CLN’S LAB 2006: SUPPORTING CLINICAL DECISIONS SERIES Oxygen Saturation A Guide to Laboratory Assessment BY SHANNON HAYMOND, PHD

uman life depends on the oxygen transport by . In healthy patients, the major-

ity of molecular oxygen (O2) is bound to hemoglobin and only a small fraction is dissolved in blood. But in patients with respiratory problems or certain metabolic and genetic disorders, the fraction of oxygenated hemoglobin can fall to dangerously low values. Therefore, labora-

tory assessment of oxygen saturation (SO2)—the percentage of hemoglobin saturated with oxygen—providesH an important indicator of a patient’s cardio-respiratory status and is frequently used in the emergency department, during general and regional anesthesia, and in intensive care settings. Although the measured parameters are quite different for each, the three major analytical methods for mea- suring oxygen saturation—arterial blood gas analyzers, pulse oximetry, and CO-oximetry—are frequently used interchangeably by health care workers. Arterial blood gas analyzers calculate estimated oxygen saturation

(O2sat) in a blood sample based on empirical equations using pH and PO2 values, while pulse oximeters moni- tor arterial blood oxygen saturation, commonly referred to as SpO2 or SaO2, noninvasively by passing selected wavelengths of light through an area of the body, such as a finger. Both are measures of oxygen saturation.

The Clinical Laboratory Standards In- leading results, so health care professionals mutations, such as the thalessemias, reduce stitute (CLSI) defines O2sat as an estimated need to understand the differences between the quantity of α- or ß-chain synthesis in the value based on a calculation, whereas SaO2 these methods and the limitations of each. hemoglobin subunit or lower the solubility and SpO2 refer to the arterial saturation This article will review the basics of oxygen- of hemoglobin—as occurs with HbS (sickle cell hemoglobin) or HbC (hemoglobin C), for example—but gene mutations rarely al- ter the O2 affinity of hemoglobin. can be divided into two classes: normal hemoglobin that is capable of binding O2, and dyshemoglobins—he- moglobin derivatives that are incapable of binding O2. The normal hemoglobins in- clude oxyhemoglobin (O2Hb) and deoxy- hemoglobin (HHb), while the dyshemoglo- bins include (COHb), (MetHb), and sulfhemo- (SHb). COHb forms when a person is exposed to CO fumes and CO replaces O2 in hemoglobin, which can result in death. SHb forms through a reaction of sulfa-contain- ing compounds with the moiety; cases of are rare and usually result from extensive use of sulfa-containing drugs. MetHb represents the oxidized, deoxy form (Fe(III)-Hb) of hemoglobin to which O2 cannot bind. The likely etiologies of met- hemoglobinemia include exposure to highly oxidizing drugs or the presence of genetic such as HbM. To understand how hemoglobin carries and releases oxygen, the oxygen dissociation curve (ODC) serves as an important tool (Figure 1). In the lungs, where partial pres- measured spectrophotometrically. (To avoid ation of hemoglobin and will provide guid- sures of O2 are high, O2 binds to hemoglo- further confusion, I will use only O2sat ance for laboratorians on how to accurately bin to form O2Hb. Erythrocytes carrying and SO2 for the remainder of this article.) assess oxygenation saturation values. O2Hb then circulate in the blood and release CO-oximetry, a more complex and reliable O2 in response to decreased partial pressures An Oxygen Saturation Primer method, measures the concentration of he- of O2 in the tissues. The cooperativity of O2 moglobin derivatives in the blood, the results Hemoglobin, the O2 transport in binding—an allosteric phenomenon where- of which are then used to calculate various blood, is comprised of four subunits: two by binding of oxygen by one hemoglobin parameters such as hemoglobin derivative α subunits and two non-α subunits, for subunit enhances the ability of the remain- fractions (e.g., FO2Hb), total hemoglobin, example ß, γ, or δ. Each subunit contains a ing subunits to bind oxygen—produces the and oxygen saturation. porphyrin heme iron (Fe) moiety and seven sigmoidal shape of the curve. As O2 binds to In most patients, the results—O2sat, SO2, helices. The heme Fe exists in the Fe(II) or the second and third subunits of hemoglo- and FO2Hb—from these three methods are Fe(III) oxidation state, but only the Fe(II) bin, binding increases incrementally so that virtually identical. But in cases of dyshemo- state is capable of binding O2. Most clini- the four subunits of hemoglobin all become globinemia, some methods can yield mis- cally important dyshemoglobinemia gene fully saturated at the normal O2 tension in 10 CLINICAL LABORATORY NEWS FEBRUARY 2006 Figure 1 Figure 2 Oxygen Dissociation Curve Extinction Curves of Purified

Of Hemoglobin Hemoglobin Derivatives OF OHMEDA COURTESY 10 100 R IR t n e i 1 MetHb 80 c i f f e

o O Hb C 2 n n o i t o HHb c i

60 n t i .1 t a x E r u t a

S 40 COHb .01

% 600 640 680 720 760 800 840 880 920 960 1000 Wavelength (nm)

20 The graph shows the light absorption relationship of HHb, O2Hb , COHb, and MetHb. Vertical lines indicate the red (R) and infrared (IR) monitor- P50 ing wavelengths used in most pulse oximeters, at 660 nm and 940 nm, respectively. Modified from Journal of Clinical Monitoring, 4(4), 1988, p. 0 292. Pulse Oximetry: Analysis of Theory, Technology, and Practice. M.W. 0 20 40 60 80 100 Wukitsch et al. With permission from Kluwer Academic Publishers. Oxygen tension (mmHg) The percent saturation of hemoglobin with oxygen at different oxygen SO2, expressed as a percentage, is typically tion, the altered molecular structure of the 94%–98%. Blood gas analyzers report an heme moiety in the various hemoglobin tensions is depicted by the sigmoidal curves. The P50, indicated by the dashed lines, is about 27 mm Hg in normal erythrocytes. Modifications of estimated saturation, O2sat, which is based derivatives gives rise to unique absorption hemoglobin function that increase oxygen affinity shift the curve to the on measurement of pH, PO2, and hemoglo- spectra, making it possible to determine the left, whereas those that decrease oxygen affinity shift the curve to the bin values and utilization of empirical equa- concentrations of each derivative present in right. Reprinted with permission from Kelley’s Textbook of Internal Medi- tions. blood. (Figure 2) cine, 4th ed., 2000, figure 241.2. Lippincott Williams & Wilkins. Fractional oxyhemoglobin. Only instru- Pulse oximetry. Pulse oximeters assess arte- ments with a multi-wavelength spectro- rial oxygen saturation (SO2) by measuring photometer, such as CO-oximeters or some light transmission through a well-perfused lung alveoli. The same process works in re- temperature and 2,3-DPG concentration, modern blood gas analyzers, are capable of area of the body such as a finger or an ear verse in the tissues; once fully loaded hemo- or decreases in pH, can shift the ODC to the measuring FO2Hb. The value, as calculated lobe. Light sources, typically light-emit- globin releases one O2 molecule, it releases right, increasing the P50 and indicating de- from the formula below, represents the frac- ting diodes, shine two wavelengths of light the next more easily. creased O2 affinity. tion of oxyhemoglobin in relation to the to- through the tissue —visible red (R) light The ODC also explains what happens to tal hemoglobin present, including the non- (600 to 750 nm) and infrared (IR) light (850 patients when oxygen cannot bind hemoglo- Definitions of Oxygenation Status oxygen-binding hemoglobins. to 1000 nm). Deoxygenated hemoglobin bin. For example, in the presence of CO or CLSI has defined the three key terms used Fractional oxyhemoglobin allows more infrared light to pass through when the heme Fe is oxidized to the Fe(III) to describe oxygenation status: oxygen sat- FO2Hb = cO2Hb and absorbs more red light than oxygen- state, O2 cannot bind one of the hemoglo- uration (SO2), fractional oxyhemoglobin ctHb ated hemoglobin, while highly oxygenated bin subunits. These conditions decrease O2 (FO2Hb), and estimated oxygen saturation FO2Hb is usually expressed as a percent- hemoglobin allows more red light to pass capacity in addition to inhibiting O2 trans- (O2sat). The Institute recommends the use age and typically ranges from 90%–95% in through and absorbs more infrared light. port by blood. In addition, CO and oxidized of the term “oxygen saturation” to indi- healthy individuals. Reporting FO2Hb alone After calculating the absorption at the two heme Fe alter hemoglobin’s conformation cate the amount of hemoglobin capable of is of limited value because the dyshemoglo- wavelengths, the instrument uses a calibra- in a way that decreases the O2 affinity for transporting O2 and “fractional O2Hb” to bin fractions also play an important role in tion curve programmed into the device to the remaining heme Fe groups, shifting the represent the fraction of hemoglobin that the analysis of oxygen carrying capacity. The compute the SO2. ODC to the right. The net biological effect is oxygenated. The terms “fractional satura- fraction of any of the hemoglobin deriva- For example, on most pulse oximeters is decreased O2 delivery to tissues. Tem- tion” and “functional saturation” refer to the tives may be calculated in the same manner an absorbance ratio (AR/AIR) of 0.43 cor- perature, pH, and 2,3-diphosphoglycerate FO2Hb and SO2, respectively. as that used for oxyhemoglobin. responds to 100% oxygen saturation, and a (2,3-DPG) concentration also affect the O2 Oxygen saturation. The following empiri- As demonstrated by the above equations, ratio of 3.4 corresponds to 0% oxygen satu- affinity of hemoglobin, shifting the ODC as cal equations are used to determine oxygen SO2 and FO2Hb are not equivalent terms. In ration. In the absence of a dyshemoglobin, shown in Figure 1. saturation, SO2, from measured hemoglo- healthy individuals, the values obtained for an absorbance ratio of 1.0 corresponds to an An important aspect of the ODC is the bin concentrations: SO2 and FO2Hb are approximately equal, oxygen saturation of approximately 85%. As P50, the partial pressure of oxygen in the Total hemoglobin concentration due to the absence of dyshemoglobins. For mentioned previously, pulse oximetry SO2 blood at which the hemoglobin is 50% satu- ctHb = cO2Hb + cHHb + cMetHb + patients with dyshemoglobinemias, how- values can be clinically misleading in the rated. The value for P50—typically about cCOHb + cSHb ever, FO2Hb will be considerably lower than presence of dyshemoglobins. COHb and 26.6 mm Hg for a healthy person—is used Hemoglobin oxygen saturation SO2. Although the SO2 typically remains MetHb greatly decrease the oxygen carrying as an indicator of the affinity of hemoglobin cO2Hb within the normal range in the presence of capacity of blood, but not the oxygen satu- SO2 = for O2. In the presence of disease or other cO2Hb + cHHb elevated COHb or MetHb, the O2 capacity ration. Consequently, the SO2 value will be conditions that change the hemoglobin’s Pulse oximetry and CO-oximetry report may be severely decreased, leading to fatal close to normal in these cases. For example, affinity for O2, the curve shifts to the right SO2, but with these methods, the values are outcomes. a patient with fractional carboxyhemoglo- or to the left and the P50 changes accord- often referred to as SaO2 or SpO2. Regardless bin (FCOHb) of 20% may have a SO2 value Spectrophotometric Assessment ingly. An increased P50 indicates a rightward of the method used, SO2 is a measure of the of 95%, although the FO2Hb will be near shift of the standard curve and means that a fraction of oxygenated hemoglobin in rela- Of Oxygenation Status 75%. Therefore, during the initial evaluation higher partial pressure is necessary to main- tion to the amount of hemoglobin capable Hemoglobin molecules are easily measured of oxygen status in an unconscious, dys- tain 50% oxygen saturation. Increases in of transporting O2. The normal range for by spectrophotometric methods. In addi- pneic, or otherwise impaired patient, health CLINICAL LABORATORY NEWS FEBRUARY 2006 11 care professionals should use CO-oximetry to rule out the presence of high fractions of Table 1 dyshemoglobins. Similarly, the use of pulse Analytical Methods for Measuring Oxygen Saturation oximetry to monitor SO2 in patients with elevated MetHb is not recommended. When MetHb concentration increases above 35%, Device Specimen Measurement Reported Data Advantages Disadvantages Notes the pulse oximetry response reaches a plateau Arterial Blood Partial pressure PO2, SO2 Also mea- Invasive, O2sat If the ABG and of 84%–86% saturation. At this point, the SO2 Blood Gas of oxygen dis- (O2sat) sures pH and may be inaccu- pulse oximetry becomes virtually independent of the MetHb (ABG) solved in whole Some models PCO2 rate in hospital- are discrepant, an concentration; such situations have been Analyzer blood at an elec- available with ized patients abnormal Hb may noted in several case reports of symptomatic trode CO-oximetry and if dysHb be present. methemoglobinemia in the literature. capabilities present The effect on SO2, if any, of increased (see below) fractional sulfhemoglobin (FSHb) or FCO- Hb is much less pronounced than for ele- Pulse Trans- Absorption at SO2 Non-invasive, Inaccurate When MetHb vated fractional methemoglobin (FMetHb). oximeter cutaneous two wavelengths continuous when interfer- >25% the pulse This phenomenon may be explained by (660 nm and bedside ing substances oximeter will examining the spectroscopic signatures of 940 nm) in blood monitoring are present: read saturation of the hemoglobin derivatives. MetHb absorbs MetHb, certain 75%–85%. light almost equally at the red and infrared dyes wavelengths used by pulse oximeters (Figure 2). Although MetHb absorption at 660 nm CO- Blood Absorption of Hb SO2, FO2Hb, Measures the Invasive, Most accurate is similar to HHb absorption at that wave- oximeter derivatives using FHHb, FMetHb, concentra- performed in method; even in length, MetHb’s absorption at 940 nm is multiple wave- FCOHb, FSHb, tion of Hb laboratory; not cases of CO-poi- markedly greater than that of either HHb or lengths of light ctHb species all instruments soning and met- O2Hb. As a result, MetHb will contribute to report SHb, hemoglobinemia. the perceived absorption of both HHb and total bili May be inaccurate O2Hb. This increases the numerator and the in cases of HbM. denominator of the ratio between absorp- tion at 660 nm and absorption at 940 nm, wavelengths improve the accuracy of the Unfortunately, this approach to calculating cal Chemistry. 3rd ed. Burtis CA, Ashwood causing the ratio to approach unity. As previ- spectrum, minimize or eliminate the effects O2sat assumes a normal ODC, which is fre- ER, eds. Philadelphia, Pa.: WB Saunders, ously mentioned, a ratio of 1.0 corresponds of interfering substances, and enable report- quently not the case in hospitalized patients. 1999: 1642–1710. to a saturation of 85% on many pulse oxim- ing of additional components. More com- To circumvent this problem, some labs cus- Holbek CC. New developments in the mea- eter calibration curves, which in the case of a plex CO-oximeters are also available that tomize the output of blood gas analyzers surement of CO-oximetry. Anesth Analg patient with methemoglobinemia could be measure absorbance at 128 wavelengths and so that they do not report O2sat; however, 2002; 94: S89–92. dangerously misleading. can report total hemoglobin concentration some POC devices do not offer this option NCCLS. Blood gas and pH analysis and re- CO-oximetry. Early CO-oximeters were (ctHb) and SO2 in addition to fractional de- and could potentially mislead some health lated measurements; Approved guideline simplified spectrophotometers that mea- oxyhemoglobin (FHHb), FO2Hb, FCOHb, care professionals. To avoid such errors, the C46-A. Wayne, Pa.: National Committee sured light absorbance at four or more dif- FMetHb, and FSHb. most recent approved guideline from CLSI for Clinical Laboratory Standards; 2001. ferent wavelengths chosen to correspond to The assumption that FO2Hb is equiva- discourages the use of estimated values. Report. the specific absorbance characteristics of the lent to SO2 does not hold in cases of cyano- NCCLS. Fractional oxygen content and satu- hemoglobin derivatives (Figure 2). By the sis that are due to increased dyshemoglobin Best Practice for Oxygen Saturation Measurement ration, and related quantities in blood: mid-1980s, CO-oximeters were capable of fractions. Consequently, in these cases, labs Terminology, measurement, and reporting; measuring fractions of HHb, O2Hb, COHb, should not rely solely on SO2, but should use Despite the limitations discussed above, Approved guideline C25-A. Wayne, Pa.: MetHb, and SHb using six wavelengths. a CO-oximeter that has been programmed measurement of the oxygen saturation of National Committee for Clinical Labora- Current models measure absorbance at over to report FO22Hb, FCOHb, FMetHb, and hemoglobin remains a valuable tool for as- tory Standards; 1997. Report. 100 wavelengths and are called continuous FSHb until the presence of dyshemoglobins sessing a patient’s respiratory status, and lab- NCCLS. Definitions of quantities and conven- wave spectrophotometers. The additional is eliminated. Furthermore, laboratorians oratorians can enhance the quality of patient tions related to blood pH and gas analysis; need to be aware that neither SO nor FO Hb care by being aware of these limitations and 2 2 Approved standard C12-5. Wayne, Pa.: reflects the total amount of O bound to he- educating other health care workers about 2 National Committee for Clinical Labora- Oxygen Saturation moglobin, as this depends on the PO and them. Table 1 summarizes the advantages 2 tory Standards; 1994. Report. Some Common the total Hb concentration. and disadvantages of the three analytical methods for assessing oxygen saturation. Sinex JE. Pulse oximetry: principles and limi- Abbreviations Non-Spectrophotometric Methods of Analysis The most important point to note is that the tations. Am J Emerg Med 1999; 17: 59–67. Most blood gas analyzers are comprised of a commonly reported parameters from the O2 Molecular oxygen ACKNOWLEDGMENTS series of electrodes that provide information three methods—O sat, SO , and FO Hb— O Hb Oxyhemoglobin 2 2 2 2 regarding a patient’s acid/base status (pH), are quite different and therefore should not The author would like to acknowledge the CO Carbon dioxide respiratory function (PCO2), and oxygen- be used interchangeably. efforts of Drs. Mitch Scott, Chuck Eby, and HHb Deoxyhemoglobin ation status (PO2). PO2 measurements are In addition, laboratorians and other Rohit Cariappa, who were co-authors on the SO2 Oxygen saturation based on changes in electrical current mea- health care workers need to be aware that case conference publication that inspired this COHb Carboxyhemoglobin sured at a Clark electrode, whereas pH and estimated O2sat values should be interpreted article (Clin Chem 2005; 51: 434–444). PCO measurements are determined from with caution as the algorithms used to cal- O Sat Estimated oxygen 2 2 voltage changes at high-impedance elec- culate this value assume that the patient has Shannon Haymond, PhD, is saturation trodes. Newer, point-of-care (POC) blood normal O2 affinity, normal 2,3-DPG con- a Senior Scientist and Project MetHb Methemoglobin gas analyzers employ dry slide technology centration, and no dyshemoglobin or he- Leader for the Cystic Fibrosis SaO2 Arterial oxygen with optical detection. moglobinopathy. IVD application at Nano- or saturation, as reported Some of the parameters measured by Finally, it is important to note that SO2 sphere, Inc. in Northbrook, Ill. SpO2 by pulse oximeters blood gas instruments are different than has limited value as the sole indicator of Email: [email protected]. SHb Sulfhemoglobin those measured by the spectrophotomet- oxygen status in cases of suspected dysh- HbS Sickle cell hemoglobin ric methods. The PO2 value determined by emoglobinemia, because the presence of these instruments refers to dissolved oxygen dyshemoglobins affects O capacity and FO2Hb Fractional 2 This article is available as an oxyhemoglogin gas in blood. The concentration of dissolved affinity but not saturation. In these cases, 1 HbC Hemoglobin C oxygen (cdO2) is linearly related to the par- information from CO-oximetry is neces- 8 /2” x 11” reprint on the tial pressure of oxygen in blood according to sary. In addition to the FO Hb, CO-oxim- 2 AACC Web site cO2Hb Oxyhemoglobin Henry’s law where the solubility constant of eters should be programmed to report the concentration O2 (αO2) is 0.03 mL O2/L/mm Hg: various dyshemoglobin fractions in addi- (www.aacc.org). HbF Hemoglobin F cdO = O PO tion to FO Hb , as FO Hb alone has limited 2 α 2 × 2 2 1 2 Click on ctHb Total hemoglobin However, the relationship between the utility. CLN concentration estimated SO2, often referred to as O2sat, “Clinical Laboratory News,” and PO may not always be linear. O sat is SUGGESTED READINGS FCOHb Fractional 2 2 then “Series Articles.” carboxyhemoglobin calculated from the pH and PO2 values and Fairbanks VF, Klee GG. Biochemical aspects the standard ODC for oxygen saturation. of . In: Tietz Textbook of Clini- 12 CLINICAL LABORATORY NEWS FEBRUARY 2006