Whole Blood Lysates Research Articles

Estimated Plasmodium 18S ribosomal RNA prevalence in asymptomatic blood donors from three African countries.

The World Health Organization (WHO) African Region accounts for 94% of malaria cases globally, with variability recognized within endemic regions. To determine the detection rate of Plasmodium RNA in blood donors resident in malaria-endemic areas, samples from three African countries were tested using a Plasmodium nucleic acid test. Whole blood (WB) samples collected from routine donors in Cameroon, Madagascar and Mali were shipped frozen to the United States. Samples were tested individually from WB lysates with the Procleix Plasmodium assay (transcription-mediated amplification [TMA]). Reactive samples were considered either repeat reactive or initial reactive only, depending on TMA-retest results. When available, matching plasma samples were tested for Plasmodium antibodies by enzyme immunoassay (EIA). Plasmodium repeat reactivity ranged from 41% (91/223 tested) in Cameroon to 12% (26/216) in Mali and 1% (3/249) in Madagascar. Initially reactive samples, where reactivity did not repeat, were identified from Cameroon (5/223; 2%) and Mali (2/216; 1%). The matched-plasma subgroup had EIA reactivity ranging from 86% (113/131 tested) in Cameroon to 59% (10/17) in Mali and 27% (68/248) in Madagascar. Plasmodium ribosomal RNA (rRNA) detection and antibody rates varied greatly in the three countries studied. Detection of Plasmodium rRNA can provide an additional tool to address malaria risk in blood donors.

Addition of exogenous γ-glutamyl hydrolase eliminates the need for lengthy incubation of whole blood lysate for quantitation of folate vitamers by high performance liquid chromatography-tandem mass spectrometry

BackgroundMeasurement of folate monoglutamates by HPLC-MS/MS in whole blood lysate (WBL) requires lengthy incubation prior to analysis, risking degradation of labile folate vitamers. ObjectiveWe explored whether addition of a commercially available recombinant exogenous γ-glutamyl hydrolase (exoGGH) enzyme reduced the required incubation time of WBL for measurement of folate as monoglutamates. MethodsFor conventional deglutamylation of polyglutamates, WBL was incubated 4 h at 37°C. Alternatively, we added exoGGH to WBL at varying concentrations (1–10 µg/mL) and incubation times (0–90 min). We also investigated modifications to the sample diluent (pH, ascorbic acid vs. sodium ascorbate, and ascorbate concentration). Finally, we tested the effect of the enzyme in different sample types: WBL from frozen whole blood vs. frozen WBL or vs. frozen washed red blood cells (RBC). Samples (n ≤ 15 per experiment) were analyzed by HPLC-MS/MS for 6 folate monoglutamates and 5-methyltetrahydrofolate diglutamate. ResultsOptimal deconjugation of folate polyglutamates was achieved using 1% ascorbic acid and 5 µg enzyme/mL WBL, requiring ≤ 30 min incubation time to achieve complete folate recovery as monoglutamates. This treatment resulted in similar folate concentrations as conventional deglutamylation (4 h at 37°C). The exoGGH enzyme was effective in samples stored frozen as whole blood and as WBL. However, extended thaw time of whole blood resulted in 5-methyltetrahydrofolate loss and unacceptable changes to non-methyl folate concentration. Total folate (with exoGGH) measured in washed RBC was ∼15% lower than RBC folate calculated from WBL concentrations (conventional deglutamylation). ConclusionsUse of exoGGH minimized incubation time and thus may avoid degradative losses of labile folate forms during sample preparation. The lower folate results in washed RBC may be due to inadequate packing of RBC among other unidentified factors. A larger study is required to confirm lack of differences in folate concentrations determined with and without the use of exoGGH.

Robustness of the CDC Folate Microbiologic Assay Kit During Simulated Delayed Shipping

ObjectivesThe WHO has recommended the accurate and practical microbiologic assay (MBA) to assess population folate status. To help laboratories in low-resource countries conduct folate measurements, the CDC developed a folate MBA kit containing critical reagents: calibrator, microorganism, quality control (QC), and reagent stock solutions to prepare culture medium. Shipping frozen kits internationally is complex and sometimes delayed, potentially causing materials to thaw. We investigated whether refrigerated or ambient kit temperatures affect folate results.MethodsWe conditioned 5-methyltetrahydrofolate calibrator stock solution, L. rhamnosus microorganism inoculation, serum and whole blood lysate (WBL) QC samples, and reagent stock solutions (ascorbic acid, chloramphenicol, and manganese sulfate) at 6°C ≤ 3 d and 20°C ≤ 2 d protected from light; we then compared conditioned vs. optimally stored materials (-70°C) by analyzing serum and/or WBL samples (≥3 runs).ResultsThe conditioned calibrator showed a mean folate difference (range from 3 runs for 43 WBL samples) of −0.3% (−4.0 to 4.1%) at 6°C/3 d and 0.9% (−1.6% to 2.7%) at 20°C/2 d. Similarly, the conditioned microorganism showed a mean difference of 4.3% (0.7% to 8.3%) at 6°C/3 d and 0.6% (−3.5% to 4.9%) at 20°C/2 d. The conditioned reagents showed a mean difference (range from 3 runs for 5 serum and 5 WBL samples) of 0.3% (−7.4% to 9.0%) at 6°C/3 d and 3.6% (0.1% to 9.0%) at 20°C/2 d. The 2 conditioned serum QC samples (∼13 and ∼40 nmol/L) showed a mean difference (range from 6 runs) of −0.6% (−6.0% to 2.3%) at 6°C/1 d, but higher differences of −5.8%, −5.9%, and −20.7% at 6°C/3 d, 20°C/1 d, and 20°C/2 d, respectively. The conditioned low WBL QC sample (∼300 nmol/L) showed small mean differences (−1.5%, −2.4%, −1.7%, and −0.3% at 6°C/1 d, 6°C/3 d, 20°C/1 d, and 20°C/2 d, respectively), while the conditioned high WBL QC sample (∼600 nmol/L) showed higher mean differences (−3.1%, −12.4%, −6.9%, and −23.7%, respectively).ConclusionsAll kit components except for the QC materials demonstrated adequate stability (<5% change) at 6°C ≤ 3 d or at 20°C ≤ 2 d. The QC materials should not be exposed to 6°C for > 1 d to ensure stability. These findings provide important and practical information for kit shipment management.Funding SourcesNone.

Robustness of the CDC Folate Microbiologic Assay Kit During Simulated Delayed Shipping

ObjectivesThe WHO has recommended the accurate and practical microbiologic assay (MBA) to assess population folate status. To help laboratories in low-resource countries conduct folate measurements, the CDC developed a folate MBA kit containing critical reagents: calibrator, microorganism, quality control (QC), and reagent stock solutions to prepare culture medium. Shipping frozen kits internationally is complex and sometimes delayed, potentially causing materials to thaw. We investigated whether refrigerated or ambient kit temperatures affect folate results.MethodsWe conditioned 5-methyltetrahydrofolate calibrator stock solution, L. rhamnosus microorganism inoculation, serum and whole blood lysate (WBL) QC samples, and reagent stock solutions (ascorbic acid, chloramphenicol, and manganese sulfate) at 6°C ≤ 3 d and 20°C ≤ 2 d protected from light; we then compared conditioned vs. optimally stored materials (-70°C) by analyzing serum and/or WBL samples (≥3 runs).ResultsThe conditioned calibrator showed a mean folate difference (range from 3 runs for 43 WBL samples) of −0.3% (−4.0 to 4.1%) at 6°C/3 d and 0.9% (−1.6% to 2.7%) at 20°C/2 d. Similarly, the conditioned microorganism showed a mean difference of 4.3% (0.7% to 8.3%) at 6°C/3 d and 0.6% (−3.5% to 4.9%) at 20°C/2 d. The conditioned reagents showed a mean difference (range from 3 runs for 5 serum and 5 WBL samples) of 0.3% (−7.4% to 9.0%) at 6°C/3 d and 3.6% (0.1% to 9.0%) at 20°C/2 d. The 2 conditioned serum QC samples (∼13 and ∼40 nmol/L) showed a mean difference (range from 6 runs) of −0.6% (−6.0% to 2.3%) at 6°C/1 d, but higher differences of −5.8%, −5.9%, and −20.7% at 6°C/3 d, 20°C/1 d, and 20°C/2 d, respectively. The conditioned low WBL QC sample (∼300 nmol/L) showed small mean differences (−1.5%, −2.4%, −1.7%, and −0.3% at 6°C/1 d, 6°C/3 d, 20°C/1 d, and 20°C/2 d, respectively), while the conditioned high WBL QC sample (∼600 nmol/L) showed higher mean differences (−3.1%, −12.4%, −6.9%, and −23.7%, respectively).ConclusionsAll kit components except for the QC materials demonstrated adequate stability (<5% change) at 6°C ≤ 3 d or at 20°C ≤ 2 d. The QC materials should not be exposed to 6°C for > 1 d to ensure stability. These findings provide important and practical information for kit shipment management.Funding SourcesNone.

Folate Forms in RBC and Whole-Blood Lysates Appear Stable When Stored Frozen for 2 Years

BackgroundThe use of RBC lysate (RBC-Lys) eliminates the need for serum folate and hematocrit (Hct) measurement to calculate RBC folate. Information on the long-term frozen storage stability of RBC-Lys is missing. ObjectivesWe aimed to assess the comparability of RBC folate forms in whole-blood lysate (WB-Lys) and RBC-Lys and the folate stability in both matrices. MethodsWe prepared conventional WB-Lys (1:11 dilution with 1% ascorbic acid) and RBC-Lys (1:11 dilution of washed and saline-diluted RBCs with 1% ascorbic acid) from EDTA blood (n = 60 adult donors) and stored lysates at −70°C until analysis at baseline (1 wk), 3, 6, 12, and 24 mo. Before analysis by HPLC–tandem MS, we incubated the WB-Lys (4 h at 37°C) and treated the RBC-Lys with human recombinant γ-glutamyl hydrolase for folate polyglutamate deconjugation. We analyzed RBC-Lys samples for hemoglobin (Hb) (same aliquot) to normalize for the preanalytical dilution; Hb-folate was converted to RBC folate for each folate form using the mean corpuscular Hb concentration. We analyzed Hct as well as folate forms in matching serum samples for traditional RBC folate calculation. We conducted descriptive data analyses (correlation, Bland–Altman plot, Deming regression). ResultsAt baseline, results for RBC folate forms derived from WB-Lys compared with RBC-Lys samples showed excellent correlation (Pearson r ≥ 0.97). Mean ± SD concentrations compared well for total folate (WB-Lys: 886 ± 255 compared with RBC-Lys: 899 ± 271 nmol/L), 5-methyltetrahydrofolate (WB-Lys: 831 ± 258 compared with RBC-Lys: 843 ± 276 nmol/L), and nonmethyl folate (WB-Lys: 53.3 ± 74.4 compared with RBC-Lys: 52.9 ± 70.7 nmol/L), but were 17% higher in RBC-Lys for pyrazino-s-triazine derivative of 4α-hydroxy-5-CH3-H4folate (MeFox) (WB-Lys: 147 ± 44.1 compared with RBC-Lys: 172 ± 53.5 nmol/L). Frozen storage of WB-Lys and RBC-Lys samples for ≤24 mo showed ≤5%, ≤5%, ≤13%, and ≤11% change in total folate, 5-methyltetrahydrofolate, nonmethyl folate, and MeFox, respectively. ConclusionsErythrocyte folate forms appear to be stable in RBC-Lys samples stored frozen at −70°C for ≤2 y. The relatively small changes in folate concentrations over time were comparable between RBC-Lys and conventionally prepared WB-Lys samples.

Addition of Exogenous γ-Glutamyl Hydrolase Eliminates the Need for Lengthy Incubation of Whole-Blood Lysate for Quantitation of Folate Vitamers by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

BackgroundMeasurement of folate monoglutamates by HPLC–tandem mass spectrometry (HPLC-MS/MS) in whole-blood lysate (WBL) requires lengthy incubation before analysis, risking degradation of labile folate vitamers. ObjectiveWe explored whether the addition of a commercially available recombinant exogenous γ-glutamyl hydrolase (exoGGH) enzyme reduced the required incubation time of WBL for measurement of folate as monoglutamates. MethodsFor conventional deglutamylation of polyglutamates, WBL was incubated for 4 h at 37°C. Alternatively, we added exoGGH to WBL at varying concentrations (1–10 µg/mL) and incubation times (0–90 min). We also investigated modifications to the sample diluent (pH, ascorbic acid compared with sodium ascorbate, and ascorbate concentration). Finally, we tested the effect of the enzyme in different sample types: WBL from frozen whole blood compared with frozen WBL or with frozen washed RBCs. Samples (n ≤ 15/experiment) were analyzed by HPLC-MS/MS for 6 folate monoglutamates and 5-methyltetrahydrofolate diglutamate. ResultsOptimal deconjugation of folate polyglutamates was achieved by using 1% ascorbic acid and 5 µg enzyme/mL WBL, requiring ≤30 min incubation time to achieve complete folate recovery as monoglutamates. This treatment resulted in similar folate concentrations as conventional deglutamylation (4 h at 37°C). The exoGGH enzyme was effective in samples stored frozen as whole blood and as WBL. However, the extended thaw time of whole blood resulted in 5-methyltetrahydrofolate loss and unacceptable changes to the non-methyl folate concentration. Total folate (with exoGGH) measured in washed RBCs was ∼15% lower than RBC folate calculated from WBL concentrations (conventional deglutamylation). ConclusionsThe use of exoGGH minimized incubation time and thus may avoid degradative losses of labile folate forms during sample preparation. The lower folate results in washed RBCs may be due to inadequate packing of RBCs, among other unidentified factors. A larger study is required to confirm the lack of differences in folate concentrations determined with and without the use of exoGGH.

Caspase‐3 Activation, Bcl‐2 Contents, and Soluble FAS‐Ligand Are Not Related to the Inflammatory Marker Profile in Patients with Sepsis and Septic Shock

The current comparative investigation analyses markers of inflammation and apoptosis in peripheral blood of intensive care unit (ICU) patients with postoperative/posttraumatic SIRS (systemic inflammatory response syndrome), sepsis, severe sepsis, or septic shock. Inflammatory markers (C-reactive protein [CRP], cytokines, metalloproteinases [MMPs]) and soluble FAS-Ligand (sCD178) were determined in plasma, and apoptosis-relevant antigens such as active caspase-3, Bcl-2, and sCD178 were quantified in whole-blood cell lysates. These parameters were analyzed daily in 20 postoperative/posttraumatic patients: 2 patients had SIRS, 5 suffered from sepsis (2 died), and 13 had septic shock (5 died). Active caspase-3, Bcl-2, and sCD178 were determined by ELISA and by fluorescence-activated cell sorting (FACS)-array kits using bead-assisted flow cytometry. Cytokines and MMPs were quantified by Luminex-assisted Beadlyte assays. Active caspase-3 was identified in defined samples of whole-blood lysates covering, for example, 5/7, 8/18, and 6/11 consecutive days during the patients' stay on the ICU. Also, sCD178 was detected on successive days. Peaks of active caspase-3 antigen contents in whole blood occurred independently of CRP and inflammatory cytokines such as tumor necrosis factor (TNF)-alpha and IL-6. In addition, high MMPs 1-3, 7-10, and 13 concentrations were detected. Interestingly, active caspase-3 and cell-associated sCD178 were either elevated simultaneously or in a close time window. The same was true for Bcl-2. In conclusion, activation of apoptosis can be determined in whole blood of postoperative/posttraumatic patients by active caspase-3 and by Bcl-2. Pro- and antiapoptotic effects during sepsis may occur independently of peaks in inflammatory markers. Apoptosis could explain modeling and remodeling of leukocyte subpopulations.

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.

Cell-associated interleukin-8 in cord blood of term and preterm infants.

To assess the effect of gestational age and labor on the interleukin-8 (IL-8) concentration in whole cord blood and serum, IL-8 levels were determined simultaneously in cord blood serum and lysate in 134 infants. Following the elimination of some of the samples due to exclusion criteria, the data for 99 uninfected infants (71 term and 28 preterm) and 9 infants with neonatal bacterial infection delivered either vaginally or by elective or emergency cesarean section were analyzed. The effects of labor and gestational age were tested by analysis of variance. IL-8 was not detectable in the serum of 25 infants, whereas IL-8 levels in whole blood were measurable in all of the samples. The median IL-8 conncentrations in whole cord blood lysate were 106 pg/ml (range, 20 to 415 pg/ml) in preterm infants and 176 pg/ml (range, 34 to 1,667 pg/ml) in term infants. In contrast to the IL-8 levels in serum, IL-8 levels in whole blood were reduced after ECS. Gestational age had no independent effect on the IL-8 concentrations in either serum or whole blood; these concentrations increased in infected infants after labor. We conclude that the neonatal proinflammatory response to labor stress was more evident in the concentrations of IL-8 in whole blood than in serum. The levels of IL-8 in whole-blood lysate reflect proinflammatory stimulation in neonates and may be a useful diagnostic tool for the early diagnosis of neonatal infection.

Microbiological assay for serum, plasma, and red cell folate using cryopreserved, microtiter plate method

This chapter presents microbiological assay for serum, plasma, and red cell folate, using cryopreserved, microtiter plate method. In the 500-ml beaker a solution of 0.5% (w/v) sodium ascorbate is prepared by dissolving 2.5 g of sodium ascorbate in 500 ml of water. This solution is used for preparing the working standard and for all assay dilutions. The working standard is prepared in the following way—a universal tube containing stock standard solution is brought to room temperature and a 50- μ l aliquot is taken and diluted to 100 ml with 0.5% (w/v) sodium ascorbate in a volumetric flask. This solution is mixed thoroughly, then 5 ml is taken and diluted to 100 ml with 0.5% (w/v) sodium ascorbate in a second volumetric flask to give a final working standard concentration of 500 ng/liter. Duplicate 50- μ l aliquots of serum or plasma or duplicate 25- μ l aliquots of whole-blood lysate are pipetted into labeled 4-ml polypropylene tubes. Using the adjustable repetitive sampling pipette the aliquots are diluted to a total of 1 ml with 0.5% (w/v) sodium ascorbate.

Comparison of CD4 cell count by a simple enzyme-linked immunosorbent assay using the TRAx CD4 test kit and by flow cytometry and hematology

Measurement of CD4 T-lymphocyte levels is clinically useful in monitoring immune status in a number of conditions, including human immunodeficiency virus (HIV) infection, in which the absolute CD4 count is used to guide therapy. The absolute CD4 count is obtained by multiplying the results of the leukocyte count and the differential with a hematology cell counter and the percentage of CD4+ T lymphocytes determined by flow cytometry. These techniques require expensive, complex instrumentation, and interlaboratory results are difficult to standardize and reproduce. The rapid growth of HIV infection worldwide has increased the need for more-reproducible and cost-effective methods for CD4 T-cell monitoring. The TRAx CD4 test kit is based on a novel adaptation of conventional enzyme-linked immunosorbent assay (ELISA) and permits the simple quantitation of total CD4 protein from whole-blood lysates. In this study, the relationship between total CD4 protein measured in units per milliliter (TRAx) and in cells per microliter (flow cytometry and hematology) was defined in a multisite clinical study using linear regression analysis. Data from 230 HIV-seronegative and 321 HIV-seropositive specimens were used to calibrate the TRAx assay recombinant CD4 standards and controls in equivalent CD4 T lymphocytes per microliter (cells per microliter). The calibration of the TRAx CD4 assay in cells per microliter was validated with a second group of specimens from 17 healthy volunteers and 20 HIV-seropositive patients which were collected and tested under strictly controlled conditions intended to minimize the effects of specimen aging on the results of the reference method. These data were also used to estimate the variability of absolute CD4 count by cytometric methods as well as the precision of the TRAx CD4 result after it was calibrated in cells per microliter. Overall, correlations between the two methods ranged from 0.87 to 0.95. Additional studies demonstrated that the contribution of CD4 protein from monocytes and any soluble CD4 in sera are negligible in the TRAx assay and do not significantly affect results. This new method represents a promising alternative to absolute CD4 T-cell enumeration by flow cytometry and hematology.