The Evaluation of APTT Reagents in Reference Plasma, Recombinant FVIII Products; Kovaltry® and Jivi® Using CWA, Including sTF/7FIX Assay

The FVIII activity in patients treated with several extended half-life FVIII (EHL-FVIII) agents different when various activated partial thromboplastin time (APTT) reagents were used. The present study examined the difference in clot waveform analysis (CWA) findings and FVIII activity when various APTT reagents and CWA were used. The CWA including FVIII activity was measured using 12 APTT reagents, and the FIX activation based on a small amount of tissue factor assay (sTF/FIX) were examined in reference plasma (RP), EHL-FVIII (Jivi®) and Kovaltry®. The 3 APTT reagents were associated with high variation in the peak time and height in the CWA when analyzing low concentrations of FVIII. The peak time and height could not be measured with one APTT reagent, and there were marked differences in the CWA findings between Jivi® and Kovaltry® among APTT reagents. Several APTT reagents showed a markedly lower FVIII activity with Jivi® than with Kovaltry®. In the FVIII assay, the peak time measured with sTF/FIX did not differ markedly between Jivi® and Kovaltry®; however, the FVIII activity in Jivi® (as measured by the peak height) tended to be higher than in Kovaltry®. The CWA findings for monitoring Jivi® varied for monitoring Jivi® depending on the APTT reagents used, and sTF/FIX assay may be able to measure the EHL-FVIII.

Evaluation of Rosners index vs Brandt correction and Chang’s %, in the interpretation of mixing studies at varying dilutions

Background: For evaluation of unexplained prolongation of PT and PTT, mixing tests forms a great diagnostic tool. On mixing equal volume of patient plasma with normal pooled plasma, if there is correction it indicates factor deficiency and non-correction indicates inhibitors.Methods: Sysmex CS-5100 Coagulometer with Pathrombin SL APTT reagent, LA1 and LA2 reagents supplied by siemens were used. All data were expressed as Mean±SD. Statistical analysis was done using unpaired students t test. A p value of <0.05 was used to indicate statistical significance in all analyse.Results: APTT with (1:1) and (4:1) mixing study for detection of factor deficiency showed a sensitivity of 91% and 92% for RI, 88% and 90% for Changs %, and 75% for Brandt correction PNP aPTT + 5 secs respectively. For Inhibitors, RI shows a sensitivity of 79% and 89%, Changs 71 and 80% and Brandt test 50% for APTT (1:1) and (4:1) mix, respectively.Conclusions: Mixing tests forms an important diagnostic tool in differentiating factor deficiency from inhibitors especially in LAC patients. This study recommends mandatory use of mixing tests in LAC cases as also advocated by BSH, ISTH and CLSI. Rosners Index is more sensitive than changes % and Brandt correction in the interpretation of mixing studies. It can be safely concluded that RI can be used as a reference method for evaluation of mixing studies and its sensitivity is greatly increased by using PP4:1 PNP. It’s a matter of debate that whether these indices can be effective with other Analysers and reagents?

Evaluation of N8-GP Activity Using a One-Stage Clotting Assay: A Single-Center Experience

N8-GP (ESPEROCT®; turoctocog alfa pegol; Novo Nordisk A/S, Bagsvaerd, Denmark) is an extended half-life recombinant factor VIII (FVIII) molecule. FVIII-deficient plasma spiked with N8-GP can be accurately measured using many activated partial thromboplastin time (aPTT)-based one-stage clotting assay reagents with normal human plasma calibrators. To date, there are few data on the measurement accuracy of samples from patients treated with N8-GP. Here, we measure patient samples during routine treatment monitoring. Three previously treated patients with severe hemophilia A (HA) without inhibitors (baseline FVIII activity <0.01 IU/mL) received 50 IU/kg N8-GP every fourth day or twice weekly over 5 years as part of the pathfinder2 trial. Patient samples were monitored using the Pathromtin® SL aPTT reagent (Siemens Healthcare GmbH, Erlangen, Germany), a BCS® XP System analyzer (Siemens), and Standard Human Plasma (Siemens) or product-specific calibrators. Patient age ranged from 36 to 62 years. Overall, measurements performed using product-specific or Standard Human Plasma calibrators were in good agreement, with ratios randomly distributed around 1.0. Peak ratios tended to be closer to 1.0 than trough samples. Pathromtin® SL with Standard Human Plasma calibrator consistently and accurately measured FVIII activity in samples from severe HA patients receiving N8-GP prophylaxis.

Case Report: A 19-Years Old Patient with Haemophilia a with High Response Inhibitor Screening and Quantitation and the Bethesda Method

A severe haemophiliac patient, high response inhibitors, 19 years, who was diagnosed at 8 months old and since then he begins to administer factor VIII concentrates. However, a year later after the start of treatment, they were detected inhibitors that behave as high response inhibitors from the start. He discontinues treatment with Factor VIII concentrate and instead he begins to administer Factor IX concentrate and prothrombin complex as an alternative treatment. Through his illness, the patient has made significant bleeding at the level of joint and other muscles such as the psoas. It is a bleeding patient at rest up for what is currently administered prophylactically Prothrombin Concentrate 3 times a week. This case, the comment will be directed towards laboratory diagnosis and its evolution since he was diagnosed in 1994 to date. From 2013, screening protocols and quantification of both factors and inhibitors were modified in the Specialized Hematology laboratory of Hospital México, due to problems in the sensitivity and specificity of the method and reagents we were using. The results from these patient specific inhibitors are described in the following paragraphs. Observations With the experience and current knowledge of the following it is concluded, according to a literature review that was performed (see Table 1): 1) This time period has persisted inhibitor high title, which is evidenced of the study of mixtures which do not clearly show a potentiation by incubating 2 hours at 37 ° C, since the values of the Control Mix and patient give very similar high values. 2) We were shown only on the date of 14.02.2013, there was a real interference Lupic Anticoagulant (LA), which is confirmed by the method of Russel viper venom. 3) The dates high titer inhibitors were reported against factor VIII (from 6 November 2013 to date), both screening and for the Bethesda, aPTT reagent was used with Kaolin activator which is low sensitivity to LA. 4) On November 20, 2015 there was an error in the interpretation and anti factor VIII inhibitors as negative were reported as potentiation at 37 ° C is not evidenced, but rather was interpreted as interference of an LA, which was communicated to the medical and preventive measures cited in paragraph corrections were made. 5) The last date that the sample was processed, on February 1, 2016, the inhibitor against factor VIII did not affect dilution of silica APTT, so the index ROSNER was not affected (<12) and it was not necessary to mount the LA test, according to the request in identifying protocols inhibitors. 6) Factor VIII deficient plasma is currently being used, which contains von Willebrand factor, as recommended by international guidelines quantization factors. Conclusions According to an experience as support center Reference Center in our country, we conclude and recommend the following: 1) Registration of haemophiliac patients with high antibody titer is essential as the description of the protocol to be followed in these patients. 2) The behavior of this inhibitor in the screening test of time and temperature dependence, it was decided to directly mount the Bethesda assay, following the recommendations of the literature on when the use of a reagent with low sensitivity to lupus inhibitor. 3) Within the protocol and as far as we can, we will process the purchase for quantification of factor VIII chromogenic by ELISA methodology, as a confirmatory method. Disclosures No relevant conflicts of interest to declare.

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

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.

Evaluating the use of commercial drug-specific calibrators for determining PT and APTT reagent sensitivity to dabigatran and rivaroxaban

SummarySuitable laboratory methodologies for quantifying the non-vitamin K oral anticoagulants (NOAC) include liquid chromatography-tandem mass spectrometry (LC-MS/MS) or drug-calibrated assays such as the dilute thrombin time for dabigatran or anti-Xa measurements for rivaroxaban. In situations when these tests are unavailable, it has been suggested that using commercial drug calibrators on APTT and PT assays would theoretically provide reagent sensitivity to these drugs. The purpose of this study was to determine whether commercial drug calibrators deliver similar reagent sensitivity information as samples from patients receiving dabigatran or rivaroxaban as part of their routine care. Two laboratory sites tested commercial calibrator material for dabigatran and rivaroxaban (Hyphen Biomedical) using PT and APTT reagents and data was compared to samples collected from patients taking NOACs that were quantified by LC-MS/MS. Correlation statistics and calculating the amount of drug required to double the clotting time of normal plasma were performed. All drug calibrator material correlated more strongly (R2> 0.95) for any reagent/drug combination than patient samples (R2 ranged from 0.29–0.86). Dabigatran calibrator results and patient data were equivalent for SynthASil and PTT-A APTT reagents. The dabigatran and rivaroxaban calibrator material over-estimated drug sensitivity for all PT reagents when compared to sensitivity data calculated based on drug levels obtained by LC-MS/MS from patient samples. In conclusion, drug-specific calibrators overestimated reagent sensitivity which may underestimate in vivo drug concentration in a given patient. Further studies are required to assess whether this method of determining relative sensitivity of NOAC on routine coagulation assays should be recommended.

Use of an Automated Coagulation Analyzer to Perform Heparin Neutralization With Polybrene in Blood Samples for Routine Coagulation Testing: Practical, Rapid, and Inexpensive

Context.—Heparin contamination in blood samples may cause false prolongation of activated partial thromboplastin time (aPTT) and prothrombin time results. Polybrene can neutralize heparin, but it affects coagulation by itself. Objectives.——To determine the optimal concentration of polybrene to neutralize heparin, to determine the suitable sequence of reagents for the neutralization method performed on the analyzer at the same time as prothrombin time and aPTT testing, and to detect the heparin contamination in blood samples for coagulation tests in our hospital using this method. Design.—Various concentrations of heparin were added to 10 normal and 76 abnormal plasma samples to study the efficacy of polybrene. Two programs of reagent sequencing for aPTT with polybrene performed on the analyzer were tested. Samples suspected of heparin contamination according to our criteria were selected for neutralization during a 3-month period. Results.——The optimal final concentration of polybrene was 25 μg/mL. Polybrene should be added after the aPTT reagent to minimize its interference effect. Even though results of prothrombin time and aPTT after neutralization did not equal those before the spike of heparin, the differences might not be clinically significant. Eighty-one of 4921 samples (1.6%) were selected for aPTT with the neutralization method, and the detection rate of heparin contamination was 84% (68 of 81), giving an overall incidence of 1.4% (68 of 4921). Conclusions.—This method is inexpensive and can be performed rapidly with prothrombin time and aPTT on the automated analyzer, which makes it easy to practice with no need for extra plasma volumes.

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

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.