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Determination of Hb mass by CO rebreathing Christoph Siebenmann Outcome CO rebreathing Total number of hemoglobin molecules Total (g/dl) hemoglobin mass (g) Total red cell Plasma volume Total blood volume (l) (l) volume (l) Changes

  1. Determination of Hb mass by CO rebreathing Christoph Siebenmann

  2. Outcome CO rebreathing Total number of hemoglobin molecules Total (g/dl) hemoglobin mass (g) Total red cell Plasma volume Total blood volume (l) (l) volume (l)

  3. Changes in intravascular volumes Anemia Heat acclimatization Endurance training EPO Diuretics RCV ↓ RCV → RCV ↑ RCV ↑ RCV → PV → PV ↑ PV ↑ PV ↓ PV ↓

  4. The dilution principle 2. 1. Volume of water = x Volume of sirup = n Sirup concentration = c c = n / x ? x = n / c Sirup x = 500 ml / 50 ml/l = 10 l 500 ml 3. Sirupometer (ml of sirup / l) 50 ml / l

  5. For solid objects 1. Tag a known number of fish 20 2. Release fish (” mixing ”) 3. Count the fraction of marked fish 4/20 Volume of water = x Total number of fish = x Volume of sirup = n Number of tagged fish = n ? Sirup concentration = c Fraction of tagged fish = c c = n / x x = n / c x = 20 / 0.2 = 100

  6. For hemoglobin

  7. CO rebreathing 1. Tag 2. Mix 3. Count tagged fraction 10 min Change FCOHb pre (%) FCOHb post (%)

  8. The setup Siebenmann et al., 2017

  9. Measurement routine 1. Subject supine 2. Open circuit breathing 3. Closed circuit rebreathing (20 min) (4 min) (10 min) => Standardize conditions => Wash N 2 from airways => Take up/mix CO • • • Place catheter Fill CO syringe Collect blood (Pre) • • Provide 0.5 l of water Administer CO • Collect blood (Post)

  10. Calculations After the rebreathing you know: • ∆ FCOHb (=FCOHb post - FCOHb pre ) • Volume of absorbed CO (VCO) • Hct • [Hb] • Temperature • Barometric pressure Molar weight of Hb (6.44 × 10 -4 g/mol) •

  11. Hb mass • Number of absorbed CO molecules (nCO) => pV = nRT = barometric pressure × VCO / (R × T) • Number of tagged Hb molecules (nHb tagged ) = nCO / 4 • Total number of Hb molecules (nHb tot ) = (nHb tagged / ∆ FCOHb) × 100 % • Hb mass = nHbtot × 6.44 × 10 -4 g/mol

  12. Deriving volumes • Blood volume = Hbmass/[Hb] • Total red cell volume = Blood volume × Hct • Plasma volume = Blood volume – Red cell volume Hct = RCV / BV

  13. Circulatory CO distribution ? Overestimation Underestimation

  14. Distribution concerns CO → Mixing time ”Pockets”

  15. Mixing time Arterial Capillary Venous Garvican et al., 2010

  16. ”Pockets” Blood pooling Splenic storage • • Underestimation Underestimation (up to 10 %) • • Variability Variability

  17. Exercise on CO distribution p = 0.017 Keiser et al., 2013

  18. Mixing with tilt table

  19. Safety ➢ Subject safety ➢ Room contamination

  20. Subject safety • Serious symptoms start at %HbCO of 20 – 25 % • %HbCO post usually < 10% 4 h %HbCO = 8% Heavy smokers: 10 – 15% • Washes out within 24 hours BUT: • Limited exercise capacity • Provide washout time

  21. Prevent room contamination To the To the CO window cylinder Filling button Emptying button Stopper

  22. Thank you

  23. Accuracy and precision Accuracy Precision Comparison to reference method Typical measurement error 24 h (Standard deviation of differences) / √2 Usually < 2 % Thomsen et al., 1991

  24. Determination of unabsorbed CO 1. Subject exhales completely 2. Subject is disconnected 3. Determine remaining volume • Pulmonary dead space (formula) • Closed circuit dead space (measured) • Volume in rebreathing bag (extract) 4. Measurement of CO concentration in remaining volume 5. Multiply remaining volume with CO concentration Alternatively: use 2.2 % as estimate

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