In house anti-factor Xa kit for determination of heparin amount coated inside available plastic and glass hematocrit tubes in Thailand

Kit interno antifator Xa para determinação da quantidade de heparina revestida dentro de tubos de hematócrito de plástico e vidro disponíveis na Tailândia

Abstract

A company successfully developed heparin-coated plastic hematocrit tubes; however, they have no appropriate determination method to perform quality control and assurance of the heparin amount coated inside the tubes. Here, we aimed to develop a kit using the anti-factor Xa principle. After the kit was fabricated, we compared the efficiency of our in-house kit with other commercially available kits. Furthermore, we determined the sensitivity of our developed kit with a variety of anti-coagulated drugs, which are usually used for treating cardiovascular diseases. The results showed that our in-house kit could detect similar amounts of heparin to other commercial kits. Our in-house kit could also detect anti-coagulated drugs at the ppm level. According to the results, the collaborator company uses our in-house kit for the determination of heparin amounts coated inside the plastic hematocrit and blood collecting tube. Future development of the kit also has a high potential for the detection of anti-coagulated drugs inside human and animal blood samples, instead of aPTT, which is generally used in Thailand’s healthcare facilities.

Keywords:
anti-factor Xa; commercial kit; LMWH; hematocrit tube; heparin

Resumo

Uma empresa desenvolveu com sucesso tubos de hematócritos de plástico revestidos com heparina. No entanto, eles não possuem um método adequado para determinar a quantidade de heparina revestida nos tubos, o que é essencial para o controle de qualidade. O objetivo deste estudo foi desenvolver um kit baseado no princípio do antifator Xa. Após a fabricação do kit, comparou-se sua eficiência com a de outros kits comerciais disponíveis no mercado. Além disso, avaliou-se a sensibilidade do kit desenvolvido em relação a uma variedade de medicamentos anticoagulantes comumente utilizados no tratamento de doenças cardiovasculares. Os resultados demonstraram que o kit interno foi capaz de detectar quantidades de heparina semelhante às dos kits comerciais. Além disso, o kit desenvolvido foi capaz de detectar medicamentos anticoagulantes em níveis de ppm (partes por milhão). Com base nesses resultados, a empresa colaboradora atualmente passou a utilizar o kit interno para determinar a quantidade de heparina revestida nos tubos de hematócritos de plástico e nos tubos de coleta de sangue. O desenvolvimento futuro do kit apresenta também um elevado potencial para a detecção de fármacos anticoagulantes em amostras de sangue humano e animal, em substituição do aPTT, que é geralmente utilizado nas unidades de saúde da Tailândia.

Palavras-chave:
antifator Xa; kit comercial; HBPM; tubo de hematócrito; heparina

1. Introduction

There are several methods for the determination of heparin or related compounds, which are called Low Molecular Weight Heparins (LMWH) class, depending on the type of sample. For blood samples, in Thailand, we usually use the activated Partial Thromboplastin Time (aPTT) method. The aPTT analysis between 2 time points allows us to predict and calculate the decreasing rate of the LMWH amount inside the collected blood. As a result, physicists or veterinarians will be able to use the information for adjusting an appropriate personalized drug dosage (Bronić et al., 2021; Lardinois et al., 2022; Maeckelbergh and Acierno, 2008). ELISA can also specifically determine amounts of heparin, but not the other LMWH compounds, inside any form of biological samples, in addition to serum. The sandwich assay using a specific antibody against heparin is available commercially, and the manufacturer claims that the entire process can be performed within 90 minutes (Zwicker et al., 2004; Engelmaier et al., 2020; Attah et al., 2024). Another faster method, 6-minute anti-factor Xa analysis, was more recently developed. This method involves the inhibition of factor Xa by antithrombin conjugated with the LMWH drug. The activity of leftover factor Xa from the inhibition is determined by supplementation of the chromogenic substrate of factor Xa. Comparing with a standard curve, the amount of heparin or LMWH can be reported in units (Lehman et al., 2006; Newall, 2013; Pirahanchi et al., 2024; Lanoiselée et al., 2024).

The aPTT method can be used to determine the depletion of LMWH over time, but the result cannot be reported in units. Furthermore, the aPTT method cannot be used with other biological samples in addition to the blood from cardiovascular disease human patients and animals that were collected by hematocrit capillary tube (McLaughlin et al., 2019; Williams-Norwood et al., 2020). ELISA also has several limitations and flaws, even though it can be applied to any biological sample. The antibody used in the ELISA kit can be degraded easily at high temperatures during transportation. We need to order the kit from a foreign country and ship it with a special low-temperature delivery courier, which is costly and still has a risk of reducing the kit’s quality (Nagler et al., 2016; Sahu et al., 2020). ELISA is more time-consuming and requires more specialized materials for in-house kit fabrication than the anti-factor Xa method. However, anti-factor Xa commercial kits are available from only one company. We need to purchase the kit and then have it shipped overseas to Thailand. The kit is also expensive because there are lyophilized proteins, chromogenic substrates, and enzymes in the kit, which should be transported at low temperatures. Moreover, the kit provides only 3 vials of the mentioned reagents, but the user needs to prepare the rest of the instruments and buffers themselves. Considering the advantages and disadvantages of all the methods available here, it is likely that the anti-factor Xa method is the highest potential one to be developed in-house for the determination of LMWH in any form of sample with cheaper expense (Samuel et al., 2016; Lessire et al., 2018; Amiral et al., 2021).

A company in Thailand developed a plastic hematocrit blood collecting capillary tube. It is their objective to provide safe capillary tubes for customers when collecting blood from patients. They claimed that their plastic tube can’t be broken in the user’s hand during the blood collection process. It reduces contamination from possibly available pathogens in the patient’s blood to the operational physicist, nurse, medical technologist, or veterinarian. They have 2 types of plastic capillary tubes: plain plastic capillary tubes and heparin-coated plastic capillary tubes. According to quality control standardization for heparin-coated capillary tubes, a single tube should contain heparin inside at least 4 IUs (DIN, 2007). However, the company has no such quality control method to determine the amount of heparin inside their tubes, and they are interested in developing an in-house protocol to solve this problem. Together with their collaboration, we aim to develop an in-house anti-factor Xa kit for the determination of heparin amounts inside the heparin-coated capillary tube. The developed kit should have a similar capacity to the gold-standardized kits available in the market to determine the heparin amount. Moreover, we provide all the proteins, enzymes, reagents, and some materials for the user’s convenience in the kit. Nowadays, the company uses our kit as standard quality control protocol for all sizes of their heparin coated blood-collecting tubes in addition to the capillary hematocrit tubes.

2. Materials and Methods

2.1. Chemicals

Heparin, human anti-thrombin, bovine factor Xa, and chromogenic substrate specific for factor Xa (N-α-Z-D-Arg-Gly-Arg-pNA, 2HCl; C₂₈H₃₉N₁₁O₇, 2HCl) (5-Dagnostics, Austria) were bulky and purchased from Zigma Biotech (Thailand). We bought other general chemicals (acetic acid, EDTA, NaCl, PEG, and tris) from Sigma (Thailand). For Biophen’s anti-factor Xa kit (Hyphen Biomed, France), we purchased online from Aniara’s (USA) website. Both heparin-coated glass and plastic hematocrit tubes were purchased from responsible brand distributors. We defined 6 of them as brand A to F. Furthermore, we purchased other LMWH drugs, including apixaban (Eliquis), edoxaban (Lixiana), fondapalinux (Arixtra), and rivaroxaban (Xarelto) from their sales representatives for testing of kit sensitivity.

2.1.1. Heparin standards for calibration curve

We prepared 8 concentrations of heparin aqueous solution at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 IU/mL. One hundred microliters of each concentration of heparin solution was aliquoted into a 96-well plate strip (8 wells). Two heparin standard strips were given for 1 kit.

2.1.2. Antithrombin (At solution)

We provided 10 IU/vial antithrombin in powder form for 2 vials/kit. The user must dissolve antithrombin with 2 mL sterile water to obtain a 5X stock solution. The 5X stock solution must be diluted 5 times before use by the Ab solution (working concentration of 1 IU/mL).

2.1.3. Factor Xa (Xa solution)

We provided 30 μg/vial purified bovine factor Xa in powder form for 2 vials/kit. The user must dissolve purified bovine factor Xa with 750 μL sterile water to obtain a 5X stock solution. The 5X stock solution must be diluted 5 times before use by the Ab solution (working concentration of 8 μg/mL).

2.1.4. Chromogenic substrate (Ch solution)

We provided 4 mg/vial chromogenic substrate in powder form for 2 vials/kit. The user must dissolve the chromogenic substrate in 1 mL of sterile water to obtain a 5X stock solution. The 5X stock solution must be diluted 5 times before use by the Ab solution (working concentration of 1.2 mM).

2.1.5. Assay reaction buffer (Ab solution, ready to use)

The final concentration of each chemical in sterile, ready-to-use Ab solution is 50 mM Tris, 175 mM NaCl, 7.5 mM EDTA, 0.1% PEG, and pH at 8.40. We gave 25 mL/vial of Ab solution for 4 vials/kit.

2.1.6. Stop buffer (St solution, ready to use)

The ST solution is sterile 20% acetic acid. We gave the 10 mL/vial of St solution for 2 vials/kit.

2.2. Hematocrit tubes sample preparation

Three bottles of hematocrit tubes were purchased from each supplier. We randomly chose 3 hematocrit tubes from each bottle of each brand. Heparin coated inside the tube was dissolved out by pipetting 40 μL sterile water flow through the hematocrit tube into PCR tube. The 40 μL sample was used for the analysis.

2.3. Large-scale prototype

We performed a large-scale prototype analysis to confirm that all the purchased chemicals can produce appropriate result values. Five-point standards at 0, 0.25, 0.5, 0.75, and 1 IU/mL were prepared together with a mocking sample at 0.8 IU/mL as an unknown. The analysis steps were performed as mentioned in the general procedure (Table 1).

Table 1
General procedure.

Solution or step	96 wells plate	Large scale prototype
Sample or heparin standard	40 μL	500 μL
Preincubated At solution at 37 °C	40 μL	500 μL
Mixing	2 minutes	2 minutes
Preincubated Xa solution at 37 °C	40 μL	500 μL
Mixing	2 minutes	2 minutes
Preincubated Ch solution at 37 °C	40 μL	500 μL
Mixing	2 minutes	2 minutes
St solution	80 μL	1 mL

• Mixing and measure the O.D. at 405 nm against the corresponding blank.

2.4. Construction of standard curve

We plot the standard curve on a semi-logarithmic graph with the -(log) O.D.₄₀₅ nm along the Y-axis and the heparin concentration, expressed as IU/mL, along the X-axis. The concentration of heparin in the sample is directly inferred from the standard curve. Results are expressed in IU/mL, if the dilution is used; multiply with the concentration measured by the dilution factor.

2.5. Statistical analysis

The 3 independent triplicate assay values in all hematocrit tube analyses were expressed in the form of mean ± SEM. We used one-way ANOVA followed by Tukey HSD, and Friedman tests to confirm that there is no difference between the analyses of Biophen’s and our kit. We considered the presence of a statistically significant difference when the p-value > 0.05. We performed all statistical analyses using Microsoft Excel, Statistics Kingdom (2025), and Statology (2025) online tools.

3. Results

3.1. Large-scale prototype fabrication and minimization into a convenient 96-wells plate platform

First, we needed to confirm that all the supplied chemicals can properly perform the analysis. We designed a large-scale analysis using standard spectrophotometers, which require 3 mL samples for the determination of O.D. at 405 nm. The direct O.D.₄₀₅ plot of the standard values produced an exponential graph (Y = 0.4457e^-1.875X) (Figure 1A).

Figure 1
The raw and exponential analysis of 3 mL large scale prototype. (A) Plot between O.D.₄₀₅ and 8 points standard heparin concentration; (B) Plot between -log O.D.₄₀₅ and 8 points standard heparin concentration.

It was very difficult to compare and interpret the O.D.₄₀₅ result of the sample to the O.D.₄₀₅ plot of the standards because of the curve behavior. After we did the linear regression by replacing normal standards O.D.₄₀₅ with -log O.D.₄₀₅ values, the graph became linear, and the new linear equation was calculated (Y = 0.8142X + 0.351) (Figure 1B). Using mock sample -log O.D.₄₀₅ value (Determined O.D.₄₀₅ = 0.10 ± 0.01), the calculated mocking sample concentration is 0.80 ± 0.02 IU/mL (Actual mocking sample concentration = 0.80 IU/mL).

We reduced the reaction size from 3 mL in the test tubes to 240 μL in a 96-well plate as stated in the kit specifications. Using the new 8-point standard, the new small-scale standard curve was plotted, and the linear equation was calculated (Y = 0.3818X + 0.4386) (Figure 2). Putting the mocking samples -log O.D.₄₀₅ (Determined O.D.₄₀₅ = 0.18 ± 0.01) value into the equation, the calculated mocking samples concentration is 0.80 ± 0.06 IU/mL (Actual mocking sample concentration = 0.800 IU/mL).

Figure 2
The plot of 96-well small-scale analysis between -log O.D.₄₀₅ and 8-points standard heparin concentration.

3.2. Determination of the heparin amount inside 6 brands of heparin-coated hematocrit tubes available in Thailand

To reduce bias from changes in environmental factors, we recommended that the user always determine the new standard curve together with the sample(s) (Figure 3). We prepared the sample solution by flowing 1 mL of water through the hematocrit tube at least 10 times to dissolve the coated heparin inside the tube. The concentration of the eluted heparin was unknown IU/mL. According to the international standard indication, the coated amount of heparin inside a hematocrit tube must be at least 4 IU per tube. We expected that the concentration of heparin inside our A - F hematocrit tube samples should be around the international standard indication. Before doing the analysis, we diluted all the samples 4 times to fit the standard concentration.

Figure 3
The plot between -log O.D.₄₀₅ and 8-points standard heparin concentration in the same 96-wells plate with 6 brands hematocrit tube analysis.

Brand A and B hematocrit tubes are plain tubes with no heparin coating. Brand C is our collaborator’s heparin-coated tubes. Brands D to F are the heparin-coated tubes that are famously imported and used in Thailand. The results revealed the amount of heparin coated inside each brand of hematocrit tubes is as expected (Table 2). For uncoated brands, the coated heparin values are 0 IU/mL. Our collaborative brand C has the coated heparin value at 6.041 ± 0.403 IU/mL, which is very close to brand D at 6.075 ± 0.329 IU/mL. For brands E and F, they also have near-coated heparin values at 4.557 ± 0.696 IU/mL and 4.583 ± 0.224 IU/mL, respectively.

Table 2
Raw O.D.₄₀₅ value, -log O.D.₄₀₅ value, and calculated heparin concentration in 6 brands hematocrit tube available in Thailand’s market.

Brand	Material	O.D.405	-log O.D.405	Calculated concentration (IU/tube)
Brand A Cap1	Plastic	1.467 ± 0.078	-0.166 ± 0.023	-0.491 ± 0.153
Brand B Yuj	Glass	1.359 ± 0.028	-0.133 ± 0.009	-0.272 ± 0.060
Brand C Cap2	Plastic	0.155 ± 0.022	0.813 ± 0.060	6.041 ± 0.403
Brand D Vit	Glass	0.153 ± 0.018	0.818 ± 0.049	6.075 ± 0.329
Brand E Glo	Glass	0.263 ± 0.065	0.590 ± 0.104	4.557 ± 0.696
Brand F Hir	Glass	0.255 ± 0.020	0.594 ± 0.034	4.583 ± 0.224

3.3. Sensitivity of our in-house kit toward other LMWH drugs

All of the chemicals used in this study were purchased as powder directly from the manufacturers. After dissolving them in water, we created the 8-point of chemical concentrations by doing general serial dilution in ppm (μg/mL) to ng/mL. We first prepared the drug concentrations according to the heparin information at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 μg/mL. The appropriate set of drug concentrations should give an analysis result with O.D.₄₀₅ in between 0.2-0.6 (As the acceptable accuracy of the microplate reader). If the set of drug concentrations gives analysis results exceeding the mentioned accuracy value, the concentrations should be further adjusted by more dilution until the result is in the expected range. Our kit has the highest sensitivity against Edoxaban (Lixiana) with the detection range between 0-0.035 ppm (Figure 4A). The second rank sensitivity is Fondaparinux (Arixtra), which gives the detection range between 0-0.175 ppm (Figure 4B). For Apixaban (Eliquis), the sensitivity is similar to heparin, that is, 0-1.75 ppm (Figure 4C). But for Rivaroxaban (Xarelto), the appropriate detection concentration is higher than heparin, which is 0-7 ppm (Figure 4D).

Figure 4
Sensitivity analysis of our in-house kit with 4 cardiovascular disease drugs, which are usually used in Thailand’s hospitals: (A) Plot of Edoxaban; (B) Plot of Fondapalinux; (C) plot of Apixaban; (D) plot of Rivaroxaban.

3.4. Comparison of our in-house kit’s accuracy with Biophen’s anti-factor Xa kit

We chose our collaborator’s hematocrit tubes to do the comparison analysis of our kit with Biophen’s anti-factor Xa kit. Using the same protocol, we constructed an 8-point standard heparin concentration (which is not provided in Biophen’s anti-factor Xa kit) and performed analysis together with 5 randomly chosen hematocrit tube samples. The standards analysis gave the equation as Y = 0.5992X – 0.0924 and Y = 0.6660X – 0.0951 for our in-house kit and Biophen’s anti-factor Xa kit, respectively (Figure 5). Putting the same sample -log O.D.₄₀₅ values into the equation and multiplying by the dilution factor, the calculated concentration of heparin coated inside brand C tubes from our kit and Biophen’s anti-factor Xa kit were 6.041 ± 0.403 and 6.103 ± 0.350 IU/mL, respectively (Table 3).

Figure 5
The plot between -log O.D.₄₀₅ and 8-points standard heparin concentration of our in-house kit comparing with Biophen’s anti-factor Xa kit.

Table 3
Raw O.D.₄₀₅ value, -log O.D.₄₀₅ value, and calculated heparin concentration in brand C hematocrit tubes analyzed by our in-house kit comparing with Biophen’s anti-factor Xa kit.

Kit	O.D.405	-log O.D.405	Calculated concentration (IU/tube)
In-house kit	0.155 ± 0.022	0.813 ± 0.060	6.041 ± 0.403
Biophen’s	0.120 ± 0.016	0.923 ± 0.058	6.117 ± 0.350

4. Discussion

We first checked that the transported kit’s components from the provider company worked well. We created the mock heparin sample at 8 IU/mL and then performed the analysis using our kit. Using both large- and small-scale kits, they gave very close analytical values to each other and to the actual value of the mock sample concentration. The results confirmed that all the enzymes, proteins, and chemicals were in good condition after transportation to us.

To achieve our goal, we then used the kit to analyze our collaborator’s plastic hematocrit tubes, brand C, together with 3 other heparin-coated glass tubes brands and 2 non-heparin-coated glass tubes brands as a negative control. As expected, the 2 glass tubes negative control, brand A and B, analysis gave the heparin-coated concentration values as 0. These negative control analyses confirmed that there is no artifact occurring from our kit’s determination. Quantitative analysis of heparin inside our collaborator’s plastic tubes, brand C, gave a similar concentration to the brand D glass tubes, which is higher than the international standard indication at 4 IU/mL. Additionally, it is a fact that the brand D glass tubes are the cheapest among all the tubes analyzed in this study. Brand E and F glass tubes, which are more expensive than the others, contain the approximate heparin amount near the international standard indication. According to these results, we could recommend that our collaborator reduce the concentration of heparin used during the tube’s manufacturing process to save the operation cost.

From several previous studies, Biophen kits were used for analyzing other LMWH drugs with very high sensitivity (Samama et al., 2013; Johnson et al., 2013; Willekens et al., 2021). To confirm that our kit has the same capability to analyze those LMWH drugs, we designed the drug’s concentration and then performed analyses to obtain the most appropriate standard curve. We found that our kit could determine the concentration of the LMWH drugs at the ppm level, similar to those previously analyzed by Biophen’s kit (Samama et al., 2012; Ebner et al., 2018; Riahi et al., 2023). We then analyzed and compared the efficiency of our kit to determine the amount of heparin coated inside our collaborator’s plastic tubes, brand C, with Biophen’s kit. Consequently, the comparative results showed that there is no significant difference between our kit’s and Biophen’s analyses. Altogether, we confirmed that our new in-house anti-factor Xa kit (Supplementary Material 1) could accurately and precisely evaluate anti-coagulated blood substances inside any aqueous sample with a reliable value. Nowadays, our collaborator uses this kit to analyze the amount of heparin coated inside their tube products as quality control before selling these tubes to the customer.

Abbreviations

ANNOVA: Analysis of variance, aPTT: Activated partial thromboplastin time, EDTA: Ethylenediaminetetraacetic acid, ELISA: Enzyme linked immunosorbent assay, IU: International unit, LMWH: Low molecular weight heparins, mg: Milligram, mL: Milliliter, mM: Millimolar, O.D.: Optical density, PEG: Polyethylene glycol, ppm: parts per million, SEM: Standard error of the mean, μg: Microgram, μL: Microliter.

Supplementary Material

Supplementary material accompanies this paper.

Supplementary material 1.

The look of the in-house kit, which is fabricated in this work. The kit contains all solutions and plasticwares needed for the LMWH analysis.

This material is available as part of the online article from https://doi.org/10.1590/1519-6984.294499

Acknowledgements

We would like to acknowledge the following institutes, companies, and people for supporting our work. We received the research grant from Thailand’s National Innovation Agency (NIA), passing through our collaborator’s company, Zigma Biotech (Thailand). We would like to show our best gratitude to Mr. Noppanut Nilnam, who arranged and prepared all the requested chemicals and plasticware for us. Finally, we would like to acknowledge Marko Zizek, who edited the whole paper. This work was supported by Thailand’s National Innovation Agency (NIA) [grant number P2103-04-62-02-004].

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

• AMIRAL, J., AMIRAL, C. and DUNOIS, C., 2021. Optimization of heparin monitoring with anti-fXa assays and the impact of dextran sulfate for measuring all drug activity. Biomedicines, vol. 9, no. 6, pp. 700. https://doi.org/10.3390/biomedicines9060700 PMid:34205548.
  » https://doi.org/10.3390/biomedicines9060700
• ATTAH, A., PETERSON, C., JACOBS, M., BHAGAVATULA, R., SHAH, D., KAPLAN, R. and SAMHOURI, Y., 2024. Anti-PF4 ELISA-negative, SRA-positive heparin-induced thrombocytopenia. Hematology Reports, vol. 16, no. 2, pp. 295-298. https://doi.org/10.3390/hematolrep16020029 PMid:38804282.
  » https://doi.org/10.3390/hematolrep16020029
• BRONIĆ, A., MARGETIĆ, S., COEN HERAK, D., MILIĆ, M., KREŠIĆ, B., RADIŠIĆ BILJAK, V. and LENIČEK KRLEŽA, J., 2021. Reporting of activated partial thromboplastin time (aPTT): could we achieve better comparability of the results? Biochemia Medica, vol. 31, no. 2, pp. 020708. https://doi.org/10.11613/BM.2021.020708 PMid:34140831.
  » https://doi.org/10.11613/BM.2021.020708
• DEUTSCHES INSTITUT FÜR NORMUNG – DIN, 2007. DIN ISO 12772: laboratory glassware: disposable microhaematocrit capillary tubes. Berlin.
• EBNER, M., BIRSCHMANN, I., PETER, A., HÄRTIG, F., SPENCER, C., KUHN, J., RUPP, A., BLUMENSTOCK, G., ZUERN, C.S., ZIEMANN, U. and POLI, S., 2018. Limitations of specific coagulation tests for direct oral anticoagulants: A critical analysis. Journal of the American Heart Association, vol. 7, no. 19, e009807. https://doi.org/10.1161/JAHA.118.009807 PMid:30371316.
  » https://doi.org/10.1161/JAHA.118.009807
• ENGELMAIER, A., ZLABINGER, C. and WEBER, A., 2020. Establishment and qualification of a competitive enzyme-linked immunosorbent assay for the measurement of heparin in plasma products. Journal of Analytical Techniques and Research, vol. 2, no. 3, pp. 110-122. https://doi.org/10.26502/jatri.015
  » https://doi.org/10.26502/jatri.015
• JOHNSON, P.N., BENEFIELD, E.C., BUI, P.Y., GAUSMAN, J.N., MARLAR, R.A. and GESSOUROUN, M.R., 2013. Fondaparinux monitoring: need for a local fondaparinux-calibrated anti-factor Xa assay. The Journal of Pediatric Pharmacology and Therapeutics, vol. 18, no. 4, pp. 318-319. https://doi.org/10.5863/1551-6776-18.4.318 PMid:24719594.
  » https://doi.org/10.5863/1551-6776-18.4.318
• LANOISELÉE, J., MOURER, J., JUNGLING, M., MOLLIEX, S., THELLIER, L., TABAREAU, J., JEANPIERRE, E., ROBIN, E., SUSEN, S., TAVERNIER, B., VINCENTELLI, A., OLLIER, E. and MOUSSA, M.D., 2024. Heparin dosing regimen optimization in veno-arterial extracorporeal membrane oxygenation: A pharmacokinetic analysis. Pharmaceutics, vol. 16, no. 6, pp. 770. https://doi.org/10.3390/pharmaceutics16060770 PMid:38931891.
  » https://doi.org/10.3390/pharmaceutics16060770
• LARDINOIS, B., HARDY, M., MICHAUX, I., HORLAIT, G., ROTENS, T., JACQMIN, H., LESSIRE, S., BULPA, P., DIVE, A. and MULLIER, F., 2022. Monitoring of unfractionated heparin therapy in the intensive care unit using a point-of-care aPTT: A comparative, longitudinal observational study with laboratory-based aPTT and anti-Xa activity measurement. Journal of Clinical Medicine, vol. 11, no. 5, pp. 1338. https://doi.org/10.3390/jcm11051338 PMid:35268436.
  » https://doi.org/10.3390/jcm11051338
• LEHMAN, C.M., RETTMANN, J.A., WILSON, L.W. and MARKEWITZ, B.A., 2006. Comparative performance of three anti-factor Xa heparin assays in patients in a medical intensive care unit receiving intravenous, unfractionated heparin. American Journal of Clinical Pathology, vol. 126, no. 3, pp. 416-421. https://doi.org/10.1309/8E3U7RXEPXNP27R7 PMid:16880140.
  » https://doi.org/10.1309/8E3U7RXEPXNP27R7
• LESSIRE, S., DOUXFILS, J., POCHET, L., DINCQ, A.S., LAROCK, A.S., GOURDIN, M., DOGNÉ, J.M., CHATELAIN, B. and MULLIER, F., 2018. Estimation of rivaroxaban plasma concentrations in the perioperative setting in patients with or without heparin bridging. Clinical and Applied Thrombosis/Hemostasis : Official Journal of the International Academy of Clinical and Applied Thrombosis/Hemostasis, vol. 24, no. 1, pp. 129-138. https://doi.org/10.1177/1076029616675968 PMid:27811211.
  » https://doi.org/10.1177/1076029616675968
• MAECKELBERGH, V.A. and ACIERNO, M.J., 2008. Comparison of prothrombin time, activated partial thromboplastin time, and fibrinogen concentration in blood samples collected via an intravenous catheter versus direct venipuncture in dogs. American Journal of Veterinary Research, vol. 69, no. 7, pp. 868-873. https://doi.org/10.2460/ajvr.69.7.868 PMid:18593235.
  » https://doi.org/10.2460/ajvr.69.7.868
• MCLAUGHLIN, K., RIMSANS, J., SYLVESTER, K.W., FANIKOS, J., DORFMAN, D.M., SENNA, P., CONNORS, J.M. and GOLDHABER, S.Z., 2019. Evaluation of antifactor-Xa heparin assay and activated partial thromboplastin time values in patients on therapeutic continuous infusion unfractionated heparin therapy. Clinical and Applied Thrombosis/Hemostasis, vol. 25, pp. 1076029619876030. https://doi.org/10.1177/1076029619876030 PMid:31530176.
  » https://doi.org/10.1177/1076029619876030
• NAGLER, M., BACHMANN, L.M., TEN CATE, H. and TEN CATE-HOEK, A., 2016. Diagnostic value of immunoassays for heparin-induced thrombocytopenia: a systematic review and meta-analysis. Blood, vol. 127, no. 5, pp. 546-557. https://doi.org/10.1182/blood-2015-07-661215 PMid:26518436.
  » https://doi.org/10.1182/blood-2015-07-661215
• NEWALL, F., 2013. Anti-factor Xa (anti-Xa) assay. Methods in Molecular Biology (Clifton, N.J.), vol. 992, pp. 265-272. https://doi.org/10.1007/978-1-62703-339-8_19 PMid:23546719.
  » https://doi.org/10.1007/978-1-62703-339-8_19
• PIRAHANCHI, Y., SALOTTOLO, K., BURRELL, C., TANG, X., BAR-OR, D. and BARTT, R., 2024. Pilot study examining anti-factor Xa levels for heparin monitoring and outcomes in patients with cerebral venous thrombosis. Frontiers in Medicine, vol. 11, pp. 1317246. https://doi.org/10.3389/fmed.2024.1317246 PMid:38343640.
  » https://doi.org/10.3389/fmed.2024.1317246
• RIAHI, N., ROZEN, L. and DEMULDER, A., 2023. Usefullness of heparin calibrated anti-Xa activity to assess anticoagulant activity of apixaban and rivaroxaban in emergency patients scheduled for acute interventions. Journal of Clinical Medicine, vol. 12, no. 21, pp. 6785. https://doi.org/10.3390/jcm12216785 PMid:37959250.
  » https://doi.org/10.3390/jcm12216785
• SAHU, K.K., JINDAL, V., ANDERSON, J., SIDDIQUI, A.D. and JAIYESIMI, I.A., 2020. Current perspectives on diagnostic assays and anti-PF4 antibodies for the diagnosis of heparin-induced thrombocytopenia. Journal of Blood Medicine, vol. 11, pp. 267-277. https://doi.org/10.2147/JBM.S232648 PMid:32884385.
  » https://doi.org/10.2147/JBM.S232648
• SAMAMA, M.M., CONTANT, G., SPIRO, T.E., PERZBORN, E., GUINET, C., GOURMELIN, Y., LE FLEM, L., ROHDE, G. and MARTINOLI, J.L., 2012. Evaluation of the anti-factor Xa chromogenic assay for the measurement of rivaroxaban plasma concentrations using calibrators and controls. Thrombosis and Haemostasis, vol. 107, no. 2, pp. 379-387. https://doi.org/10.1160/TH11-06-0391 PMid:22187012.
  » https://doi.org/10.1160/TH11-06-0391
• SAMAMA, M.M., CONTANT, G., SPIRO, T.E., PERZBORN, E., LE FLEM, L., GUINET, C., GOURMELIN, Y., ROHDE, G. and MARTINOLI, J.L., 2013. Laboratory assessment of rivaroxaban: a review. Thrombosis Journal, vol. 11, no. 1, pp. 11. https://doi.org/10.1186/1477-9560-11-11 PMid:23822763.
  » https://doi.org/10.1186/1477-9560-11-11
• SAMUEL, S., ALLISON, T.A., SHARAF, S., YAU, G., RANJBAR, G., MCKAIG, N., NGUYEN, A., ESCOBAR, M. and CHOI, H.A., 2016. Antifactor Xa levels vs. activated partial thromboplastin time for monitoring unfractionated heparin: a pilot study. Journal of Clinical Pharmacy and Therapeutics, vol. 41, no. 5, pp. 499-502. https://doi.org/10.1111/jcpt.12415 PMid:27381025.
  » https://doi.org/10.1111/jcpt.12415
• STATISTICS KINGDOM [online], 2025 [viewed 23 February 2025]. Available from: https://www.statskingdom.com
  » https://www.statskingdom.com
• STATOLOGY [online], 2025 [viewed 23 February 2025]. Available from: https://www.statology.org
  » https://www.statology.org
• WILLEKENS, G., STUDT, J.D., MENDEZ, A., ALBERIO, L., FONTANA, P., WUILLEMIN, W.A., SCHMIDT, A., GRAF, L., GERBER, B., BOVET, C., SAUTER, T.C. and NAGLER, M., 2021. A universal anti-Xa assay for rivaroxaban, apixaban, and edoxaban measurements: method validation, diagnostic accuracy and external validation. British Journal of Haematology, vol. 193, no. 6, pp. 1203-1212. https://doi.org/10.1111/bjh.17470 PMid:33954979.
  » https://doi.org/10.1111/bjh.17470
• WILLIAMS-NORWOOD, T., CASWELL, M., MILNER, B., VESCERA, J.C., PRYMICZ, K., CISZAK, A.G., INGLE, C., LACEY, C. and STAVROU, E.X., 2020. Design and implementation of an anti-factor Xa heparin monitoring protocol. AACN Advanced Critical Care, vol. 31, no. 2, pp. 129-137. https://doi.org/10.4037/aacnacc2020132 PMid:32526007.
  » https://doi.org/10.4037/aacnacc2020132
• ZWICKER, J.I., UHL, L., HUANG, W.Y., SHAZ, B.H. and BAUER, K.A., 2004. Thrombosis and ELISA optical density values in hospitalized patients with heparin-induced thrombocytopenia. Journal of thrombosis and haemostasis. Journal of Thrombosis and Haemostasis, vol. 2, no. 12, pp. 2133-2137. https://doi.org/10.1111/j.1538-7836.2004.01039.x PMid:15613017.
  » https://doi.org/10.1111/j.1538-7836.2004.01039.x

Edited by

• Editor:
  Marcelo A.M. Esquisatto

Publication Dates

• Publication in this collection
  10 July 2026
• Date of issue
  2026

History

• Received
  23 Feb 2025
• Accepted
  15 Apr 2026