Improved accuracy and extended flow range for a Fleisch pneumotachograph

A large linear flow range and a small instrumental dead space volume are incompatible properties for a pneumotachometer (PTM). The linearity of a Fleisch number 2 PTM is studied for flows up to 6 litre s⁻¹ (nominal range 0–2 litre s⁻¹) with various up- and downstream geometries. It is hypothesised that using an array of calibration factors (conductance; flow/pressure), instead of a single calibration factor over the entire flow range, could improve accuracy and also extend the applicable flow range. The conductance against pressure characteristics are calculated with a previously described weighted averaging technique based on multiple strokes from a precision syringe. A single conductance value gives stroke volume errors in the range of −5 to 3% (0–2 litre s⁻¹) and −6 to 11% (0–6 litre s⁻¹) for validation using the same geometry as for calibration. The pressure dependent conductance improves accuracy to within −3% and 1% independent of flow range. However, for validation using a different geometry than for calibration, errors range from −5% to +8%. The degree of non-linearity varies between the geometries (range 3–15%) and is highest when using a one-directional valve upstream of the PTM and a Y-shaped connector. In conclusion, a pressure-dependent conductance improves accuracy and can also be used to extend the applicable flow range up to at least three times the nominal flow range.     

Design and construction of a linear shear stress flow chamber

A new paralle plate flow chamber that has a linear variation of shear stress, starting from a predetermined maximum value at the entrance and falling to zero at the exit, has been designed and tested. This is in contrast to the usual rect-angular channel plan which produces a constant shear stress over the entire length. The new design is based on the theory of Hele-Shaw flow between parallel plates. To verify the efficacy of the flow channel, the effect of fluid shear stress on platelet adhesion to a fibrinogen-coated glass surface was tested. The percentage of attached platelets after 5 min of shear stress is shown to be a function of shear stress. With this new flow chamber, cell-cell interactions can be studied efficiently over a wide range of shear stress using a single run at constant discharge.     

Stabilization of white blood cells and immunologic markers for extended analysis using flow cytometry

We evaluated whole blood samples drawn from 25 healthy donors and 20 HIV-infected donors into K3EDTA and Cyto-Chex BCT blood collection tubes for CD4, CD8, and CD3 cell counts (HIV Panel). Samples collected in Cyto-Chex BCT were stored at room temperature and tested by 4-color flow cytometry at 6 h, 3 days, and 7 days after isolation for CD4, CD8, and CD3 absolute cell counts/microl and compared with samples collected in K3EDTA tubes and tested at 6 h. Regardless of donor type, the samples collected in Cyto-Chex BCT and tested on day 7 yielded results that were statistically indistinguishable (with correlation coefficients of 0.96 or greater) from samples collected in K3EDTA tubes and tested at 6 h. We conclude that whole blood samples collected in Cyto-Chex BCT are stabilized for their marker phenotype for at least 7 days after phlebotomy.     

Renal blood flow in isolated kidneys measured with an electromagnetic flowmeter and by xenon-133 and krypton-85 wash-out techniques

Summary By use of an electromagnetic flowmeter and the inert gas desaturation method 43 comparative measurements of the blood flow were performed in 6 isolated dog kidneys. For the measurements by use of the latter method krypton-85 and xenon-133 were employed, for a fast, and respectively, a slow injection of the indicator into the renal artery. Calculation of the blood flow from the wash-out curves was made by use of the formulas advanced by Kety (1951) and by Zierler (1965).A good accordance was found between the flowmeter values and those obtained with the desaturation methods. Recirculation and resorption of indicator in fat tissue surrounding the kidney explain that previous examinations in kidneys in situ have resulted in very low perfusion values, when Zierler's formula has been applied. Extrapolation of the final slope to obtain the area of the curve may be a major source of error by calculation of the blood flow with Zierler's formula.     

Method validation and reference range values for a peripheral blood immunophenotyping assay in non-human primates

A peripheral blood immunophenotyping assay was developed and validated for determination of total T-lymphocytes, helper T-lymphocytes, cytotoxic T-lymphocytes, B-lymphocytes, and natural killer cells in cynomolgus monkeys. Validation parameters included assessment of precision, linearity, antibody optimization, stability of peripheral blood samples, and stability of fixed immunophenotyping samples. Total lymphocyte populations were determined using a heterogeneous lymphocyte gating strategy consisting of CD45 fluorescent staining and side-scatter demarcation. Relative lymphocyte subset values were determined using antigen-specific gating strategies. Absolute subset concentrations for each lymphocyte subset were subsequently determined using a dual-platform methodology wherein relative lymphocyte subset values (via flow cytometry analyses) were multiplied by the absolute total lymphocyte (via hematology analyses) values. Reference ranges are presented for cynomolgus monkey, rhesus monkey, and baboon. Additional 1-year longitudinal immunophenotyping values are presented for the cynomolgus monkey. The method validation and reference ranges presented in this research provide a robust analytical methodology for determination of peripheral blood lymphocyte subsets in various non-human primate species.     

Improved flow cytometry based cytotoxicity and binding assay for clinical antibody HLA crossmatching

The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM). This study was initiated to further evaluate our recently developed flow cytometry based procedure for determination of both cytotoxicity of and IgG binding to donor-derived lymphocytes by HLA antibodies. Highly enriched immuno-magnetic bead purified T and B lymphocytes were used as target cells for patient sera using 96-well plates. Importantly, the assay shows high sensitivity and specificity as determined by HLA typed donor cells and serum with defined HLA antibody IgG and C1q. Based on this and additional data generated in this paper, such as evaluation of appropriate serum and complements incubation times and assay reproducibility and stability, will enable us to more rapidly implement this assay in our clinical laboratory routines. In addition, we demonstrate that FCtox crossmatching of deceased donor cells has superior specificity compared to conventional CDC assay especially regarding high frequencies of false-positive reactions.     

A new flow cell for platelet adhesion studies

A flow cell has been designed and tested for the purpose of exposing platelets to various substrates. The design adopts the principle of flow relaminarization by acceleration to dissipate secondary fluid motions and turbulence so that platelet diffusion will be controlled by Brownian diffusion coefficients. To assess the effectiveness of this flow mechanism two critical tests were undertaken. First, hot film anemometry signals were obtained to observe visually the local fluid velocity in the flow cell. Second, in vitro platelet adhesion results were obtained by exposing glass and silane coated glass to platelet suspensions. Surface platelet concentrations for varying exposure times were compared to a theoretical model which is based on a laminar diffusion model. Both tests confirm that hydrodynamically the flow cell behaves in a manner which is consistent with that of a fully developed, laminar flow with a constant diffusion coefficient.     

血管生物反应器流场的数值分析

目的 研究一种新型小口径血管生物反应器在不同工况下的流场分布。方法 采用数值方法模拟反应器外筒单独旋转、内筒单独旋转和内外筒同向等速旋转情况下其内部的流场分布,对流速和切应力等参数进行比较分析。结果 血管生物反应器工作时,培养液随内外筒的旋转而旋转流动,速度分布均匀;可为培养液内的细胞提供非破坏性的低切应力环境;并且反应器内部的切应力大小与转速呈二次函数关系。结论 该反应器能为血管培养提供良好的培养环境,本研究可为其实验研究提供指导意见和理论基础。     

A new pulsatile flow visualization method using a photochromic dye with application to Doppler ultrasound

A nonintrusive method for the visualization of pulsatile flow velocity profiles is described. The method is based on the use of a photochromic dye that is added to the fluid being studied and a nitrogen laser which excites the dye producing a marker “line” whose movement can be photographed. A microcomputer is used as a system controller, to coordinate the system timing and to manage the data transfer. The method used for analysis of the photographs to determine the velocity profiles is described. Examples are presented of instantaneous velocity profiles obtained from velocity waveforms that are similar to those of the femoral artery. In addition, application of the system for studying the relationship between Doppler ultrasound spectral recordings and flow velocity profiles is discussed.     

Computer-controlled positive displacement pump for physiological flow simulation

A computer-controlled pump for use both in the study of vascular haemodynamics and in the calibration of clinical devices which measure blood flow is designed. The novel design of this pump incorporates two rack-mounted pistons, driven into opposing cylinders by a micro-stepping motor. This approach allows the production of nearly uninterrupted steady flow, as well as a variety of pulsatile wave-forms, including waveforms with reverse flow. The capabilities of this pump to produce steady flow from 0·1 to 60 mls⁻¹, as well as sinusoidal flow and physiological flow, such as that found in the common femoral and common carotid arteries are demonstrated. Cycle-to-cycle reproducibility is very good, with an average variation of 0·1 mls⁻¹ over thousands of cycles.     

Use of a personal computer for fast acquisition of cardiovascular data over an extended period

This paper describes data collection during open chest cardiovascular research based on an IBM compatible personal computer. Data from eight analogue data channels are collected at a rate of 500 Hz per channel for a period of more than 40 sec per run. General analysis functions include the integration of the data obtained from any channel as well as an exponential curve fitting routine. Special functions are available for the calculation of cardiac parameters. This includes the automatic determination of end-diastole and end-systole, as well as maximum and minimum values of all curves for both the systolic and diastolic phases of contraction.     

Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus MHV-A59

The murine coronavirus [murine hepatitis virus (MHV)] is limited to infection of susceptible mice and murine cell lines by the specificity of the spike glycoprotein (S) for its receptor, murine carcinoembryonic antigen cell adhesion molecule 1a (mCEACAM1a). We have recently shown that 21 aa substitutions and a 7-aa insert in the N-terminal region of S are associated with the extended host range of a virus variant derived from murine cells persistently infected with the A59 strain of MHV (MHV-A59). We used targeted RNA recombination (TRR) to generate isogenic viruses that differ from MHV-A59 by the 21 aa substitutions or the 7-aa insert in S. Only viruses with both the 21 aa substitutions and the 7-aa insert in S infected hamster, feline, and monkey cells. These viruses also infected murine cells in the presence of blocking anti-mCEACAM1a antibodies. Thus, relatively few changes in the N-terminal region of S1 are sufficient to permit MHV-A59 to interact with alternative receptors on murine and non-murine cells.     

Blood flow velocity in the common carotid artery in humans during graded exercise on a treadmill

Cerebral blood volume flow and flow velocity have been reported to increase during dynamic exercise, but whether the two increase in parallel and whether both increases occur as functions of exercise intensity remain unsettled. In this study, blood flow velocity in the common carotid artery was measured using the Doppler ultrasound method in eight healthy male students during graded treadmill exercise. The exercise consisted of stepwise progressive increases and decreases in exercise intensity. The peak intensity corresponded to approximately 85% of maximal oxygen consumption. During this exercise, the heart rate (f _c), mean blood pressure (BP) in the brachial artery and mean blood flow velocity (_cc) in the common carotid artery increased as functions of exercise intensity. At the peak exercise intensity, (f _c), BP and _cc increased by 134.5%, 20.5% and 51.8% over the control levels before exercise (P < 0.01), respectively. The resistance index (RI) and pulsatility index (PI) were determined from the velocity profile and were expected to reflect the distal cerebral blood flow resistance. The RI and PI increased during the graded exercise, but tended to decrease at the highest levels of exercise intensity. As _cc increased with increases in exercise intensity it would be expected that cerebral blood flow would also increase at these higher intensities. It is also suggested that blood flow velocity in the cerebral artery does not proportionately reflect the cerebral blood flow during dynamic exercise, since the cerebral blood flow resistance changes.     

动脉血流旋动原理在人造血管研制和血管移植术中的应用

目的 将血液流动的旋动流原理用于心血管介入治疗,以期解决小口径人造血管的急性血栓堵塞问题和搭桥手术后下游处血管内膜增生引起的血管再狭窄问题。方法 使用计算流体力学（CFD）分别研究具有旋动流特性的新型小口径、S型搭桥和偏心搭桥模型中的流场以及壁面剪切力的分布。同时研究在旋动流下血小板的黏附情况和不同角度的S型搭桥下的血管内膜增生。结果 旋动流能明显提高壁面剪切力,抑制血小板的黏附以及血管内膜增生。结论 在心血管介入治疗和器械设计中引入旋动流确实可明显改善这些器械中的血流流场,达到抑制小口径人造血管的急性血栓形成和搭桥手术后血管内膜增生的目的。     

恶性肿瘤细胞DNA倍体分类方法的改进及其临床应用

采用流式细胞术建立一种改良的细胞DNA倍体分类方法，对100例恶性肿瘤患者瘤组织进行了DNA倍体分类，并对其倍体类型和瘤组织细胞生物学特性、患者临床生物学行为之间的关系进行了分析。结果表明瘤组织细胞的DNA倍体类型分为：二倍体、近二倍体、四倍体、非整倍体和多异倍体细胞，后4种倍体类型统称为DNA异倍体。患者瘤组织细胞DNA倍体分类不同，其细胞生物学特性明显不同，患者临床生物学行为也明显不同。改良后的DNA倍体分类方法，不仅能客观地反映肿瘤细胞的生物学特性，而且还能准确地反映患者肿瘤的恶性程度和不同的临床生物学行为特征，可在临床推广应用。