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Sources of error associated with sample collection and preparation of nucleated blood cells for flow cytometric analysis.

Analysis of cellular DNA content by flow cytometry has been used to detect genetic changes associated with exposure to environmental contaminants. In lower vertebrates, nucleated red blood cells can be collected for analysis without harm to the animal. Because erythrocytes sampled from an individual should have identical amounts of DNA, the coefficient of variation (CV) around the G0/G1 peak should be small. Increases in CV can indicate genetic aberrations, but may also be caused by sample handling and preparation or problems with instrumentation. To increase confidence in associating increases in CV with external causes, artifactual changes in CV due to sample treatment and instrument parameters should be identified and minimized. We assessed the effects of various sampling and handling protocols on the CV of nucleated blood cells collected from largemouth bass (Micropterus salmoides). We also compared the distribution of cells among the G0/G1, S, and G2/M phases of the cell cycle to see whether these were affected by sampling or treatment protocols. Groups of 7 fish were bled on 7 consecutive days, and blood from each fish was analyzed by flow cytometry when freshly collected, and after freezing for 1 hour or 10 days. The same fish were bled again over a consecutive 7-day period, and the experiment was repeated. CV and cell cycle distribution were not affected by our freezing protocol. Repeat sampling from the same individual did not affect CV, but altered the distribution of cells in the cell cycle, suggesting increased hemopoiesis in response to blood sampling.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Changing measurement instrument at follow-up: a potential source of error.

The purpose of this investigation was to determine whether headache activity information collected over the phone can be directly compared with headache activity information collected by systematic self-observation without jeopardizing internal validity because of calibration differences between the two measurement methods. A number of headache studies have relied on phone information for long-term follow-up data, while using systematic self-observation to collect all other data. Twenty-six headache sufferers participating in a tension headache study reported their headache activity over the phone and subsequently charted their headaches. Correlations were computed between the two measures. Results indicated that differences exist in the calibration of the two measurement methods. This seriously limits the conclusions of studies that used phone information to obtain follow-up data. Other recommendations concerning follow-up methodologies are discussed.

Adult

Storage of irradiated human blood; a source of error in quantitative chromosome analysis.

Human whole blood was irradiated with 2.5 Gy of 220 k Vp X-rays and stored before culture with 9.7 microM BrdU and 19.4 or 38.7 microM BrdU for 0, 24, 48 and 72 h. The frequency of dicentrics and ring chromosomes was determined in cells staining as first division (M1) metaphases with the fluorescence plus Giemsa technique. Storage had no influence on the observed aberration yields in 44 h cultures containing 9.7 microM BrdU. In 66 h cultures at 19.4 microM BrdU the observed yields after 2 and 3 days' storage were significantly lower as compared to cultures from fresh blood. No storage effect was revealed in 66 h cultures containing 38.7 microM BrdU. In cases where cytogenetic radiation dosimetry has to be carried out using blood samples which have been in transit for 2-3 days, the findings are of relevance for a correct determination of the chromosome damage in M1 cells.

Blood Preservation

Major pitfalls in Doppler investigations with particular reference to the cerebral vascular system. Part I. Sources of error, resulting pitfalls and measures to prevent errors.

Major pitfalls in Doppler investigations are presented based on 340 evaluated cerebral Doppler examinations in infants. Substantial pitfalls may result from: A. Physics of sound waves and Doppler instruments (errors due to high pass filter cut off, aliasing, rapid image update). B. Quality and adjustment of the Doppler instrument (errors due to low sensitivity, inappropriate adjustment of Doppler controls, inadequate wall filter). C. Examination technique (errors due to an unfavourable angle of incidence or due to transducer-induced pressure: decrease predominantly in diastolic flow velocity-increase in maximum flow velocity in the straight sinus). D. Hemodynamics (errors due to spatial or temporal variations of the flow profile, pulsatility, non-uniform distribution of cerebral blood flow/CBF). E. Cerebral vascular anatomy (errors due to an unfavourable probe position as related to the three-dimensional arrangement of vessels, inadequate separation of closely adjacent vessels). F. Interpretation (flow velocity or Resistance Index/RI is taken to equal CBF, RI is taken to equal peripheral vascular resistance, one artery is taken to represent the cerebral circulation). Pitfalls may be avoided by using adequate means (low wall filter adjustment, high Doppler frequency, critical assessment of velocity spectra) to reduce the likelihood of errors occurring.

Adolescent

Increased left ventricular ejection fraction after a meal: potential source of error in performance of radionuclide angiography.

The effect of a standardized meal on left ventricular (LV) ejection fraction (EF) was determined by equilibrium radionuclide angiography in 16 patients with stable congestive heart failure but without pulmonary or valvular heart disease. LVEF was determined in the fasting state and 15, 30, and 45 minutes after a meal. Patients with moderately depressed fasting LVEF (30 to 50%), Group I, had a mean increase of 6.9 +/- 2.9% (p less than 0.005) in the LVEF at 45 minutes after the meal. Patients with severely depressed fasting LVEF (less than 30%), Group II, had no change after the meal. It is concluded that significant increases in LVEF may occur after meals in patients with moderate but not severe left ventricular dysfunction. Equilibrium radionuclide angiography studies that are not standardized for patients' mealtimes may introduce an important unmeasured variable that will affect the validity of data in serial studies of left ventricular function.

Adult