Small Amounts Of Whole Blood Research Articles

Simplified Cell Magnetic Isolation Assisted SC2 Chip to Realize "Sample in and Chemotaxis Out": Validated by Healthy and T2DM Patients' Neutrophils.

Neutrophil migration in tissues critically regulates the human immune response and can either play a protective role in host defense or cause health problems. Microfluidic chips are increasingly applied to study neutrophil migration, attributing to their advantages of low reagent consumption, stable chemical gradients, visualized cell chemotaxis monitoring, and quantification. Most chemotaxis chips suffered from low throughput and fussy cell separation operations. We here reported a novel and simple "sample in and chemotaxis out" method for rapid neutrophils isolation from a small amount of whole blood based on a simplified magnetic method, followed by a chemotaxis assay on a microfluidic chip (SC2 chip) consisting of six cell migration units and six-cell arrangement areas. The advantages of the "sample in and chemotaxis out" method included: less reagent consumption (10 μL of blood + 1 μL of magnetic beads + 1 μL of lysis buffer); less time (5 min of cell isolation + 15 min of chemotaxis testing); no ultracentrifugation; more convenient; higher efficiency; high throughput. We have successfully validated the approach by measuring neutrophil chemotaxis to frequently-used chemoattractant (i.e., fMLP). The effects of D-glucose and mannitol on neutrophil chemotaxis were also analyzed. In addition, we demonstrated the effectiveness of this approach for testing clinical samples from diabetes mellitus type 2 (T2DM) patients. We found neutrophils' migration speed was higher in the "well-control" T2DM than in the "poor-control" group. Pearson coefficient analysis further showed that the migration speed of T2DM was negatively correlated with physiological indicators, such as HbA1c (-0.44), triglyceride (-0.36), C-reactive protein (-0.28), and total cholesterol (-0.28). We are very confident that the developed "sample in and chemotaxis out" method was hoped to be an attractive model for analyzing the chemotaxis of healthy and disease-associated neutrophils.

An insight into crystallization test

Crystallization test is based on the principle that, when a salt crystallizes out of an aqueous solution, the crystal growth is influenced by the presence of other substances in the solution, such as blood or plant extracts. If a mixture of copper chloride solution with a small amount of whole blood is allowed to crystallize under controlled experimental conditions, an aggregate of crystals forms. Crystallization method can be used as a diagnostic aid to provide information about the systemic conditions and general health of the patient. This study aims to study the patterns of crystallization and to further determine the efficacy of crystallization test as a screening modality in premalignant lesions and oral squamous cell carcinoma (OSCC). Fifty patients of OSCC, 50 patients of premalignant lesions, and 50 healthy individuals were selected. One drop of blood was collected from the study groups to perform crystallization using cupric chloride. Statistical analysis was performed using Chi-square test, Student's t-test (two-tailed), and analysis of variance. The different patterns of crystals formed were studied and statistically analyzed. Based on the study, it was concluded that Crystallization test can be used as an effective screening modality for detection of premalignant lesions and OSCC.

Determination of free G-type nerve agents in blood: in situ derivatization on a dried blood spot (DBS) paper followed by LC–MS/MS analysis

G-type nerve agents, including sarin, soman and cyclosarin are potent neurotoxic chemical weapons, sarin of which is the most used chemical warfare agent of our time. These compounds have limited stability, are highly reactive in the body and thus require the detection of stable metabolites, mainly their hydrolysis products or adducts to proteins. The aim of this study was to develop a new method for the determination of trace amounts of intact G-type nerve agents from small amounts of whole blood. The G-agents were derivatized on a dried blood spot (DBS) paper with 2-((dimethylamino)methyl)phenol. After drying, they were extracted from the paper and analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) or Orbitrap LC–MS/MS. Recoveries of ~ 34% were obtained in the range of 3–300 ng/mL with good linear response. A detection limit as low as 0.7 ng/mL in whole blood was demonstrated. Derivatized sarin was stable on the DBS paper for 19 days at room temperature before analysis. The method was applied for the short-time-course determination of free sarin in mouse blood after in vivo intranasal exposure to 3LD50 sarin. A fast-time-course decay (t1/2: ~ 2 min) was observed, with blood sarin concentrations in the range of 6−60 ng/mL in the post-exposure time range of 1−7 min. This method is simple and unique, enabling the monitoring of G-agents before their rapid conversion in the body. The technique can serve for the study of very fast decay mechanisms of drugs and toxic materials in the body.

High accuracy detection for T-cells and B-cells using deep convolutional neural networks

Providing an accurate count of total leukocytes and specific subsets (such as T-cells and B-cells) within small amounts of whole blood is a rather challenging ordeal due to the lack of techniques that enable the separation of leukocytes from a limited amount of whole blood. In a previous study we designed a microfluidic chip utilizing a micropillar array to isolate T-cells and B-cells from the sub-microliter of whole blood. Due to the variability of cells in size, morphology and color intensity, a Histogram of Oriented Gradients (HOG) based Support Vector Machine (SVM) classifier was proposed with an average accuracy of 94%, specificity of 99% and sensitivity of 90%. The HOG can separate the cells from the background with a high accuracy rate however, some noise is similar in shape and size to the actual cells and this results in misclassification. To alleviate this situation, in this study a convolutional neural network is trained and used to distinguish T-cells and B-cells with an accuracy rate of 98%, a specificity of 99% and a sensitivity of 97%. We also propose an HOG feature based SVM classifier to preselect the detection windows to accelerate the detection to process images in less than 10 min. The proposed on-chip cell detecting and counting method will be useful for numerous applications in diagnosis and for monitoring diseases.

A pillar-based microfluidic chip for T-cells and B-cells isolation and detection with machine learning algorithm

Absolute counting of total leukocytes and specific subset (such as T-cells and B-cells) within small amounts of whole blood is difficult due to the lack of techniques that enables separation of leukocytes from limited volume of whole blood. In this study, a microfluidic chip equipped with a size controlled micropillar array for highly separation of T-cells and B-cells from sub-microliter of whole blood was studied. Based on the difference in size and deformability, leukocytes were separated from other blood cells by micropillar arrays. However, the variability of cells in size, morphology and color intensity along with the spectrum crosstalk between fluorescence dyes make cell detection among pillars extremely difficult. In this paper, an support vector machine supervised machine learning classifier based on both Histogram of Oriented Gradients (HOG) and color distribution features was proposed to distinguish T-cells and B-cells fast and robustly. HOG features were utilized to detect cells from background and noise; color distribution features were employed to alleviate the effect of fluorescence spectrum crosstalk. Experiment showed we achieved average detection accuracy of 94% for detecting T-cells and B-cells from the background. Furthermore, we also got 96% accuracy with cross validation to detect T-cells from B-cells. Both theoretical analysis and experiments demonstrated the proposed method and system has high performance in T-cells and B-cells counting. And our microfluidic cell counting system has great potential as a tool for point-of-care leukocyte analysis system.

Cryopreservation of human blood for alkaline and Fpg-modified comet assay

The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1 mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at −20 °C and −80 °C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at −20 °C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at −20 °C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at −80 °C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay.

A fast and reliable method for GHB quantitation in whole blood by GC–MS/MS (TQD) for forensic purposes

Gamma-hydroxybutyric acid (GHB) is an endogenous compound with a story of clinical use since the 1960s. However, due to its secondary effects, it has become a controlled substance, entering the illicit market.A fully validated, sensitive and reproducible method for the quantification of GHB by methanolic precipitation and GC–MS/MS (TQD) in whole blood is presented. Using 100μL of whole blood, obtained results included a LOD and LLOQ of 0.1mg/L and a recovery of 86% in a working range between 0.1 and 100mg/L. This method is sensitive and specific to detect the presence of GHB in small amounts of whole blood (both ante-mortem or post-mortem), and is, to the authors’ knowledge, the first GC–MS–MS TQD method that uses different precursor ions and product ions for the identification of GHB and GHB-D6 (internal standard). Hence, this method may be especially useful for the study of endogenous values in this biological sample.

Use of Humanized Rat Basophilic Leukemia Reporter Cell Lines as a Diagnostic Tool for Detection of Allergen-Specific IgE in Allergic Patients: Time for a Reappraisal?

The interaction between allergens and specific IgE is at the heart of the allergic response and as such lies at the center of techniques used for diagnosis of allergic sensitization. Although serological tests are available, in vivo tests such as double-blind placebo-controlled food challenges (DBPCFC) and skin prick test (SPT) associated to the patients' clinical history are still the main guides to clinicians in many practices around the world. More recently, complex protein arrays and basophil activation tests, requiring only small amounts of whole blood, have been developed and refined, but are yet to enter clinical practice. Similarly, the use of rat basophilic leukemia (RBL) cell lines for detection of allergen-specific IgE has been made possible by stable transfection of the human FcεRI α chain into this cell line more than 20years ago, but has not found widespread acceptance among clinicians. Here, we review the perceived limitations of diagnostic applications of humanized RBL systems. Furthermore, we illustrate how the introduction of reporter genes into humanized RBL cells is able to overcome most of these limitations, and has the potential to become a new powerful tool to complement the armamentarium of allergists. A demonstration of the usefulness of humanized RBL reporter systems for elucidation of complex IgE sensitization patterns against wheat proteins and a section on the use of fluorescence-based reporter systems in combination with allergen arrays close the review.

Research note: methodology for high-quality RNA extraction from poultry whole blood for further gene expression analysis

1. There are no published methods for RNA isolation from avian whole blood where nucleated red blood cells prevent the use of established mammalian protocols. The aim of this study was therefore to develop a protocol for total RNA extraction using avian whole blood by defining the effect of anticoagulants and sample purification protocols on RNA yield and quality.2. Blood collections from the cutaneous ulnar or medial metatarsal veins of birds yielded adequate blood volume (2–3 ml) draws. The experiment was a 2 × 2 × 3 factorial arrangement of treatments, with two levels of DNase (0 and TURBO DNA-free Kit), two levels of Cleanup (0 and RNeasy MinElute Cleanup Kit), and three anticoagulants (no anticoagulant, EDTA, or sodium citrate).3. RNA was isolated successfully by adding TRIzol LS to 0.25 ml of chicken whole blood at 3:1 ratio. From 0.125 ml of avian whole blood, 2–3 µg of RNA with RNA integrity number values of 7.75 was successfully isolated with the TRIzol LS extraction and an RNeasy MinElute Cleanup Kit.4. This reliable protocol can be used to extract high yield and quality of total RNA from a small amount of whole blood.

Measurement of In Vivo Therapeutic Mab Concentrations: Comparison of Conventional Serum/Plasma Collection and Analysis to Dried Blood Spot Sampling

Development of an alternative sampling method that uses small amounts of whole blood, such as dried blood spots (DBS), would be an advance in the quantitative assay field. Previously, we assessed the ability to quantitate therapeutic monoclonal antibodies present in DBS compared with a typical serum sample-based method, and concluded that measurements in DBS were reproducible and yielded methods that met requirements for precision, accuracy and sensitivity. The goal herein was to assess the measurement of therapeutic antibodies in DBS compared with serum and plasma in vivo. Comparison of DBS versus serum in Sprague-Dawley rats and DBS versus plasma in cynomolgus monkeys for measurement of antibody concentrations revealed a two- to three-fold difference in exposure between the samples. Overall, there was good correlation between DBS versus serum and DBS versus plasma, but there was a discrepancy in DBS exposures, presumably attributable to hematocrit and recovery effects.

Point-of-care testing of portable blood coagulation detectors using optical sensors

A portable optical blood coagulation detector is proposed for point-of-care testing for determining prothrombin time (PT). The detector is applicable to sending the results to a computer via wireless transmission. The integrated apparatus comprises an optical module, a signal processing module, and a communication module for directly sensing real-time changes of transmittance during the blood coagulation process. The efficacy of the proposed device was validated with patients’ whole blood specimens. Only a small amount of whole blood (approximately 60 μl) is required. The mean relative error of the PT values obtained using the proposed device is 4.8 + 3.5% (less than one second) compared with the PT values obtained using the standard manual PT method (n = 26). The two testing platforms have highly correlated results (r = 0.997, p ＜ 0.001). The proposed device has advantages of portability, ease of operation, real-time results, accuracy, carrying a potential for remote monitoring in the personalized long-distance healthcare industry.

Leukocyte counting from a small amount of whole blood using a size‐controlled microcavity array

Absolute counting of total leukocytes or specific subsets within small amounts of whole blood is difficult due to a lack of techniques that enable separation of all leukocytes from limited amounts of whole blood. In this study, a microfluidic device equipped with a size-controlled microcavity array for highly efficient separation of leukocytes from submicroliters of whole blood was developed. The microcavity array can separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered over 90% of leukocytes in 1 µL of whole blood without pretreatment such as density gradient centrifugation or erythrocyte lysis. In addition, the proposed system successfully performed absolute enumeration of human CD4(+) and CD8(+) leukocytes from 1 µL of whole blood, and the obtained data showed good correlation with conventional flow cytometric analysis. Our microfluidic device has great potential as a tool for a point-of-care leukocyte analysis system.

Developmental hemostasis: recommendations for laboratories reporting pediatric samples

Developmental hemostasis: recommendations for laboratories reporting pediatric samples

Single blood drop diagnostic activation test (DAT) for food allergies

Background Quantitative measurement of basophil cell surface markers, CD63 and CD203c, by flow cytometry has been developed as useful tool for the diagnosis of IgE mediated allergies. Available methods require large volume of blood and include extra steps such as prior cell purification, priming with IL-3, stopping cell activation and fixing. We have developed a new basophil based test which can be performed with small amount of whole blood directly from skin prick or from small amount of the whole blood where multiple allergens can be tested at the same time for high-throughput testing.

Rapid diagnosis of FHL3 by flow cytometric detection of intraplatelet Munc13-4 protein

AbstractFamilial hemophagocytic lymphohistiocytosis (FHL) is a potentially lethal genetic disorder of immune dysregulation that requires prompt and accurate diagnosis to initiate life-saving immunosuppressive therapy and to prepare for hematopoietic stem cell transplantation. In the present study, 85 patients with hemophagocytic lymphohistiocytosis were screened for FHL3 by Western blotting using platelets and by natural killer cell lysosomal exocytosis assay. Six of these patients were diagnosed with FHL3. In the acute disease phase requiring platelet transfusion, it was difficult to diagnose FHL3 by Western blot analysis or by lysosomal exocytosis assay. In contrast, the newly established flow cytometric analysis of intraplatelet Munc13-4 protein expression revealed bimodal populations of normal and Munc13-4–deficient platelets. These findings indicate that flow cytometric detection of intraplatelet Munc13-4 protein is a sensitive and reliable method to rapidly screen for FHL3 with a very small amount of whole blood, even in the acute phase of the disease.

Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots

Kaletra® (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for therapeutic drug monitoring of lopinavir and ritonavir concentrations in whole blood and in plasma from HIV-1-infected children. Whole blood was blotted onto dried blood spot (DBS) collecting cards, and plasma was collected simultaneously. DBS collecting cards were extracted by an acetonitrile/water mixture while plasma samples were deproteinized with acetone. Drug concentrations were determined by matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (MALDI-QqQ-MS/MS). The application of DBS made it possible to measure lopinavir and ritonavir in whole blood in therapeutically relevant concentrations. The MALDI-QqQ-MS/MS plasma assay was successfully cross-validated with a commonly used high-performance liquid chromatography (HPLC)–ultraviolet (UV) assay for the therapeutic drug monitoring (TDM) of HIV-1-infected patients, and it showed comparable performance characteristics. Observed DBS concentrations showed as well, a good correlation between plasma concentrations obtained by MALDI-QqQ-MS/MS and those obtained by the HPLC-UV assay. Application of DBS for TDM proved to be a good alternative to the normally used plasma screening. Moreover, collection of DBS requires small amounts of whole blood which can be easily performed especially in (very) young children where collection of large whole blood amounts is often not possible. DBS is perfectly suited for TDM of HIV-1-infected children; but nevertheless, DBS can also easily be applied for TDM of patients in areas with limited or no laboratory facilities.

Prostate‐specific antigen assay using whole blood samples spotted on filter paper and its application to mass screening for prostate cancer

The disc assay system for prostate-specific antigen (PSA) is a novel technique using a small amount of whole blood on filter paper. The accuracy of this assay system and its feasibility for use in prostate cancer mass screening were evaluated. In the first arm of the study, to evaluate the accuracy of the disc assay system, PSA values were determined by both a disc assay system and a standard serum assay system using the same blood samples obtained from 420 outpatients. In the second arm of the study, the feasibility and reliability of the disc assay system were examined in prostate cancer mass screening. A total of 2475 men were screened by the disc assay (disc group) and 3348 men were screened by the standard serum assay (serum group) in the first step of mass screening. In the second step of the screening in the disc group, 101 men underwent PSA tests by a standard serum assay, then the first PSA values determined by the disc assay were compared with the second PSA values determined by the standard serum assay. In the second step of the screening in the serum group, 94 men underwent additional PSA tests by a serum assay, and then the first PSA values were compared with the second PSA values. Two men in each group were excluded from analysis because the true PSA values of the first step were not available (more than 50 ng/mL). The PSA values determined by the disc assay closely correlated with those obtained by the standard assay (r = 0.987) in 295 outpatients with PSA levels between 1.0 and 20 ng/mL. In the PSA mass screening, the PSA values determined in the first step closely correlated with those in the second step both in the disc group (r = 0.916) and in the serum group (r = 0.845). A significant dissociation of the two PSA values was observed in seven of 99 men in the disc group and in three of 92 men in the serum group. However, there was no statistical significance in the incidence of dissociation in the two PSA values between the disc group and the serum group. The disc assay system seems to be a sensitive and accurate assay system. The feasibility and reliability of the disc assay system were well demonstrated in the field during prostate cancer mass screening.

Sensitive and Quantitative Measurement of Gene Expression Directly from a Small Amount of Whole Blood

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Rapid Identification of Hemoglobin Variants by Electrospray Ionization Mass Spectrometry

ABSTRACTThe precise identification of human hemoglobin variants, over 700 human hemoglobin variants are known, is essential for prediction of their clinical and genetic significance. A systematic approach to their rapid identification is described. Traditionally this requires protein or DNA characterization which entails lengthy analytical procedures. To overcome these obstacles a rapid approach to variant hemoglobin identification has been developed using conventional phenotypic methods combined with electrospray ionization–mass spectrometry (ESI–MS). The latter requires only a small amount of whole blood (10 μl) but in most cases 2 μl would have been sufficient and no preanalytical steps, such as separation of red cells or globin chains, are necessary. Aged, hemolyzed blood samples can also be analyzed. This approach has been used to positively identify 95% of the variants in over 250 samples. The remaining 5% in which a variant was detected by phenotypic techniques were not resolved by mass spectrometry. Ninety-nine different abnormalities comprising 36 α-chain variants, 59 β-chain variants (including 2 extensions), and 4 hybrid hemoglobins were identified. These include 15 novel variants. The application of ESI–MS described requires approximately 1 h to prepare and analyze each sample and has minimal reagent costs. The turnaround time on a single sample can be as little as 2 h. This technique can now be considered a useful additional tool for reference laboratories.

Histamine release test and measurement of antigen-specific IgE antibody in the diagnosis of allergic conjunctival diseases.

Although systemic allergic laboratory tests for the quantification of allergen-specific serum IgE antibody have been widely used, in these tests a high titer of serum specific IgE does not necessarily indicate evidence of allergy. We evaluated the diagnostic value of the glass microfiber-based histamine release test (HRT) using small amounts of whole blood, in 36 cases of allergic conjunctival diseases: 17 cases of allergic conjunctivitis and 19 of atopic keratoconjunctivitis. The patients were evaluated by HRT, capsulated hydrolic carrier polymer (CAP)-RAST, and conjunctival provocation test (CPT) against ten allergens. The positive rates for all allergens were higher in CAP-RAST than in HRT. The mean concordance of HRT with CAP-RAST results was 0.789. The mean concordance of HRT with CPT was 0.892 and that of CAP-RAST with CPT was 0.693. A significantly higher concordance was observed in HRT than CAP-RAST for Japanese cedar and mite antigen. The mean sensitivity, specificity, and efficiency of HRT were higher than those of CAP-RAST. These results indicate that CAP-RAST is good for the screening of allergens and that HRT has an advantage in the confirmation of clinical allergens in allergic conjunctival diseases because of its high sensitivity, specificity, efficiency, and higher concordance with CPT.