From Barbara Schmidtz BSA, MSc, MLT, Regina General Hospital, Sask Health Authority (SHA), 12/6/2024:
Hi, I found your page while googling. I’m an MLT in a hospital lab. I was wondering if you could tell me why Bethesda titers are read at 50% residual activity. I understand the curve is linear from 25-75%, but I can’t find an explicitly stated reason for 50% being where it’s read. Was it just the midpoint or an arbitrary convention? Thank you for your time, Barb
On 12/7/2024 I (Geo) sent Barb’s question to my colleague Ali Sadeghi-Khomami, PhD, Scientist, Precision BioLogic Inc. Here is Ali’s response:
Hi George, The 50% residual activity threshold is embedded in the definition of a Bethesda Unit, making it an arbitrary convention: “One Bethesda unit (BU) is defined as the amount of inhibitor that results in 50% residual FVIII:C activity in a defined test mixture.”
Although residual activities between 25% and 75% could theoretically be used for inhibitor titer estimation, I believe the most reliable approach is to use the patient sample dilution that yields a residual activity closest to 50%. Some practitioners calculate the average BU value from all dilutions with residual FVIII activities within the 25%–75% range. However, this approach tends to be less reproducible and exhibits greater variability between patients and FVIII assays. The underlying reason is that the linearity of the inhibitor antibody response differs among individuals and assays. For instance, one patient’s sample may produce three dilutions with residual FVIII activities between 25% and 75%, while another sample may only yield one dilution in this range. For this reason, it is preferable to adhere to the conventional BU definition, which equates 50% residual FVIII activity to 1 unit of FVIII inhibitor. If the averaging approach is used, the number of results included in the calculation needs to be disclosed in the report to ensure transparency.
The 25% and 75% thresholds are derived from the logarithmic formula used in titer calculations and are unrelated to the assay’s linearity. Specifically, 25% residual activity corresponds to 2 BU (maximum), while 75% corresponds to 0.5 BU (limit of quantification, LoQ). These values are then multiplied by the dilution factors used during sample preparation to determine the inhibitor titer in the patient sample.
Ultimately, the linearity of the Bethesda assay depends on the FVIII:C assay/reagent system and the sample type used in the investigation. For example, a bovine chromogenic assay may exhibit linearity from 0 to 100 BU/mL, while a one-stage clotting assay, such as Actin FSL, may have a linear range of 0.5 to 60 BU/mL. These linearity ranges are provided here as illustrative examples. Best regards, Ali.
I also sent Barb’s question to colleague David McGlasson, who on 12/8-2024 directed me to this post provided by Connie H. Miller, PhD. previously director of the Division of Blood Disorders National Center on Birth Defects and Developmental Disabilities Centers, Center for Disease Control and Prevention, USA.
FVIII inhibitors are time-dependent. The Bethesda assay is performed by mixing one part of patient plasma with one part of normal pooled plasma (NPP), incubating at 37°C for 2 hours, and measuring the VIII:C remaining in the mixture. This activity is divided by the activity remaining in a control 1:1 mixture of NPP with imidazole buffer and multiplied by 100 to give the % residual activity (RA). The RA is converted to BUs using a graph plotting the logarithm of RA against BU…
Calculation of Bethesda units in the Bethesda assay for inhibitors.
For RA=100%, BU=0. For RA 25–100%, BU of the lowest dilution in that range is reported. If no dilution falls above 25%, patient plasma is tested at higher dilutions, with the first dilution falling between 25% and 75% RA multiplied by the dilution factor and reported. For inhibitors with complex kinetics, which do not respond linearly to dilution, the dilution closest to an RA of 50% is reported. For increased sensitivity, the assay may be performed using a higher proportion of patient plasma (e.g. a 3 : 1 mix).
Modifications to increase the sensitivity and reproducibility of the Bethesda method have been widely adopted. Substitution of FVIII-deficient plasma for buffer in patient dilutions and the control mix and buffering of NPP with imidazole to pH 7.4 serve to maintain the protein concentration and the pH of the mixtures during incubation. This has been called the Nijmegen-Bethesda (NB) method, and the results are Nijmegen-Bethesda units (NBU).
Dr. Miller’s chapter appears in Transfusion Medicine and Hemostasis, 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA, 2009; https://doi.org/10.1016/B978-0-12-374432-6.00156-1.
Here is a direct communication from Dr. Connie H. Miller received on 12-9-2024:
Hi George, The use of 50% is arbitrary to assure assay standardization, and I am not aware of its origin. The choice of which dilution to use makes a difference only in inhibitors with Type 2 kinetics. For the more usual Type 1 inhibitors, each dilution should provide a similar result. For Type 2 inhibitors, the dilution closest to 50% residual activity or the first dilution falling below 75% residual activity is usually reported, because the result for each dilution will vary, as shown below. We no longer graph the results of multiple dilutions and read the BU at 50%, primarily for ease of calculation, although that was done in the past.
I addressed the inhibitor calculations in the attached review, as follows:
Inhibitors with Type 1 kinetics are expected to show a negative slope within that range, and multiplication of the calculated NBU by the dilution factors produces approximately equivalent results for each dilution (Table). Multiplication by a large dilution factor, however, magnifies small errors, and even acceptable variation in the performance of the factor assays can result in differences between dilutions and between different assays when multiplied by 10 or higher. Inhibitors with Type 2 kinetics show similar %RA at multiple dilutions, which can result in progressively higher inhibitor results with multiplication by dilution factors (Table). It is usually recommended to report the dilution having RA closest to 50%; however, some laboratories prefer to use the first dilution falling below 75% RA to avoid error introduced by use of larger multiplication factors. For monitoring change in titre over time, it may be most useful to follow the same dilution in each subsequent assay to detect rise or fall in the NBU.
Examples of calculation of Nijmegen-Bethesda units (NBU) in assays of factor VIII (FVIII) inhibitors with Type I and Type II kinetics.
| Pre-dilution | Patient Mix
U/dL FVIII* |
Control Mix
U/dL FVIII* |
% Residual Activity† | Calculated
NBU‡ |
Total NBU§ | |
| Type 1 inhibitor | Undiluted | 19 | 45 | 42.2 | 1.24 | 1.2 |
| 1:2 | 30 | 45 | 66.7 | 0.58 | 1.2 | |
| 1:4 | 35 | 45 | 77.8 | 0.36 | 1.4 | |
| Type 2 Inhibitor | Undiluted | 27 | 45 | 60.0 | 0.74 | 0.7 |
| 1:2 | 27 | 45 | 60.0 | 0.74 | 1.5 | |
| 1:4 | 27 | 45 | 60.0 | 0.74 | 3.0 |
*units per deciliter of FVIII activity
†patient mix units per deciliter FVIII activity/control mix units per deciliter FVIII activity X 100
‡NBU read from graph or calculated as NBU = (2-log %residual activity)(0.301)-1
- calculated NBU X pre-dilution factor
Regards, Connie
Connie H. Miller, Ph.D.
Agility Consulting Group
Consultant to…
Division of Blood Disorders and Public Health Genomics
National Center on Birth Defects and Developmental Disabilities
Centers for Disease Control and Preventions
Click here for Miller Laboratory testing for inhibitors a review
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