The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four years.

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from -0.8 log10 to +0.8 log10, the majority of dPCR results were between -0.2 log10 to +0.2 log10. Only 4 out of 45 results exceeded this interval with the maximum deviation of -0.542 log10. In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than ±0.2 log10 from the target value, while more than 95% deviated less than ±0.4 log10 from the target value. Evaluation of intra- and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing.

Function of Hemoglobin-Based Oxygen Carriers: Determination of Methemoglobin Content by Spectral Extinction Measurements.

Suspensions of hemoglobin microparticles (HbMPs) are promising tools as oxygen therapeutics. For the approval of clinical studies extensive characterization of these HbMPs with a size of about 750 nm is required regarding physical properties, function, pharmaco-kinetics and toxicology. The standard absorbance measurements in blood gas analyzers require dissolution of red blood cells which does not work for HbMP. Therefore, we have developed a robust and rapid optical method for the quality and functionality control of HbMPs. It allows simultaneous determination of the portion of the two states of hemoglobin oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) as well as the content of methemoglobin (metHb). Based on the measurement of collimated transmission spectra between 300 nm and 800 nm, the average extinction cross section of HbMPs is derived. A numerical method is applied to determine the composition of the HbMPs based on their wavelength-dependent refractive index (RI), which is a superposition of the three different states of Hb. Thus, light-scattering properties, including extinction cross sections can be simulated for different compositions and sizes. By comparison to measured spectra, the relative concentrations of oxyHb, deoxyHb, metHb are accessible. For validation of the optically determined composition of the HbMPs, we used X-ray fluorescence spectrometry for the ratio of Fe(II) (oxyHb/deoxyHb) and Fe(III) (metHb). High accuracy density measurements served to access heme-free proteins, size was determined by dynamic light scattering and analytical centrifugation and the shape of the HbMPs was visualized by electron and atomic force microscopy.

Extinction spectra of suspensions of microspheres: determination of the spectral refractive index and particle size distribution with nanometer accuracy.

A method is presented to infer simultaneously the wavelength-dependent real refractive index (RI) of the material of microspheres and their size distribution from extinction measurements of particle suspensions. To derive the averaged spectral optical extinction cross section of the microspheres from such ensemble measurements, we determined the particle concentration by flow cytometry to an accuracy of typically 2% and adjusted the particle concentration to ensure that perturbations due to multiple scattering are negligible. For analysis of the extinction spectra, we employ Mie theory, a series-expansion representation of the refractive index and nonlinear numerical optimization. In contrast to other approaches, our method offers the advantage to simultaneously determine size, size distribution, and spectral refractive index of ensembles of microparticles including uncertainty estimation.

Determining the refractive index of human hemoglobin solutions by Kramers-Kronig relations with an improved absorption model.

The real part of the refractive index of aqueous solutions of human hemoglobin is computed from their absorption spectra in the wavelength range 250-1100 nm using the Kramers-Kronig (KK) relations, and the corresponding uncertainty analysis is provided. The strong ultraviolet (UV) and infrared absorbance of the water outside this spectral range were taken into account in a previous study employing KK relations. We improve these results by including the concentration dependence of the water absorbance as well as by modeling the deep UV absorbance of hemoglobin's peptide backbone. The two free parameters of the model for the deep UV absorbance are fixed by a global fit.

Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.

Microflow cytometers with integrated hydrodynamic focusing.

This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex) principle. The sample--A suspension of micro particles or blood cells--is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel. Particle velocities of a few m/s are achieved as required for high throughput blood cell analysis. The stability of hydrodynamic particle positioning was evaluated by measuring the pulse heights distributions of fluorescence signals from calibration beads. Quantitative assessment based on coefficient of variation for the fluorescence intensity distributions resulted in a value of about 3% determined for the micro-device exploiting cascade hydrodynamic focusing. For the spin focusing approach similar values were achieved for sample flow rates being 1.5 times lower. Our results indicate that the performances of both variants of hydrodynamic focusing suit for blood cell differentiation and counting. The potential of the micro flow cytometer is demonstrated by detecting immunologically labeled CD3 positive and CD4 positive T-lymphocytes in blood.

A microflow cytometer exploited for the immunological differentiation of leukocytes.

In this article, we demonstrate the potential of a microfluidic chip for the differentiation of immunologically stained blood cells. To this end, white blood cells stained with antibodies typically applied for the determination of the immune status were measured in the micro-device. Relative concentrations of lymphocytes and subpopulations of lymphocytes are compared to those obtained with a conventional flow cytometer. The stability of the hydrodynamic focusing and the optical setup was determined by measuring the variation of the signal pulse height of fluorescence calibration beads, being about 2% for the micro-device. This value and the overall performance of the micro-device are similar to conventional flow cytometers. It follows from our results that such microfluidic structures are well suited as modules in a compact, portable read-out instrument. The production process of the microflow cytometers, which we exploited for immunological cell differentiation, is compatible with mass production technology like injection molding and, hence, low cost disposable chips could be available in the future.

Refractive index of human red blood cells between 290 nm and 1100 nm determined by optical extinction measurements.

The knowledge of optical properties of biological cells is essential to interpret their interaction with light and to derive morphological information and parameters associated with cell function like the oxygen transport capacity of human red blood cells (RBCs). We present a method to determine the dependence between the refractive index (RI) of human RBCs and their intracellular hemoglobin (Hb) concentration from spectral extinction measurements of a cell suspension. The procedure is based on the analysis of the corresponding ensemble averaged extinction cross section [Formula: see text]. Thus far two complementary approaches have been taken to derive RIs of RBCs. The first one uses homogeneous macroscopic samples prepared by hemolysis for the destruction of the RBCs' membranes and subsequent centrifugation. A second approach is the determination of RIs of single intact cells by microscopic investigation. These techniques are limited to a few discrete wavelengths or a rather narrow wavelength range. In addition most of these techniques require additional information about the concentration dependence. In contrast, our approach yields the RI increment with Hb concentration of intact, reversibly isovolumetrically sphered, oxygenated RBCs over a wide wavelength range from 290 nm to 1100 nm from macroscopic measurements.

Assessment of deformation of human red blood cells in flow cytometry: measurement and simulation of bimodal forward scatter distributions.

Light scattering by single cells is widely applied for flow cytometric differentiation of cells. However, even for human red blood cells (RBCs), which can be modeled as homogeneous dielectric particles, the potential of light scattering is not yet fully exploited. We developed a dedicated flow cytometer to simultaneously observe the forward scattering cross section (FSC) of RBCs for orthogonal laser beams with incident wave vectors k → 1 and k → 2 . At a wavelength λ = 632.8 nm , bimodal distributions are observed in two-dimensional dot plots of FSC( k → 1 ) vs. FSC( k → 2 ), which result from the RBCs' random orientation around the direction of flow, as well as from the distributions of their size and their optical properties. Typically, signals of 7.5 × 10 4 RBCs were analyzed. We actively oriented the cells in the cytometer to prove that orientation is the main cause of bimodality. In addition, we studied the wavelength dependence of FSC( k → 1 ) using λ = 413.1 nm , 457.9 nm , 488 nm and 632.8 nm, covering both weak and strong light absorption by the RBCs. Simulations of the light scattering by single RBCs were performed using the discrete dipole approximation (DDA) for a range of sizes, orientations and optical properties to obtain FSC distributions from RBC ensembles. Using the axisymmetric biconcave equilibrium shape of native RBCs, the experimentally observed distributions cannot be reproduced. If, however, an elongated shape model is employed that accounts for the stretching of the cell by hydrodynamic forces in the cytometer, the features of the strongly bimodal measured frequency distributions are reproduced by the simulation. Elongation ratios significantly greater than 1 in the range of 1.5 to 2.5 yield the best agreement between experiments and simulated data.

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

BACKGROUND: Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, volumetric flow cytometers have the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations. METHOD: In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated. RESULTS: The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method. CONCLUSIONS: Overall, this study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Label-free whole blood cell differentiation based on multiple frequency AC impedance and light scattering analysis in a micro flow cytometer.

We developed a microfluidic sensor for label-free flow cytometric cell differentiation by combined multiple AC electrical impedance and light scattering analysis. The measured signals are correlated to cell volume, membrane capacity and optical properties of single cells. For an improved signal to noise ratio, the microfluidic sensor incorporates two electrode pairs for differential impedance detection. One-dimensional sheath flow focusing was implemented, which allows single particle analysis at kHz count rates. Various monodisperse particles and differentiation of leukocytes in haemolysed samples served to benchmark the microdevice applying combined AC impedance and side scatter analyses. In what follows, we demonstrate that AC impedance measurements at selected frequencies allow label-free discrimination of platelets, erythrocytes, monocytes, granulocytes and lymphocytes in whole blood samples involving dilution only. Immunofluorescence staining was applied to validate the results of the label-free cell analysis. Reliable differentiation and enumeration of cells in whole blood by AC impedance detection have the potential to support medical diagnosis for patients with haemolysis resistant erythrocytes or abnormally sensitive leucocytes, i.e. for patients suffering from anaemia or leukaemia.

Sensitivity of hemozoin detection by automated flow cytometry in non- and semi-immune malaria patients.

BACKGROUND: Cell-Dyn automated blood cell analyzers use laser flow cytometry technology, allowing detection of malaria pigment (hemozoin) in monocytes. We evaluated the value of such an instrument to diagnose malaria in febrile travelers returning to Berlin, Germany, the relation between the instrument's performance and the patients' immune status, and the capacity to increase its sensitivity. METHODS: Malaria diagnosis was routinely established by thick-film microscopy. The patients' immune status was determined by an indirect fluorescent antibody test. Hemozoin detection was performed with a Cell-Dyn 3000. To assess the capacity for sensitivity increase, the relative frequencies of pigment-containing monocytes were determined for a subgroup of patients with the Cell-Dyn 3000 in comparison with a flow cytometer specifically adapted to rare-event analysis. RESULTS: Of 403 patients screened microscopically, 107 had malaria. Overall sensitivity with the Cell-Dyn 3000 reached 48.6% (73.7% in semi-immune and 28.6% in nonimmune individuals; P < 0.0001). Specificity was 96.2%. The detection limit was at a relative concentration of 2 x 10(-4) pigment-containing monocytes (PCMs). By employing rare-event flow cytometry, the detection limit decreased to 3.25 x 10(-5), thus yielding a considerably increased sensitivity for the subgroup studied. CONCLUSIONS: The correlation between immune status and relative concentration of PCMs explains the failure of the routine instrument for nonimmune patients and its significantly higher sensitivity for semi-immune individuals. The technique can be significantly improved by rare-event flow cytometry.

Concept for the traceability of fluorescence (beads) in flow cytometry: exploiting saturation and microscopic single molecule bleaching.

We have determined the fluorescence yield of stained micro beads, used for calibration purposes in flow cytometry, as function of the irradiance of the exciting laser beam. A rate equation model has been applied to derive the number of fluorescence molecules carried by each micro bead. To derive in situ photo-physical properties of the specific dye, required for the rate equation model, we discuss an approach based on flow cytometric sorting of micro beads, which have passed two laser beams with properly chosen different irradiances, and subsequent observation of single molecule bleaching employing high sensitivity microscopy. The feasibility of our approach is demonstrated presenting first results concerning saturation of fluorescence of beads in flow and single molecule bleaching by high sensitivity microscopy.

Angular distribution of light scattered by single biological cells and oriented particle agglomerates.

We used a flow cytometer together with an intensified CCD camera to record spatially resolved light scattering from micrometer-sized single particles and single oriented particle agglomerates. Experimental differential cross sections of an oriented dumbbell made from two identical polystyrene spheres were compared with theoretical values calculated within the discrete dipole approximation, and good agreement was achieved. Furthermore, characteristic two-dimensional patterns of the scattered-light intensity were recorded for single blood cells, yielding information on the cells' shape and volume. Besides flow cytometry, we observed and analyzed differential light scatter of particle clusters of known size, shape, and orientation located within an optical trap.