Red Blood Cell Contamination Research Articles

Abstract B1-46: How collection, processing and storage of PBL samples impact the measurement of telomerase activity

Abstract Introduction: Activation of telomerase and telomere lengthening in patient peripheral blood leukocytes (PBL) is associated with longer survival in cancer patients. There are few studies to date that have measured telomerase activity in clinical PBL samples, and it is unclear how initial collection and processing of PBL samples impact the measurement of telomerase activity. In this study, we assessed if telomerase activity measurement varies based on the method of blood collection, processing and storage. Methods: We first collected whole blood drawn in EDTA tubes from two healthy patients that were then processed three different ways. The first whole blood sample was spun down and the buffy coat was pipetted from the top of the red blood cells, and then placed directly into the -80°C freezer. The next two whole blood samples were isolated using an ACCUSPIN™ tube, which utilizes density gradient separation to recover the PBL without contamination of red blood cells. The resulting PBL from these two samples were suspended in freezing media containing RPMI and DMSO, with one sample also containing penicillin streptomycin glutamine, and then both samples were stored in liquid nitrogen following a slow freeze. We then measured enzymatic telomerase activity in each of the three PBL samples from the two different patients. Results: We detected a comparable level of telomerase activity in all three PBL samples. These findings indicate that telomerase activity can be measured in buffy coat samples from patients independent of how the samples were processed and stored. Conclusions: These findings are important because many biorepositories may not have collected or processed PBL samples by identical processes, therefore not allowing direct comparison across samples. Additionally, our finding that telomerase activity levels are comparable between PBL samples containing red blood cells and those processed using an ACCUSPIN™ tube is valuable because it eliminates the necessity for the extra cost of the ACCUSPIN™ tube, freezing media, antibiotics and use of liquid nitrogen storage tanks. Future studies will focus on the determining the relationship of telomerase activity between the PBL, normal colon and cancer tissue in colorectal cancer patients. Citation Format: Ruth A. Johnson, Brooke R. Druliner, Jill Washechek Alleto, Donna Felmlee Devine, Xiaoyang Ruan, Russell Vanderboom, Lisa A. Boardman. How collection, processing and storage of PBL samples impact the measurement of telomerase activity. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-46.

A novel cytologic sampling technique to diagnose subclinical endometritis and comparison of staining methods for endometrial cytology samples in dairy cows

The present article describes a study of the diagnosis of subclinical endometritis in dairy cows having two principal aims: first, to validate a novel technique for taking endometrial cytology samples to diagnose subclinical endometritis in dairy cows. Second, to compare the percentage of polymorphonuclear cells (PMNs) in cytology samples stained with Diff-Quik versus a specific staining method for PMNs, naphthol-AS-D-chloroacetate-esterase (CIAE). In the first experiment, Holstein-Friesian cows (n = 204) were used to take two cytology samples at the same time using the conventional cytobrush (CB) and the new cytotape (CT). Both devices were assembled within the same catheter allowing sampling at the same time, and approximately at the same location. Cytotape consisted of a 1.5-cm piece of paper tape rolled on the top of an insemination catheter covered with a double guard sheet. Parameters used to evaluate both methods were: PMNs percentage, total cellularity, quality of the smears, and red blood cell contamination. The concordance correlation coefficient analysis was used to assess agreement between continuous and Pearson chi-square tests for categorical variables. Agreement between the percentage of PMNs in both methods was good ρ = 0.84 (0.79, 0.87) with a minor standard error of 2%. Both methods yielded similar total cellularity (P = 0.62). Cytotape yielded better quality smears with more intact cells (P < 0.01) while samples that were taken by CB were more likely to be bloody (P < 0.01). Hence, CT and CB methods yielded smears with a similar PMNs percentage and a total number of cells, but CT provided smears with higher quality and significantly less blood contamination. For the second experiment, 114 duplicate cytology slides were stained using both Diff-Quik and CIAE. Agreement between PMNs percentage in both staining techniques was good ρc = 0.84 (0.78, 0.89) with a standard error of only 2%. Hence, Diff-Quik was confirmed as an easy, fast, and high-quality staining technique, which can be routinely used to stain endometrial cytology samples satisfactorily.

Generation of Autologous Platelet-Rich Plasma by the Ultrasonic Standing Waves.

Platelet-rich plasma (PRP) is a volume of autologous plasma that has a higher platelet concentration above baseline. It has already been approved as a new therapeutic modality and investigated in clinics, such as bone repair and regeneration, and oral surgery, with low cost-effectiveness ratio. At present, PRP is mostly prepared using a centrifuge. However, this method has several shortcomings, such as long preparation time (30min), complexity in operation, and contamination of red blood cells (RBCs). In this paper, a new PRP preparation approach was proposed and tested. Ultrasound waves (4.5MHz) generated from piezoelectric ceramics can establish standing waves inside a syringe filled with the whole blood. Subsequently, RBCs would accumulate at the locations of pressure nodes in response to acoustic radiation force, and the formed clusters would have a high speed of sedimentation. It is found that the PRP prepared by the proposed device can achieve higher platelet concentration and less RBCs contamination than a commercial centrifugal device, but similar growth factor (i.e., PDGF-ββ). In addition, the sedimentation process under centrifugation and sonication was simulated using the Mason-Weaver equation and compared with each other to illustrate the differences between these two technologies and to optimize the design in the future. Altogether, ultrasound method is an effective method of PRP preparation with comparable outcomes as the commercially available centrifugal products.

Difficulties in hematopoietic progenitor cell collection from a patient with TEMPI syndrome and severe iatrogenic iron deficiency

Collection of hematopoietic progenitor cells by apheresis (HPC-A) requires separation of cells by density. Previous studies highlighted the challenges of HPC-A collection from patients with abnormal red blood cells (RBCs). TEMPI syndrome is a recently described condition defined by teleangiectasias, elevated erythropoietin and erythrocytosis, monoclonal gammopathy, perinephric fluid collections, and intrapulmonary shunting. Patients with TEMPI syndrome have responded to therapies used to treat plasma cell dyscrasias and may benefit from autologous HPC transplantation. We report HPC-A collection from a patient with TEMPI syndrome that was complicated by severe iron deficiency. The patient received granulocyte-colony-stimulating factor (G-CSF) and plerixafor for HPC mobilization and underwent 3 days of HPC-A collection. The patient presented for collection with a microcytic erythrocytosis. Over 3 days, approximately 50 L of whole blood was processed, and 2 × 10(8) CD34+ cells were collected (2.8 × 10(6) CD34+ cells/kg). The mean collection efficiency (CE), percentage of mononuclear cells, hematocrit (Hct), and RBC count were 18%, 90%, 14%, and 9 × 10(11) , respectively. Altering collection variables to avoid RBC contamination reduced CE. Ficoll preparations of the products after freeze-thaw showed RBC contamination and hemolysis. Postthaw viability exceeded 95%. The products were not RBC reduced or washed. There were no adverse reactions during or after infusion. HPC-A collection from a patient with TEMPI syndrome was complicated by microcytic erythrocytosis, leading to RBC contamination and hemolysis in the product. Adequate HPCs were collected and the patient tolerated infusion without RBC depletion or washing. Our report highlights difficulties of HPC-A collection from iron-deficient patients.

Recent advances in the diagnosis and treatment of acute myocardial infarction.

The Third Universal Definition of Myocardial Infarction (MI) requires cardiac myocyte necrosis with an increase and/or a decrease in a patient's plasma of cardiac troponin (cTn) with at least one cTn measurement greater than the 99(th) percentile of the upper normal reference limit during: (1) symptoms of myocardial ischemia; (2) new significant electrocardiogram (ECG) ST-segment/T-wave changes or left bundle branch block; (3) the development of pathological ECG Q waves; (4) new loss of viable myocardium or regional wall motion abnormality identified by an imaging procedure; or (5) identification of intracoronary thrombus by angiography or autopsy. Myocardial infarction, when diagnosed, is now classified into five types. Detection of a rise and a fall of troponin are essential to the diagnosis of acute MI. However, high sensitivity troponin assays can increase the sensitivity but decrease the specificity of MI diagnosis. The ECG remains a cornerstone in the diagnosis of MI and should be frequently repeated, especially if the initial ECG is not diagnostic of MI. There have been significant advances in adjunctive pharmacotherapy, procedural techniques and stent technology in the treatment of patients with MIs. The routine use of antiplatelet agents such as clopidogrel, prasugrel or ticagrelor, in addition to aspirin, reduces patient morbidity and mortality. Percutaneous coronary intervention (PCI) in a timely manner is the primary treatment of patients with acute ST segment elevation MI. Drug eluting coronary stents are safe and beneficial with primary coronary intervention. Treatment with direct thrombin inhibitors during PCI is non-inferior to unfractionated heparin and glycoprotein IIb/IIIa receptor antagonists and is associated with a significant reduction in bleeding. The intra-coronary use of a glycoprotein IIb/IIIa antagonist can reduce infarct size. Pre- and post-conditioning techniques can provide additional cardioprotection. However, the incidence and mortality due to MI continues to be high despite all these recent advances. The initial ten year experience with autologous human bone marrow mononuclear cells (BMCs) in patients with MI showed modest but significant increases in left ventricular (LV) ejection fraction, decreases in LV end-systolic volume and reductions in MI size. These studies established that the intramyocardial or intracoronary administration of stem cells is safe. However, many of these studies consisted of small numbers of patients who were not randomized to BMCs or placebo. The recent LateTime, Time, and Swiss Multicenter Trials in patients with MI did not demonstrate significant improvement in patient LV ejection fraction with BMCs in comparison with placebo. Possible explanations include the early use of PCI in these patients, heterogeneous BMC populations which died prematurely from patients with chronic ischemic disease, red blood cell contamination which decreases BMC renewal, and heparin which decreases BMC migration. In contrast, cardiac stem cells from the right atrial appendage and ventricular septum and apex in the SCIPIO and CADUCEUS Trials appear to reduce patient MI size and increase viable myocardium. Additional clinical studies with cardiac stem cells are in progress.

Dietary methanol regulates human gene activity.

Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.

Fully immunomagnetic isolation of untouched purified lymphocyte populations directly from whole blood without the need for red blood cell sedimentation, hypotonic lysis or density gradient centrifugation in just 25 minutes. (TECH2P.871)

Abstract Human whole blood is made up of 93-96% red blood cells (RBCs), 4-7% platelets and 0.1-0.2% leukocytes consisting mainly of granulocytes, lymphocytes and monocytes. Due to the low frequency of lymphocyte subsets within whole blood, their isolation typically requires a pre-processing step such as Lymphoprep™ or hypotonic lysis to deplete RBCs prior to cell isolation. We have therefore developed a new, fully immunomagnetic method for the negative selection of untouched cells directly from whole blood, without the need for any pre-processing steps. The EasySep™ Direct procedure involves labeling RBCs, platelets and unwanted leukocytes present in human whole blood with antibody complexes and magnetic particles. The magnetically labelled unwanted cells are separated from the untouched desired cells using an EasySep™ magnet by simply pouring or pipetting the desired cells into a new tube. In just 25 minutes, purities of 92-99% total lymphocytes, 93-98% T cells, 87-97% CD4+ T cells, 75-91% CD8+ T cells, 91-99% B cells, 87-98% naïve B cells and 65-84% NK cells can be achieved with little to no residual RBC contamination. The isolation of cells directly from whole blood will enable rapid access to highly purified cells immediately ready for downstream functional assays with minimal sample handling.

Novel flow cytometric screening method for bacterial contamination of red blood cells: a proof‐of‐principle evaluation

Platelet concentrates (PCs) are the major source of transfusion-transmitted bacterial infection but the significantly higher number of transfused red blood cells (RBCs) has resulted in a similar occurrence of possible transfusion-associated bacterial infections as those of recipients transfused with platelets. The aim of this study was adaption of the BactiFlow (BF) flow cytometric rapid PC screening method for the detection of bacterial contamination in RBCs. The BF assay was used to detect and count bacteria based on esterase activity in viable cells in RBCs. The initial sample preparation for RBCs included the lysis of RBCs, followed by enrichment of bacteria by centrifugation and subsequent standard PC sample preparation. Two-hundred RBC units were analyzed by BF and the BacT/Alert automated culture system. Furthermore, RBCs were spiked with eight different bacteria to monitor bacterial growth kinetics. The BF showed an excellent correlation to conventional plate count results after RBC sample processing. The diagnostic sensitivity of the assay was determined to fewer than 500 counts/mL. Analysis of RBC units showed concordant negative results by BF and culture. Growth kinetics of bacteria were successfully monitored using flow cytometry, showing that Yersinia enterocolitica or Serratia liquefaciens had the capability to grow in RBCs to high counts. Our study demonstrates the successful modification and application of a rapid flow cytometric screening method for bacterial contamination in RBCs. The BF assay represents a powerful basic tool to study bacterial contamination, potential screening strategies of RBCs, and the clinical outcome of screened RBCs prepared for transfusion.

Unstimulated leukapheresis in patients and donors: comparison of two apheresis systems

Unstimulated mononuclear cell (MNC) apheresis plays a role in the generation of donor lymphocytes (DLIs; healthy donors) and in extracorporeal photopheresis (ECP; patients). The new apheresis system Spectra Optia MNC has been shown in small studies to be capable of performing the desired cell collections, but larger data sets from real-life clinical apheresis procedures are lacking. Presented are comparative data from DLI collections randomly performed with either the new technology or a clinical standard technology, COBE Spectra MNC, as well as data from patients with chronic graft-versus-host disease undergoing MNC collections alternating between the two apheresis systems to generate products for ECP. Target cell yield and collection efficiency, product volume, nontarget cell contamination, platelet (PLT) attrition, and some process variables such as process volume and time were analyzed. For most relevant apheresis outcomes, differences between the devices were at best marginal. Spectra Optia MNC collections in patients, but not in donors, took 10% longer to achieve the target process volume. Not unexpectedly, given previous observations for granulocyte-colony-stimulating factor-stimulated leukapheresis, the novel device collected smaller products with less red blood cell contamination. PLT attrition with Spectra Optia MNC was markedly lower in donors. ECP apheresis outcome variability was, to a significant degree, donor dependent, irrespective of the device used. Based on more than 200 unstimulated apheresis procedures, we conclude that both apheresis systems are safe, robust, and equally suitable for unstimulated MNC collections. Both can be successfully run with manufacturer-recommended settings and algorithms.

Traumatic Lumbar Punctures in Infants Hospitalized in the Neonatal Intensive Care Unit

Traumatic lumbar punctures occur frequently in the neonatal intensive care unit, making the interpretation of cerebrospinal fluid values difficult. We report correction factors for cerebrospinal fluid protein and white blood cells in the face of red blood cell contamination. These correction factors should facilitate the diagnosis of bacterial meningitis in highrisk hospitalized infants.

A high throughput method for enrichment of natural killer cells and lymphocytes and assessment of in vitro cytotoxicity

In vitro assessment of lymphocyte and natural killer (NK) cell cytotoxicity typically employs density gradient centrifugation and magnetic cell separation to isolate effector cells, and chromium release to assess cytotoxicity. In order to improve the rapidity and scalability of in vitro cytotoxicity assessment, we evaluated the efficacy of a protocol utilizing tetrameric antibody complexes and SepMate™ isolation tubes to negatively select NK cells (TACs/Sep), and calcein-AM release to measure cytotoxicity. We compared the efficiency and accuracy of this protocol to a conventional approach employing density gradient centrifugation and magnetically labeled antibodies (DG/MACS) to isolate NK cells and chromium release to measure cytotoxicity.The TACs/Sep method significantly decreased the time required for NK cell isolation (1h vs. 4h), but resulted in higher red blood cell contamination. NK cell activation marker expression (including CD94, NKG2D, NKp30, NKp46, DNAM-1, 2B4, KIR2DL1/S1, KIR2DL2/L3, intracellular granzyme B, and perforin) was similar when comparing NK cells isolated by the TACs/Sep or DG/MACS methods, but the TACs/Sep method induced higher expression of CD16. In vitro cytotoxicity against HT29 colon cancer and K562 leukemia cells was not affected by the isolation method. Lastly, by combining the TACs/Sep NK cell isolation method with calcein-acetoxymethyl diacetylester (calcein-AM) release, the time required to assess in vitro cytotoxicity was reduced by 33% (4h) compared to protocols employing DG/MACS and chromium release. Altogether, these results provide the foundation for the development of a rapid, high throughput functional assay, and make it practical for the multiplexing of downstream applications, such as flow cytometric analysis and enzyme-linked immunosorbent assays (ELISAs).

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error (P3233)

Abstract Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method.

Human blood-derived raw material: enabling controlled, consistent collection (P4415)

Abstract Human cells are critical raw materials for manufacturing cell therapy products, but often introduce significant variability. Rigorous operational controls and quality systems, however, enable optimal collection of high-quality, consistent cellular material. HemaCare, a long-standing supplier of human-derived blood components, controls apheresis procedures and collection sites under a formal quality system, with GMP-compliant, validated procedures and equipment, and GTP-compliant donor screening and tracking. HemaCare performed 69,658 cellular apheresis collections in the last five years, including patient and normal-donor MNCs, G-CSF-mobilized PBPCs and plateletpheresis products, for use in research, clinical trials, and commercial products. Expanded capabilities include bone marrow, umbilical cord blood, and cord tissue collection, immunomagnetic selection, cryopreservation, and analysis by flow cytometry. HemaCare unmobilized apheresis products showed consistently high MNC purity, with 93.8% of products containing ≥75% MNC, and an average of 85.2% MNC ± 6.6% (mean ± 1 SD). Red blood cell contamination was low, with hematocrit averaging 1.8% ± 0.8%. Approximately 85% of HemaCare donors have donated apheresis products 5 or more times, and this repeat-donor pool also contributes to product consistency, as MNC content of individual donor apheresis products had an average coefficient of variation of 3.5%, compared to a CV of 7.7% for all apheresis products.

Mononuclear cell collection for extracorporeal photochemotherapy: a study comparing an automatic and a semiautomatic apheresis device

Extracorporeal photochemotherapy (ECP) is an effective cell therapy employed in several diseases, including graft-versus-host disease (GVHD) and organ rejection after transplantation. When ECP is performed using the off-line technique, mononuclear cell (MNC) collection by leukapheresis is necessary for further manipulation. Semiautomated apheresis systems require experienced personnel to obtain a good MNC collection; an automated device, able to efficiently collect MNCs with high purity, is desirable. We compared the semiautomated COBE Spectra MNC and the new automated Spectra Optia v.5.0 MNC (Terumo BCT) devices in terms of efficacy and safety. Adult patients with GVHD or bronchiolitis obliterans syndrome (BOS) after lung transplant undergoing ECP at our center were alternatively assigned, within the same ECP cycle (composed by two procedures each), to MNC collection with either device. Patients' characteristics, procedure, and product-related variables were compared. Thirty-nine patients (24 with GVHD and 15 with BOS) underwent a total of 126 ECP procedures, with good compliance to both devices. Product volume and platelet (PLT) and red blood cell contamination were significantly lower with the Spectra Optia. MNC collection efficiency (CE), purity, and PLT loss were similar between the two devices, while white blood cells CE was in favor of the COBE Spectra. The Spectra Optia device proved to be a good option for MNC collection in the difficult ECP setting, since it ensures high-quality MNC collection, while at the same time saving personnel's time, guaranteeing optimal monitoring and care to this frail patient population.

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error

Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs, and (5) AO/PI dual staining method. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method.

ULTRASOUND‐GUIDED CERVICAL CENTESIS TO OBTAIN CEREBROSPINAL FLUID IN THE STANDING HORSE

Horses with intracranial lesions and severe ataxia are not good anesthesia candidates; however, only one method to obtain cerebrospinal fluid (CSF) from the cervical region in a standing horse has been reported. This method is not performed routinely due to the difficulty for sample acquisition. Our hypothesis is that standing cervical centesis can be performed in horses without complication. Ultrasound-guided centesis of the CSF between C1 and C2 in 11 clinically normal horses and two horses with neurologic signs were performed. Horses were sedated and ultrasound was used to identify the subarachnoid space and spinal cord between C1 and C2. With ultrasound guidance, a needle was introduced into the dorsal aspect of the subarachnoid space using a lateral approach. Ten milliliters of CSF was obtained and analyzed. Two normal horses in this study had moderate red blood cell contamination in the CSF (940 and 612 RBC/microl). One horse had 11 RBC/microl and the remaining horses had < 4 RBC/microl. The total procedure time was approximately 2 min. No reaction was observed and no complications were detected up to 48 h after the procedure. Ultrasound-guided centesis between C1 and C2 is a rapid procedure that causes minimal to no reaction in standing, sedated horses used in this study. The use of ultrasound to guide a standing C1-2 centesis of the subarachnoid space provides an additional route to obtain CSF for analysis in the equine patient.

Comparison of different procedures to prepare platelet-rich plasma for studies of platelet aggregation by light transmission aggregometry

Light transmission aggregometry (LTA), the gold standard for the study of patients with defects of platelet function, is a poorly standardized technique. The guidelines that have been produced so far are largely based on consensus of experts, due to the absence of studies directly comparing different procedures. Therefore, ad hoc studies are needed to gather scientific evidence on how to choose the most appropriate procedures for LTA measurement. In this study, we aimed at evaluating the most appropriate conditions for preparing samples of platelet-rich plasma (PRP) for studies of platelet aggregation by LTA. Citrate-anticoagulated blood from 32 individuals was centrifuged at 150, 200, 250 or 300×g at room temperature for 10 min. Red blood cells contamination was highest in PRP prepared at 150×g; mean platelet volume (MPV) was lowest in PRP prepared at 300×g. The extent of platelet aggregation measured by LTA was lower and more variable in PRP prepared at 300×g. Therefore, centrifugation of blood at 200×g or 250×g for 10 min appears to be the best condition for preparing PRP for LTA studies.

Identification of lipids that accumulate during the routine storage of prestorage leukoreduced red blood cells and cause acute lung injury

Lipids accumulate during the storage of red blood cells (RBCs), prime neutrophils (PMNs), and have been implicated in transfusion-related acute lung injury (TRALI). These lipids are composed of two classes: nonpolar lipids and lysophosphatidylcholines based on their retention time on separation by high-pressure liquid chromatography. Prestorage leukoreduction significantly decreases white blood cell and platelet contamination of RBCs; therefore, it is hypothesized that prestorage leukoreduction changes the classes of lipids that accumulate during storage, and these lipids prime PMNs and induce acute lung injury (ALI) as the second event in a two-event in vivo model. RBC units were divided: 50% was leukoreduced (LR-RBCs), stored, and sampled on Day 1 and at the end of storage, Day 42. Priming activity was evaluated on isolated PMNs, and the purified lipids from Day 1 or Day 42 were used as the second event in the in vivo model. The plasma and lipids from RBCs and LR-RBCs primed PMNs, and the LR-RBC activity decreased with longer storage. Unlike RBCs, nonpolar lipids comprised the PMN-priming activity from stored LR-RBCs. Mass spectroscopy identified these lipids as arachidonic acid and 5-, 12-, and 15-hydroxyeicsotetranoic acid. At concentrations from Day 42, but not Day 1, three of four of these lipids individually, and the mixture, primed PMNs. The mixture also caused ALI as the second event in a two-event model of TRALI. We conclude that the nonpolar lipids that accumulate during LR-RBC storage may represent the agents responsible for antibody-negative TRALI.

Development of a Novel Protocol for Isolation and Purification Of Human Granulosa Cells

OBJECTIVE(S): To develop an optimal method of purification of human granulosa cells from heterogenous follicular fluid and a method to account for red blood cell (RBC) contamination in samples for accurate comparison of results between individual patients.

Red Blood Cell Contamination of the Final Cell Product Impairs the Efficacy of Autologous Bone Marrow Mononuclear Cell Therapy

The aim of this study was to identify an association between the quality and functional activity of bone marrow-derived progenitor cells (BMCs) used for cardiovascular regenerative therapies and contractile recovery in patients with acute myocardial infarction included in the placebo-controlled REPAIR-AMI (Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction) trial. Isolation procedures of autologous BMCs might affect cell functionality and therapeutic efficacy. Quality of cell isolation was assessed by measuring the total number of isolated BMCs, CD34+ and CD133+ cells, their colony-forming unit (CFU) and invasion capacity, cell viability, and contamination of the final BMC preparation with thrombocytes and red blood cells (RBCs). The number of RBCs contaminating the final cell product significantly correlated with reduced recovery of left ventricular ejection fraction 4 months after BMC therapy (p = 0.007). Higher numbers of RBCs in the BMC preparation were associated with reduced BMC viability (r = -0.23, p = 0.001), CFU capacity (r = -0.16, p = 0.03), and invasion capacity (r = -0.27, p < 0.001). To assess a causal role for RBC contamination, we coincubated isolated BMCs with RBCs for 24 h in vitro. The addition of RBCs dose-dependently abrogated migratory capacity (p = 0.003) and reduced CFU capacity (p < 0.05) of isolated BMCs. Neovascularization capacity was significantly impaired after infusion of BMCs contaminated with RBCs, compared with BMCs alone (p < 0.05). Mechanistically, the addition of RBCs was associated with a profound reduction in mitochondrial membrane potential of BMCs. Contaminating RBCs affects the functionality of isolated BMCs and determines the extent of left ventricular ejection fraction recovery after intracoronary BMC infusion in patients with acute myocardial infarction. These results suggest a bioactivity response relationship very much like a dose-response relationship in drug trials. (Reinfusion of Enriched Progenitor cells and Infarct Remodeling in Acute Myocardial Infarction [REPAIR-AMI]; NCT00279175).