Applications

Microparticle Testing

Microparticles are present in most biological fluids and elevated concentration levels are often an indicator of disease. Microparticles range in diameter from 100 nm to 1 µm. In circulation, microparticles are produced by platelets in the presence of strong agonists; in addition to releasing chemokines that induce angiogenesis, microparticles have been implicated in thrombosis occurring in cardiovascular disease, cancer, diabetes, and infection1. In platelet products, microparticle concentration has been shown to increase during platelet concentrate aging2. Aged platelets are considered to have inferior quality as a result of the storage lesion. The increase in microparticle levels concurrent with platelet aging may therefore be inversely related to platelet quality.

The importance of microparticles

Microparticles have been implicated in cardiovascular disease, cancer, diabetes, and inflammation3. Elevated levels of microparticles may be indicative of a disease state4 and if so, it would be beneficial to identify elevated microparticle levels in patients, blood donors and in platelet concentrates.

Microparticle concentration in platelet products has been shown to increase as the platelet concentrate ages3, leading to product heterogeneity. Pathogen inactivation was shown to add additional stress and affect platelet viability over time5. An increase in microparticle content may be a measure of decreased platelet viability.

Donor microparticles were shown to carry-over into finished apheresis product6. Thus, microparticle content measured in donor samples might be a useful predictor of the microparticle content in the final blood product, and therefore a predictor of product viability.

It is estimated that platelet refractoriness occurs in 10-20% of patients who require platelet transfusions. HLA/HPA matching is the go-to solution for refractoriness, however only 3-5% of cases are actually caused by HLA/HPA antibodies7. Ineffective matching results in increased and unnecessary cost to both patients and healthcare facilities, and does not address the problem. What is the cause of refractoriness for the 15% of cases that do not respond to HLA/HPA matching? Microparticle content in the product and in the recipient may play a role. The variability of microparticle levels in donor samples, final product, and recipient samples highlights the possibility that there could be additional factors to consider when matching products to recipients. Thus, microparticle screening might aid in providing the right product to the right patient.

What does ThromboLUX do?

ThromboLUX® can characterize the microparticles in platelet samples based on size distribution, and content. ThromboLUX is a non-invasive, optical test utilizing dynamic light scattering that does not require any reagents or sample manipulation. ThromboLUX can provide a relative concentration and size distribution of microparticles in blood products without the need for centrifugation or filtering.

Why use ThromboLUX?

ThromboLUX can be used as a tool to detect and characterize microparticles in biological fluid samples without the need for isolation from other particles.

ThromboLUX can be used to:

  • Pre-test donors for microparticles
  • Predict microparticle content of products
  • Measure changes/differences in concentration.
  • Screen for conditions where high microparticles may be a risk factor or indicator of a disease state

When can ThromboLUX be used?

ThromboLUX can be used:

  • To test donors prior to donation
  • At any stage of platelet production
  • At any stage of process development
  • For process validation
  • For process monitoring
  • For any type of platelet product (ie Plasma, PAS, frozen, artificial)
  • Research for optimizing product composition
  • Research for optimizing product storage
  • To screen patients for microparticle content in blood or blood components

Output

ThromboLUX performs measurements at 37°C, then 20°C, then again at 37°C; and calculates a size distribution for each temperature. ThromboLUX then uses a proprietary algorithm to analyze the size distribution at the three test temperatures. The data that is gathered during the test is used to output the following parameters: photon rate, mean particle size, gated microparticle size and intensity, gated platelet size and intensity, MP factor and the ThromboLUX Score.

The gated parameters are calculations of the size and percent intensity within two windows that represent platelets and microparticles. The window from 50nm to 550nm is used for Gated MP parameters, and the window from 550nm to infinity is used for Gated PLT parameters.

The MP Factor parameter is an estimate of the relative concentration of microparticles to platelets based on the gated parameters. If a platelet count of the sample is available, then the product of MP Factor and Platelet count gives an approximate microparticle count.

Figure 5.4 ThromboLUX Score Card

Figure 5.4 ThromboLUX Score Card

References

  1. Burnier L, Fontana P, Kwak BR, Angelillo-Scherrer A. Cell derived microparticles in haemostasis and vascular medicine. Thromb Haemost 2009;101:439-51.
  2. Bode AP, Orton SM, Frye MJ, Udis BJ. Vesiculation of platelets during in vitro aging. Blood 1991; 77: 887-95.
  3. Simak J, Gelderman, P. Cell Membrane Microparticles in Blood and Blood Products: Potentially Pathogenic Agents and Diagnostic Markers. Transfus Med Rev 2006; 20: 1-26

  4. Diamant M, Tushuizen E, Sturk A, Nieuwland R. Cellular microparticles: new players in the field of vascular disease? Eur J Clin Invest 2004; 34: 392-401

  5. Bashir S, Cookson P, Wiltshire M, et al. Pathogen inactivation of platelets using ultraviolet C light: effect on in vitro function and recovery and survival of platelets. Transfus 2012; 53: 990-1000

  6. Rank A, Nieuwland R, Liebhardt S et al. Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles. Vox Sang 2011; 100: 179-86

  7. The Trial to Reduce Alloimmunization to Platelets Study Group. Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. N Engl Med 1997; 337: 1861-1870