blood hematocrit effect with dried blood spots: what is the big deal?
What Is Blood Hematocrit?
Hematocrit (also spelled haematocrit) is a volume measurement of red blood cells in blood, expressed as a percentage. In the laboratory, this can be determined manually through a "packed cell volume" measurement or in an automated fashion by determining the red cell count and then multiplying by the mean cell volume (i.e. erythrocyte volume fraction). Hematocrit can be estimated by tripling the measured hemoglobin concentration (in grams per deciliter or g/dL) and removing the units.
The term is derived from the Greek words "haima," meaning "blood," and "krites," meaning "judge." This word combination means "to separate blood." Hematocrit goes by these additional names:
- Packed cell volume (this is generally viewed as being less accurate than the erythrocyte volume fraction measurement due to the plasma found in the interstitial space of the packed cells)
- Volume of packed red cells
- Erythrocyte volume fraction
The effect of blood hematocrit on dried blood samples is a large topic of discussion. The hematocrit level can vary depending on multiple factors that affect the red blood cells. The level of body hydration can affect hematocrit. As the volume of water in the body decreases the percent volume of red blood cells increases. Additionally, studies indicate that arterial blood has a slightly lower hematocrit than venous blood.
Why Does It Matter?
Blood viscosity can alter results of dried blood spotting (DBS) because the viscosity determines how far the blood spreads on the dried blood spotting paper. The spot area of the sample typically has a linear, inverse relationship to the blood hematocrit. Therefore, blood with a high hematocrit level results in a smaller dried blood sample; a lower hematocrit value causes a larger dried blood sample. The properties of the paper substrate can also affect how the blood sample spreads.
The blood hematocrit effect (or hematocit bias) is known to greatly affect data reliability and quality. If blood spreads unevenly on the sample card, the area that's punched for analysis might vary despite the widespread assumption that punches of the same size contain the same amount of blood. Furthermore, the blood cells themselves can cause variations in the amount of analyte that is extractable from the surface of the DBS card itself.
Ways to Address Volumetric Blood Hematocrit Bias
Whole Spot Analysis, Perforated and Precut DBS (PDBS)
Whole spot analysis is a way to avoid the variation that results from non-homogeneity and the different rates of blood spreading due to hematocrit levels. Typically, the entire blood spot is cut from the paper, though this analysis method has evolved with perforated and precut DBS methods. With perforated DBS, an area of the card is defined and the blood is limited to the area, which is then punched from the card for analysis. With precut DBS, discs are precut from DBS paper and the blood is sampled directly onto the disc eliminating the hematocrit effect.
Volumetric Absorptive Microsampling (VAMS™)
The volumetric absorptive microsampling technique involves dipping an absorbent tip into a pool or drop of blood. After a few seconds, the tip is filled with a precise volume of blood (e.g. 20 µL) with a variation of less than 4%. The entire sample is then dried, extracted, and analyzed. Due to the fact that the whole tip is extracted makes this analogous to whole spot analysis in DBS with the added benefits of a more convenient sample collection and a simpler, automatable workflow. Studies have shown that VAMS effectively eliminates the hematocrit effect.
Share with us how you overcome the hematocrit effect with DBS.