Rapid measurement of formed blood component sedimentation rate from small sample volumes
Devices and methods are described for measuring formed blood component sedimentation rate. Some of the methods may use (1) centrifugal techniques for separating red blood cells from plasma and (2) video and/or still imaging capability. Both may be used alone or in combination to accelerate formed blood component sedimentation and to measure its rate. In one example, the method may advantageously enable rapid measurement of sedimentation rate using small blood sample volumes. Automated image analysis can be used to determine both sedimentation rate and hematocrit. Automated techniques may be used to compensate for effects of hematocrit on uncorrected sedimentation rate data.
1. A method comprising:
centrifuging a blood sample in a vessel for a period of time;
establishing a time-related compaction curve for at least one formed blood component in said blood sample after centrifuging has begun;
correcting for impact of hematocrit on sedimentation rate of the formed blood component using a hematocrit correction factor based on the formula:
=
U
uncorr
(
1
-
φ
φ
max
)
γ
,
where U uncorr and U corr are the uncorrected (raw) and corrected sedimentation rates, φ is the volume fraction of cells (hematocrit), and φ max and γ are empirical parameters obtained by curve fitting.
2. The method of claim 1 , wherein curve fitting for the hematocrit correction factor comprises calibrating sedimentation rates from centrifuge based technique with sedimentation rates from a reference technique.
3. The method of claim 1 , wherein the reference technique is the Westergren technique.
4. The method of claim 1 , wherein fibrinogen levels as high as 15 mg/ml does not impact sedimentation rate measurement.
5. The method of claim 1 , wherein said blood sample is about 100 uL or less.
6. The method of claim 1 , wherein said blood sample is about 50 uL or less.
7. The method of claim 1 , wherein said blood sample is about 25 uL or less.
8. The method of claim 1 , wherein centrifugation occurs at a first speed for a first period of time and then at a second, faster speed for a second period of time.
9. The method of claim 1 , wherein centrifugation uses a centrifuge configured to allow the blood sample to be visually observed during centrifugation to establish interface positions of one or more formed blood components in the blood sample.
10. The method of claim 1 , wherein centrifugation uses a centrifuge having a window thereon to enable visual observation of the blood sample to establish erythrocyte/plasma interface positions over time.
11. The method of claim 1 , wherein centrifugation uses a centrifuge, a light source, and an image capture device to enable visual observation of the blood sample to establish formed blood component/plasma interface positions over time.
12. The method of claim 1 , wherein compaction curve data is collected by capturing a plurality of images of interface positions of one or more formed blood components in the centrifuge vessel over the time period.
13. The method of claim 1 , wherein compaction curve data is collected while the sample is being centrifuged.
14. The method of claim 1 , wherein centrifugation is used to obtain accurate values for the hematocrit and to correct for hematocrit impact on sedimentation rate measurement.
15. The method of claim 1 , wherein correcting for hematocrit comprises calculating a mathematical function for a plurality of formed blood component interface positions occurring in said curve, said function being operative to correct for sedimentation rate variations due to hematocrit.
16. The method of claim 1 , wherein hematocrit correction factor is determined without using data from a non-linear portion of the compaction curve.
17. The method of claim 1 , wherein hematocrit level in the sample is derived from a technique separate from centrifugation.
18. The method of claim 1 , wherein φ max and γ are for fit optimization and do not relate directly to physical parameters.
19. The method of claim 1 , further comprising image transformation for conversion of a curved interface to a flat interface.
20. The method of claim 1 , wherein hematocrit correction is capable of essentially eliminating the effects of hematocrit on formed blood component sedimentation rate.
21. The method of claim 1 , wherein image transformation parameters are selected, video of formed blood component interface position is put through image transformation, and then a region of interest is chosen that covers both the whole range of positions for both air/plasma interface and erythrocyte interface.
22. The method of claim 1 , wherein for each timepoint in the video, pixel intensity values for each row across the sample vessel within the region of interest are averaged to produce a single column representing the intensity radially down the sample vessel.
23. The method of claim 1 , wherein columns for each timepoint are then assembled into a kymograph.
24. The method of claim 23 wherein positions of the two local maxima of the image, one representing the air/plasma interface and other the plasma/erythrocyte interface are determined.
25. The method of claim 23 comprising converting pixel positions into volume occupied by the whole sample and volume occupied by red blood cells, wherein the y-position of the top and bottom of the centrifuge vessel are used as reference locations together with knowledge of the shape of the centrifuge vessel.
26. The method of claim 1 , comprising converting plasma/erythrocyte interface position to the volume fraction occupied by red blood cells and plotted against time as a centrifuge sedimentation curve.
27. The method of claim 1 , wherein a linear region of a sedimentation profile is used to extract a sedimentation rate.
28. The method of claim 1 , further comprising deriving an estimate of the sedimentation rate linearly related to the Westergren ESR, the centrifuge-derived, hematocrit corrected data further corrected using the formula: Estimated Westergren ESR=10^(((LOG(HCT corrected ESR)−LOG(a))/b)).
29. The method of claim 1 , further comprising hematocrit-correcting and linearly-transforming Log(ESR) values to establish a linear graph of sedimentation rate.
30. The method of claim 1 , wherein the blood sample is whole blood.
31. The method of claim 1 , wherein the blood sample is an anti-coagulated sample.
32. The method of claim 1 , wherein the formed blood component is white blood cells.
33. The method of claim 1 , wherein the formed blood component is platelets.
34. The method of claim 1 , further comprising determining white cell sedimentation rate after centrifugation has begun, wherein measuring white cell sedimentation rate characterizes at least one of the following regarding the white blood cells: cell density, shape, and aggregation state.