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Erythrocyte sedimentation rate (ESR) is a physical parameter, often used in routine health checks and medical diagnosis. A theoretical model that allows to extract physically-meaningful parameters from the whole sedimentation curve, based on modern colloidal knowledge, has recently been developed. Here, we present a protocol to automatically collect the ESR over time, and extract the parameters of this recent model from this automated data collection. These refined parameters are also likely to improve the medical testimony.
Erythrocyte (or red blood cell) sedimentation rate (ESR) is a physical derived parameter of blood which is often used in routine health checks and medical diagnosis. For instance, in the case of inflammation, a higher ESR is observed due to the associated increase in fibrinogen and other plasma proteins. It was believed that this increase was due to the formation of larger aggregates of red blood cells (RBCs) caused by the increase in fibrinogen. Indeed, fibrinogen is an agent-fostering aggregation of RBCs and in the Stokes regime-assumed to be observed in blood-larger aggregates sediment faster. However, all models of ESR measurements based on this hypothesis require further specific physical assumptions, not required in any other system. Besides, modern studies in the field of colloidal suspensions have established that attractive particles form percolating aggregates (i.e. aggregates as wide as the container). The sedimentation of these colloids then follows a so-called "colloidal gel collapse". Recently, it has been shown that RBCs actually follow the same behavior. This hypothesis also allows to efficiently and analytically model the sedimentation curve of RBCs, from which robust and physically-meaningful descriptors can be extracted. This manuscript describes how to perform such an analysis, and discusses the benefits of this approach.
The erythrocyte sedimentation rate (ESR) is a medical in vitro clinical tool, formally introduced in evidenced-based medicine during the twentieth century1,2,3,4. It is currently used worldwide as a nonspecific inflammatory test, or to monitor the evolution of some specific conditions5,6,7,8. This is mainly due to an increase in the fibrinogen concentration, but also in other plasma components such as IgM1,9,10,11. According to the current Westergren standard protocol, ESR values are reported as the measurement of the cell-free plasma layer at a given time point (30 min or 1 h) after leaving a vertical tube of a typical size of 20 cm vertically at rest12. However, this measurement method has been criticized since qualitatively different stages in the sedimentation process have been reported, including a delay before reaching the maximum settling velocity13. This delay lasts more than 1 h in approximately half of healthy samples14. The velocity during this phase obeys a different scaling than during the second, faster phase of the sedimentation15. Restricting the readout to the average settling velocity during the first hour then compares a different mix of various blood properties between different individuals.
Moreover, it has recently been demonstrated that the usual theoretical considerations behind this protocol were erroneous16,17,18. At physiological hematocrit (above approximately 25%), red blood cells (RBCs) do not sediment as separate aggregates, but rather as a continuous, so-called percolating, network of RBCs17,18, obeying a different set of physical equations than the usually mentioned Stokes sedimentation16,17. It has been shown that considering a physical description based on the time-resolved measurements of the sedimentation (whole curve) was more robust in some novel medical contexts19,20. Moreover, these measurements could be used to shed light on the physical mechanisms altering the ESR in pathologies in which cell shapes are altered19,20. Additionally, a slow ESR can have a useful medical interpretation, as indicated in the measurements of a cohort of neuroacanthocytosis syndrome patients19,20. This article reviews how to practically implement the measurement of physically-meaningful parameters, based on the whole ESR kinetics. More accurately, the method presented here extracts the maximum sedimentation speed Um, the value of which can be corrected to consider the effect of the hematocrit of the donor16,17. This parameter is more accurate and thus more reliable than the traditional measurement16,17,19,20.
In addition, in some fundamental research, instead of monitoring the inflammation state of a given patient, it is interesting to exclude the effect of hematocrit on the ESR21,22,23, or to investigate the role of RBCs in a modified ESR19,20,24,25 between different donors. It might be useful to compare samples which are not directly full blood samples from patients. Therefore, resuspending RBCs with a controlled hematocrit in the autologous plasma, or in a plasma-substituent, might be used as the first step of ESR measurement. For instance, solutions of Dextran 70 kDa with a concentration of 55 mg/mL in phosphate buffered saline (PBS) produces a sedimentation range within the control range for healthy cells19. This manuscript also shows how such steps should be conducted, and that the presented analysis is also relevant in these cases.
Blood sample collection and experiments were approved by the "Ärztekammer des Saarlandes", ethics votum 51/18, and performed after informed consent was obtained according to the Declaration of Helsinki. Standard measurements should be performed with ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood (standard EDTA concentration of 1.6 mg/mL blood, European norm NF EN ISO 6710), in Westergren tubes. The volume required to fill the Westergren tube depends on the manufacturer (as lower parts sometimes contain a wider reservoir); The volume should be about 1 mL of full blood, and 800 µL for the tubes indicated in the Table of Materials. The method described below is however valid no matter the specific suspension and container shape, as long as the hematocrit of the probed samples is higher than 25%16. Volumes, containers, suspending medium, and additives should therefore be selected according to the specific objectives of the performed research.
1. Experiments and measurements
NOTE: Record the sedimentation rate of the sample every minute.
2. Image analysis
NOTE: Once the images are recorded, extract the ESR curve. An example of Matlab code is provided as Supplementary File 1 (MatlabCodeImageAnalysisSampled.m).
3. Fitting of the physical model
An example of an image sequence correctly acquired is provided as Supplementary Movie 1 (MovieS1.avi). A series of characteristic fits of the model is shown for various conditions in Figure 2. Fibrinogen concentration was determined from the fibrinogen concentration in the plasma Fib0, assuming that the serum does not have any fibrinogen at all. Hence, Fib = C Fib0, where C is the plasma volume fraction in the plasma-...
For the automated protocol to work efficiently, it is important to have a clear background and proper illumination. A dark background might prevent the existence of an efficient binarization threshold. For samples with some hemolysis, which usually occurs (increases) over time, it is important to verify first that the chosen binarization threshold is relevant for both the initial and final pictures.
When it comes to the binarization process of the picture, the choice of the ROI and binarizatio...
The authors have no conflicts of interest to declare relevant to the content of this article.
This work was supported by the research unit FOR 2688 - Wa1336/12 of the German Research Foundation and by the Marie Skłodowska-Curie grant agreement No. 860436-EVIDENCE. T. J. and C. W. acknowledge funding from French German University (DFH/UFA). A. D. acknowledges funding by the Young Investigator Grant of Saarland University.
Name | Company | Catalog Number | Comments |
Anticoagulant (EDTA or Heparin) tube (for blood sample) | SARSTEDT | 267001 or 265 | Anticoagulated blood sample to characterize |
Camera EOS M50 | Canon | Kit EF-M18-150 IS STM | Any camera should work, provided that sector alimentation, connection to computer for automated shooting and adapted objective are available |
Centrifuge | HERMLE | 302.00 V03 - Z 36 HK | Requirements: at least 3000 x g ofr 7 min. |
Micro-centrifuge | MLW | TH21 | or any other way to determine the hematocrit |
Micro-hematocrit capilaries | Fisher scientific | 11884040 | or other capillaries/containers for hematocrit determination |
Phosphate Buffered Saline (PBS) | ThermoFisher | 10010023 | 1x PBS, pH 7.4, 298 Osm |
Pipettes (e.g. positive displacement pipette) | Gilson | FD10006 | Pipette required to manipulate blood and/or packed cells.Other models are of course suitable, but be careful to treat blood and pakced cells as highly viscous fluids. |
Wax sealing plate | Hirschmann | 9120101 | Sealing wax for the micro-hematocrit capillaries |
Westergren tubes | Praxindo | A9244560 | Any other standard Wetsergren tube should work too |
White background with illumination | / | / | White sheet(s) of paper behind the samples, with usual room light is perfcetly sufficient. |
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