Establishing reference values for cervical spine range of motion in pre-pubescent children

Medical professionals, physical therapists, product designers, and computational models all use cervical spine range of motion reference values. To support these functions, researchers have collected a plethora of data to determine the normal range of motion of the cervical spine of adult subjects. However, little to no data exists for subjects under the age of 14. This study utilized the cervical range of motion device, referenced with respect to the Frankfort Plane, to measure the active cervical spine range of motion in all three cardinal planes of the human body, for 106 subjects whose ages ranged from 8 to 10 years. The active range of motion for flexion, extension, lateral extension, and rotation was calculated as 66+/-13 degrees , 85+/-14 degrees , 58+/-8 degrees , and 77+/-7 degrees , respectively, using linear statistics. The observed data significantly differed from the published American Medical Association guidelines for adults but fell within the range of the reference values for 10 year olds. Stratifying and analyzing the range of motion data with respect to gender yielded no significant effect. Appendix A analyzes the data using angular statistics, and produces virtually identical results as those from linear statistics.     

An adapter for defined sample volumes makes it possible to count absolute particle numbers in flow cytometry.

A device is described which makes it possible to count absolute particle (cell) numbers per volume by flow cytometry. It can easily by adapted to several types of flow cytometers, especially to the Coulter EPICS V and EPICS 750 series. A volume adapter has been installed in place of the normal sample handling system without any further modifications of the instrument or the data acquisition program. The adapter consists of a special pipette with two opto-electronic detectors for the beginning and end of the measuring period. These switch on/off a shutter for the illuminating laser beam so that acquisition of the data is controlled indirectly. Sample volumes of 50 microliters were measured at flow rates up to 10(3) particles/s. Calibration beads as well as blood cells were enumerated according to FALS (forward angle light scatter), to SSC (90 degrees light scatter), and to fluorescence parameters. The results were compared to the evaluation made on a Coulter counter or in a Neubauer chamber of a light microscope. Using a concentration of 1 x 10(5)-5 x 10(5) particles/ml, the absolute numbers of particles were determined with a high reproducibility and an estimated error rate of 2-5%.     

Interrelation of formalin fixation, chromatin compactness and DNA values as measured by flow and image cytometry

The severity and consistency of the effect of formalin fixation on the quantitation of DNA by flow cytometry (FCM) and image cytometry (ICM) were studied. As compared to ethanol, formalin fixation substantially decreased the propidium iodide fluorescence from mouse hepatocyte nuclei analyzed by FCM; it was also associated with an altered 4n-to-2n signal ratio and with false aneuploid peaks by FCM, but not by ICM (microspectrophotometry). ICM, on the other hand, suffered from a dependence of the DNA signal on nuclear size, which was not seen with FCM. The DNA signal variation was related to variations in the chromatin state, as shown by differences between monocytes and lymphocytes, and between RAJI cells fixed under various ionic strengths. The dependence of the DNA signal on the chromatin state indicates a need for caution in interpreting aneuploidy in formalin-fixed cells. For FCM, pseudoaneuploidy appears avoidable by using a Feulgen fluorescence staining technique. New imaging modes may be necessary to solve the problem of cell size dependence for ICM DNA determination.     

Cardiac two-dimensional imaging and reference values for blood flow velocities in the ovine fetus.

Since the ovine model is the most commonly used for foetal haemodynamic investigation it was felt important 1) to investigate the technical difficulties involved with ultrasound fetal cardiac imaging in this species and 2) to establish normal reference values for ovine cardiac and umbilical blood flow velocity measurements. Both two-dimensional and pulsed Doppler techniques were used for this assessment in 25 unsedated ewes. All morphological features described in human features to identify the ventricular cavities could be found in the ovine fetus with the two-dimensional echocardiogram. Specific differences included a flatten thorax as visualized from the lateral position, the mesocardial position of the heart, and a large left azygos vein behind the left atrium draining blood into a dilated coronary sinus. The mean peak velocities (cm/sec) of the early diastolic wave (E) and the atrial wave (A), along with their calculated ratio, were not statistically different between the two atrio-ventricular valves (E: 30.6 +/- 6.6, 31.2 +/- 6.1; A: 43.0 +/- 8.3, 41.6 +/- 8.0; E/A: .72, .76 for mitral and tricuspid valves respectively). A significant difference was observed between the acceleration times of the blood ejected into the aorta and the pulmonary artery, with the time interval being shorter in the pulmonary artery (Aorta: 0.052 +/- 0.011; Pulmonary artery: 0.037 +/- 0.009 s). A mean pulsatility index of the umbilical artery of 0.89 was recorded. The data recorded in this study should serve as a reference base for further non-invasive studies of the ovine foetal circulatory system using the ultrasound technique.     

Hematology and plasma biochemistry reference range values for free-ranging desert tortoises in Arizona

Baseline values and ranges for 10 hematologic and 32 plasma chemistry parameters were analyzed for 36 free-ranging Sonoran desert tortoises (Gopherus agassizzi) collected in Yavapai and La Paz Counties (Arizona, USA) from 1990 to 1995. Tortoises were radio tagged from 1990 to 1994, and attempts were made to recapture them three times a year. Tortoises were weighed, measured, and chemically immobilized to collect blood for hematology and blood chemistry assessments. Tortoise biochemistry differed (P < 0.01) between sites and sexes and among seasons and years. Normal reference ranges for hematologic and plasma biochemistry parameters were determined. Seasonal and annual differences in hematology and blood chemistry were related to rainfall patterns, forage availability, and physiological condition.     

Multicenter study of reference stabilized human blood for lymphocyte immunophenotyping quality control in flow cytometry. GEIL.

The development of automated methods and normative rules, as well as the dependence of some therapeutic approaches on lymphocyte subsets counts, has led to the appearance of calibration reagents. Such reagents are expected to perform equally well in very different settings. We developed a multicenter trial to evaluate the performance of a new quality control reagent, i.e., stabilized blood to be used in immunophenotyping laboratories. Aliquots of the same batch of stabilized blood were shipped to 45 French and Belgian laboratories on a Monday and had to be tested for percentages and absolute counts of at least CD3-, CD4-, and CD8-positive lymphocytes on 4 consecutive days. The percentages and absolute counts obtained on each assay were recorded, as well as the type of lysis used, the trademark of reagents, and the brand of flow cytometer. The mean values collected did not differ significantly from those expected by the manufacturer. Absolute counts generated through one-step techniques displayed lower CVs. This new reagent therefore appears to be a robust product, liable to yield consistent results in the array of different conditions represented by routine laboratories, and it could be useful for quality control procedures.     

Effects of thermal exposure on immunophenotyping combined with in situ PCR, measured by flow cytometry.

BACKGROUND: The combination of in situ PCR and cell phenotyping by antibody labeling (ISPCR/Flow) allows for the identification of cell subsets carrying a particular genetic sequence. ISPCR utilizes thermal cycling for genetic amplification, which can reduce the effectiveness of surface antibody labeling. This study explored and characterized the effects of thermal exposure on antibody labeling using CD4 and CD45. METHODS: Single temperature incubations and thermal cycling exposures were performed on leukocytes labeled with either direct antibody conjugates or with biotinylated antibodies and PE-streptavidin. RESULTS: Fluorescence emission decreased above 70 degrees ( )C when cells were stained with directly conjugated antibodies or a biotinylated antibody and PE-streptavidin prior to high heat exposure. If counter stained with PE-streptavidin after heat, fluorochrome fluorescence was detectable. We tested a second CD4 clone, that provided poor results under similar labeling conditions, suggesting the combination of fixation and heat may have an epitope specific effect for the same cellular antigen. CONCLUSIONS: Immunophenotyping can be combined with ISPCR, but each antibody must be tested to determine its efficacy. The denaturation of protein above 70 degrees C appears to be the main reason for loss of fluorescence. The best procedure is to first stain cells with a biotinylated antibody to an epitope that survives fixation and thermocycling. The cells are then subjected to the desired PCR procedure. Finally they are stained with a fluorochrome conjugated streptavidin.     

新生儿凝固酶阴性葡萄球菌败血症血降钙素原、C反应蛋白、白细胞及血小板计数变化及其意义

目的观察新生儿凝固酶阴性葡萄球菌(CNS)败血症患儿血降钙素原(PCT)、C反应蛋白(CRP)、白细胞(WBC)及血小板(PLT)水平的变化,探讨其与新生儿CNS败血症的关系。方法选择2013年至2014年住院的新生儿CNS败血症患儿38例作为观察组,另选同时期收住无感染症状的32例新生儿为对照组,比较两组患儿入院后血PCT、CRP、WBC及PLT水平和阳性率的差异,并观察这些指标诊断新生儿CNS败血症灵敏度和特异度。结果观察组PCT水平、阳性率显著高于对照组(P0.05),两组患儿CRP、WBC、PLT水平及阳性率比较差异无统计学意义(P0.05),PCT诊断新生儿CNS败血症灵敏度高于CRP、WBC及PLT,但特异度低于后三者。结论新生儿CNS败血症无特异性实验室检查方法,PCT对新生儿CNS败血症早期诊断有一定的价值,联合检测PCT、CRP、WBC及PLT能提高诊断的准确率,血培养阳性仍是诊断新生儿CNS败血症的金标准。     

Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations.

BACKGROUND: Previous studies of intracellular expression of phospho-epitopes in human leukocytes using flow cytometry have used erythrocyte removal or lysis before fixation. Because many of the phospho-epitopes of interest are part of signaling networks that respond to the environment and turn over rapidly, the interval and manipulations used to eliminate erythrocytes from samples have the potential to introduce artifacts. We report a procedure to fix samples containing red blood cells with formaldehyde and then remove erythrocytes by lysis. Detection of phospho-Thr 202/Tyr 204-p44/42 extracellular-regulated kinase (ERK) after phorbol ester acetate (PMA) stimulation was used as a model to measure phospho-epitopes in leukocyte populations in whole blood. METHODS: Normal blood samples were activated with PMA followed by formaldehyde fixation and subsequent treatments with detergents and protein denaturants. The effects of each treatment were monitored by light scatter, selected CD expression intensity, and phosphorylated ERK (pERK) expression. RESULTS: Red cells could be lysed using 0.1% Triton X-100 after brief fixation of whole blood with 2% or 4% formaldehyde. Light scatter improved as a function of formaldehyde concentration and inversely with MeOH concentration. CD3 signal intensity increased when MeOH concentration was reduced. The ratio of pERK immunofluorescence in PMA-stimulated versus nonstimulated (control) samples was highest with high MeOH (90%) and lowest without MeOH treatment. This pattern is consistent with epitope unmasking by alcohol. The pERK epitope could also be unmasked by treatment with high salt, urea, acid, or heat, but none of these produced the level of unmasking of MeOH and each of these was associated with degradation of light scatter and CD3 staining intensity. The final procedure employed 4% formaldehyde, 0.1% Triton X-100, followed by 50% methanol denaturation. Samples prepared in this way demonstrated good preservation of light scatter and surface immunophenotypic patterns, similar to those obtained using a commercial whole blood/red blood cell lysing system (Q-Prep) and an acceptable PMA-stimulated pERK signal (essentially 100% of CD3+ cells that are pERK positive). CONCLUSIONS: Brief fixation of whole blood in 4% formaldehyde followed by treatment with Triton X-100 results in erythrocyte lysis and leukocyte light scatter and immunophenotypic features equivalent to those of other commercial lysis reagents. Intracellular pERK staining is significantly improved by treatment with methanol, but levels of MeOH above 50% degrade light scatter and CD3 expression. This protocol (formaldehyde/Triton X-100/MeOH) circumvents potential artifactual changes in phospho-epitopes due to removal of erythrocytes or erythrocyte lysis followed by fixation, and results in a pERK signal that resolves positive from negative cell populations.     

Triploidization in the streaked prochilod Prochilodus lineatus inferred by flow cytometry,blood smears and karyological approaches

The streaked prochilod Prochilodus lineatus is an important fish species from the Neotropical region. In this study, a protocol of triploidization was established by temperature shock. Fertilized eggs were shocked at 2 min post-fertilization at 0, 38, 40 and 42°C. At 0°C, the embryos were maintained for 30 min, while the rest for 2 min. Heat shocked embryos and control (during all experiment) were incubated at 27°C. The ploidy status was confirmed by flow cytometry, erythrocyte diameter, conventional cytogenetics (Giemsa staining), chromosome banding and fluorescence in situ hybridization using 5S and 18S rDNA probes. Heat-shock at 40°C produced 96.7% of triploids and such a procedure did not reduce the fertilization, hatching rate and the percentage of normal embryos. The use of chromosome banding and FISH gave rise to effective procedure to identify triploids. The data obtained here is innovative for the species and bring new information for basic and applied studies.     

Acquisition of improved reference values for cesium,iodine, strontium,thorium, and uranium in selected NIST reference materials

As part of a study on the ingestion and organ content of some trace elements of importance in radiological protection, additional work has been undertaken to acquire improved reference values for cesium, iodine, strontium, thorium, and uranium in four selected reference materials provided by the US National Institute of Standards and Technology. The materials are SRM-1548 Total Diet, SRM-1548a Typical Diet, SRM-1486 Bone Meal, and RM-8414 Bovine Muscle. A coordinated study was undertaken with the help of seven selected laboratories in five countries. Instrumental and radiochemical neutron activation analysis and inductively coupled plasma-mass spectrometry were the analytical main techniques used.     

A small-volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four-color flow cytometry

BACKGROUND: Cell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood. METHODS: In 20 microl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8alpha) and a DNA binding dye (TO-PRO-3). RESULTS: After a 24-h incubation of whole blood with 1 microM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone-induced percentage of apoptotic cells was 58.9 +/- 4.6 for CD4+ and 77.4 +/- 2.9 for CD8+ T cells, compared with 12.6 +/- 2.7 for CD4+ and 17.2 +/- 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples). CONCLUSIONS: We have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood.     

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted.     

Relationships among Doppler-derived umbilical artery absolute velocities, cardiac function, and placental volume blood flow and resistance in fetal sheep

We hypothesized that umbilical artery (UA) absolute blood flow velocities measured by Doppler ultrasonography reflect placental volume blood flow (Q(UA)) and placental vascular resistance (R(UA)) in a late gestation fetal sheep model. In addition, we examined the relationships between umbilical artery absolute blood flow velocities and parameters of fetal cardiac function. Twenty-six sheep fetuses were instrumented at 112-132 days of gestation. After a 5-day recovery period, experiments were performed under general anesthesia in 16 normal fetuses, in 5 fetuses after maternal administration of phenylephrine, and in 5 fetuses after placental embolization. The Q(UA) and arterial blood pressures were measured using a transit-time ultrasonic flow probe and a catheter placed into the descending aorta, respectively. UA peak systolic velocity (PSV), end-diastolic velocity (EDV), time-averaged maximum velocity (TAMXV), pulsatility index (PI), mean velocity (V(mean)), fetal cardiac output, ventricular ejection forces, and the proportion of isovolumetric relaxation time (IRT%) in the cardiac cycle were measured with the use of Doppler ultrasonography. Significant positive linear correlations were found between UA EDV, TAMXV, and V(mean) versus Q(UA), whereas UA PI had a significant negative correlation with Q(UA). Significant negative correlations were shown between UA EDV, TAMXV, and V(mean) versus R(UA). A significant positive correlation was present between UA PI and R(UA). Doppler-derived UA parameters did not correlate with fetal arterial blood pressures, cardiac output, ventricular ejection forces or IRT%. In fetal sheep, Doppler-derived UA PI and absolute velocities, except PSV, are closely related to directly measured Q(UA) and R(UA), validating the use of noninvasive Doppler velocimetry in the assessment of placental circulation.     

Phagocytic cells in blood from rainbow trout, Salmo gairdneri (Richardson), characterized by flow cytometry and electron microscopy

An in vitro assay for estimating the proportion of phagocytic cells among peripheral leucocytes from rainbow trout by flow cytometry (FCM) and fluorescence microscopy was evaluated. Data from FCM were compared with fluorescence microscopic observations and good correlation ( r = 0.87) was found. The influence of various culture conditions, such as serum type, duration of incubation and temperature, on the in vitro phagocytic assay was investigated. Cultures supplemented with brown trout serum and incubated for 18 h at 19° C were considered to give optimal conditions for phagocytosis. The proportion of phagocytic cells detected in the peripheral blood leucocyte preparation was 3.3 ± 1.5% with FCM and 5.5 ± 2.4% with fluorescence microscopy. The applicability of the method was demonstrated in a preliminary study with arsenic. In a concentration of 1 μg ml⁻¹, arsenic increased the proportion of actively phagocytic cells, but, at a high concentration, 100 μg ml⁻¹, it decreased the phagocytic activity. Electron microscopy was used for morphological classification of the peripheral leucocytes throughout the study.     

p53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer

The aim of this study was to quantify p53 expression by flow cytometry. A panel of three monoclonal antibodies: NCL-p53-240, NCL-p53-1801 and NCL-p53-DO7, was tested on breast cell lines and primary breast cancers. The relationships between ploidy, tumour grade and p53 expression for each antibody, were examined. Methodology was assessed using a variety of breast cell lines. Staining patterns were confirmed and the quantification technique qualified. Cytokeratin-positive cells from 58 samples obtained from patients with breast cancer were assayed for DNA content and p53 expression. p53 quantification was performed using calibrated fluorescent beads on cytokeratin-positive cells. Bloom and Richardson grading revealed 20 grade I and 38 grade II/III breast cancers. Examination of fluorescence thresholds showed a positive correlation between grade and DO7 (P=0.003) at a level of 8900 molecules, 240 (P=0.005) at a level of 2900 molecules and 1801 (P=0.005) at a level of 1850 molecules. These levels equated with 34% (DO7), 43% (240) and 43% (1801) of the samples being classified as p53-positive. Examination of ploidy revealed 23 diploid and 35 aneuploid breast cancers. Application of p53 threshold levels on diploid and aneuploid tumours showed correlation between aneuploidy and p53 expression for DO7 at a level of 9000 molecules, 240 at a level of 1900 molecules and 1801 at a level of 1800 molecules. These levels equated with 34% (DO7), 52% (240) and 52% (1801) of the samples being classified as p53-positive. We conclude that measurement of p53 by flow cytometry may be of clinical importance by indicating levels of positivity using fluorescence thresholds. p53 expression has been shown to correlate with both grade and ploidy. Flow-cytometric measurement of p53 may be a useful prognostic assay.This study was supported by the North of England Cancer Research Campaign