special communication Determination of hemoglobin mass and blood volume with CO: evaluation and application of a method CAROLINE M. BURGE AND SANDFORD L. SKINNER Department of Physiology, University of Melbourne, Parkville, Victoria 3052, Australia Burge, Caroline M., and Sandford L. Skinner. Deter- mination of hemoglobin mass and blood volume with CO: evaluation and application of a method. J. Appl. Physiol. 79(2): 623-631, 1995.—An improved protocol was applied to the use of carboxyhemoglobin (HbCO) saturation for the estimation of body hemoglobin (Hb) mass, red blood cell vol- ‘ume, and blood volume and was appraised in normal volun- teers. A body size-related dose of CO (50-90 mb) estimated to change HbCO saturation by 6.5%, was introduced into a low-volume (2.8 liters) closed-circuit rebreathing system and allowed to equilibrate over 10 min. Plots of venous HbCO were characterized by a plateau after 8 min, which remained stable for at least 40 min. No loss of CO from the vascular space was evident. Three estimations of Hb mass at weekly intervals in seven subjects produced a coefficient of variation of 0.8% such that, in the absence of physiological influences, changes as little as 1.5% in Hb mass are detectable. Venesec- tion (498 = 16 (SE) ml] in seven subjects was associated with a measured decrease in Hb mass after 1 wk equal to the calculated loss. Blood volume was, however, largely restored by plasma expansion. The method is sensitive and precise. I€ can be used safely and repeatedly in normal volunteers and hospital patients. plasma volume; red blood cell volume; venesection; hemato- crit; carbon monoxide MEASUREMENTS OF BLOOD (BV) or plasma volume (PV) involve indicator-dilution techniques with either la- beled autologous red blood cells (RBC; *'Cr, °"Tc, “P, or Fe), labeled albumin (!*I or the dye Tiszs), or 1I- labeled fibrinogen. The primary measurements are to- tal red blood cell volume (VrB¢)' and PV. Repeat tests using unstable isotopic tracers are of concern, espe- cially in the reproductive age group. Plasma markers have the additional disadvantage that multiple blood samples must be taken over a period of 60 min to iden- tify the rapid disappearance phase and thereby avoid overestimation of the space (6). Carbon monoxide (CO) labeling of hemoglobin (Hb) offers an alternative approach because the primary de- terminant is total Hb mass, which has a more direct application to certain questions than VRBC or PV but which, nevertheless, can be derived with widely ac- "Vasc is the entire circulating volume of RBCs as distinguished from the volume of an individual RBC. Attempts to refer to Vasc as “RBC mass” are to be avoided because it implies that VaBc has been multiplied by the specific gravity of a RBC, which, in many recent publications, has not been the ease. (0161-7567/95 $3.00 Copyright © 1995 the American Physiological Society cepted assumptions. The first use of CO for BV determi- nation was over 100 yr ago (29), but the method did not gain wide acceptance because carboxyhemoglobin (HCO) could not be measured accurately. Sjéstrand (45) subsequently developed a respiratory technique where the CO saturation of Hb was estimated in rectly from a measured value of the partial pressure of CO in the expired air before the after the administra- tion of CO, assuming a standard dissociation curve for Hb and CO (Haldane M factor). The method was again not widely adopted because the results were variable. Using the Van Slyke-Neill apparatus, Myhre et al. (39) clearly demonstrated the feasibility of direct blood HbCO measurements for the estimation of BV, but the analyses were time consuming and the method is not acceptable for routine clinical estimations. Hb mass has also been estimated by using injected ''CO-labeled RBCs (27), which are reliable and convenient but have the disadvantage of being radioactive. An accurate and convenient method for directly estimating blood HbCO with the diode-array spectrophotometer is now avail- able (24), and this has been developed for the measure- ment of BV in humans (47). In the method of Thomsen et al. (47), a bolus dose of stable CO gas (50 ml) is administered via a low-volume rebreathing system con- taining pure oxygen, with blood sampling before and 10 min after the administration of CO. The method demonstrated good agreement with *"Tc-labeled RBCs for BV determination. But Thomsen et al. did not give precise details of the apparatus, nor did they formally appraise precision and sensitivity of the Hb mass esti- mation. Based on their description, we have developed a simple apparatus and a standard protocol and identi- fied the main sources of error. MATERIALS AND METHODS ‘The CO rebreathing apparatus. The open-circuit breathing system (Fig. 1) consists of a Douglas bag with ~60 liters pure ‘oxygen connected via tubing and a respiratory vaive (2700, Hans Rudolph, Kansas City, MO) to a small mouthpiece (plus nose clip). The subject's breathing is switched between the open- and closed-circuit systems via a slide valve (series 2870 three-way manual sliding valve, Hans Rudolph). The re- breathing (closed-circuit) system (Fig. 1) consists of a Y-tube and flexible disposable tubing (22-mm Flexitube cuffed at 400 ‘mm; 1574, Commonwealth Industrial Gases (CIG) Health Care] connecting to a cross valve (Medical Developments Aus: tralia, Melbourne) with a polypropylene anesthetic bag (Chmeda antistatic BS.3353 2 liter, no. 372762) and a CO: 623624 dosed cuit ‘pen cicuit Mouth nde ree Flexible disposble tg er anesthetic bag tached 10 ros valve respiratory valve Continaous O; ape eee ‘50-90 mi CO (bolus). Pure CO reservoir {250 mt) Removable for ling CO syringe {ener tock CO port ‘Stopeock configurations Line closed tn diredios af the andle Ret] Fil | Flesh Inject Conti | eying |seaepac| dose os] ef a [is se lee] gd [ad Fic. 1. Open- and closed-circuit breathing systems connected via respiratory and manual 3-way slide valves, respectively. CO port consists of a CO reservoir, a syringe, and three 3-way stopeocks. ‘Bottom right: stopcock configurations (Config) for filling CO syringe, Aushing CO port dead space, and injecting bolus CO dose into re- breathing system. absorber (Dryden, Medical Developments Australia) con- taining ~300 g fresh soda lime (Vivalyme 524753, CIG Health Care). All components are static resistant or electri- ‘ally earthed. Before each test, air is blown through the re- newed CO; absorber to remove any free dust that can be otherwise irritating in the airways and provoke coughing. The closed-circuit system is then tested for leaks at 40 mmHg under water. Oxygen flow into the rebreathing system is reg- ulated with a calibrated flow valve (range 50-2,500 ml/min). ‘The CO (CIG 99.5% f-grade) is withdrawn from a previously filled second polypropylene bag (CO reservoir) into a cali- brated plastic syringe (capacity 60-100 ml), and the bolus dose is injected via a series of three-way Luer lock stopcocks iscofix stopcock 40990170, B. Braun Melsungen) (Fig. 1), At all times, the subject is isolated from exposure to the CO in the reservoir by at least two closed stopcocks. Because the accuracy of the technique is critically dependent on the volume and purity of CO administered, extreme care must be taken to avoid all dead space and leak errors. Removable plugs and Luer lock taps in the CO port permit the dead space to be flushed thoroughly with CO from the CO reservoir. €O port and preliminary procedures. The CO reservoir “with its stopcock is first detached from the CO port and evacu- ated with a vacuum pump. The bag’s stopcock is then attached to the regulator of a CO cylinder stored under secu- rity (not shown), and the plug on the stopcock side arm is removed. The regulator dead space and any tubing are BLOOD VOLUME DETERMINATION WITH CARBON MONOXIDE, flushed by exhausting CO through the unplugged side arm by venting to the exterior of the building. While the CO is still flowing, the stopcock tap is turned and the Lliter bag is filled to no more than 250 mi. The stopcock tap is returned to the closed position (toward the bag), and the CO flow is interrupted. The sidearm plug is replaced, and the bag and stopeock are reattached to the CO port on the breathing sys- tem. The CO port dead space is then flushed once with CO by withdrawing 15-20 ml of CO into the syringe see stopcock configurations in Fig. 1) and venting it to the room through the loosened lower plug that is then immediately tightened. All stopoock taps are then returned to the rest configuration. CO administration protocol and subjective aspects. The subject is seated for 20 min before the procedure to allow PV to stabilize. With the nose clip comfortably attached, the subject first breathes pure oxygen from the open circuit for 4 min to flush nitrogen from the airways (17). The subject is then switched, at the end of a normal expiration, to the rebreathing system that has been previously oxygen flushed and left with the 2-liter bag three-quarters filled. This proce- dure ensures that the oxygen partial pressure in the re- breathing system is as high as possible. As the subject breathes into the oxygen-filled closed circuit, further oxygen is continuously added at a rate that maintains the bag nei- ther over- or underinflated during tidal breathing (200-240 ml/min). After a 2 to 4-min adjustment period, duplicate con- trol antecubital venous blood samples (2 ml each) are col- lected via an indwelling 22-gauge catheter (Jelco) without stasis or dilution into.dry heparinized syringes (Bard-Parker Drihep Plus kit 375396). A bolus of pure CO (see HbCO deter- mination, predicted precision, sensitivity, and safety below) is then added to the rebreathing system over a 15- to 30-5 period. Immediately ater the CO is injected, the stopcock closest to the oxygen inlet is closed (rest position), the clock is started, and then the middle stopcock tap is also returned to its rest position. The subject continues rebreathing for 10 min, at which time in the routine protocol a single post-CO bblood sample is collected. All syringes are capped (without air bubbles) and placed horizontally in ice before analysis (within 4 h) for %HbCO and Hb concentration (HbI. Aliquots of the blood (I ml each) are stored at room temperature for estimation of hematocrit (Het) (average of 4~6 replicates! sample) and analyzed within 20 min. ‘When initially switched to the rebreathing system, a per- ceived small increase in resistance to breathing raises tidal volume, and possibly because of this, early during the re- breathing period, some subjects experience a desire to fully inhale, but this is restricted by the capacity of the 2-liter anesthetic bag. During an initial preamble, before rebreath- ing, subjects are told of this possibility and are encouraged to avoid deep breaths and to consciously breathe normally. The sensation is unrelated to CO administration and typi- cally occurs during the first few minutes of rebreathing. All new subjects are taken to beyond such a stage (viz. 4 min) before the CO administration, the addition of which is not consciously perceived. It is crucial that the subject cooperates in ensuring that no gas escapes from around the mouthpiece or through the nose clip because even small leaks in the rebreathing system were associated with appreciable de- creases in HbCO. The operator must pay striet attention to the oxygen flow rate that must equal metabolic uptake over several breaths to avoid under- or overfilling of the bag and ‘consequent disturbance to tidal volume. The subjects tolerated the technique well. The volume of the rubber bag was sufficient for our largest volunteer (male, 110 kg, 195 cm) to breathe with comfort. To prevent neck soreness or fatigue from developing during the test, the canis- ter and the valves/mouthpiece were suspended on lightBLOOD VOLUME DETERMINATION WITH CARBON MONOXIDE chains from above and the Douglas bag and oxygen cylinder were placed on a trolley next to the subject. The subjects were seated in a comfortable chair, and the height of the mouthpiece was adjusted by using Velcro inserts in the chain, ‘The CO port was located beyond the subject's peripheral range of vision to deliver the CO bolus discretely. A paper tissue was needed to wipe any saliva accumulating around the mouth. A simple mouthpiece was preferred to a face mask because of increased dead space and the difficulty of ensuring a perfect seal with a mask. pLietdations.Cirelatory volumes were ealeulated using a Correcting nCO by ~2.2% to account for CO remaining in the rebreathing apparatus (see below) nCO x 25 ‘AHBCO 644 x Het (Hb) Vasc x 100 Het X Fa ratio PV = BV - Vrec © where nCO is the molar amount of CO added to the rebreath- ing system (in mmol), Ps is the barometric pressure (in mmHg), Veo is the volume of CO (ateD) added to the re- breathing system (in liters), T is the room temperature (in “C), nb is the Hb mass (in mmol), AHbCO is the difference in HbCO levels estimated before and after rebreathing (in 4%), VBC is in milliliters, Hct (corrected for trapped plasma) is in percent, (Hb is in grams per liter, BV is in milliliters, PV is in milliliters, and Fay ratio is the venous-to-body Hct correction factor. ‘The molar amount of CO administered is calculated from the volume (ATeD) injected (Eq. 1) and then corrected for the amount of CO remaining in the system after rebreathing. This correction has been estimated to be ~2.2% (range 1.8— 2.7%) (47) but can be calculated for each subject from the lung plus the rebreathing cireuit volume (~5 liters) by mea- suring the concentration of CO remaining in the circuit at the end of the rebreathing. CO binds to Hb in the ratio of one CO molecule per Hb monomer. The value 25 in the numerator of Eq. 2 is 100 x 0.25 to account for AHbCO being entered as a percentage and to convert millimoles of monomeric Hb to tetrameric Hb. HCO is calculated and displayed by a diode-array spectro- photometer (OSM3 hemoximeter, Radiometer, Copenhagen, Denmark) as a percentage of the total amount of Hb present in the sample aliquot, making the calculation of Hb mass (Eq. 2) independent of (Hb) or Het. This also permits blood samples low in Hb to be concentrated if necessary. Previous workers using the CO rebreathing method (12, 22, 23, 41, 47) have neglected to convert monomeric Hb to tetrameric HD such that their results must be divided by four to obtain mass. Estimation of Vac requires the inverse calculation of mean corpuscular (Hb), i.e. HeU/[Hb], multiplied by Hb mass (Eq. 3). This estimation is independent of the whole body Het (see below) because mean corpuscular [Hb] is a measure of the amount of Hb present in an aliquot of pure RBCs, and, as such, no correction for its effect should be made. The nb = @) Vrec | x nHb 3 @ 625 Het should be corrected for trapped plasma if the Het is estimated by using the microcapillary centrifuge method, The value 0.96 is a commonly applied correction for trapped plasma (30), although we have adopted 0.98 as nearer to the true value (26, 32, 42). The bufly coat is not included in the measurement of the RBC column. The value 644 in the numerator is 1,000/100 x 64.4 to correct for the Het being entered as a percentage, to express VaBC in milliliters, and to convert (Hb] from grams per liter to millimoles per liter Ge, 16.1 x 4). BV is derived from VRBC and Het corrected for the whole body phenomenon (Fa ratio; Eq. 4). The correction is re- quired because the Het averaged over the entire body is less than the Het of a blood sample obtained from a peripheral vein. A value of 0.91 is commonly adopted for subjects with normal-sized spleens, but values approaching unity are used if the spleen is grossly enlarged (25). Because the true value for the Fu ratio is unknown (see DISCUSSION), a value of tunity has been adopted for all estimates of BV reported here. PV is derived from the difference between BV and Vesc (Eq. 5). In conditions such as leukemia, the volume of circulating leukocytes may constitute a substantial fraction of the BV, and it may be preferable to calculate its individual contribu: tion to the total BV from the buffy coat volume. HCO determination, predicted precision, sensitivity, and safety. %HbCO-and (Hb] were measured spectrophotometri- cally with the diode-array spectrophotometer OSM3 hemoxi- meter. The OSM3 was calibrated with respect to HbCO satu. ration as recommended by Fogh-Andersen et al. (23). In pre liminary experiments, repeated determinations of HbCO and [Hb] were made on a single normal blood sample to determine the precision of the OSM3 in our hands. Four consecutive replicates demonstrated a mean + SD of 137 = 0.5 g/l and 1.7 + 0.05% for (Hb] and HbCO saturation, respectively. In indicator-dilution techniques, it is important to give a dose of indicator large enough to measure the space with sufficient precision. Figure 2A shows the calculated relation- ship among %AHbCO, Hb mass, and CO dose under typical ambient conditions experienced in our laboratory (769 mmHg, 22°C), The OSM3 gives %HbCO to one decimal place; thus %AHDCO is plotted at 0.1% intervals. Figure 2B shows the calculated precision or the detection limit for the observa- tion of absolute changes in Hb mass for a given AHbCO and CO dése. Each point is the difference between adjacent points in Fig. 2A with respect to Hb mass plotted against CO dose and AHbCO. Figure 2C shows the sensitivity of the method or the detection limit for the observation of relative changes in Hb mass for a given AHbCO. The points were obtained by dividing every point in Fig. 28 by the corresponding Hb mass from Fig. 24. Thomsen et al. (47) used a dose of 50 ml CO irrespective of body weight, and, consequently, at larger Hb mass, the 0.1% interval in %AHBCO represented exponen- tially larger errors in the estimation of Hb mass. However, at the other end of the curve, increasing doses of CO are associated with exponential rises in HbCO saturation. A dose of CO that induces an ~6.5% AHbCO provides a satisfactory margin of safety and a good degree of sensitivity (1.5% of Hb mass). Most nonsmoking normal city residents have resting HDCO levels of 1.5-2.0%, and a 6.5% change will raise HbCO levels to ~8%, which has no subjective or untoward effects Approximate HbCO levels or other causes of reduced oxygen ‘carriage should be determined or anticipated before per: forming CO rebreathing (e.g., heavy smoking, engine exhaust ‘contamination, methemoglobinemia, or exposure to methyl- ‘ene chloride vapors), and appropriate adjustments should be made. CO toxicity is not consciously perceived and can be raised to 15% without untoward effects in normal individuals (46), allowing a wide margin of safety.