Erythrocytes, Red Blood Cell Lysis, Hemolytic Assay in Whole Blood

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Hemolytic Assay in Whole Blood: A relevant assay that is reflective of the in vivo cytotoxic potential of a drug [e.g., antimicrobial peptides (AMPs)] when administered systemically

Keywords: Hemolytic assay in whole blood, erythrocytes lysis assay, red blood cells lysis assay in whole blood

 Background:

 Hemolytic assay in phosphate buffered saline (PBS) is routinely used to examine toxic effects of a particular drug on human erythrocytes (1). This assay is widely accepted for determining potential toxic effects on red blood cells, particularly in the context of structure-function studies. While informative, the hemolytic property of a drug [e.g., antimicrobial peptides (AMPs)] in PBS is not a realistic measure of its toxic potential in vivo. As we have demonstrated (2), the antimicrobial selectivity of antimicrobial peptides in serum or whole blood is different from that determined in PBS. Both antimicrobial and hemolytic effects in blood substantially decrease. In fact, it was known for years that AMPs are inactivated in serum; their antimicrobial activity and their hemolytic activity are dampened in blood when compared to PBS. Hence, as a general principle, both antimicrobial activity and host toxicity should be examined in the context of an environment that is reflective of the ultimate application of the drug.

It is because of the observation of this serum or blood effect that the hemolytic assay in whole blood was developed. Interestingly, this assay is not very different from the standard assay in PBS, except for one or two steps. However, here I will describe the assay completely.

 Materials:
  • Heparinized blood or uncoagulated blood
  • Red blood cell lysis buffer or just deionized water (recommended)
  • A U-bottom 96-well plate
  • 1.5 ml eppendorf tubes
  • A flat-bottom 96-well plate
  • A 98-well plate reader
  • The drug you are studying
Methods:
  1. Treat the blood with your drug(s) of interest
  2. Prepare the drug in a U-bottom 96-well plate using serial dilutions from an 8x maximum test concentration (80, 40, 20 μM, etc.)
  3. Be sure that the drug is prepared in 50 μL volume prior to serial dilutions. After the dilutions, there will be 25μL in each well at 8x the ideal test concentrations. Please be sure that there are 2 wells in each column with no drug (only PBS)
  4. To each well, add 175μL of uncoagulated blood and incubate for at least one hour at 37⁰C. Please note that, in standard red blood cells lysis in PBS, the RBCs are isolated from the blood and resuspended in PBS at 5-10% prior to drug exposure. Here, we simply use the erythrocytes in their natural environment to perform the assay.
  5. Prepare your standard samples
    1. Using 8 eppendorf tubes, add 450 to 500 uL of dH20 (or RBC lysis buffer)
    2. Transfer 0, 5 (10% lysis), 10, 20, 30, 40 (80% lysis), and 50 μL of whole blood from a well-mixed sample to each tube up to a volume of 500μL. Incubate at room temperature for at least 15 minutes and be sure to vortex before use.
    3. Add 200μL of standard to one column of flat-bottom 96-well plate for future spectrophotometric analysis with your test samples
  6. Prepare your test samples for measurement of hemoglobin release
    1. Centrifuge your test plate (the one with the blood treated with the drug) at 500G for 5-10 minutes. Your supernatants contain the red hemoglobin if your drug is hemolytic.
    2. In the flat-bottom plate, add 178 uL of dH20. This step is critical, and here is why: The concentration of RBC in your standard is 0-10% (you could argue 5% since RBC is about 50% of the blood volume).  Remember the blood was diluted up to 1:10. Then when the drug lyses the RBCs (hypothetically), the hemoglobin released from your sample was only diluted about 9:10 (175/200). These samples are up to 9x more concentrated compared to your standard. This means that you have to dilute your samples 1/9 so that the concentrations of hemoglobin released in your test samples are comparable to the blood or RBC concentrations used for your standards. If this is not clear, please send me a question.
    3. Carefully remove 22μL from the top layer of each of your test wells and add it to 178μL in corresponding wells of the flat-bottom plate already containing your standards (feel free to read all samples in duplicates)
    4. Place the plate in a plate reader and read at a wavelength between 550 and 570nm
  7. Data analysis
    1. Standard curve: Below is an example of an actual standard curve I generated from an RBC lysis assay in  whole blood:

 

hemolytic assay: rbc lysis assay standard curve

 

STD RBC (% lysis) ABS570
100 0.5905
80 0.5145
60 0.3855
40 0.264
20 0.167
10 0.0895
0 0.037

All you have to do now is to replace the ABS570 for each of your test sample in the derived equation to find the percent hemolysis.

% hemolysis (test) = [(Abs570 -0.0416)/0.0057];
(some  use ABS550 as well)

I hope I did not leave any step unclear. If I did, please send me a message from the comment box.

Best of luck with your experiment!

 Selected References

1.  Rational design of engineered cationic antimicrobial peptides consisting exclusively of arginine and tryptophan, and their activity against multidrug-resistant pathogens.

Deslouches B, Steckbeck JD, Craigo JK, Doi Y, Mietzner TA, Montelaro RC.

Antimicrob Agents Chemother. 2013 Jun;57(6):2511-21. doi: 10.1128/AAC.02218-12. Epub 2013 Mar 18.

PMID: 23507278 [PubMed - indexed for MEDLINE] Free PMC Article

2. Activity of the de novo engineered antimicrobial peptide WLBU2 against Pseudomonas aeruginosa in human serum and whole blood: implications for systemic applications.

Deslouches B, Islam K, Craigo JK, Paranjape SM, Montelaro RC, Mietzner TA.

Antimicrob Agents Chemother. 2005 Aug;49(8):3208-16.

PMID: 16048927 [PubMed - indexed for MEDLINE] Free PMC Article
Keywords: Hemolytic assay in whole blood, erythrocytes lysis assay, red blood cells lysis assay in whole blood
hemolytic assay: rbc lysis standard curve

Hemolytic Assay in Whole Blood: A relevant assay that is reflective of the in vivo cytotoxic potential of a drug [e.g., antimicrobial peptides (AMPs)] when administered systemically Keywords: Hemolytic assay in whole blood, erythrocytes lysis assay, red blood cells lysis assay in whole blood  Background:  Hemolytic assay in phosphate buffered saline (PBS) is routinely used […]