Detection of Mild Clotting Factor Deficiencies. Critical Role of the Activated Partial Thromboplastin Time (aPTT) Reagent

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3281-3281
Author(s):  
Pierre A. Toulon ◽  
Yaelle Eloit ◽  
Motalib Smahi ◽  
Didier Jambou ◽  
Anny Appert-Flory ◽  
...  
Keyword(s):  
Partial Thromboplastin Time ◽  
Activated Partial Thromboplastin Time ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Plasma Samples ◽  
Factor Activity ◽  
Single Factor ◽  
Clotting Factors ◽  
The Common ◽  
Aptt Reagent

Abstract Activated partial thromboplastin time (aPTT) is a routine clotting assay that is widely used to globally screen for coagulation abnormalities, particularly in the pre-operative period. It is commonly admitted that a prolonged test result, may trigger the need for specific assays to be performed and particularly factor assay. However, the responsiveness of aPTT reagents to deficiencies of clotting factors varies because of differences in the type of activator and in the composition/concentration in phospholipids. To investigate the suitability of 5 commercially available aPTT reagents to detect mild/moderate deficiencies of clotting factors, we assessed their responsiveness at increasing concentrations of factors involved not only in the intrinsic pathway of the coagulation system, but also in the common pathway, as very few data were available. The sensitivity of 5 aPTT reagents i.e. HemosIL APTT SP (Instrumentation Laboratory, IL), HemosIL SynthASil (IL), STA-CK Prest (Stago), TriniCLOT aPTT HS (TCoag), and TriniCLOT Automated aPTT (TCoag) to clotting factor was assessed according to the recommendation of the CLSI H47-A2 guideline, by using factor-deficient plasmas spiked with a calibration plasma (all from IL) to produce individual FVIII:C, FIX, FXI, FXII, FV, FX, or FII activities ranging from <1% to ~100 %. Each of the spiked plasma samples was used for the determination of aPTT after being assayed to confirm the activity of the single factor. Tests were simultaneously performed in duplicate on the ACL TOP 700 analyzer (IL) and the values were averaged. Test results were expressed as the sample-to-control ratio, the latter was defined as the clotting time obtained in the calibration plasma containing ~100 % factor activity. The factor activity producing a prolongation of aPTT above 1.20 (patient-to-control ratio) was assigned as the factor responsiveness in % for that reagent. The level (in %) of a given clotting factor responsible for the prolongation of aPTT above 1.20 was highly variable from one aPTT reagent to another (Table). Moreover, for one given aPTT reagent, its sensitivity was very different depending on the specific factor. Actually, the aPTT responsiveness to FVIII:C ranged from 33 % with TriniCLOT aPTT HS to 46 % with HemosIL APTT SP. For FIX, the range was from 18 % with TriniCLOT Automated aPTT to 57 % with HemosIL SynthASil. The aPTT responsiveness to FXI ranged from 38 % with HemosIL SynthASil to 50 % with HemosIL APTT SP, and that to FXII was from 27 % with STA-CK Prest to 48 % with TriniCLOT aPTT HS. So, HemosIL APTT SP showed a good sensitivity (above 42%) to all 3 clotting factors which mild deficiencies are known to be associated with an hemorrhagic tendency (FVIII:C, FIX, and FXI). The same applied to HemosIL SynthASil, which was very sensitive to FIX deficiency (57%), and to STA-CK Prest and TriniCLOT aPTT HS with borderline sensitivity to FIX (29 % for both). The responsiveness to FIX of TriniCLOT Automated APTT was found to be very low (18%). Concerning coagulation factors involved in the common coagulation pathway, the sensitivity to FV was between 38 % with STA-CK Prest and 61 % with TriniCLOT Automated aPTT and that to FX was between 7.0 % with TriniCLOT aPTT HS and 49 % with HemosIL SynthASil. The sensitivity to FII was very low and quite similar for the 5 tested reagents in the range from 9.1 % to 10.7 %. Table. HemosIL APTT SP HemosIL SynthASil STA-CK Prest TriniCLOT APTT HS TriniCLOT Automated aPTT Factor VIII (%) 46 38 43 33 41 Factor IX (%) 42 57 29 29 18 Factor XI (%) 50 38 44 48 46 Factor XII (%) 31 42 27 48 41 Factor V (%) 45 44 38 44 61 Factor X (%) 17 49 16 7.0 9.2 Factor II (%) 9.7 9.1 10.0 9.5 10.7 The difference between reagents responsiveness to FIX was confirmed using plasma samples from patients with hemophilia B either treated with FIX concentrates or not. These results suggested that the sensitivity of the 5 tested aPTT reagents to single factor deficiency was highly variable. If the responsiveness to FVIII:C and FXI of the tested aPTT reagents was accurate, it was not the case for FIX with borderline sensitivity of STA-CK Prest and TriniCLOT aPTT HS (29 %), and more importantly a too low sensitivity of Triniclot Automated aPTT (18%). As this was confirm in clinical materials, this must be considered when analyzing clinical materials, particularly plasma from hemophilia B patients, as mild deficiency states might be undiagnosed with the less sensitive reagent. Disclosures No relevant conflicts of interest to declare.

The effect of dabigatran on the activated partial thromboplastin time and thrombin time as determined by the Hemoclot thrombin inhibitor assay in patient plasma samples

2013 ◽  
Vol 110 (08) ◽  
pp. 308-315 ◽  
Author(s):  
Jenny Butler ◽  
Erica Malan ◽  
Sanjeev Chunilal ◽  
Huyen Tran ◽  
Greg Hapgood
Keyword(s):  
Partial Thromboplastin Time ◽  
Activated Partial Thromboplastin Time ◽  
Thrombin Inhibitor ◽  
Therapeutic Drug ◽  
Clinical Settings ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Aptt Reagent ◽  
The Relationship ◽  
Modest Correlation

SummaryDabigatran is an oral direct thrombin inhibitor that does not require routine laboratory monitoring. However, an assessment of its anticoagulant effect in certain clinical settings is desirable. We examined the relationship between dabigatran levels, as determined by the Hemoclot thrombin inhibitor assay (HTI), the thrombin time (TT) and the activated partial thromboplastin time (aPTT) using different reagents. We describe these parameters with the clinical outcomes of patients receiving dabigatran. Seventy-five plasma samples from 47 patients were analysed. The HTI assay was established to measure dabigatran level. aPTTs were performed using TriniCLOT aPTT S reagent (TC) and three additional aPTT reagents. From linear regression lines, we established the aPTT ranges corresponding to the therapeutic drug levels for dabigatran (90–180 ng/ml). The aPTT demonstrated a modest correlation with the dabigatran level (r= 0.80) but the correlation became less reliable at higher dabigatran levels. The therapeutic aPTT ranges for reagents were clinically and statistically different compared with our reference reagent (46–54 s (TC) vs 51–60 s (SP), 54–64 s (SS) and 61–71 s (Actin FS) (p<0.05)). The TT was sensitive to the presence of dabigatran with a level of 60 ng/ml resulting in a TT > 300 s. In conclusion, the aPTT demonstrated a modest correlation with the dabigatran level and was less responsive with supra-therapeutic levels. aPTT reagents differed in their responsiveness, suggesting individual laboratories must determine their own therapeutic range for their aPTT reagent. The TT is too sensitive to quantify dabigatran levels, but a normal TT suggests minimal or no plasma dabigatran.

How to Optimize Activated Partial Thromboplastin Time (APTT) Testing: Solutions to Establishing and Verifying Normal Reference Intervals and Assessing APTT Reagents for Sensitivity to Heparin, Lupus Anticoagulant, and Clotting Factors

2019 ◽  
Vol 45 (01) ◽  
pp. 022-035 ◽  
Author(s):  
Geoffrey Kershaw ◽  
Soma Mohammed ◽  
Giuseppe Lippi ◽  
Emmanuel Favaloro
Keyword(s):  
Molecular Weight ◽  
Partial Thromboplastin Time ◽  
Lupus Anticoagulant ◽  
High Sensitivity ◽  
Activated Partial Thromboplastin Time ◽  
Factor Xii ◽  
Reference Ranges ◽  
Clotting Factors ◽  
Hospital System ◽  
Normal Reference

AbstractThe activated partial thromboplastin time (APTT) assay is a very common coagulation test, used for several reasons. The test is conventionally used for assessing the contact factor (intrinsic) pathway of blood coagulation, and thus for screening deficiencies in this pathway, most typically factors VIII, IX, and XI. The APTT is also sensitive to contact factor deficiencies, including factor XII, prekallikrein, and high-molecular-weight kininogen. The APTT may also be elevated in a variety of conditions, including liver disease, vitamin K deficiency, and disseminated intravascular coagulation. The APTT can also be used for monitoring unfractionated heparin (UFH) therapy, as well as for screening lupus anticoagulant (LA) or for assessing thrombosis risk. Which of these separate uses is important to a given laboratory or clinician depends on the laboratory and the clinical context. For example, UFH sensitivity is important in hospital-based laboratories, where UFH therapy is used, but not in hospital-based laboratories where low-molecular-weight heparin (LMWH) is largely employed or where UFH may be assessed by anti-factor Xa testing, or in private/community laboratories not associated with a hospital system. High sensitivity to (low levels of) factors VIII, IX, and XI is generally preferred, as their deficiencies are clinically significant. Also preferred, but not usually achieved, is low sensitivity to factor XII and other contact factors, as these deficiencies are usually asymptomatic. Nevertheless, a good knowledge of factor sensitivity is usually needed, if only to help explain the reasons for a prolonged APTT in a given patient, or whether factor testing or other investigation is required. A good working knowledge of reagents sensitivity to LA is also advisable, especially when the reagent is used as part of a LA test panel, or else as a “general-purpose screening reagent.” The current report is aimed at providing some guidance around these questions, and is intended as a kind of “how to” guide, that will enable laboratories to assess APTT reagents in regard to their sensitivity to heparin, LA, and clotting factors. The report also provides some advice on generation of normal reference ranges, as well as solutions for troubleshooting prolonged APTTs, when performing factor testing or searching for inhibitors.

Extravascular Clotting Factor Activity within Joint Tissues Protects Against Progression of Hemophilic Arthropathy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 496-496 ◽  
Author(s):  
Junjiang Sun ◽  
Narine Hakobyan ◽  
Leonard A. Valentino ◽  
Paul E. Monahan
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Joint Capsule ◽  
Factor Ix ◽  
Clotting Factor ◽  
Needle Puncture ◽  
Factor Activity ◽  
Clotting Factors ◽  
Hemophilic Arthropathy ◽  
Human Factor Viii

Abstract Hemophilic arthropathy is the major morbidity of congenital factor VIII and IX deficiency. Therapies localized to hemophilic joints could provide adjunctive protection, in addition to that provided by systemic factor replacement. However, the ability of extravascular clotting factors to contribute to hemostatic protection within joint tissue is unknown. We hypothesized that replacing deficient factor VIII or IX within the injured joint capsule of mice with hemophilia A (FVIII −/ −) or hemophilia B (FIX −/ −), respectively, would decrease the progression of synovitis. We developed a bleeding model consisting of a unilateral knee joint capsule needle puncture to induce hemorrhage in hemophilic mice. Pathology of the joint at two weeks after the injury is graded 0 to 10 using a murine hemophilic synovitis grading system (Valentino, Hakobyan. Haemophilia, 2006). Hemostatically normal mice do not develop synovitis following this injury, but > 95% of FIX −/ − mice develop bleeding and synovitis with a mean grade of 3–4 or greater. Coincident with needle puncture, recombinant human coagulation factor doses ranging from 0 to 20 IU/kg body weight of factor IX or 0 to 25 IU/kg of factor VIII were instilled intraarticularly (I.A.). Comparison groups received the same injury and intravenous (I.V.) factor IX or VIII doses of 25 IU/kg to 100 IU/kg (n= 4–7 mice per study group). Joint bleeding phenotype of the two strains of mice was similar. Mice receiving only saline injection at the time of needle puncture developed mean synovitis scores of 5 ±0.5 in the FVIII −/ − mice and 6 ±0.5 in the FIX −/ − mice. Protection by human clotting factor in the mouse coagulation system was incomplete; mice receiving 100 IU/kg I.V. of factor VIII or factor IX developed synovitis scores of 2.6 ± 1.7 and 2.1 ± 0.2, respectively. In contrast, pathology grade of FVIII −/ − mice dosed with 25 IU/kg I.A. was 0.67 ± 0.3 (p = 0.05 for comparison of 25 IU/kg I.A. with 100 IU/kg IV); FIX−/ − mice receiving 20 IU/kg I.A. had synovitis scores of 0.45 ± 0.58 (p < 0.01 for comparison of 25 IU/kg I.A. with 100 IU/kg I.V.). We next ruled out the possibility that I.A. factor was entering the circulation, and via that route resulting in joint protection, either through technical error at the time of injection, or from a depot effect in the joint with late equilibration into the circulation. Additional groups of mice received factor VIII or IX intravenously at 100 IU/kg, or intraarticularly at 4 times the doses used in the hemarthrosis challenge (80 IU/kg FIX or 100 IU/kg FVIII), and factor activity assays were performed at 1, 4, 12, 24, and 48 hours. Expected circulation kinetics were seen following I.V. dosing; no increase in circulating factor VIII or IX activity were seen in the intraarticular dosing groups at any timepoint. In considering the potential immunogenicity of an intraarticular therapy approach for hemophilic joint therapy, factor VIII −/ − mice were treated with three doses of human factor VIII 100 IU/kg at five day intervals either I.V. or I.A. At two weeks after exposure, 5/5 I.V.-treated mice developed inhibitor antibodies with titers ranging 0.8–7.2 BU; 2/5 I.A.-treated mice had detectable low-titer antibodies (1.3 BU), indicating no greater immunogenicity in the I.A. model. Extravascular factor VIII and factor IX can contribute to protection against blood-induced joint deterioration; enhancing local tissue hemostasis with protein or gene therapy may prove a useful adjunct to systemic replacement.

scholarly journals Isocitrate as Calcium Ion Activity Buffer in Coagulation Assays

1999 ◽  
Vol 45 (8) ◽  
pp. 1176-1180 ◽  
Author(s):  
Mats RÅnby ◽  
Tony Gojceta ◽  
Kerstin Gustafsson ◽  
Kenny M Hansson ◽  
Tomas L Lindahl
Keyword(s):  
Partial Thromboplastin Time ◽  
Calcium Ion ◽  
Activated Partial Thromboplastin Time ◽  
Plasma Samples ◽  
Healthy Individuals ◽  
Physiological Concentration ◽  
Diagnostic Efficacy ◽  
Coagulation Assays ◽  
Coagulation Tests ◽  
Ion Activity

Abstract Background: Ca2+ activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca2+ activity at ∼1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca2+ buffering improves diagnostic efficacy in global diagnostic coagulation tests. Methods: Buffering was investigated by mixing CaCl2 and 11 candidate compounds and determining Ca2+ activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca2+ activity to ∼1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved. Results: Two suitable Ca2+ buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca2+ buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca2+ activity of 1.60 ± 0.07 mmol/L, compared with 2.73 ± 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 ± 5.7, compared with 3.6 ± 5.0 (P &lt;0.005) for the original APTT. Conclusions: Isocitrate can be used to buffer Ca2+ activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca2+ activity shows signs of improved diagnostic efficacy.

scholarly journals A Multicenter Evaluation of an Activated Partial Thromboplastin Time Test, Cepho-Test

1977 ◽  
Author(s):  
D. Collen ◽  
H.C. Godal ◽  
P.M. Mannucci ◽  
I.M. Nilsson ◽  
C. Gilhuus-Moe ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Partial Thromboplastin Time ◽  
High Sensitivity ◽  
Mean Value ◽  
Activated Partial Thromboplastin Time ◽  
Satisfactory Precision ◽  
Time Test ◽  
The Mean ◽  
Aptt Reagent ◽  
Control Plasma

To compare the sensitivity and precision of the Activated Partial Thromboplastin Time (APTT) test Cephotest to that of APTT methods in current use, Cepho-test and current APTT method (Leuven and Milan: Locally modified Thrombofax/kaolin procedures; Malmoe: Automated APTT; Oslo: APTT of human brain/kaolin) were performed in parallel (20 tests) on lyophilized standard plasmas of 4 levels of factors VIII. The mean value (1 standard deviation) of Cephotest on Control Plasma Normal was 36.3 (2.21) s in Leuven, 31.7 (1.13) s in Oslo, 35.0 (1.36) s in Milan and 35.0 (1.16) s in Malmoe. The corresponding values of the local APTT methods were 50.2 (I.58) s, 34.5 (1.27) s, 51.9 (1.17) s and 38.8 (1.23) s, respectively. In Oslo, Milan and Malmoe, the sensitivity of Cephotest was superior to that of the local APTT reagent at all levels of factor VIII. In Leuven, the local APTT method had a higher ratio than Cephotest. There was no statistical significant differences between the standard deviation of Cephotest and the local APTT methods. The study indicates that Cephotest has a high sensitivity, satisfactory precision and is subjected to only minor interlaboratory variations.

The Control Of Heparin Administration By The Activated Partial Thromboplastin Time(APTT)

1981 ◽  
Author(s):  
Jean M Thomson
Keyword(s):  
Quality Control ◽  
Partial Thromboplastin Time ◽  
Activated Partial Thromboplastin Time ◽  
Reference Laboratory ◽  
Plasma Samples ◽  
Heparin Administration ◽  
National Quality ◽  
Fresh Plasma ◽  
Low Levels ◽  
The Uk

The UK National Quality Control Trials have previously shown that the various APTT methods differ in their ability to detect low levels of heparin (Poller et al 1980). UK and US proficiency surveys have also shown lack of linearity of some APTT methods over a range of heparin concentrations. A further, recent collaborative exercise, using lyophilised plasma from a heparinised donor, has confirmed that most of the commonly-used commercial reagents have a higher threshhold of sensitivity to heparin than the reference reagent provided by the National (UK) Reference Laboratory. Additional studies on fresh plasma samples obtained from heparinised patients, have demonstrated considerable variations in the detection of heparin by widely-used commercial APTT techniques.

Control of Heparin Treatment with three Different Methods. Behaviour of Antithrombin III

1979 ◽  
Author(s):  
H. Bounameaux ◽  
G.A. Marbet ◽  
H. Airenne ◽  
E. Grossmann ◽  
B. Stanojevic ◽  
...  
Keyword(s):  
Correlation Coefficient ◽  
Partial Thromboplastin Time ◽  
Antithrombin Iii ◽  
Heparin Therapy ◽  
Activated Partial Thromboplastin Time ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Heparin Concentration ◽  
The Mean ◽  
Antithrombin Iii Activity

In 63 plasma samples from patients under heparin we determined the heparin concentration using the chromogenic substrats S-2222 (COATEST Heparin). Thrombin time (TT), activated partial thromboplastin time (APTT), antithrombin III activity (ATIIIact) and concentration (ATIIIimm) were also measured. A good correlation was found between heparin concentration and TT (r= .850, p< .001), heparin concentration and APTT (r =669, p < .001) while the correlation coefficient r between TT and APLT was .896 (p< .001).We found a statilttically significant reduction of ATIIIact with increasing APTT (p < .05. The ATIIIact and ATIIIimm values were also lower (p < .001) in the overanticoaculated group (n=ll) than in the group with insufficient heparinisation (0.18). The mean (±SO) heparin concentration in 12 plasmas with both TT and APTT in the therapeutic range was .54 (±.15) USP-U/al, very similar to that of 13 plasma (.68 ± .46 U/al) insufficiently heparinised accordirig to the APTT. However, the TT recognised then as correctly anticoagulated. Regarding these findings and our good experience without complication by monitoring heparin therapy with TT we assume that TT is more accurate than APTT for this aim.

scholarly journals Aromatic Amines Exert Contrasting Effects on the Anticoagulant Effect of Acetaldehyde upon APTT

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
La'Teese Hall ◽  
Sarah J. Murrey ◽  
Arthur S. Brecher
Keyword(s):  
Partial Thromboplastin Time ◽  
Aromatic Amines ◽  
Room Temperature ◽  
Activated Partial Thromboplastin Time ◽  
Anticoagulant Effect ◽  
Pharmacological Effects ◽  
Independent Action ◽  
Clotting Factors ◽  
Selective Interaction ◽  
Procoagulant Effect

The pharmacological effects of amphetamine, procaine, procainamide, DOPA, isoproterenol, and atenolol upon activated partial thromboplastin time in the absence and presence of acetaldehyde have been investigated. In the absence of acetaldehyde, amphetamine and isoproterenol exhibit a procoagulant effect upon activated partial thromboplastin time, whereas atenolol and procaine display anticoagulant effects upon activated partial thromboplastin time. DOPA and procainamide do not alter activated partial thromboplastin time. Premixtures of procaine with acetaldehyde produce an additive anticoagulant effect on activated partial thromboplastin time, suggesting independent action of these compounds upon clotting factors. Premixtures of amphetamine with acetaldehyde, as well as atenolol with acetaldehyde, generate a detoxication of the anticoagulant effect of acetaldehyde upon activated partial thromboplastin time. A similar statistically significant decrease in activated partial thromboplastin time is seen when procainamide is premixed with acetaldehyde for 20 minutes at room temperature. Premixtures of DOPA and isoproterenol with acetaldehyde do not affect an alteration in activated partial thromboplastin time relative to acetaldehyde alone. Hence, a selective interaction of atenolol, procaine, and amphetamine with acetaldehyde to produce detoxication of the acetaldehyde is suggested, undoubtedly due to the presence of amino, hydroxyl, or amide groups in these drugs.

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