Goals of Outline

• Introduce theory and concepts of measuring regional organ perfusion with microspheres.
• Highlight necessary conditions and assumptions invoked when using microspheres.
• Provide details of microsphere use.
• Review statistical considerations.
• Discuss the advantages and disadvantages of microspheres.

Theory

• Indicator dye dilution curves primary tool for measuring blood flow in early 1900 - 1960

• Microspheres (10 - 60 µm in diameter) are particulate indicator dyes with signal integration accomplished by entrapment in organ
• Microspheres injected "up-stream" from organ(s) of interest
• Microspheres labeled with nuclide that can be quantitated
• Able to measure blood flow in ml/min to entire organ or multiple regions within organ using "reference organ"

History and Evolution

• Introduced by Rudolf and Heyman (Rudolf and Heyman 1967) carbonized particles
• Reference withdrawal method (artificial organ)
• Multiple labels required stripping overlap
• Matrix inversion method (Schosser, Arfors, et al. 1979)
• Validation vs. molecular microspheres (Bassingthwaighte, Malone, et al. 1987)

Physical properties of microspheres

• NEN Research Products
• 10-µm (11.3 ± 0.1 µm)
• 15-µm (16.5 ± 0.1 µm)
• diameter uniformity (CV) = 13.94%
• specific gravity = 1.3 (1.05 whole blood)
• radiolabels - ¹⁴¹Ce, ⁵⁷Co, ¹¹³Sn, ⁴⁶Sc, ¹⁰³Ru, ⁹⁵Nb, ⁵¹Cr, ¹¹⁴In

Necessary conditions

• Uniform mixing in blood
  
  • concentration in all downstream arteries should be equal
• Complete entrapment during first circulation
• Single blood supply to organ(s) of interest
• Must remain trapped until tissue counted
• Does not affect physiology

Assumptions

• Microspheres follow blood flow, distribution = RBC distribution

Details of Use

"poor techniques and inattention to details may result in inaccurate and misleading results" (Heyman, Payne, et al. 1977)

• Determining number of microspheres to inject
  
  • Dependent on goal(s) of study
    
  • Determine minimum flow of interest relative to mean, Q_min (e.g. Q_min = 0.5 of mean).
    
  • Estimate number of pieces expected, N_pieces.
    
  • Estimate fraction of cardiac output going to organ of interest, Q_organ/Q_total
  
• Preparation of microspheres
  
  • surfactant (Tween-80®), dextran
    
  • vortexing and sonicating
• Injection (insure uniform mixing)
  
  • mixing chambers

• multiport catheters
• as far upstream as possible to provide optimal mixing
  • LA injections for myocardial studies
  • LV injections for other left sided organs
  • IVC for pulmonary studies

• Reference organ method
  • withdrawal pump is "reference organ", known blood flow (pump rate)
  • blood flow to any organ in ml/min can be calculated from
  Q_i(ml/min) = (C_i/C_ref)*R(ml/min)

  where the Q_i is the flow to piece i, C_i and C_ref are the radioactivity in piece i and the reference sample, respectively, and R is the withdrawal rate of the reference blood flow sample in ml/min.

• withdrawal pump must be initiated prior to microsphere injection and be continued until last microsphere is past sampling site.
• any upstream site prior to microsphere entrapment is adequate

Radioactivity

• Most commonly use up to 6 different nuclides per study
  • able to use 8 to 9 using a least squares separation technique (Baer, Payne, et al. 1984)
  • up to 12 labels with gallium crystal

• Scintillation counters crystal geometry
  • all samples must have similar spatial relationship with crystal
• Calibration using ¹³⁷Cs source
• Correct for background, decay, and spillover
• Stripping vs. matrix inversion
  • stripping - errors of determination accumulate in lower energy nuclides
  • matrix inversion - preferred method with advent of computers

• Poisson statistics
  

• Method error
  • Simultaneous injections

• Buckberg (Buckberg, Luck, et al. 1971)
  • 95% confidence interval on absolute flow is <=10% variation if >=384 microspheres/piece therefore some authors exclude all pieces with <= 400 microspheres/piece
  • however, confidence in flow relative to other pieces is quite high depending on focus of study, pieces with less than 400 microspheres should not necessarily be excluded.
• Measures of heterogeneity
  • if interested in heterogeneity of flow (CV) can use far fewer
  • as the number of microspheres counted becomes small, the noise due to counting will increase. The CV for the flow distribution can be corrected for counting noise by the following equation:

where is the corrected CV, CV_obs is the observed CV, is the mean number of microspheres per piece, and N is the number of pieces

Sources of error - Austin (Austin, Hauck, et al. 1989)

• Validation using molecular microsphere - Bassingthwaighte [Am. J. Physiol. 1987]
  • 2-iododesmethylimipramine (IDMI)
  • High correlation (0.95) vs. nuclide-labeled 16 µm microspheres
  • Systematic bias: 16 µm microspheres over estimate flow at higher rates and underestimate flow at lower rates
    • therefore microspheres are adequate for estimating regional flows via arterioles of diameter 25 - 50 µm
• Effects of size difference
  • increasing size produces increasing endo:epi myocardial blood flow
    > 60 µm diameter microspheres concentrate centripetally in arterioles
    
    therefore 10µm microspheres best for determining differences in endo:epi blood flow
• Size vs. entrapment
  • - 15 µm microspheres - 100% entrapment in all organs
  • - 10 µm microspheres - 80 - 95% entrapment
• Effects of density difference
• Number of microspheres that can be injected
• Serial injections of 15-µm diameter microspheres, totaling 48 x 10⁶ caused no change in systemic hemodynamics or regional myocardial flows (Baer, Payne, et al. 1984) .
  • - 250 x 10⁶ can depress myocardial function in dogs [personal experience].
• Chronic studies using radiolabeled microspheres
  Medvedev transplanted rats hearts that had been injected with radiolabeled microspheres into rats that did not have microspheres. Concluded that microspheres remain lodged but radiolabel came off of microspheres.

A number of studies have looked at retention of microspheres in ischemic regions but have been unable to draw any conclusions because of swelling and scaring in infarcted regions.
• Tween and Dextran can cause systemic hypotension in certain animal models.

Advantages of Microspheres

• Inter- and intra-organ determination of blood flow
• Multiple measurements within same organ
• Measurements are highly accurate and precise
• Well validated method

Disadvantages of Microspheres

• Requires terminal preparation
• Radioactivity
  • increasing costs of microspheres
  • disposal costs
• Smallest region of interest probably limited to regions supplied by 50 µm arterioles (Bassingthwaighte, Malone, et al. 1987)

References

Austin, R. E., W. W. Hauck, G. S. Alsea, A. E. Flynn, D. L. Coggins, and J. I. E. Hoffman. (1989). "Quantitating error in blood flow measurements with radioactive microspheres." Am. J. Physiol. 257: H280-H288.

Baer, R. W., B. D. Payne, E. D. Verrier, G. J. Vlahakes, D. Molodowitch, P. N. Uhlig, and J. I. E. Hoffman. (1984). "Increased number of myocardial blood flow measurements with radionuclide-labeled microspheres." Am. J. Physiol. 246: H418-H434.

Bassingthwaighte, J. B., M. A. Malone, T. C. Moffett, R. B. King, S. E. Little, J. M. Link, and K. A. Krohn. (1987). "Validity of microsphere deposition for regional myocardial flows." Am. J. Physiol. 253: H184-H193.

Buckberg, G. D., J. C. Luck, D. B. Payne, J. I. E. Hoffman, J. P. Archie, and D. E. Fixler. (1971). "Some sources of error in measuring regional blood flow with radioactive microspheres." J. Appl. Physiol. 31(4): 598-604.

Heyman, M. A., B. D. Payne, J. I. Hoffman, and A. M. Rudolf. (1977). "Blood flow measurements with radionuclide-labeled particles." Prog Cardiovasc Dis. 20: 55-79.

Rudolf, A. M., and M. A. Heyman. (1967). "The circulation of the fetus in utero: Methods for studying distribution of blood flow, cardiac output and organ blood flow." Circ. Res. 21: 163-184.

Schosser, R., K. E. Arfors, and K. Messmer. (1979). "MIC-II - A program for the determination of cardiac output, arterio-venous shunt and regional blood flow using the radioactive microsphere method." Comp. Prog. Biomed. Res. 9: 19-38.