The Nijmegen Hemostasis Assay: Simultaneous Fluorogenic Measurement of Thrombin and Plasmin Generation in a Single Well

Summary

This protocol describes the Nijmegen Hemostasis Assay, which enables simultaneous, time-resolved measurement of thrombin and plasmin generation to provide an integrated assessment of coagulation and fibrinolysis. The assay aims to improve characterization of hemostatic balance in research and clinical settings beyond the scope of conventional hemostatic tests.

Abstract

Thrombin and plasmin both play a central role in hemostasis, regulating coagulation and fibrinolysis, respectively. Conventional hemostatic assays often focus on isolated components or limited phases of hemostasis; for example, activated partial thromboplastin time and prothrombin time evaluate only the initiation of fibrin formation, while factor activity assays assess the function of individual coagulation factors. Although valuable, these assays lack a comprehensive view of the overall process of secondary hemostasis and fibrinolysis, which unfolds dynamically over time. The Nijmegen Hemostasis Assay (NHA) addresses this limitation by enabling simultaneous, time-resolved measurement of thrombin and plasmin generation in a single microplate well. Using two synthetic fluorogenic substrates with distinct excitation and emission spectra and no cross-reactivity, the assay allows accurate quantification of both enzymes in parallel. Hemostasis is initiated in platelet-poor plasma using tissue factor, calcium and tissue-type plasminogen activator, and fluorescence is recorded every 30 s over 70 min at 37 °C. Enzyme activity curves are generated using calibration standards, from which parameters such as lag time, thrombin potential, fibrin lysis time, and plasmin peak height are derived. The assay reliably captures simultaneous thrombin and plasmin generation with good intra- and inter-assay precision, and detects modulation of coagulation and fibrinolysis by effectors such as heparin, activated protein C, and epsilon-aminocaproic acid. In clinical samples, significantly reduced thrombin generation was observed in patients with coagulation factor deficiencies, while patients with fibrinolytic disorders showed elevated plasmin generation profiles. By combining thrombin and plasmin generation measurements, the NHA provides a comprehensive insight into the hemostatic balance beyond conventional assays. This integrated approach holds promise for research and clinical applications, such as the characterization of hemostatic phenotypes, assessment of bleeding and thrombotic risk, and monitoring of targeted therapeutic interventions. This protocol outlines the NHA procedure that aims to enhance the understanding and assessment of hemostatic balance.

Introduction

Hemostasis is the result of a dynamic balance between coagulation and fibrinolysis. Thrombin and plasmin are the key enzymes driving these processes: Thrombin converts fibrinogen into fibrin, while plasmin degrades fibrin to dissolve clots. Disruption of this balance can lead to bleeding or thrombotic events^1,2. However, the laboratory tests used to evaluate hemostasis in clinical practice typically focus on isolated steps of coagulation or fibrinolysis, providing limited insight into the overall hemostatic balance. In patients with a suspected bleeding tendency, routine screening tests such as prothrombin time (PT) and activated partial thromboplastin time (aPTT) are commonly used to screen for coagulation factor deficiencies. However, these assays measure the time to initial fibrin formation and do not capture thrombin generation beyond clotting onset, where most physiologically relevant thrombin activity occurs^1,2,3. Consequently, they may yield normal results in patients with mild coagulation factor deficiencies^3,4,5,6,7,8. To identify these specific deficiencies, coagulation factor activity assays are used. While informative for diagnosis, these assays assess individual proteins in isolation and do not reflect the overall hemostatic balance. As a result, in patients with mild deficiencies, factor levels often correlate poorly with the bleeding phenotype^9,10. This complicates clinical risk assessment and the optimization of patient management, including the timing and dosing of therapy, as well as interventions in response to thrombotic or severe hemorrhagic events. These limitations highlight the need for assays that offer a more comprehensive view of the hemostatic process over time and may help bridge the gap between laboratory results and clinical presentation.

The Nijmegen Hemostasis Assay (NHA) was developed to address these limitations by enabling the simultaneous, time-resolved measurement of thrombin and plasmin generation in platelet-poor plasma^8,9. It uses two synthetic fluorogenic substrates with distinct excitation and emission spectra to quantify both enzymes in the same microplate well without cross-reactivity. Enzyme generation curves allow derivation of quantitative parameters that reflect the dynamics of secondary coagulation and fibrinolysis, including lag time, thrombin peak height, fibrin lysis time and plasmin peak height. The NHA provides several advantages over existing thrombin generation assays, such as the Calibrated Automated Thrombogram (CAT), Ceveron s100, and ST Genesia, which provide valuable information on thrombin dynamics but do not assess fibrinolytic activity^10,11,12,13. Clot lysis assays, in turn, capture aspects of fibrinolysis but lack information on thrombin generation¹⁴. This limits their usefulness in evaluating the complex interactions that determine overall hemostatic balance. While some laboratory-developed approaches have combined thrombin and plasmin generation measurements, these typically require separate wells or parallel assays, limiting temporal alignment and throughput¹⁵. Global assays such as thromboelastography (TEG) and rotational thromboelastometry (ROTEM) provide complementary information by capturing primary hemostasis, secondary coagulation, and fibrinolysis^16,17. Flow chamber systems represent another emerging yet experimental approach to assess total hemostasis under flow conditions¹⁸. While these global assays offer a broader physiological context, they are generally less sensitive to mild bleeding disorders^19,20,21. By simultaneously measuring thrombin and plasmin generation in the same reaction well, the NHA combines the sensitivity of conventional TGAs with the integrated view of global assays, enabling direct assessment of secondary hemostasis, fibrinolysis and their interactions.

Thrombin generation assays have already shown considerable promise for translational applications, such as individualized bleeding and thrombotic risk profiling and therapy monitoring^22,23,24,25. In clinical research, the NHA has demonstrated reduced thrombin generation in patients with hemophilia A, hemophilia B, and rare coagulation factor deficiencies, as well as elevated plasmin generation in hyperfibrinolytic conditions such as α2-antiplasmin deficiency^26,27,28,29. Differences in both pathways have also been observed in patients with hypofibrinogenemia, a disorder associated with both bleeding and an increased risk of thrombosis. In this population, lower fibrinogen levels correlated with reduced plasmin generation, likely due to the lack of plasminogen-binding sites, while patients with a history of thrombosis showed increased thrombin generation³⁰. Additionally, the assay detects the effects of therapeutic agents, including anticoagulants, procoagulants, and antifibrinolytics⁸. In a previous study, we proposed a standardization and normalization approach to improve reproducibility and enable comparison of results across runs and laboratories, an essential step towards clinical implementation²⁸. This protocol outlines the full procedure and analytical approach of the NHA for implementation in clinical research or translational settings.

Protocol

All procedures involving human plasma samples were conducted in accordance with institutional guidelines and approved by the medical ethics committee of Arnhem-Nijmegen. Written informed consent was obtained from all donors prior to sample collection.

1. Preparation of buffers, reagents and plasma samples

1. Prepare buffer solutions
   1. Weigh 1.2 g Tris (p.a.) and dissolve in 180 mL of 0.9% NaCl.
   2. Adjust the pH to 7.4 using HCl.
   3. Bring the final volume to 200 mL with 0.9% NaCl to obtain 50 mM Tris-150 mM NaCl buffer (TBS).
   4. Aliquot the TBS into two 50 mL tubes.
   5. Use the remaining 100 mL TBS to prepare the calcium-containing buffer. Dissolve 7.34 g CaCl₂·2H₂O in 100 mL of TBS to obtain Tris-NaCl-CaCl₂ buffer (TBS-Ca, pH 7.4).
   6. Aliquot the TBS-Ca into two 50 mL tubes.
   7. Store both buffers at 4 °C and use within one week.
2. Prepare fluorogenic substrates
   1. Dissolve 53.2 mg of the thrombin substrate (Bz-beta-Ala-Gly-Arg-AMC) in 4 mL of Tris-NaCl buffer.
      NOTE: The box contains a leaflet with a certificate of analysis. The volume of buffer to add depends on the specified concentration and should be adjusted so that 1 µL of added human thrombin in 120 µL reagent yields a final concentration of 76 nmol/L.
   2. Aliquot 100 µL in Eppendorf tubes and store at −80 °C.
   3. Thaw an aliquot of thrombin-specific fluorogenic substrate for 10 min at room temperature before use. Vortex and keep on ice during the experiment.
   4. Dissolve the contents of one vial of plasmin substrate (CBZ-L-Phe-Arg-rhodamine) in 1,955 µL of DMSO.
      NOTE: The box contains a leaflet with a certificate of analysis. The volume of buffer to add depends on the specified concentration and should be adjusted so that 1 µL of added human plasmin in 120 µL reagent yields a final concentration of 200 nmol/L.
   5. Aliquot 60 µL in Eppendorf tubes and store at −80 °C.
   6. Thaw an aliquot of plasmin-specific fluorogenic substrate for 10 min at room temperature before use. Vortex and keep at room temperature during the experiment.
      NOTE This substrate is dissolved in dimethylsulfoxide (DMSO) and must not be cooled to avoid precipitation.
3. Prepare trigger reagents
   1. Reconstitute the lyophilized cephalin by adding 1 mL of distilled water. Vortex thoroughly until fully dissolved.
      NOTE: Cephalin is reconstituted to provide phospholipids, which are essential to catalyze multiple coagulation reactions enabling physiologically relevant coagulation in the assay.
   2. Aliquot 60 µL in Eppendorf tubes (sufficient for 20 wells) and store at −20 °C.
   3. Thaw an aliquot before use and keep it on ice. Vortex briefly before pipetting.
   4. Reconstitute the contents of one vial of recombinant human tissue factor (TF) in 4 mL of distilled water.
   5. Aliquot 60 µL in Eppendorf tubes and store at −80 °C.
   6. Thaw an aliquot of TF and vortex briefly.
   7. Prepare a 1:500 dilution of TF by sequential dilution:
      1. Add 6 µL of TF to 54 µL of TBS buffer. Vortex.
      2. Take 6 µL of this dilution and add to 54 µL of TBS. Vortex.
      3. Take 20 µL of this second dilution and add to 80 µL of TBS to reach a final concentration of 0.28 pM. Vortex.
   8. Reconstitute the contents of one vial (20 mg) of tissue-type plasminogen activator (tPA) in 20 mL of distilled water to obtain a final concentration of 580,000 IU/mL (verify batch-specific concentration).
   9. Aliquot 50 µL in Eppendorf tubes and store at −80 °C.
   10. Thaw an aliquot of tPA and vortex briefly.
   11. Prepare a 1:50 dilution of tPA by adding 20 µL of tPA to 980 µL of Tris-NaCl buffer, reaching a final concentration of 11,600 IU/mL. Vortex.
4. Prepare plasma samples
   1. Collect venous blood into 3 mL citrate tubes containing 3.2% citrate, ensuring at least 2.7 mL (90% fill) is collected.
   2. Do not use the first tube after venipuncture; discard it to avoid activation by tissue factor.
   3. Invert the tube at least 10 times immediately after collection.
   4. Centrifuge within 2 h at room temperature, first at 2,500 × g for 5 min, followed by 10,000 × g for 10 min.
   5. Use a disposable pipette to collect the plasma just above the cell layer.
   6. Aliquot plasma into labeled cryovials, snap-freeze in liquid nitrogen, and store at −80 °C.
      NOTE: Ensure the sample is free of clots. Use only fresh or once-thawed plasma. One CTAD tube is sufficient for duplicate analysis.

2. Instrument preparation and software setup

1. Prepare the fluorescence plate reader
   1. Turn on the plate reader and allow it to equilibrate to 37 °C.
   2. Set the following basic parameters:
      1. Greiner 96-well, flat-bottom, black.
      2. General settings: Positioning delay: 0.1 s; Number of kinetic windows: 1.
      3. Kinetic window: Number of cycles: 140; Measurement start time: 0.0 s; Number of flashes per well per cycle: 10; Cycle time: 30 s.
      4. Optic: Top optic
      5. Filter settings: Number of monochromatics: 2. Excitation filter: 355 nm for thrombin; 485 nm for plasmin. Emission filter: 460 nm for thrombin, 520 nm for plasmin. Gain: 600.
   3. Define the layout with a maximum of 20 wells per plate.
   4. Navigate to Concentrations/Volumes/Shaking and confirm the following settings. Pump 1 = 20 µL; Pump 2 = 0 µL; Speed = 420 µL/s.
2. Prepare the injection system
   1. Remove and rinse the injection needles in a container with buffer.
   2. Set the rinse volume to 2,500 µL and rinse the pumps with 37 °C Tris/NaCl buffer.
   3. Reset the rinse volume to 500 µL.

3. Assay setup in microplate format

1. Microplate shaker setup
   1. Set the microplate shaker to 37 °C and verify the temperature before use.
   2. Set the shaking intensity to 1100 rpm.
   3. Use this shaker for all mixing steps in the assay to ensure homogeneous reagent distribution and consistent enzymatic activity.
2. Add reagents to the wells
   1. Thaw patient plasma and normal pooled plasma (NPP) in a 37 °C water bath for up to 10 min.
   2. Mix thoroughly and verify complete thawing (no cryoprecipitate).
   3. Add 80 µL of patient plasma or NPP to the preselected wells. Use a fresh pipette tip for each sample.
      NOTE: Include a NPP sample in every assay run to account for between-run variability (see further in step 5.6).
   4. Add 2 µL of cephalin.
   5. Add 2 µL of tissue factor (1:500 dilution). Mix briefly using a plate shaker.
   6. Add 10 µL of Tris-NaCl buffer. Mix briefly using a plate shaker.
   7. Add 4 µL of thrombin substrate.
   8. Add 2 µL of plasmin substrate. Mix briefly using a plate shaker.
3. Pre-incubation
   1. Insert the microplate into the plate reader.
   2. Allow the plate to equilibrate at 37 °C for several min.

4. Preparation and injection of the starting reagent

1. Prepare the reagent for Pump 1
   1. Combine 672 µL of Tris/NaCl buffer, 192 µL of Tris/NaCl/CaCl₂ buffer, and 96 µL of tPA (from step 1.2.5). Vortex thoroughly.
   2. Pierce two holes in the cap of the reagent tube.
   3. Insert the pump tubing through one hole to the bottom of the tube.
2. Prime and start the assay
   1. Use the software to prime the pump with 500 µL of the reagent.
   2. Reinsert the injection needles into their designated positions.
   3. Immediately start the fluorescence measurement.
3. Manual initiation of the assay
   NOTE: This manual initiation procedure provides an alternative to the automated injection workflow described in steps 2.2, 4.1, and 4.2.
   1. Prepare the starting reagent as described in 4.1.1 immediately before use.
   2. Remove the microplate from the plate reader and quickly add 20 µL of the starting reagent manually to each well using a calibrated pipette or multichannel pipette for faster handling.
   3. Immediately return the plate to the reader and start the fluorescence measurement without delay.
      NOTE: Minimize the time between reagent addition and measurement start (<10 s per row) to reduce variability.
4. Kinetic readout
   1. Record fluorescence every 30 s for 70 min.
   2. Maintain the plate at 37 °C during measurement.

5. Data analysis

1. Prepare the data for analysis
   1. Export the kinetic fluorescence data from the plate reader as a table with timepoints and fluorescence values per well.
   2. Convert the raw fluorescence data into enzyme generation curves by calculating the first derivative using Excel or another program.
   3. Plot the enzyme generation curves for each well to visualize the enzymatic reaction.
      NOTE: These curves represent the rate of thrombin or plasmin generation over time and correspond to the graphical output shown in the figures.
2. Analyze thrombin generation curves
   1. Identify the following parameters from the thrombin generation curve (see Figure 1):
      1. Lag time: The time point where the thrombin generation curve rises consistently and reaches 8% of the thrombin peak height.
      2. Thrombin peak height: The maximum signal value on the y-axis (i.e., maximal thrombin generation rate).
      3. Thrombin Peak time: The time from the assay start to the thrombin peak height.
      4. Thrombin potential (area under the thrombin generation curve): The area under the curve from lag time until the thrombin signal returns to baseline.
         NOTE: The thrombin potential reflects the total thrombin-generating capacity of the sample under assay conditions.
3. Analyze plasmin generation curves
   1. Identify the following parameters from the plasmin generation curve (see Figure 2):
      1. Fibrin lysis time: The time from onset of plasmin generation (i.e., the time point where the plasmin generation curve rises consistently and reaches 8% of the plasmin peak height) to the point at which the plasmin peak is reached.
      2. Plasmin peak height: The first distinct maximum in the curve, corresponding to the initial burst in plasmin generation, is determined at the point where the second derivative of the plasmin generation curve (based on a three-minute moving average) reaches zero after the onset of plasmin generation.
      3. Plasmin potential (area under the plasmin generation curve): The area under the curve from the onset of plasmin generation to the time of the plasmin peak.
         NOTE: Signal fluctuations do occur after the initial plasmin peak, and are not included in the analysis of the plasmin generation parameters.
4. Extract parameters manually or using custom software
   1. Use Microsoft Excel, GraphPad Prism, or a custom script (e.g., in Python or R) to extract kinetic parameters.
   2. Save the raw data, plotted curves, and calculated parameters in a structured format for archiving and future reference.
      NOTE: If using macros, verify the correctness of all parameters visually and manually before relying on automated output. Our macro for data conversion is available upon request.
5. Standardization and normalization
   1. Establish a physiological reference by measuring 40 individual samples from healthy donors in parallel with the NPP sample used for normalization. Perform this step once per NPP batch.
   2. Calculate the mean thrombin and plasmin generation parameter values of the healthy donors, and determine their ratio to the corresponding NPP values obtained in the same run.
   3. For each patient sample in a given run, first normalize the thrombin or plasmin generation parameter to the NPP value measured in that same run.
   4. Divide this result by the healthy donor-to-NPP ratio previously established for the same NPP batch (see step 5.6.2). This yields a relative value normalized to the average healthy individual, with standardization for inter-run variation using NPP.
   5. Express parameter values as percentages relative to the average value of healthy individuals (defined as 100%). Present patient sample results accordingly.
      NOTE: The relative parameter values for each individual patient sample are calculated using the following formula, where run a refers to the measurement run of the patient sample, and run b refers to the reference run in which healthy donor samples were measured in parallel with the same NPP batch. Our Excel file containing the calculations for data normalization is available upon request.
      

6. Generation of calibration curves

1. Prepare the thrombin calibration curve
   1. Reconstitute the lyophilized human α-thrombin as instructed in the manufacturer's datasheet.
      NOTE: For example, if the datasheet specifies reconstitution in 100 µL water to yield 3.34 mg/mL, this corresponds to 0.334 mg in 100 µL.
   2. Dilute the reconstituted thrombin 1:10 in Tris/NaCl buffer.
      NOTE: This yields a stock concentration of approximately 9.1 nmol/mL (0.0091 nmol/µL), based on a molecular weight of 36,700 g/mol.
   3. Prepare a dilution series in Tris/NaCl buffer to achieve final thrombin concentrations in the assay ranging from 0 to 152 nmol/L.
   4. Pipette thrombin into selected wells already containing buffer to reach a total volume of 100 µL per well.
   5. Prepare the substrate solution for Pump 1 by mixing 300 µL of Tris/NaCl buffer with 200 µL of thrombin substrate from step 1.2.2.
   6. Further dilute this solution by combining 400 µL of the mixture with 360 µL of Tris/NaCl buffer.
   7. Add 20 µL of this substrate mix to each calibration well, bringing the total assay volume to 120 µL.
   8. Gently mix the plate and insert it into the plate reader. Run the kinetic assay for 25 cycles, using the same fluorescence settings described in section 2.
   9. Export the raw fluorescence data and determine the initial slope (rate of fluorescence increase per cycle) for each thrombin concentration using GraphPad Prism or equivalent software.
   10. Generate a standard curve by plotting the initial slope versus thrombin concentration.
       NOTE: Ensure that all buffers are preheated to 37 °C and handle enzymes on ice. Always prepare fresh dilutions and mix thoroughly. The resulting standard curve is used to calculate the relative thrombin activity in unknown samples using the macro described in section 5.
2. Preparation of the plasmin calibration curve
   1. Reconstitute lyophilized plasmin in distilled water (e.g., 0.543 mL) according to the manufacturer's instructions.
      NOTE: Plasmin has a molecular weight of 75,400 g/mol. The resulting stock concentration is approximately 24.3 nmol/mL (0.024 nmol/µL), assuming 1 mg in 0.543 mL.
   2. Prepare a dilution series in Tris/NaCl buffer to achieve final plasmin concentrations in the assay ranging from 66 to 532 nmol/L.
   3. Pipette plasmin into wells containing Tris/NaCl buffer to reach a final volume of 100 µL per well.
   4. Prepare the substrate solution for Pump 1 by mixing 680 µL of Tris/NaCl buffer with 80 µL of plasmin/rhodamine substrate (pre-diluted 1:1 if required) from step 1.2.5.
   5. Add 20 µL of this substrate solution to each calibration well, bringing the total volume to 120 µL.
   6. Gently mix the plate and insert it into the plate reader. Run the kinetic assay for 25 cycles using the same fluorescence settings as described in Section 2.
   7. Export the raw fluorescence data and determine the initial slope of the fluorescence increase for each plasmin concentration by applying linear regression to the linear phase of the kinetic curve using GraphPad Prism or equivalent software.
   8. Generate a standard curve by plotting the initial slope versus plasmin concentration.
      NOTE: Due to batch-to-batch variation in plasmin activity, calibration should be repeated with each new lot. If unexpectedly high activity is observed, a 1:2 dilution of the stock is recommended before preparing the calibration series.
3. Use the calibration curve for quantification
   1. Use the calibration curves generated in Section 6 to convert fluorescence slope values into relative enzyme activity.
   2. Use only the linear portion of the calibration curve for interpolating unknown samples.

Results

Representative thrombin and plasmin generation curves obtained with the Nijmegen Hemostasis Assay (NHA) are shown in Figure 3. The signal intensity of each curve is expressed as a percentage relative to the concurrently measured normal pooled plasma (NPP) sample. A sample with NPP is included in every run as an internal control to verify assay validity and ensure inter-run comparability.

Figure 3A,B display results fr...

Discussion

The Nijmegen Hemostasis Assay (NHA) is a dual-enzyme fluorogenic assay that enables the simultaneous, time-resolved measurement of thrombin and plasmin generation in platelet-poor plasma. By capturing both processes in a single well, the method facilitates high-resolution kinetic analysis of secondary hemostasis and fibrinolysis. This dual readout offers distinct methodological advantages over conventional assays and has been successfully applied in the study of coagulation factor deficiencies, hyperfibrinolytic conditio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
Calcium chloridedihydrate (CaCl2.2H2O)	Merck	1.02382
Cephalin (C.K. Prest 5 APTT reagent kit)	Stago	00847	Stable for 6 months at -20 °C.
Distilled water			Record the date of opening. Use within 1 day after first opening.
Dimethylsulfoxide (DMSO)	Merck	1.02950.0500
Human alpha-thrombin	Enzyme Research Laboratories	HT 1002a	The box contains a leaflet with a certificate of analysis. The volume of buffer to add depends on the specified concentration and should be adjusted so that 1 µL of added human thrombin in 120 µL reagent yields a final concentration of 76 nmol/L.
Human plasmin	Enzyme Research Laboratories	HPlas	The box contains a leaflet with a certificate of analysis. The volume of buffer to add depends on the specified concentration and should be adjusted so that 1 µL of added human plasmin in 120 µL reagent results in a final concentration of 200 nmol/L.
Normal pooled plasma	In-house preparation	Not applicable	Stable for 3 hours after thawing if kept on ice.
Patient plasma	Not applicable	Not applicable
Plasmin substrate (CBZ-L-phenylalanyl-L-arginine amide)-rhodamine-morpholino urea	Symeres		Stable for 6 months at -80 °C.
Recombinant human tissue factor (TF): Innovin	Dade Behring	B4212	Stable for 6 months at -80 °C.
Recombinant tissue-type plasminogen activator (tPA): Alteplase/Actilyse	Boehringer Ingelheim		Stable for 12 months at -80 °C.
Tris(hydroxylmethyl)aminomethane (H2NC(CH2OH)3)	Merck	1.08382.2500
Tris-NaCl buffer, pH 7,4 (TBS)	In-house preparation	Not applicable
Tris-NaCl-CaCl2 buffer, pH 7,4 (TBS-Ca)	In-house preparation	Not applicable
Thrombin substrate Bz-beta-Ala-Gly-Arg-7-amino-4-methylcoumarin	Symeres		Stable for 6 months at -80 °C.
Equipment
Fluostar Optima fluorimeter / microplate reader with temperature control and reagent injectors	BMG Labtech
96-well microtiter plate, polystyrene, flat-bottom, black, medium binding surface (Microlon equivalent)	Greiner BIO-ONE	655076
Analytical balance (top-loading balance)
Blue pipette tips (for 100-1000 µL)
Citrate Blood Collection Tubes 3mL (3.2% Citrate)	BD Vacutainer
Ice bucket
Ice machine
Incubator / microplate shaker
Magnetic stirrer
Microcentrifuge tube rack
Microcentrifuge tubes, closable
pH-meter
Pipette tips 0.1-10 µL, non-sterile
Pipettes (2, 10, 20, 100, and 1000 µL)
Screwdriver (used as a probe)
Tube mixer
Water bath
Yellow pipette tips (for 20-200 µL)