WBC Depletion Research Articles

Effects of Hemorrhage on Hematopoietic Cell Depletion after a Combined Injury with Radiation: Role of White Blood Cells and Red Blood Cells as Biomarkers.

Combined radiation with hemorrhage (combined injury, CI) exacerbates hematopoietic acute radiation syndrome and mortality compared to radiation alone (RI). We evaluated the effects of RI or CI on blood cell depletion as a biomarker to differentiate the two. Male CD2F1 mice were exposed to 8.75 Gy γ-radiation (60Co). Within 2 h of RI, animals were bled under anesthesia 0% (RI) or 20% (CI) of total blood volume. Blood samples were collected at 4-5 h and days 1, 2, 3, 7, and 15 after RI. CI decreased WBC at 4-5 h and continued to decrease it until day 3; counts then stayed at the nadir up to day 15. CI decreased neutrophils, lymphocytes, monocytes, eosinophils, and basophils more than RI on day 1 or day 2. CI decreased RBCs, hemoglobin, and hematocrit on days 7 and 15 more than RI, whereas hemorrhage alone returned to the baseline on days 7 and 15. RBCs depleted after CI faster than post-RI. Hemorrhage alone increased platelet counts on days 2, 3, and 7, which returned to the baseline on day 15. Our data suggest that WBC depletion may be a potential biomarker within 2 days post-RI and post-CI and RBC depletion after 3 days post-RI and post-CI. For hemorrhage alone, neutrophil counts at 4-5 h and platelets for day 2 through day 7 can be used as a tool for confirmation.

Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer.

Simple SummaryCirculating tumour cells (CTC) are metastatic seeds that arise from solid tumours and drive the metastatic spread of malignancy. A better understanding of the biology of CTCs is critical to enable its clinical application for diagnosis and as a therapeutic target. Currently, CellSearch is the only method approved by the U.S. Food and Drug Administration (FDA) for CTC enrichment. However, it isolates only epithelial CTCs and is resource-intensive, which has led to an interest in developing simpler size-based CTC isolation methods. The MetaCell CTC enrichment kit is a size-based method which has been previously used for enriching and culturing CTCs in vitro from several cancer types. Herein, we evaluate the MetaCell platform on its efficacy for retaining colorectal cancer (CRC) cells spiked into blood and the extent of purity of the enriched CTC fraction, which has not been described in previous studies utilising MetaCell. We subsequently applied this method to colorectal cancer patient blood samples and successfully enriched CTCs.Circulating tumour cells (CTC) from solid tumours are a prerequisite for metastasis. Isolating CTCs and understanding their biology is essential for developing new clinical tests and precision oncology. Currently, CellSearch is the only FDA (U.S. Food and Drug Administration)-approved method for CTC enrichment but possesses several drawbacks owing to a reliance on the epithelial cell adhesion molecule (EpCAM) and a resource-intensive nature. Addressing these shortcomings, we optimised an existing size-based method, MetaCell, to enrich CTCs from blood of colorectal cancer (CRC) patients. We evaluated the ability of MetaCell to enrich CTCs by spiking blood with CRC cell lines and assessing the cell recovery rates and WBC depletion via immunostaining and gene expression. We then applied MetaCell to samples from 17 CRC patients and seven controls. Recovery rates were >85% in cell lines, with >95% depletion in WBCs. MetaCell yielded CTCs and CTC clusters in 52.9% and 23.5% of the patients, respectively, without false positives in control patients. CTCs and cluster detection did not correlate with histopathological parameters. Overall, we demonstrated that the MetaCell platform enriched CRC cells with high recovery rates and high purity. Our pilot study also demonstrated the ability of MetaCell to detect CTCs in CRC patients.

Continuous centrifugal microfluidics (CCM) isolates heterogeneous circulating tumor cells via full automation.

Understanding cancer heterogeneity is essential to finding diverse genetic mutations in metastatic cancers. Thus, it is critical to isolate all types of CTCs to identify accurate cancer information from patients. Moreover, full automation robustly capturing the full spectrum of CTCs is an urgent need for CTC diagnosis to be routine clinical practice.Methods: Here we report the full capture of heterogeneous CTC populations using fully automated, negative depletion-based continuous centrifugal microfluidics (CCM).Results: The CCM system demonstrated high performance (recovery rates exceeding 90% and WBC depletion rate of 99.9%) across a wide range of phenotypes (EpCAM(+), EpCAM(-), small-, large-sized, and cluster) and cancers (lung, breast, and bladder). Applied in 30 lung adenocarcinoma patients harboring epidermal growth factor receptor (EGFR) mutations, the system isolated diverse phenotypes of CTCs in marker expression and size, implying the importance of unbiased isolation. Genetic analyses of intra-patient samples comparing cell-free DNA with CCM-isolated CTCs yielded perfect concordance, and CTC enumeration using our technique was correlated with clinical progression as well as response to EGFR inhibitors.Conclusion: Our system also introduces technical advances which assure rapid, reliable, and reproducible results, thus enabling a more comprehensive application of robust CTC analysis in clinical practice.

Coagulopathy, Hypoxemia, and Mortality Outcomes in Newly Diagnosed Acute Myeloid Leukemia with Hyperleukocytosis Treated with Large Volume Leukapheresis

Background: Patients with acute myeloid leukemia (AML) presenting with hyperleukocytosis have frequent complications and early mortality. Pulmonary, central nervous system, and cardiovascular complications are common. Attempts to improve outcomes in the 10% of AML patients arriving with hyperleukocytosis have included leukapheresis. No prospective randomized study has supported the use of leukapheresis, and retrospective reports have revealed persistently poor outcomes as well as emerging concerns of acquired coagulopathy and worsening hypoxemia after leukapheresis. While many centers use a leukapheresis protocol processing two blood volumes, Johns Hopkins protocol routinely processes three-five blood volumes. This study aimed to assess coagulopathy, hypoxemia, and mortality with large volume leukapheresis. Methods: 32 patients with newly diagnosed AML treated with large volume leukapheresis for WBC depletion are included in this report. Demographic, clinical, laboratory, and apheresis-related data were collected. Coagulopathy and hypoxemia-related metrics were evaluated within 6 hours before leukapheresis and within 6 hours of completion of leukapheresis. Descriptive and inferential statistics (chi square and Mann-Whitney U test) were used to compare pre and post-leukapheresis findings and assess clinical outcomes. Results: Twenty-nine of 32 (93.8%) patients presented with symptomatic leukostasis (with pulmonary and/or CNS symptoms in 26/29). Median blood volume processed was 14.8 liters (range 4-23.4L). Mean platelet count decreased from 60x109/L to 37x109/L after leukapheresis (p<0.001). Comparing supplemental oxygen requirements before and after leukapheresis, 1 patient had decreased requirements, 6 increased, and 25 were unchanged. Median change in prothrombin time (PT) was an increase of 1 second (range: -2.4 to +7.7 seconds; p=0.13). Twenty-six of 26 evaluable patients had a decline in fibrinogen post-pheresis, with median reduction of 84 mg/dL (range: -12 to -483 mg/dL; p=0.04). Twelve of 18 (66.7%) patients 65 years and older died by day 30, compared with 3/14 <65 years-old (p=0.01). Fifteen of 32 (46.9%) arrived with acute renal failure on admission, 10 of whom died by day 30. All 8 patients requiring hemodialysis during their initial admission died prior to day 30. 12/32 (37.5%) required mechanical ventilation, 10 of whom died by day 30. Overall, 10/32 (31.3%) died by day 7, and 15/32 (46.9%) died by day 30. The most common primary cause of death was multiorgan failure including both renal failure and hypoxemia in 8 patients. Two patients died of confirmed intracranial hemorrhage, and 2 died with clinical suspicion for intracranial hemorrhage but were too unstable for imaging prior to death. Two deaths were attributed to ischemic stroke, and 1 patient died with isolated refractory hypoxemia. Conclusions: Patients with AML presenting with hyperleukocytosis have a very high mortality, particularly when complicated by symptomatic leukostasis. Similar to Van de Louw's report, we observed worsening coagulopathy and a subgroup with increased oxygen requirements after leukapheresis. While our sample size is too small to draw broad conclusions, we were not able to identify a group clearly benefiting from leukapheresis. We did not find evidence that larger volume leukapheresis decreased complications or mortality. These results should lead to caution when considering leukapheresis for patients with newly diagnosed AML, particularly in those presenting with a severe coagulopathy. Table Disclosures Webster: Pfizer: Consultancy; Amgen: Consultancy; Genentech: Research Funding. Gojo:Amphivena: Research Funding; Amgen Inc: Consultancy, Honoraria, Research Funding; Juno: Research Funding; Merck: Research Funding; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Pratz:Boston Biomedical: Consultancy; Millenium/Takeda: Research Funding; Agios: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Smith:Celgene: Consultancy; Jazz: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Novartis: Consultancy. DeZern:Astex Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy. Levis:Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; FUJIFILM: Consultancy, Research Funding; Menarini: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria.

Additional filtering of blood from a cell salvage device is not likely to show important additional benefits in outcome in cardiac surgery.

Several authors and manufacturers of cell salvage devices recommend additional filtering of processed blood before transfusion. There is no evidence to support this practice. Therefore, we compared the clinical outcome and biochemical effects of cell salvage with or without additional filtering. The patients, scheduled for coronary artery bypass grafting, valve replacement, or combined procedures were part of our randomized multicenter factorial study of cell salvage and filter use on transfusion requirements (ISRCTN 58333401). They were randomized to intraoperative cell salvage or cell salvage plus additional WBC depletion filter. We compared the occurrence of major adverse events (combined death/stroke/myocardial infarction) as primary outcome and minor adverse events (renal function disturbances, infections, delirium), ventilation time, and length of stay in the intensive care unit and hospital. We also measured biochemical markers of organ injury and inflammation. One hundred eighty-nine patients had cell salvage, and 175 patients had cell salvage plus filter and completed the study. Demographic data, surgical procedures, and amount of salvaged blood were not different between the groups. There was no difference in the primary outcome with a risk of 6.3% (95% confidence interval [CI], 3.34-11.25) in the cell salvage plus filter group versus 5.8% (95% CI, 3.09-10.45) in the cell salvage group, a relative risk of 1.08 (95% CI, 0.48- 2.43]. There were no differences in minor adverse events and biochemical markers between the groups. The routine use of an additional filter for transfusion of salvaged blood is unlikely to show important additional benefits.

High-throughput residual white blood cell counter enabled by microfluidic cell enrichment and reagent-containing patch integration

A residual white blood cell (rWBC) count is the remaining concentration of WBCs in leucodepleted blood products, and is an important quality-assurance parameter for blood transfusion. Current cell-counting methods rely on a small test volume that can result in the inherent statistical error involved in counting rWBCs present at low concentrations or manual sample preparation that increases experimental workload. To address these critical challenges, we present a microfluidic rWBC counter that (i) rapidly enriches rWBCs to reduce a sample volume to be examined and thus enable high-throughput cell counting, and (ii) integrates an on-chip cell staining module that contains reagent-containing hydrogel patches and allows the controlled release of a staining reagent. With these novel features, we demonstrated high-throughput rWBC counting by achieving reliable cell counting over a dynamic range of 0.1–26.8 rWBCs/μL and processing a blood sample at a high throughput of 400 μL per minute. In addition, we successfully performed sample preparation-free counting of rWBCs by integrating the on-chip staining module into the rWBC counter, offering sample-in-answer-out capability. On the basis of the results, the rWBC counter represents a promising tool for the quality control of WBC depletion and improvement in the safety of blood transfusion.

High-purity capture of CTCs based on micro-beads enhanced isolation by size of epithelial tumor cells (ISET) method

In this paper, we develop a low-cost size-based microfluidic chip using conventional polycarbonate membrane to isolate CTCs from blood, and propose a strategy to increase the capture efficiency before cell filtration by a size enlargement method utilizing modified microbeads specifically binding to CTCs. Up to 91% of target cells were isolated from whole blood samples using our microfluidic capture system at a flow rate of 1mL/min. Moreover, a WBC depletion process is introduced which greatly decreases the WBC retaining on the filter membrane. The tests of immunofluorescence analysis of cells captured on the membrane were performed, which demonstrates that the device could provide a dependable CTC identification and CTC count in whole blood samples. Finally, the device was further validated in the detection of CTCs from blood samples of cancer patients, and it indicates a promising capability to detect CTC response to treatment.

Single-Cell Isolation of Circulating Tumor Cells from Whole Blood by Lateral Magnetophoretic Microseparation and Microfluidic Dispensing.

This paper introduces a single-cell isolation technology for circulating tumor cells (CTCs) using a microfluidic device (the "SIM-Chip"). The SIM-Chip comprises a lateral magnetophoretic microseparator and a microdispenser as a two-step cascade platform. First, CTCs were enriched from whole blood by the lateral magnetophoretic microseparator based on immunomagnetic nanobeads. Next, the enriched CTCs were electrically identified by single-cell impedance cytometer and isolated as single cells using the microshooter. Using 200 μL of whole blood spiked with 50 MCF7 breast cancer cells, the analysis demonstrated that the single-cell isolation efficiency of the SIM-Chip was 82.4%, and the purity of the isolated MCF7 cells with respect to WBCs was 92.45%. The data also showed that the WBC depletion rate of the SIM-Chip was 2.5 × 10(5) (5.4-log). The recovery rates were around 99.78% for spiked MCF7 cells ranging in number from 10 to 90. The isolated single MCF7 cells were intact and could be used for subsequent downstream genetic assays, such as RT-PCR. Single-cell culture evaluation of the proliferation of MCF7 cells isolated by the SIM-Chip showed that 84.1% of cells at least doubled in 5 days. Consequently, the SIM-Chip could be used for single-cell isolation of rare target cells from whole blood with high purity and recovery without cell damage.

High-throughput malaria parasite separation using a viscoelastic fluid for ultrasensitive PCR detection.

A novel microfluidic device for high-throughput particle separation using a viscoelastic fluid, which enables the rapid detection of extremely rare malaria parasites by using PCR analysis, is proposed. Our device consists of two segments: the 1st stage for sheathless pre-alignment and the 2nd stage for separation based on size-dependent viscoelasticity-induced lateral migration. The use of a high-aspect ratio channel and a viscoelastic polymer solution with low viscosity enables high-throughput processing. The device performance was first optimized using synthetic particles. A mixture of 2 and 10 μm particles was focused at the center plane in the 1st stage. The smaller particles, serving as surrogates for malaria parasites, were subsequently separated in the 2nd stage with a recovery rate of ∼96% at 400 μl min(-1). Finally, separation of the malaria parasites from the white blood cells was performed. At 400 μl min(-1), almost all white blood cells were removed and the malaria parasites were separated with a ∼94% recovery rate and ∼99% purity. Although the initial concentration of the malaria parasites was too low to be detected by PCR analysis, WBC depletion and buffer removal increased the parasite concentration sufficiently such that PCR detection was possible.

Towards an optimal and unbiased approach for tumor cell isolation

Our current understanding of clinical significance or the lack thereof of circulating tumor cells (CTCs) is biased by the technology used to isolate these rare cells. Despite the presence of a vast number of academic and commercial technologies, the lack of a standardized and optimized platform has been widely noted. We present a negative enrichment approach, integrating WBC depletion and chemical-free RBC depletion in the same setup without the need for centrifugation, washing or multiple sample handling steps. This approach achieves an average of >90% recovery of spiked tumor cells and >99% total WBC depletion in whole blood across multiple cell lines, in a simple and easy-to-use assay. The results presented herein and ongoing improvements aim to fulfill the need for a highly reliable, unbiased, standardized, and optimized CTC isolation platform, using component technologies that are validated for cell isolation.

Leukapheresis (WBC Depletion) Treatment for Patients with Acute Leukemia and Blast Crisis Can Enable a High Percentage of Patients to Undergo Induction Chemotherapy

Abstract 4281 Introduction:Several retrospective, cohort studies have demonstrated that leukapheresis (WBC depletion) treatment in patients (pts) with acute leukemia and concurrent hyperleukocytosis and blast crisis decreases short-term mortality rate, but does not increase overall survival. However, pts selected in these studies for leukapheresis treatment are often sicker and have a higher underlying mortality rate (than pts who do not receive such treatment). No randomized controlled trials assessing the efficacy of leukapheresis in this subset of pts with acute leukemia have been performed. Methods and Clinical Presentation:Between January, 2006 and June, 2010, Apheresis Care Group (ACG) treated 1,636 pts performing 13,587 therapeutic apheresis treatments (txs). Of this patient cohort, 126 (7.7%) pts had acute leukemia with clinical and/or laboratory evidence of blast crisis and received leukapheresis treatment. 77 pts had acute myelogenous leukemia (AML) and received 174 leukapheresis txs; 49 pts had acute lymphoblastic leukemia (ALL) and received 158 txs. AML pts presented with median WBC 204 × 109/L (range 66–418 × 109/L) and 87% pts had blast crisis (defined as blast percent >75% or blast count >100 × 109/L). Median age was 54 years (6.5–85 years); 61% pts were male. Of CNS or pulmonary symptoms (sxs) of leukostasis (CNS sxs defined as: headache, lethargy, confusion, or visual abnormalities; pulmonary sxs defined as: shortness of breath, hypoxia, or chest x-ray infiltrates without evidence of pneumonia), 13% pts had no sxs, 52% pts had 1 sx, and 35% pts had 2 sxs. ALL pts presented with median WBC 338 × 109/L (104–736 × 109/L) and 82% pts had blast crisis. Median age was 22 years (4–80 years); 64% pts were male. Of sxs of leukostasis, 25% pts had no sxs, 63% pts had 1 sx, and 12% pts had 2 sxs. Treatment:All pts received a course of leukapheresis (Lp) with the following objectives: 1) decreasing the risk of thrombotic and hemorrhagic complications related to leukostasis, and 2) stabilizing pts for induction chemotherapy. WBC treatment goals were defined as: WBC count (ct) <55 × 109/L for AML pts, and WBC ct <80 × 109/L for ALL pts. AML pts received a median of 2 Lp txs (range 1–5 txs); ALL pts underwent a median of 2 Lp txs (1–7 txs). Results:Outcomes were evaluated by the percentage of pts who: 1) reached the WBC treatment goal and, 2) received induction chemotherapy. “Improved” outcome was defined as pts who reached their WBC treatment goal during leukapheresis therapy; “stabilized” was defined as pts who achieved >50% reduction in WBC ct, but did not reach their WBC goal; and “unchanged” was defined as pts who achieved neither. In the AML cohort, 76% pts improved, 23% pts stabilized, and 1% pts were unchanged. In the ALL cohort, 63% pts improved, 34% pts stabilized, and 3% pts were unchanged. For AML pts, the median final WBC ct was 53 × 109/L (range 17–133 × 109/L) and 92% pts received induction chemotherapy. For ALL pts, the median final WBC ct was 76 × 109/L (range 30–294 × 109/L) and 98% pts received induction chemotherapy. 7 (9%) AML pts and 1 (1%) ALL pt expired within 1–4 days after completing course of leukapheresis. Of the 8 expired pts, 75% had both blast crisis and 2 sxs of leukostasis; 38% had intracranial hemorrhage or CVA; and 88% were hypotensive, receiving mechanical ventilation, and unable to tolerate induction chemotherapy. Conclusion:Carefully selected patients with acute leukemia and evidence of impending thrombosis may benefit significantly from leukapheresis therapy. A limited number of treatments (median of 2 treatments) can enable a high percentage of patients to receive induction chemotherapy and may improve short-term clinical outcomes. Leukapheresis treatments are considered emergency procedures as they are often life saving. Disclosures:Hofmann: Fresenius Medical Care: Consultancy. Kiprov:Fresenius Medical Care: Employment.

Hyperconcentrated (Dry) Versus Standard Platelet Apheresis: An In Vitro Quality Study.

Introduction: Platelet concentrates (PC) collected by apheresis are effective in supporting deeply thrombocytopenic patients. The reduced risk of multiple allogeneic exposure and transmissible infectious diseases together with the high WBC depletion and diminished transfusion reactions are the main advantages offered by PC transfusion. At moment, the availability of several synthetic solutions for platelets storage permits to prepare hyperconcentrate(dry) apheresis platelets with the advantage of reducing febrile non-hemolytic transfusion reactions and, in low body weight patients the citrate toxicity, without the necessity of further manipulations. The aim of this study was to test the quality of 20 dry platelets (DP) in comparison to 20 standard plateletpheresis (SP) concentrates.Materials and methods: A total of 40 apheresis procedures were performed by the single-needle Cobe Trima separation device (Gambro BCT, Lakewood, CO, USA) collecting either DP or SP concentrates. Within 1h after collection, the bag containing DP was added with the appropriate amount (70% of DP final volume) of synthetic solution for platelets storage (SSP, MacoPharma). Both DP and SP concentrates were stored at room temperature with gentle agitation for 4 days. For both concentrates, platelet yield was calculated and in vitro studies of membrane glycoproteins expression and aggregation at day +1 and day +4 were carried out.Results: The comparison between 20 DP and 20 SP concentrates in terms of ability to aggregate in vitro and membrane glycoproteins expression at day +1 and day +4 of storage is reported in table A and B respectively.Conclusions: The in vitro tests documented a major activation of dry platelets. In particular, the ability to aggregate was reduced in the 20 DP concentrates analised and this phenomenon was more evident at day +4 of storage. The alteration of membrane glycoproteins expression (markers of storage lesion) confirms the lower in vitro quality of DP concentrates. The effectiveness of this new blood component in vivo should be evaluated in a controlled clinical trial.Table AAt collectionDay +1Day +4SPDPSPDPSPDPCollagen μg/ml 4938897828053ADP 10 μM32112416165Ristocetin 1.5 mg/ml919777817152Collagen 10 μg/ml + Adrenaline 10 μM989593949380ADP 10 μM + Adrenaline 10 μM877276615730Table BAt collectionDay +1Day +4SPDPSPDPSPDPGPIb alfa (MFI)5.065.756.316.135.264.54GPIIb-IIIa (MFI)35.4736.7134.6137.731.7640.9GP IV (MFI)11.4911.411.6710.2811.6511.38GP 5324.2327.1121.7425.9121.4631.84GMP 14021.7929.2922.6530.3820.5834.89

Supernatant from stored red blood cell primes inflammatory cells: influence of prestorage white cell reduction.

The contribution of RBC transfusion to adverse patient outcomes is controversial. There is conflicting clinical data and limited biologic data that provide an underpinning biologic rationale for any adverse impacts from RBC transfusion. This study used in-vitro measures of PMN stimulation to determine the ability of supernatant from RBCs to stimulate allogeneic WBCs and to determine the influence of residual donor WBCs and storage time on the proinflammatory potential of RBCs. Three types of RBCs were assessed: standard non-WBC-reduced RBCs (S-RBCs), buffy coat-poor RBCs (BCP-RBCs), and prestorage WBC-filtered RBC (LF-RBCs). Supernatant was collected weekly up to Day 42 of storage. PMN priming by supernatant from RBCs was determined by three methods: induction of CD11b expression on PMNs, induction of IL-8 release from PMNs, and the chemotactic effect of supernatant on PMNs. Supernatant from S-RBCs induced the expression of CD11b on PMNs, primed PMNs to release IL-8, and was chemotactic for PMNs. The magnitude of this PMN-priming progressively amplified with storage time. In contrast, supernatant from BCP-RBCs or LF-RBCs did not significantly prime PMNs. The PMN-priming effect of supernatant from RBCs correlated more closely with the level of MNCs in the RBCs than PMN content. Supernatant from stored S-RBCs prime unstimulated allogeneic PMNs in vitro. Prestorage buffy-coat WBC reduction was as effective as WBC depletion in abrogating this proinflammatory response elicited by supernatants from RBCs. The clinical consequences, if any, of these findings for transfusion recipients are unknown.

Evaluation of HTLV-I removal by filtration of blood cell components in a routine setting.

WBC depletion by filtration may prevent the transmission of HTLV-I, which requires cell-to-cell contact. The removal of HTLV-I-infected cells in routinely filtered blood cell components was measured. The study was conducted in Martinique where systematic screening for HTLV-I and -II and universal leukoreduction are mandatory. HTLV-I was quantified by use of real-time PCR in 8 RBC units and 4 PLT concentrates before and after filtration. HTLV-I proviral load in PBMNCs was determined in five of the eight HTLV-I-infected blood donors. The amount of MNC-associated HTLV-I DNA in RBC units before filtration was 21 x 10(6)+/- 29 x 10(6) copies (mean +/- SD). HTLV-I was detected in 4 of 8 RBC units after filtration, with a number of copies in the MNC fraction ranging from 20 to 140, following a 4.9 to 5.8 log reduction. Flow cytometry analysis performed in 2 of the filtered RBC units containing detectable HTLV-I showed suboptimal and out-of-range leukoreduction (0.56 x 10(6) and 1.22 x 10(6) residual WBCs). HTLV was not detected in filtered RBCs from the blood donor with the highest percentage of HTLV-I-infected PBMCs (9%). This study confirms that HTLV-I-infected cells can be detected in filtered blood cell components and shows that optimal leukoreduction is critical for HTLV-I removal.

106 depletion of WBC with CF‐2, a new leukocyte removal filter

A new leukocyte removal filter, Nipro CF-2, was evaluated. A non-woven polyester filter, gravity flow device that does not require priming and rinsing after use was developed to remove white blood cells from 400 ml of whole blood or concentrates of 400 ml of whole blood. A flow cytometric technique was developed to measure extremely low WBC counts. To evaluate the efficiency of this filter, leukocyte counts were measured by flow cytometry using a Coulter EPIC-C cytometer. After filtration through a CF-2 filter, more than 99.9999% of WBCs were depleted from 6 of 17 samples. The mean value of WBC depletion was 99.9994%. The CF-2 filter achieved nearly a million-fold depletion of WBCs from whole blood concentrates.

White cell depletion of red cell and pooled random‐donor platelet concentrates by filtration and residual lymphocyte subset analysis

This study comparing the relative white cell (WBC)-depleting efficiency of single and double filtration used two filters and new, sensitive, and reliable methods for performing WBC counts on WBC-depleted blood products. A single filtration of red cell (RBC) concentrates with a cotton-wool filter reduced WBC content by 98.64 percent, but the range of residual WBCs was wide, and many filtered units still contained more than the theoretical immunizing dose of 5 to 10 x 10(6) WBCs. A second filtration, however, always produced RBC units that had less than 5 x 10(6) WBCs. Although the degree of WBC depletion observed after a single filtration of a 6-unit pool of random-donor platelet concentrates was greater with a polyester filter than with the cotton-wool filter (98.92 vs. 98.14% reduction, respectively, when mean prefiltration WBC count was less than 600 x 10(6], in both cases, 25 percent of filtered products still contained greater than 5 x 10(6) WBCs; a second filtration (with the cotton-wool filter), however, produced units that were always below the immunizing dose. All double-filtered platelet concentrates had less than 10(6), and one-half had less than 10(4) residual WBCs. Platelet loss was similar with both filters (+/- 16% loss with one filtration). The effectiveness of the filters in producing products that were WBC-depleted below the immunizing dose was dependent on the prefiltration WBC content (but not on the age of the units), and it may be worthwhile to employ methods to ensure that total prefiltration WBC count of the product is less than 400 x 10(6).(ABSTRACT TRUNCATED AT 250 WORDS)

White cell depletion of single‐donor platelet preparations by a new adsorption filter

A newly developed adsorption filter has been evaluated for the removal of white cells (WBCs) from platelet concentrates. It consists of surface-modified polyester fibers and can be used at the patient's bedside. WBC mean removal rate was 98.8 +/- 0.9 percent, and platelet recovery was 99.0 +/- 0.7 percent. The aggregation curves (ADP, collagen) and hypotonic shock response of platelets were not influenced by the filtration, and the plasma electrolyte and lactate dehydrogenase concentrations were unaffected. Transmission and scanning electron micrographs showed no difference in platelet ultrastructure before and after filtration. Determination of posttransfusion platelet recovery did not reveal any significant difference from that of nonfiltered platelets. The tested filter seems to be a suitable device for WBC depletion of single-donor platelet concentrates at the bedside.

A rare‐event analysis model for quantifying white cells in white cell‐ depleted blood

An analysis model to detect and quantify white cells (WBCs) in red cell concentrates (RBCC) drawn from units of blood that are highly depleted of WBCs is described. WBC detection is performed by fluorescence analysis of 50 microL of RBCC labeled with propidium iodide, a DNA/RNA fluorophore. Quantification is performed by regression analysis of standard dilutions of RBCC in substantially WBC-free red cells. This RBCC diluent is obtained by filtration of blood through a new medium. The method proves to be precise (CV = 7%), efficient (+/- 30 min/aliquot), and linear (r = 0.99) to 6 log10 WBC depletion of the native product. The current technique is preferable to those suggested previously, such as ficoll concentration, which requires the sacrifice of the unit of blood for counting purposes, and to earlier fluorescence analysis techniques that do not employ WBC-free red cell diluents. The latter do not monitor extremely low concentrations of WBCs because they lack adequate signal-to-noise discrimination. The sensitivity of the described method allows for monitoring of WBC depletion procedures with greater efficiency than is currently available commercially.

Methods for measuring a 6 log10 white cell depletion in red cells

Methodology is presented for enumerating very low concentrations of white cells (WBCs) in red cells (RBCs) by two separate measurement techniques. Both techniques rely on the method of harvesting WBCs from a 300- to 350-mL unit of RBCs and concentrating them to a volume of approximately 0.5 to 1.0 mL, which is equivalent to a WBC concentration of approximately 550 to 1. The WBC separation and concentration steps require less than 3 hours to complete, and multiple RBC units can be processed in parallel. Cell counting is carried out in a fluorescence hemocytometer or by a modified cytospin technique. As few as 1000 WBCs in a unit of RBCs, which corresponds to a more than 6 log10 WBC depletion, can be measured without reaching the sensitivity limit of either technique (800 and 200 WBC/unit, respectively). The harvesting method and counting techniques are relatively simple and inexpensive.

Reduction in Bleomycin-induced Lung Hydroxyproline Content by an Iron Chelating Agent

Previous investigators have shown that long-term exposure to oxygen after administration of bleomycin can enhance early mortality and subsequent fibrosis.36 This study now demonstrates that as little as 2 minutes of oxygen exposure enhances bleomycin lung injury and that oxygen pulses can be used to titrate injury. That such very short exposures have an effect supports the probable role of free radicals in producing the initial effects of bleomycin on the lung. A second way to influence the severity of early bleomycin injury is variation in dose size. Finally, polymorphonucleated WBC depletion suppresses falls in Pa0 2. This suggests that granulocytes may play a role in the development of acute bleomycin lung injury and provides a third modality for changing the spectrum of lung injury. Granulocytes are stimulated to migrate into the lung following intratracheal bleomycin (see Haslett article, session 1.), and depletion by nitrogen mustard might protect the lung by blocking their release of free radicals, proteolytic enzymes, neutral pro­ teases, etc. Therefore, acute bleomycin lung injury can be titrated by the use of short oxygen pulses, the size of bleomycin dose given, and the depletion of granulated WBCs by nitrogen mustard. Current studies are directed at determining if these manipulations modulate subsequent chronic lung inflamma­ tion and fibrosis. If so, the relative importance of the many factors thought to regulate the transition from acute lung injury to chronic inflammation and fibrosis can be studied and evaluated.