blood samples of 30 age-matched controls. In addition, plasma carnitine levels were determined. Compared to younger adults, there was a 50% downregulation of CPT1 in elderly persons and in MDS patients. Reduction in CRAT, CPT 2, and OCTN2 was more than 85%. Reduction in microsomal CPT was more pronounced in MDS patients than in age-matched controls (96% vs. 43%). In MDS bone marrow cells there was a negative correlation of CPT1 and CRAT with the relative proportion of apoptotic cells. Plasma carnitine values were similar in all groups. The described reduction in transcription of different genes in blood cells which is well known in different tissues may reflect a systemic signaling process, associated with aging, apoptosis, and MDS.

Flow cytometric enumeration of monocytes stained with fluorescence-labelled monoclonal antibodies has been proposed as a possible reference method for monocyte counting. We compared precision and accuracy of monocyte counting of the Coulter STKS, the Cobas Argos 5 Diff, the 800-cell manual differential, and the Coulter Epics Profile II flow cytometer using double-staining with fluorescence-labelled monoclonal antibodies (CD45-F1TC and CD14-PE).Precision: STKS, Argos and Profile II achieved a precision analogous to a 3423-, 1298-, and 11089-cell differential, respectively, confirming the superiority of automated methods. Accuracy (136 normal and abnormal samples): Correlation of automated methods with the manual differential was good (STKS: r = 0.934, Argos 5 Diff: r = 0.808, Profile : r = 0.924; Spearman's rank correlation coefficient). The mean relative STKS monocyte result was 0.52 ± 1.63% (mean i SD) higher than the manual differential, whereas the Argos 5 Diff results were 1.22 ± 2.51% lower (p < 0.001). Profile II results showed a small bias against the manual differential (-0.18 ± 1.44%, p < 0.05).Analysing 135 healthy adult subjects on the Profile II, males were found to have a higher mean monocyte count (relative count: 6.95 ± 1.43% vs. 5.86 ± 0.98%; absolute count: 0.48 ± 0.15 X 10 9 /1 vs. 0.39 ± 0.11 X 10 9 /1, p < 0.001) and a higher and wider normal range than females (relative count: 4.97 to 9.78% vs. 4.26 to 7.81%, absolute count: 0.30 to 0.84 X 10 9 /1 vs. 0.25 to 0.65 X 10 9 /1).Flow cytometry based on fluorescence-labelled monoclonal antibodies for monocyte enumeration seems an efficient tool to evaluate the monocyte counting performance of haematology analysers and an ideal successor to the manual differential as reference method for monocyte counting. IntroductionEvaluations of the differential leukocyte count of haem-cannot be the main reason for this, as the less frequent atology analysers have often yielded satisfactory results eosinophils usually showed good results (1-8, 11). The for neutrophils, lymphocytes, and eosinophils, whereas morphological variety of monocytes definitely poses the performance of monocyte counting has been disap-problems for automated differentiating techniques, anpointing (1^8), even when studying only normal sam-other serious problem being lack of an appropriate referples (9, 10). The correlation with the reference method ence method. The value of the manual 400-cell difwas frequently poor and both accuracy and precision ferential, which is still used as reference in monocyte worse than for other leukocyte classes. Although mono-counting (12), is diminished by subjectivity of the examcytes represent a relatively small leukocyte class, this iner (13) and a low precision for smaller cell populations Eur J Clin Chem Clin Biochem 1995; 33 (No 11) Brought to you by | University of Arizona Authenticated Download Date | 7/20/15 8:35 PM

Background/Aim: A vegetarian diet is known to prevent a series of diseases but may influence the balance of carbohydrate and fat metabolism as well as collagen synthesis. This study compares expression patterns of relevant genes in oral mucosa of omnivores and vegetarians. Methods: Quantitative reverse transcriptase polymerase chain reaction was applied for analysis of mRNA levels from carnitine transporter OCTN2, hepatic CPT1A and nonhepatic CPT1B isoforms of carnitine palmitoyltransferase and collagen (CCOL2A1) in oral mucosa. Results: Compared with volunteers with traditional eating habits, carbohydrate consumption was significantly higher (+22%) in vegetarians. This was associated with a significant stimulation of CPT1A (+50%) and OCTN2 (+10%) and a lowered collagen synthesis (–10%). Conclusion: These novel findings provide further insight into the association of a changed fat metabolism and reduced collagen synthesis in vegetarians, which could also play a role in the aging process.