Isolation of Human Platelets (Thrombocytes)

Human peripheral blood monocytes are isolated by flotation from whole blood through a single low-density barrier prepared from OptiPrep™ at 4°C. The separation from lymphocytes depends on the more rapid rate of flotation of the monocytes because of their slightly lower density and larger size. The method works optimally only with fresh (within 2 h of drawing) EDTA-anticoagulated blood. Preliminary evidence suggests that this technique may be applicable to blood from rats.

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Drug Dependent Immune Destruction of Human Platelets in the NOD/SCID Mouse.

Blood ◽

10.1182/blood.v112.11.3419.3419 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3419-3419

Author(s):

Daniel W. Bougie ◽

Brian Boylan ◽

Dhirendra Nayak ◽

Peter J. Newman ◽

Richard H. Aster

Keyword(s):

Human Blood ◽

Blood Cells ◽

Scid Mouse ◽

Human Platelets ◽

Human Igg ◽

Human Blood Cells ◽

Rapid Clearance ◽

Human Igg1 ◽

Immune Destruction ◽

Drug Dependent

Abstract Animal models have been of limited value for studying survival and immune destruction of human blood cells because of xenoantibodies that mediate rapid elimination of the transfused cells. The NOD/SCID mouse is a genetic variant that lacks immunoglobulins, including xenoantibodies, and may therefore be useful for studying survival and immune destruction of human blood cells (Newman, PJ et al. J Thromb Haemost.2007; 5 Suppl 1:305–9). Human platelets, infused into the retroorbital plexus of NOD/SCID mice survive about three days, nearly as long as mouse platelets themselves. Injection of IgG from a patient with quinine-induced immune thrombocytopenia (TP) and a newly developed quinine-dependent murine monoclonal antibody (mAb) were without effect on platelet survival. However, both antibodies caused rapid platelet clearance after IP injection of quinine. 500 μg, but not 50 μg, of IgG from serum containing an anti-HPA-1a (PlA1) antibody also caused rapid clearance of HPA-1a positive platelets. These findings indicate that the NOD/SCID mouse can be valuable for the study of immune clearance of human platelets and perhaps other unresolved problems in transfusion medicine. However, it is not known whether mice recognize cells opsonized with human IgG or mouse IgG equally well. To examine this, we compared the survival of human platelets sensitized with 1) mAb 7E3 (IgG1) specific for an epitope in the beta A domain of GPIIIa or 2) c7E3 (from Centocor Inc), which recognizes the same epitope but possesses a human IgG1 Fc. In titration studies, we found that about four times as much c7E3 as 7E3 is needed to produce equivalent clearance of human platelets, indicating that mouse IgG is a more efficient opsonin than human IgG. However, this difference should not limit use of the model for studies of antibody-mediated blood cell destruction, since mg quantities of IgG can easily be isolated from small amounts (0.1– 0.2 ml) of human serum.

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Meeting Requirements of the California Cholinesterase Monitoring Program

International Journal of Toxicology ◽

10.1080/10915810490435613 ◽

2004 ◽

Vol 23 (2) ◽

pp. 97-100 ◽

Cited By ~ 9

Author(s):

Barry W. Wilson ◽

John D. Henderson ◽

Daniel E. Arrieta ◽

Michael A. O’Malley

Keyword(s):

Human Blood ◽

Whole Blood ◽

Blood Cells ◽

Conversion Factor ◽

Monitoring Program ◽

Plasma Samples ◽

Comparison Study ◽

Clinical Laboratories ◽

Pesticide Regulation ◽

Current List

California (CA) has a long-standing formal blood cholinesterase (ChE) monitoring program for mixers, loaders, and applicators of pesticides. When the authors found commercial clinical kits were not optimal for assaying blood ChEs, CA regulations were revised to specify use of the Ellman ChE assay or to demonstrate a conversion factor with a correlation ( r2) of 0.9 or better. The authors were enlisted to work with the clinical laboratories. Only two of seven participating laboratories generated an acceptable correlation for red blood cells (RBCs), whereas four of five laboratories had an acceptable correlation for plasma ChE. Subsequently, the CA Department of Pesticide Regulation (DPR) restated the need to meet this requirement and the authors worked with several of the clinical laboratories using a bovine ghost RBC ChE as a reference. Unfortunately, only 3 of 10 laboratories had acceptable correlations. Next, the authors provided all interested laboratories with human blood and plasma samples to perform the comparison study outlined in the regulation (Section 6728f). Fourteen laboratories participated; 9 met the ChE criteria for whole blood, 14 for plasma, and 6 for RBCs. Based on such data, on July 8, 2003, DPR notified the CA Agricultural Commissioners that nine of the participating laboratories were approved for ChE testing. Later work resulted in acceptable RBC values for two of the laboratories and their approval. The authors continue to work with laboratories interested in being on the approved list. The current list may be seen at www.cdpr.ca.gov/docs/whs/lablist.htm .

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Influence of Decalcifying Agents in Increasing Concentrations on the Stability of Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654949 ◽

1961 ◽

Vol 05 (03) ◽

pp. 559-565 ◽

Cited By ~ 1

Author(s):

R Honorato ◽

G Schindler

Keyword(s):

Red Blood Cells ◽

Human Blood ◽

Blood Cells ◽

Sodium Citrate ◽

Blood Platelets ◽

Human Platelets ◽

Sodium Oxalate ◽

Blood Samples ◽

The Stability ◽

Human Blood Platelets

Summary1. The influence of 0.025 M, 0.1 M, 0.2 M sodium oxalate and of 0.2 M sodium citrate on the stability of human blood platelets was studied.2. A diminution of the resistance of platelets to glass in the 0.025 M and 0.1.M sodium oxalate blood samples was observed.3. This effect of oxalate was not observed when red blood cells were not present.

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THE RHEOLOGY OF THE BLOOD

The Journal of General Physiology ◽

10.1085/jgp.28.2.131 ◽

1944 ◽

Vol 28 (2) ◽

pp. 131-149 ◽

Cited By ~ 6

Author(s):

Eugene C. Bingham ◽

Raymond R. Roepke

Keyword(s):

Human Blood ◽

Whole Blood ◽

Blood Cells ◽

Volume Concentration ◽

Preceding Paper ◽

Barometric Pressure ◽

The State ◽

Hemoglobin Content ◽

Healthy Human ◽

Similar Formula

In the preceding paper (1b) a formula was developed for the lowering of the fluidity of a medium by a mixture of proteins, given the volume concentration of each and its fluidity-lowering constant. Whole blood is now shown to follow an essentially similar formula, except that the hemoglobin content is taken from the literature as the best available measure of the volume of the blood cells Δ Φ = 0.24H, assuming the fluidity of the medium to be 53 rhes. Age, sex, diet, barometric pressure affect the hemoglobin content of the blood, but the formula may apply to any healthy human blood to about 3 per cent. The shape, number, and size of the blood cells, if known, might help to explain discrepancies as well as the state of oxidation of the blood. In disease the discrepancy becomes much greater, suggesting the possible use of rheology in diagnosis.

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Abstract WP108: Novel Human Acute Ischemic Stroke Blood Clot Analogues for in-vitro Testing

Stroke ◽

10.1161/str.51.suppl_1.wp108 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Seán Fitzgerald ◽

Daying Dai ◽

Andrew S Douglas ◽

Oana M Mereuta ◽

Thomas Caracena ◽

...

Keyword(s):

Ischemic Stroke ◽

Red Blood Cells ◽

Acute Ischemic Stroke ◽

Human Blood ◽

Whole Blood ◽

Blood Cells ◽

Blood Clot ◽

Buffy Coat

Introduction: Previous studies have successfully created blood clot analogues for In-vitro testing using animal blood. Blood components vary greatly among species and thus, creating clot analogues with human blood is likely a more accurate representation of thrombi formed in the human vasculature. We present a novel method of creating clot analogues from human blood and platelets that mimic the process by which clots form In-vivo . Methods: Following IRB approval from Mayo Clinic, human whole blood and platelets donations were obtained from the Blood Transfusion service. The whole blood was centrifuged at 1,200RPM for 20 minutes to separate it into its constituents. Plasma was removed and the remaining Red Blood Cells and Buffy Coat were mixed together by inverting. A total of 12 clot analogues were created with varying concentrations of components; Red Blood cells/Buffy Coat, Plasma and Platelets. Thrombin was added first to stimulate platelets activation for a total of 5 mins whilst continuously mixing by inversion. The RBC/WBC mixture was added next followed by CaCl2. The mixture was then quickly drawn into a 3cc syringe and spun overnight at 20RPM at room temperature to mimic dynamic flow conditions. Macro-photographs were taken to display the variation in texture and color between different clot analogue types. The clots were then fixed in 10% neutral buffered formalin for 24 hours prior to being processed. Histopathological analysis was performed using Hematoxylin and Eosin (H&E) and Martius Scarlet Blue (MSB) staining to confirm clot composition. Results: Red Blood cell-rich, Fibrin-rich, Platelet-rich and mixed clot analogues that accurately mimic clots retrieved from Acute Ischemic Stroke Patients were created. The range of histopathological compositions of the clot analogues is similar to that of the clinical samples. Conclusions: The addition and activation of platelets is key to creating accurate clot analogues for In-vitro testing. Spinning the clots is important to prevent natural sedimentation and mimic the In-vivo situation.

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Evaluation of a Technic for Counting Dog and Human Platelets

Blood ◽

10.1182/blood.v25.2.261.261 ◽

1965 ◽

Vol 25 (2) ◽

pp. 261-266 ◽

Cited By ~ 2

Author(s):

ROBERT I. WEED ◽

S. LEE CRUMP ◽

SCOTT N. SWISHER ◽

Norma C. Trabold

Keyword(s):

Human Blood ◽

Whole Blood ◽

Blood Platelets ◽

Ammonium Oxalate ◽

Human Platelets ◽

Blood Samples ◽

Platelet Counts ◽

Statistical Errors ◽

Human Blood Samples

Abstract 1. The addition of Na2EDTA (2.5 to 5 mg./2 ml. of whole blood) to dog or human blood samples has been shown to eliminate the problem of platelet clumping and to provide satisfactory samples for use in enumeration of blood platelets. The statistical errors of platelet counts in blood samples prepared with Na2EDTA agree well with previously published results obtained with unmodified technic. In blood samples prepared without Na2EDTA, where clumping is appreciable, the field error is inflated. 2. Variation in the time during which the platelets are permitted to settle in the chamber before counting, between limits of 30 and 90 minutes, has no effect on the count. 3. The period of standing of the diluted blood in 1 per cent ammonium oxalate, from 30 minutes to four hours, appears to have no effect on the out-come of the platelet counting results when the platelets are not clumped.

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Spectroscopic Studies of in Vitro Exposure to Low-Dose Gamma Rays from 137Cs Radioactivity on Some Human Blood Components (Plasma and Red Blood Cells)

10.21203/rs.3.rs-254110/v1 ◽

2021 ◽

Author(s):

Benjamaporn Supawat ◽

Watcharit Vorasiripreecha ◽

Sakornniya Wattanapongpitak ◽

Suchart Kothan ◽

Montree Tungjai

Keyword(s):

Red Blood Cells ◽

Gamma Rays ◽

Human Blood ◽

Whole Blood ◽

Blood Cells ◽

Low Dose ◽

Radioactive Cesium ◽

Blood Components ◽

Blood Samples

Abstract This current study was to determine the effects of in vitro exposure to radioactive cesium-137 on some human blood components (Plasma and red blood cells). Blood samples were given a radiation dose of 0.02, 0.05, 0.1, 0.2, and 0.3 mGy of gamma rays using a 137Cs radioactive standard source. The blood samples that were exposed to 0 mGy served as sham-controls. The spectrofluoroscopic technique was used to determine the autofluorescence spectrum of protein in plasma or red blood cells by using excitation wavelength and range of emission wavelengths at 280 nm and 300-550 nm, respectively. The spectrophotometric technique was used to determine the release of hemoglobin from the red blood cells to the supernatant. This data indicated no change in the ratio of fluorescence emission intensity at 340 nm of wavelength of protein extract from irradiated whole blood or red blood cells compared to the corresponding non-irradiated control. The results did not change in the absorption intensity at 415 nm of wavelength of hemoglobin leakage from in vitro irradiated red blood cells when compared to the corresponding non-irradiated red blood cells. These current results suggested that there were no harmful effects of the low-dose gamma rays from radioactive 137Cs on some blood components when human whole blood was exposed to gamma rays in an in vitro condition.

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The Effect of Red Blood Cells on the ADP-induced Aggregation of Human Platelets in Flow Through Tubes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645696 ◽

1990 ◽

Vol 63 (01) ◽

pp. 112-121 ◽

Cited By ~ 18

Author(s):

David N Bell ◽

Samira Spain ◽

Harry L Goldsmith

Keyword(s):

Platelet Aggregation ◽

Shear Rate ◽

Red Blood Cells ◽

Blood Cells ◽

Platelet Rich Plasma ◽

Mean Transit Time ◽

White Blood Cells ◽

Aggregate Size ◽

Human Platelets ◽

Induced Aggregation

SummaryThe effect of red blood cells, rbc, and shear rate on the ADPinduced aggregation of platelets in whole blood, WB, flowing through polyethylene tubing was studied using a previously described technique (1). Effluent WB was collected into 0.5% glutaraldehyde and the red blood cells removed by centrifugation through Percoll. At 23°C the rate of single platelet aggregtion was upt to 9× greater in WB than previously found in platelet-rich plasma (2) at mean tube shear rates Ḡ = 41.9,335, and 1,920 s−1, and at both 0.2 and 1.0 µM ADP. At 0.2 pM ADP, the rate of aggregation was greatest at Ḡ = 41.9 s−1 over the first 1.7 s mean transit time through the flow tube, t, but decreased steadily with time. At Ḡ ≥335 s−1 the rate of aggregation increased between t = 1.7 and 8.6 s; however, aggregate size decreased with increasing shear rate. At 1.0 µM ADP, the initial rate of single platelet aggregation was still highest at Ḡ = 41.9 s1 where large aggregates up to several millimeters in diameter containing rbc formed by t = 43 s. At this ADP concentration, aggregate size was still limited at Ḡ ≥335 s−1 but the rate of single platelet aggregation was markedly greater than at 0.2 pM ADP. By t = 43 s, no single platelets remained and rbc were not incorporated into aggregates. Although aggregate size increased slowly, large aggregates eventually formed. White blood cells were not significantly incorporated into aggregates at any shear rate or ADP concentration. Since the present technique did not induce platelet thromboxane A2 formation or cause cell lysis, these experiments provide evidence for a purely mechanical effect of rbc in augmenting platelet aggregation in WB.

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Plasma Thromboplastin Component (Christmas Factor, Factor IX) Levels in Stored Human Blood and Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654521 ◽

1960 ◽

Vol 04 (03) ◽

pp. 376-388 ◽

Cited By ~ 10

Author(s):

J Dieter Geratz ◽

John B. Graham

Keyword(s):

Human Plasma ◽

Human Blood ◽

Whole Blood ◽

Room Temperature ◽

Close Agreement ◽

Factor Ix ◽

Deficient Patient ◽

Glass Tubes ◽

Disodium Hydrogen Citrate ◽

Bank Blood

Summary1. PTC activity was assayed in 26 units of human plasma prepared from whole blood stored for 3 weeks at 4° C. The plasma had been frozen and stored at — 20° C for additional periods ranging from a few days to 4 months. High PTC activity was still present in the plasma at the end of this period, the activity averaging 95% of normal.2. The PTC activity of 19 samples of “reclaimed“ plasma stored for an additional 6 months at — 20° C decreased by an average of 23%. This decrease was statistically significant.3. Liquid plasma kept at room temperature for 5½—7½ months contained no PTC activity.4. Lyophilized plasma stored at room temperature for 6—8 years contained an average of 30% PTC activity. Lyophilized plasma stored at — 20° C for 4 years contained 68% PTC activity.5. ACD and disodium hydrogen citrate anticoagulant solutions served equally well in preserving PTC activity in whole blood stored in glass tubes over a period of 3 weeks at 4° C.6. “Reclaimed“ plasma from outdated bank blood provided effective hemostasis in two operations for the removal of 20 teeth from a severely PTC-deficient patient.7. The high PTC activity of “reclaimed“ plasma was confirmed by the close agreement between the PTC level expected in a PTC deficient patient after transfusion of such plasma and that observed.

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