Histopaque® Density Gradient Media Troubleshooting Guide

By: Mark Frei, BioFiles v6 n5, 14–16

BioFiles Volume 6, Number 5 — Centrifugation

The following recommendations for troubleshooting the use of Histopaque and ACCUSPIN™ in cell separation techniques have been compiled by our Technical Support scientists based on observations and experience from resolving customer problems. This guide addresses the most common sources of error observed when using Histopaque but is not meant to be a comprehensive list.

Typical problems observed when using Histopaque/ACCUSPIN
Blood or Cell Samples Troubleshooting Tips
Histopaque/ACCUSPIN Troubleshooting Tips
Technique Troubleshooting Tips

Typical problems observed when using Histopaque/ACCUSPIN

  • Difficulty in separating mononuclear or polymorphonuclear cells from human blood
  • Difficulty in separating mononuclear or polymorphonuclear cells from rat or mouse blood
  • Low recovery
  • Red blood cell, platelet, or neutrophil contamination in separated cells


Blood or Cell Samples Troubleshooting Tips

Cause Solution
Blood drawn >24 hours prior to separation Use blood drawn from 2–6 hours prior to separation. Blood drawn >24 hours will be more difficult to separate, and percent recovery and viability will be lower.
Blood has coagulated due to absence of anticoagulant Use of vacuum tubes with premeasured amounts of anticoagulant is suggested. Use of a syringe (without anticoagulant) is not recommended.
Blood has coagulated due to incomplete mixing Ensure vacuum tubes with anticoagulant are mixed well after blood draw.
Anticoagulant used is not suitable for use in Histopaque separation application The following anticoagulants are suitable for use with Histopaque:

      • 15–30 units heparin per mL whole blood
      • 1.25–1.75 mg EDTA per mL whole blood

ACA-A (Acid Citrate Dextrose formula A) may be used as an anticoagulant. After separation, the mononuclear cell band produced will be wider than for other anticoagulants. This is normal and the cells are acceptable for use.
Blood sample is too warm and separation attempted too soon after blood draw Red blood cell contamination may be a result of blood not being at room temperature.

After drawing, the blood should be allowed to cool at room temperature for minimum 30-45 minutes. If used immediately after being drawn, the number of mononuclear cells collected will be low. Separation procedures are optimal when both blood and Histopaque are at 18–20 °C, with an acceptable temperature range of 18–26 °C.
Blood sample diluted with high salt concentration PBS The historic cell separation technique used with Histopaque includes dilution of the blood with PBS (1:1). Subsequently, we have found not all PBS formulations are suitable for use with Histopaque.

Solutions containing 150 mM phosphate buffer and 150 mM sodium chloride are PBS solutions with salt concentrations too high for use with Histopaque. PBS molarities closer to 10 mM phosphate are more suitable for use with Histopaque. Cell viability will remain higher when high salt concentration PBS is replaced by a suitable cell culture media.
Differences in donor blood physiology (when single sample has poorer recovery) High lipid levels, rheumatoid factor, anemia, and drug treatment are all possible causes for poor separation of a specific donor’s blood.

If the plasma is not clear, this is an indication of high lipid levels.
Contamination of small animal sample with other fluids or solids Blood obtained from a non-needle bleed, e.g., through tail sampling, is often unacceptable for use due to the presence of hairs and other body fluids in the collected blood. This often results in the coagulation cascade being initiated, producing clotting.

Recommend the animal be sacrificed and the blood obtained through either the aorta or vena cava or other large blood vessel. Alternatively, a pediatric vacuum tube may be used for drawing blood.
Occlusion of white blood cells in samples with high levels of white cells Aggregates may form when red cells come in contact with the polysucrose, resulting in occlusion of white blood cells. Once trapped in these aggregates, the white blood cells will travel to the bottom of the centrifuge tube.

For routine blood specimens, percent recovery will often be higher for undiluted blood. However, bone marrow, umbilical cord blood, and samples from donors with abnormally high white cell counts should be diluted to reduce the size of the red cell aggregates and improve recovery of white cells.
Improper dilution of blood or dilution with contaminated PBS or media When blood is diluted prior to loading on the Histopaque®, there is a chance either the buffer or media formulation is incompatible with Histopaque, or that the PBS or cell culture media is contaminated.

If the blood has been diluted and yields are poor, repeat experiment without sample dilution.
Dilution using cell culture media containing FBS or other carrier protein Cell culture media without FBS can be substituted for PBS when diluting samples for use with Histopaque. If the blood is diluted with cell culture media containing FBS, the number of cells recovered will be lower.

FBS may be added to the culture media for subsequent washing or storage after the cells have been separated and washed at least once to remove the Histopaque. After the first wash, adding protein such as FBS to the wash media will further protect the cells from damage.
Isolating cells from buffy coats using Histopaque A buffy coat is prepared by collecting blood in a suitable anticoagulant and centrifuging. Red cells will pellet to the bottom of the tube. Directly on top of the red cell layer will be a gray layer. This gray layer is the buffy coat and represents all the white blood cells in the blood. Above the gray layer will be the plasma.

Buffy coats prepared from fresh blood are suitable for separation using Histopaque. If the cells are several days old, e.g., if the buffy coats are obtained from donated units of blood, cells may not provide acceptable yields.

Buffy coats must be diluted a minimum of 1:2 or 1:4 before being loaded onto the Histopaque gradient.
Isolating cells from buffy coats using ACCUSPIN™ tubes Buffy coat preparations cannot be used in ACCUSPIN tubes. The ACCUSPIN tubes require a certain quantity of red blood cells to work properly, and buffy coats lack sufficient red blood cells for use in ACCUSPIN tubes.

Histopaque/ACCUSPIN Troubleshooting Tips

Cause Solution
Bacterial contamination of Histopaque Histopaque is manufactured as a sterile solution. There are no preservatives present to reduce chance of contamination. If contamination is suspected, use fresh bottle of Histopaque.
Histopaque temperature is too cold If Histopaque or filled ACCUSPIN tubes are used cold, yields are typically very low and clumping of the cells may be observed. Red blood cell contamination may also be a result of Histopaque not being at room temperature.

Temperature is extremely important when performing the procedure. A 100 mL or 500 mL bottle of Histopaque stored at 2–8 °C may take several hours to reach 18–20 °C. When planning to use Histopaque, we recommend removing the Histopaque from the refrigerator the previous day and let the bottles stand on the bench overnight. This ensures the solution is at room temperature and ready for use.

Another option is to transfer the proper amount of Histopaque to each centrifuge or ACCUSPIN tube and allow the smaller volumes to acclimate to room temperatures.

After the cell band has been collected and the first wash performed, it is acceptable to perform the remaining wash steps at 4 °C.

Technique Troubleshooting Tips

Cause Solution
Delay in processing samples after layering blood on Histopaque When layering the blood onto the gradients intended for mononuclear cells, precise layering is not required. If a little mixing occurs between the blood and the Histopaque, the cell band will still form properly.

Once the blood has been layered, it is important to immediately place the centrifuge tubes into the centrifuge. If too much time elapses, the entire layer of Histopaque will be tinged red from the red blood cells. Depending upon the amount of “droplet formation,” this may lower the percent recovery of mononuclear cells. The number of tubes processed at one time should be limited to reduce this cell dispersal.
Improper layering of Histopaque® 1077 and Histopaque 1119 when isolating neutrophils When using both Histopaque 1077 and Histopaque 1119 to isolate neutrophils, careful technique must be used to prevent mixing at the solution interface.

Check the integrity of the interface using Schlieren optics. Any swirling or mixing at the interface between the layers should be evident when holding the tube up against the light. If the interface was properly prepared, there should be a sharply demarcated line.

If the sharply defined line is not present or if swirling is present at the interface, discard the tube and start over.

It is also important to use the gradient as soon as it is formed. There are no chemical differences between 1077 and 1119; the two solutions have the same components and will start diffusing together over time. When this happens, recoveries will be poor or nonexistent.
Contamination from powder used in powdered gloves Powdered gloves should not be used. The glove powder causes the cells to aggregate. Use powder free gloves only.
Incorrect centrifugation force used The centrifuge should be checked to ensure proper calibration. Excessive force (higher centrifugation speed) will lower yield.
Histopaque was autoclaved to sterilize prior to use Histopaque cannot be sterilized by autoclaving; autoclaving will caramelize the polysucrose in the solution, turning it brown. Sterile filtering Histopaque through a 0.22 micron filter is acceptable.
Platelet or neutrophil contamination in cells after separation Platelet contamination or neutrophil contamination is generally the result of collecting too much plasma or Histopaque 1077 or 1083 when the cell band is being collected. Reduce the amount of plasma or Histopaque collected.
Too large a volume used to wash cell pellet Washing the cell pellet typically removes platelets. If a large amount of wash solution is left over the cell pellet, this results in more platelets in the cell suspension.

Remove as much wash media as possible without disturbing the cell pellet. Platelets normally do not pellet below a centrifugal force of 1000 × g for 10 minutes. At the lower centrifugation speeds recommended for the wash steps, the platelets should stay suspended in the wash media or supernatant.
Cell pellet does not form single cell suspension When resuspending the cell pellet, only a small amount of wash solution should be used. We recommend no more than 0.5 mL wash media when performing the procedure in a 15 mL or 50 mL centrifuge tube. This wash media is gently rinsed over the pellet.

It may be necessary to draw the cell suspension into the pipet and dispense several times. Once the cells are in a single cell suspension, the remaining wash solution can be added and the solution centrifuged.
Loss of cells during washing due to adhesion to centrifuge tube wall Occasionally cells stick to the walls of the centrifuge tube. If the cells do adhere to the polystyrene tube, the tube will appear to become cloudy.

Change to another lot of polystyrene centrifuge tubes or switch to another type of plastic such as polyethylene. Polystyrene tubes are most commonly observed to cause this problem, but other types of plastic have also been reported to have cell adhesion.