Conflicting effects of haemolysis on plasma sodium and chloride are due to different haemolysis study protocols: A case for standardisation

2021 ◽  
pp. 000456322110406
Author(s):  
Tejas Kalaria ◽  
Henry Gill ◽  
Hayley Sharrod-Cole ◽  
Clare Ford ◽  
Rousseau Gama
Keyword(s):  
Shear Stress ◽  
Laboratory Experiment ◽  
Whole Blood ◽  
Osmotic Shock ◽  
Plasma Sodium ◽  
H Index ◽  
Freeze Thaw ◽  
Unit Increase ◽  
Modelling Experiment

Background Haemolysis has been reported as having a positive, negative or no effect on plasma sodium (PNa) and chloride (PCl). We investigated the haemoltytic effect of different haemolysis protocols on PNa and PCl using modelling and laboratory experiments. Methods In a modelling experiment, percentage change and recovery due to dilution in routinely ( in vitro) haemolysed samples were compared against shear stress haemolysis and samples spiked with haemolysate from whole blood freeze–thaw, packed cells freeze–thaw and osmotic shock protocols. The results were compared against a control base pool. Additionally, for the osmotic shock method, results were compared against saline- and deionised water (DIW)-spiked controls. In a laboratory experiment, percentage change and recovery were similarly compared using haemolysate from whole blood freeze–thaw and osmotic shock protocols. PNa, PCl and H-index were measured on the Abbott Architect and haemoglobin on the Sysmex XN-9000. Results In the modelling experiment, the percentage decrease in PNa and PCl was similar in in vitro haemolysis, shear stress haemolysis, whole blood freeze–thaw haemolysis and packed cells freeze–thaw haemolysis and this was lower compared to the osmotic shock method. In the laboratory experiment, the change in PNa compared to the base pool was less ( p < 0.001) per unit increase in H-index in the freeze–thaw method (−0.33 mmol, 95% CI −0.35 to −0.31) compared to the osmotic shock method (−0.65 mmol, 95% CI −0.66 to −0.64). PCl did not change with haemolysis in the freeze–thaw method and changed by −0.21 ± 0.01 mmol per unit increase in the H-index in the osmotic shock method. Recovery of PNa and PCl increased with increasing H-index in both methods. Conclusion The osmotic shock protocol is inappropriate for haemolysis studies because of dilution with DIW used for cell lysis. Recovery calculations may incorrectly compensate for genuine dilution caused by haemolysis.

Properties of Thrombolytic Agents in Vitro Using a Perfusion Circuit Attaining Shear Stress at Physiological Levels

1990 ◽  
Vol 64 (04) ◽  
pp. 550-555 ◽  
Author(s):  
N Nishino ◽  
M F Scully ◽  
M W Rampling ◽  
V V Kakkar
Keyword(s):  
Shear Stress ◽  
Whole Blood ◽  
Tissue Type ◽  
Venous Circulation ◽  
Type Plasminogen Activator ◽  
Lysis Rate ◽  
Static Conditions ◽  
Fibrin Thrombi

SummaryA perfusion circuit was designed to investigate in vitro some of the factors which may influence the success of thrombolytic treatment in vivo. The rate of lysis of clotted plasma and different types of artificial thrombi (fibrin thrombi or whole blood thrombi) was measured in citrated plasma or whole blood under static conditions or under shear stress equivalent to the arterial or venous circulation. With both streptokinase (SK) and tissue-type plasminogen activator (t-PA) the rate of lysis of fibrin thrombi and whole blood thrombi was reduced significantly, when compared to the conventional plasma gel clot model (25-fold and 8fold, respectively). This occurred particularly with SK which showed a reduction (4-fold) in potency relative to t-PA under these conditions. Lysis of thrombi by both activators was observed to be faster in plasma than whole blood, and also faster with whole blood thrombi than fibrin thrombi. High shear stress, generally, caused a reduction in the rate of lysis of fibrin thrombi and an increase in the rate of lysis of whole blood thrombi compared to lysis rates under static conditions. Under all conditions of flow the lysis rate observed at 50 units t-PA per ml was much faster than that at 500 units per ml unlike the conventional plasma gel clot model.

A Heparin-coated Circuit Maintains Platelet Aggregability in Response to Shear Stress in an In Vitro Model of Cardiopulmonary Bypass

1998 ◽  
Vol 80 (09) ◽  
pp. 437-442 ◽  
Author(s):  
I. Hioki ◽  
K. Onoda ◽  
T. Shimono ◽  
H. Shimpo ◽  
K. Tanaka ◽  
...  
Keyword(s):  
Shear Stress ◽  
Cardiopulmonary Bypass ◽  
Membrane Skeleton ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Platelet Aggregability ◽  
Vitro Model ◽  
Platelet Defects ◽  
Set Up

SummaryAlterations in platelet aggregability may play a role in the pathogenesis of qualitative platelet defects associated with cardiopulmonary bypass (CPB). We circulated fresh heparinized whole blood through tubing sets coated with heparin (C group, n = 10) and through non-coated sets (N group, n = 10) as a simulated CPB circuit. Shear stress (108 dyne/cm2)-induced platelet aggregation (hSIPA), plasma von Willebrand factor (vWF) activity and platelet glycoprotein (GP) Ib expression were measured, before, during, and after this in vitro set up of circulation. In the two groups, the extent of hSIPA significantly decreased during circulation and was partially restored after circulation. Decreases in the extent of hSIPA were significantly less with use of heparin-coated circuits. There was an equivalent reduction in plasma vWF activity, in the two groups. Expression of platelet surface GP Ib decreased significantly during circulation and recovered after circulation. Reduction of surface GP Ib expression during circulation was significantly less in the C group than that in the N group. Decrease in surface GP Ib expression correlated (r = 0.88 in either group) with the magnitude of hSIPA, in the two groups. The progressive removal of surface GP Ib was mainly attributed to redistribution of GP Ib from the membrane skeleton into the cytoskeleton. Our observations suggest that use of heparin-coated circuits partly blocks the reduction of hSIPA, as a result of a lesser degree of redistribution of GP Ib.

Effect of Dipyridamole on Spontaneous Platelet Aggregation in Whole Blood Decreases with the Time After Venepuncture: Evidence for the Role of ADP

1987 ◽  
Vol 58 (02) ◽  
pp. 744-748 ◽  
Author(s):  
A R Saniabadi ◽  
G D O Lowe ◽  
J C Barbenel ◽  
C D Forbes
Keyword(s):  
Platelet Aggregation ◽  
Correlation Coefficient ◽  
Whole Blood ◽  
Blood Platelet ◽  
Inhibitory Effect ◽  
Time Interval ◽  
Adp Receptor ◽  
Spontaneous Platelet Aggregation

SummarySpontaneous platelet aggregation (SPA) was studied in human whole blood at 3, 5, 10, 20, 30, 40 and 60 minutes after venepuncture. Using a whole blood platelet counter, SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of citrated blood at 37° C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10 μM at each time interval; (b) 0.5-100 μM at 3 and 30 minutes and (c) 15 μM in combination with 100 μM adenosine, 8 μM 2-chloroadenosine (2ClAd, an ADP receptor blocker) and 50 μM aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy with adenosine, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when 15 μM Dipy and 10 μM Ad were added together, the inhibitory effect of Dipy was not increased significantly, suggesting that Dipy inhibits platelet aggregation independent of Ad. The increase in SPA with the time after venepuncture was abolished when blood was taken directly into the anticoagulant containing 5 μM 2ClAd. It is suggested that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture and makes a serious contribution to the artifacts of in vitro platelet function studies.

The Effect of Lysed Platelets on Neutralization of Heparin In Vitro with Protamine as Measured by the Activated Coagulation Time (ACT)

1991 ◽  
Vol 66 (02) ◽  
pp. 213-217 ◽  
Author(s):  
Arthur P Bode ◽  
William J Castellani ◽  
Edna D Hodges ◽  
Susan Yelverton
Keyword(s):  
Platelet Rich Plasma ◽  
Coagulation Time ◽  
Platelet Factor ◽  
Platelet Suspension ◽  
Antiheparin Activity ◽  
Unit Increase ◽  
Heparin Anticoagulation ◽  
Heparin Activity ◽  
Activated Coagulation Time

SummaryThe effect of lysed platelets on the activated coagulation time (ACT) was studied in heparinized whole blood during titration with protamine. Frozen-thawed washed platelet suspension, or a chromatography fraction thereof, or autologous frozen-thawed platelet-rich plasma was added in various dilutions to freshly drawn blood anticoagulated with 3,000 USP units/1 heparin. After a 10 min incubation, the amount of protamine needed to restore the ACT to baseline ("protamine titration dose") was determined. We found that the protamine titration dose decreased in proportion to the amount of lysed platelet material added; expressed as a percentage of the total number of platelets present, each unit increase in lysed platelets produced a 1.7% ±0.8 (SD) reduction in the protamine dose needed to normalize the ACT. A heparin activity assay showed that this effect was not due to antiheparin activity of lysed platelets such as platelet factor 4 (PF4). Our data indicate that the procoagulant activity of platelet membranes reduced the sensitivity of the ACT to heparin. These findings suggest that membranous platelet microparticles may cause an inaccurate calculation, based on the ACT, of a protamine dose to reverse heparin anticoagulation in cardiopulmonary bypass procedures.

Haemostatic Platelet Plug Formation in the Isolated Rabbit Mesenteric Preparation - An Analysis of Red Blood Cell Participation

1980 ◽  
Vol 44 (01) ◽  
pp. 006-008 ◽  
Author(s):  
D Bergqvist ◽  
K-E Arfors
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
In Vitro Test ◽  
Pharmacological Agents ◽  
Vitro Test ◽  
Releasing Effect ◽  
Plug Formation

SummaryIn a model using an isolated rabbit mesenteric preparation microvessels were transected and the time until haemostatic plugs formed was registered. Perfusion of platelet rich plasma gave no haemostasis whereas whole blood did. Addition of chlorpromazine or adenosine to the whole blood significantly prolonged the time for haemostasis, and addition of ADP to the platelet rich plasma significantly shortened it. It is concluded that red cells are necessary for a normal haemostasis in this model, probably by a combination of a haemodynamic and ADP releasing effect.The fundamental role of platelets in haemostatic plug formation is unquestionable but there are still problems concerning the stimulus for this process to start. Three platelet aggregating substances have been discussed – thrombin, adenosine diphosphate (ADP) and collagen. Evidence speaking in favour of thrombin is, however, very minimal, and the discussion has to be focused on collagen and ADP. In an in vitro system using polyethylene tubings we have shown that "haemostasis" can be obtained without the presence of collagen but against these results can be argued that it is only another in vitro test for platelet aggregation (1).To be able to induce haemostasis in this model, however, the presence of red blood cells is necessary. To further study this problem we have developed a model where haemostatic plug formation can be studied in the isolated rabbit mesentery and we have briefly reported on this (2).Thus, it is possible to perfuse the vessels with whole blood as well as with platelet rich plasma (PRP) and different pharmacological agents of importance.

Monitoring Anticoagulant Therapy by Activated Partial Thromboplastin Time: Hirudin Assessment

1994 ◽  
Vol 72 (05) ◽  
pp. 685-692 ◽  
Author(s):  
Michael T Nurmohamed ◽  
René J Berckmans ◽  
Willy M Morriën-Salomons ◽  
Fenny Berends ◽  
Daan W Hommes ◽  
...  
Keyword(s):  
Whole Blood ◽  
Partial Thromboplastin Time ◽  
Ex Vivo ◽  
Fold Increase ◽  
Heparin Therapy ◽  
Activated Partial Thromboplastin Time ◽  
Recombinant Hirudin ◽  
Interindividual Differences ◽  
The Relationship

SummaryBackground. Recombinant hirudin (RH) is a new anticoagulant for prophylaxis and treatment of venous and arterial thrombosis. To which extent the activated partial thromboplastin time (APTT) is suitable for monitoring of RH has not been properly evaluated. Recently, a capillary whole blood device was developed for bed-side monitoring of the APTT and it was demonstrated that this device was suitable to monitor heparin therapy. However, monitoring of RH was not evaluated.Study Objectives. To evaluate in vitro and ex vivo the responsiveness and reproducibility for hirudin monitoring of the whole blood monitor and of plasma APTT assays, which were performed with several reagents and two conventional coagulometers.Results. Large interindividual differences in hirudin responsiveness were noted in both the in vitro and the ex vivo experiments. The relationship between the APTT, expressed as clotting time or ratio of initial and prolonged APTT, and the hirudin concentration was nonlinear. A 1.5-fold increase of the clotting times was obtained at 150-200 ng/ml plasma. However, only a 2-fold increase was obtained at hirudin levels varying from 300 ng to more than 750 ng RH/ml plasma regardless of the assays. The relationship linearized upon logarithmic conversion of the ratio and the hirudin concentration. Disregarding the interindividual differences, and presuming full linearity of the relationship, all combinations were equally responsive to hirudin.Conclusions. All assays were equally responsive to hirudin. Levels up to 300 ng/ml plasma can be reliably estimated with each assay. The manual device may be preferable in situations where rapid availability of test results is necessary.

Studies on Thrombolysis with Streptokinase

1971 ◽  
Vol 25 (02) ◽  
pp. 354-378 ◽  
Author(s):  
R Gottlob ◽  
L Stockinger ◽  
U Pötting ◽  
G Schattenmann
Keyword(s):  
Electron Microscopy ◽  
Cross Section ◽  
Whole Blood ◽  
Jugular Vein ◽  
Thin Sections ◽  
The Third ◽  
Morphological Findings ◽  
Blood Clots ◽  
Arteries And Veins

SummaryIn vitro whole blood clots of various ages, experimental thrombi produced in the jugular vein of rabbits and human thrombi from arteries and veins were examined in semi-thin sections and by means of electron microscopy.In all types of clots examined a typical course of retraction was found. Retraction starts with a dense excentrical focus which grows into a densification ring. After 24 hours the entire clot becomes almost homogeneously dense; later a secondary swelling sets in.Shortly after coagulation the erythrocytes on the rim of the clot are bi-concave discs. They then assume the shape of crenate spheres, turn into smooth spheres and finally become indented ghosts which have lost the largest part of their contents. In the inner zone, which makes up the bulk of the clot, we observed bi-concave discs prior to retraction. After retraction we see no crenations but irregularly shaped erythrocytes. Once the secondary swelling sets in, the cross-section becomes polygonal and later spherical. After extensive hemolysis we observe the “retiform thrombus” made up of ghosts.Experimental and clinical thrombi present the same morphology but are differentiated from in vitro clots by: earlier hemolysis, immigration of leukocytes, formation of a rim layer consisting of fibrin and thrombocytes, and the symptoms of organization. Such symptoms of organization which definitely will prevent lysis with streptokinase were found relatively late in experimental and clinical thrombi. Capillary buds and capillary loops were never found in clinical thrombi prior to the third month.The morphological findings agree with earlier physical and enzymatic investigations. The observation that phenomena of reorganization occur relatively late and frequently only in the rim areas of large thrombi explains why lytic therapy is possible in some of the chronic obliterations.

Monitoring of r-Hirudin Anticoagulation during Cardiopulmonary Bypass – Assessment of the Whole Blood Ecarin Clotting Time

1997 ◽  
Vol 77 (05) ◽  
pp. 0920-0925 ◽  
Author(s):  
Bernd Pötzsch ◽  
Katharina Madlener ◽  
Christoph Seelig ◽  
Christian F Riess ◽  
Andreas Greinacher ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
Whole Blood ◽  
Heart Surgery ◽  
Ex Vivo ◽  
Open Heart Surgery ◽  
Clotting Time ◽  
Blood Samples ◽  
Alternative Approach ◽  
Ecarin Clotting Time

SummaryThe use of recombinant ® hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor Ila assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin- spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 µg/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin- spiked blood samples obtained from 50 healthy blood donors. CV- values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 µg/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.

A Simple and Rapid Method to Determine GPIIb/IIIa-Receptor Inhibitor Activity in Whole Blood

1999 ◽  
Vol 19 (03) ◽  
pp. 134-138
Author(s):  
Gitta Kühnel ◽  
A. C. Matzdorff
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Blood Sample ◽  
Platelet Activation ◽  
Whole Blood ◽  
Receptor Occupancy ◽  
Aggregate Formation ◽  
Inhibitor Activity ◽  
Receptor Inhibitor

SummaryWe studied the effect of GPIIb/IIIa-inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GPIIb/IIIa-inhibitors (c7E3, DMP728, XJ757), then thrombin or ADP were added and after 1 min the sample was fixed. Samples without c7E3 but with 0.1 U/ml thrombin had a decrease in platelet count. Samples with increasing concentrations of c7E3 had a lesser or no decrease in platelet count. The two other inhibitors (DMP 725, XJ757) gave similar results. GPIIb/IIIa-inhibitors prevent aggregate formation and more single platelets remain in the blood sample. The agonist-induced decrease in platelet count correlates closely with the concentration of the GPIIb/IIIa inhibitor and receptor occupancy. This correlation may be used as a simple measure for inhibitor activity in whole blood.

Early Platelet-Collagen Interactions in Whole Blood and Their Modifications by Aspirin and Dipyridamole Evaluated by a New Method (Basic Wave)

1985 ◽  
Vol 54 (04) ◽  
pp. 799-803 ◽  
Author(s):  
José Luís Pérez-Requejo ◽  
Justo Aznar ◽  
M Teresa Santos ◽  
Juana Vallés
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
White Blood Cells ◽  
Reversible Aggregation ◽  
Inhibitory Compounds ◽  
Rapid Generation ◽  
Stimulation Of ◽  
Collagen Stimulation

SummaryIt is shown that the supernatant of unstirred whole blood at 37° C, stimulated by 1 μg/ml of collagen for 10 sec, produces a rapid generation of pro and antiaggregatory compounds with a final proaggregatory activity which can be detected for more than 60 min on a platelet rich plasma (PRP) by turbidometric aggregometry. A reversible aggregation wave that we have called BASIC wave (for Blood Aggregation Stimulatory and Inhibitory Compounds) is recorded. The collagen stimulation of unstirred PRP produces a similar but smaller BASIC wave. BASIC’s intensity increases if erythrocytes are added to PRP but decreases if white blood cells are added instead. Aspirin abolishes “ex vivo” the ability of whole blood and PRP to generate BASIC waves and dipyridamole “in vitro” significantly reduces BASIC’s intensity in whole blood in every tested sample, but shows little effect in PRP.

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