Laboratory 2: Hematocrit, Hemoglobin, Relative Change in Plasma Volume

Blood hemoglobin and hematocrit measures are often used to indirectly evaluate the oxygen transport capacity of blood. However, these measures have limited chronic importance in determining oxygen transport and iron status of blood due to the variability in the plasma volume of blood. For example, endurance training may increase the total blood cellular mass and hemoglobin content, but decrease hematocrit and hemoglobin concentration due to a greater relative expansion of the plasma volume. Many researchers believe that the expanded plasma volume is the most important cardiovascular adaptation to endurance exercise.

Another importance of hematocrit and hemoglobin measures is to determine the relative acute change in plasma volume. Researchers have used changes in total serum protein to reflect alterations in plasma volume, however, evidence exists to indicate protein leaching from the vascular system during exercise. Consequently, the original method proposed by Dill and Costill (1974) is still the most recognized indirect method to determine plasma volume changes. The assumptions and conditions underlying this method are adequately explained in their article.

Experimental Procedure:

Blood will be sampled from a catheter at rest (10 min. supine), at rest (10 min. seated), and every minute during incremental cycle ergometry (use subject specific Watt/min ramp protocol) to VO₂max (2 mL/sample during exercise). Blood will be prepared in the biochemistry lab for subsequent determination of hematocrit and hemoglobin, and for latter determination of serum protein. You will need to;

1) dispense ~1 mL into a heparinized tube

2) dispense the remaining blood into a plain tube so it can clot and then be centrifuged.

Materials

Gently mix the tube of blood, and then 3/4 fill three micro-hematocrit tubes with the blood and seal the red circled ends with the white putty. Place the tubes (sealed end out !!) balanced in the microcentrifuge. Spin for 5 min and when ready, read by placing against a ruler and measuring the packed cell distance vs. total distance (express as a percentage). Multiply this percentage by 0.96 to account for trapped plasma in the cell mass. Use the average of the two or three closest readings.

b. Hemoglobin

Prepare a hemoglobin-Drabkins reagent standard curve as demonstrated/discussed in prior class. See attached for the directions from Sigma.

Gently mix the tube of blood, and then pipette 20 uL into each of two tubes (13 mm) containing 5 ml of Drabkins reagent. Stopper, gently mix and stand for 20 min. Use the Drabkins reagent as the blank!! Turn on the spectrophotometer for warm up!!

Prepare to run an end-point test at 540 nm. Record the absorbances for the samples. Calculate the hemoglobin concentrations from your standard curve.

c. Calculation of % Delta plasma volume

Use the article by Dill and Costill (1974) to calculate % Delta plasma volume for each of the sample conditions.

Dill D.B. and D.L. Costill. Calculation of percentage changes in volumes of blood, plasma and red cells in dehydration. J. Appl. Physiol. 37(2):247-248, 1974.

Farjanel J., C. Denis, J.C. Chatard and A. Geyssant. An acute method of plasma volume measurement by direct analysis of Evans Blue spectra in plasma without dye extraction: origins of albumin-space variations during maximal exercise. Eur. J. APpl. Physiol. 75(1):75-82, 1997.

Haller M., H. Brechtelsbauer, U. Finsterer, H. Forst, T. Bein , J. Briegel and K. Peter. Determination of plasma volume with indocyanine green in man. Anaesthesist. 41(3):115-120, 1992.

Senay L.C. and J.M. Pivarnick. Fluid shifts during exercise. Exercise and Sport Sciences Reviews,13:385-387, 1985.