Frequently asked questions about HemaSpot-HF and HemaForm™ plate kits.
No, both products can have the same grade of absorbent paper. Both products may have grades other than the grade of paper Spot On Sciences has been recommending – TFN from Sartorius-Stedim. For example, Whatman’s 903 grade, CF12 grade of paper can both be cut to fit the HemsSpot device platform as can Ahlstrom’s 226 grade of paper.
The HemaSpot™ device will function properly up to 60 minutes after the packaging is opened. Spot On Sciences recommends applying the sample and closing the device within 15 minutes of opening the packaging. After 60 minutes, the drying capacity of the device may be compromised.
Yes the device itself is stable. HemaSpot™ has been tested in a number of extreme conditions from a baking hot car sitting in the summer sun to a biological freezer set at -80 C. The device has been submerged under water for at least 12 hours and has even been run over with the full weight of a Ford F-150. The device and the DBS sample were found to have survived all of these conditions. However, we don’t recommend any of these conditions for long term storage with the exception of the -80 C freezer.
Yes, dried blood spots are accepted by the US Postal Service as safe and may be shipped by standard mail if secured within a secondary container before placing inside the mailer. The HemaSpot™ device is considered the secondary container and renders the blood sample safe and secure after closing.
The HemaForm™-80 was designed to hold 80uL of WB. Each blade contains 11.5% of this volume or 9.2uL of WB. A total of 8 blades may be collected from each HemaForm™. A typical DBS card holds 5 circles with each circle capable of receiving a single drop of blood. A drop of blood usually means between 25 and 30uL of WB, depending upon hematocrit. And each sample might allow 3 x 4mm punches. Thus, assuming all circles are good, about 15 x 4 mm punches may be collected from each card. Each of these 4mm punches represents about 4uL of WB. Therefore, nearly 60 uL of WB may be recovered from a total of 125 uL WB collected.
Accuracy = (mean-nominal)/nominal*100
Precision = Standard Deviation/Average*100
Expected reproducibility from blade-to-blade should be less than 10% with the proper sample volume applied. Nominal sample volume is 80 µL (approximately 2-3 drops) of blood.
Yes, you can buy a single HemaSpot™ device without having to purchase the entire kit. However, the HemaSpot™ kit contains everything needed to perform a complete blood sampling from start to finish including the lancet, gauze and alcohol swabs.
The HemaForm™ kit is designed for a laboratory setting or intended for analytical validation work using our filter paper. The kit contains a 12- or 24-well culture plate with HemaForm™ paper that can handle either 80µL or 40µL target volume. This format allows for easy integration with automation systems and makes it simple to do method development and assay validation work.
Typically, we use a mixture of water and a water miscible organic solvent, such as methanol or acetonitrile, to elute analytes from the HemaForm™ blades. After vortexing or sonicating the blades in 200µL of solvent in deep well plates or conical tubes for 30 to 60 minutes followed by centrifuging at 10,000g for 10 minutes, we are able to draw off 175 – 180µL of solvent. Usually, this sample may be injected directly onto an HPLC system for mass spec analysis.
With protein or enzymatic assays, we generally use 200µL of a phosphate buffered solution or simply water to elute these components. For enzymatic assays, the enzyme substrate is generally added to the PBS solution and the deep well plate is mixed with heating for as much as 48-hours depending upon the activity of the enzyme. After incubation, the plates may be centrifuged briefly and the liquid decanted and added to an equal volume of cold methanol or acetonitrile to precipitate protein. Again, after centrifuging to pellet protein, the sample is injected onto the HPLC for analysis.
The volume will vary depending on the size of the blood drops as well as other factors related to blood consistency. The standard HemaSpot™ device is designed to absorb a target volume of 80µL of blood. A single blade is approximately 11.5% of the total fan, meaning that each blade can carry approximately 9-10µL of blood per blade.
After allowing as much of the applied blood to flow onto the HemForm™, simply closing the device will pop any bubble left on the funnel. We recommend allowing blood or sample to fall into the center of the applicator funnel and not evacuating the pipette tip into the blood on the applicator. Rather, move the tip away from the blood, evacuate the tip, then move the tip to touch the pool of blood on the device. Any blood bubble on the tip will burst at that point without forming a bubble on the funnel.
There are several established methods that can help with blood flow to the fingertips. Spot On Sciences recommends either holding the selected hand under warm running water for about 1 minute and/or vigorously massaging or rubbing the fingertip. Remember to select either the middle or ring finger on the non-dominant hand. Avoid calluses and the “pad” of the fingertip. The fleshy side of the fingertip is the ideal spot to place the lancet for the finger stick.
Hematocrit is the volume percentage of red blood cells in blood. Hematocrit (ht or HCT) is also referred to as Packed Cell Volume (PCV) or Erythrocyte Volume Fraction (EVF). Normal HCT levels for adult males is 42-52%. Normal HCT levels for adult females is 36-48%. Hematocrit is measured by centrifuging whole blood in a heparinized capillary tube (to prevent clotting) to separate the plasma from red blood cells. HCT is calculated by measuring the lengths of the layers (PCV length/total length).
Hematocrit has an effect on blood viscosity and may influence the flow and spread of a sample onto DBS filter paper. Spot On Sciences’ HemaForm™ design reduces the effect of hematocrit by controlling the flow and spread when a sample is applied. (Hematocrit poster data can be supplied)