Measurement of carbon dioxide in blood
The invention relates to the determination of the total carbon dioxide concentration in a whole blood sample. 
     BACKGROUND OF THE INVENTION 
     The measurement of total carbon dioxide concentration. (TCO 2 ) in serum or plasma is a routine analysis performed in clinical laboratories as a patient profile test. In particular, measurement of TCO 2  is useful in determining the acid-base status of a patient. For example, plasma TCO 2  can be used by a physician in the diagnosis of a patient in renal failure or acute acidosis. 
     TCO 2  in serum or plasma is comprised of three major chemical forms: dissolved CO 2  (3%); carbamino (R--NHCOO - ) derivatives of plasma protein (33%); and bicarbonate (HCO 3   - ) anion (64%). Other minor forms include carbonic acid (H 2  CO 3 ) and carbonate anion (CO 3   = ). 
     Most of the procedures that quantify TCO 2  in serum or plasma involve acidification of the sample to convert all of the CO 2  -containing species to CO 2  gas, as is summarized in equation (1): ##STR1## Once liberated, the CO 2  gas can be measured manometrically in batch analysis or by continuous-flow procedures where the CO 2  gas diffuses across a membrane into a bicarbonate-containing solution thereby lowering the pH of the solution. The change in pH can be detected either potentiometrically using a pH glass electrode or spectrophotometrically if the bicarbonate buffer contains a pH indicating dye. Additionally, there are detection methods that use enzymes or bicarbonate-selective electrodes to quantify TCO 2 . These techniques apply to serum or plasma samples. Therefore, centrifugation of the whole blood sample to separate the cells and plasma prior to analysis is required. 
     SUMMARY OF THE INVENTION 
     The invention relates to the determination of TCO 2  in plasma or serum directly from a whole blood sample. The TCO 2  in plasma is essentially equivalent to the TCO 2  in serum; TCO 2  plasma will be used herein to denote either value. The method involves adjusting TCO 2  measured in the whole blood sample (TCO 2  whole blood) using a volume dilution factor (VDF) to provide TCO 2  plasma. The VDF is determined for each whole blood sample and, preferably, can be related directly to the hematocrit (Hct) of that sample. 
     An advantage of the present invention is that TCO 2  plasma can be determined accurately and directly from a whole blood sample. Since the measurement does not depend on plasma or serum samples, the method of the invention avoids time-consuming sample preparation steps. 
     Other features and advantages of the invention will be apparent from the description of the preferred embodiment thereof, and from the claims. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     The FIGURE is a fluid flow diagram of the blood chemistry analyzer used to determine TCO 2  plasma. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The preferred method includes measuring TCO 2  whole blood in a whole blood sample, generating a VDF from the Hct of the same whole blood sample, and adjusting the TCO 2  whole blood using the VDF to provide the TCO 2  plasma of the whole blood sample. 
     Methods for measuring TCO 2  are described in U.S. Pat. Nos. 3,973,912, 4,321,545 and 5,429,930, each of which is incorporated by reference. The preferred method of measuring TCO 2  whole blood involves acidification of the whole blood sample to liberate CO 2  gas from the sample according to equation (1). The sample and acidifying agent mix as they pass through a mixing chamber. Acidifying agents include aqueous solutions of sulfuric acid, citric acid, lactic acid and combinations thereof. The preferred acidifying agent is an aqueous solution containing 90 mM citric acid, although the concentration can be varied over a wide range. 
     The preferred TCO 2  gas electrode includes a pH glass electrode along with a Ag/AgCl reference electrode. The two electrodes are covered by a CO 2  gas permeable membrane such as silicone membrane. A bicarbonate/chloride internal filling solution (IFS) is trapped between the membrane and the pH electrode. The CO 2  gas generated by acidification passes through the silicone membrane. It equilibrates with the IFS. The pH of the IFS will change with the CO 2  concentration. By measuring pH with the pH glass electrode, the CO 2  concentration can be determined. 
     The TCO 2  gas electrode is calibrated before use. Calibration involves using two standard aqueous bicarbonate solutions. Each standard solution, when mixed with acid, will produce a known CO 2  concentration which in turn will produce a known pH in the IFS trapped between the membrane and the pH electrode. Once the TCO 2  electrode is calibrated, TCO 2  whole blood is determined comparing the unknown with one calibration standard. 
     TCO 2  plasma is obtained by adjusting the measured TCO 2  whole blood using the VDF of the same sample. The preferred adjustment is shown in equation (2): 
     
         TCO.sub.2 plasma =TCO.sub.2 whole blood /VDF               (2) 
    
     The VDF represents the fractional volume of the extracellular fluid in the whole blood sample. Preferably, the VDF is determined from the Hct of the same whole blood sample according to equation (3): 
     
         VDF=1-Hct/100                                              (3) 
    
     Hct is the volume of red blood cells occupying the total volume of the whole blood sample reported as a percentage. Hct measurement is performed routinely in clinical laboratories using various methods to diagnose conditions such as anemia or polycythemia. The preferred method of determining Hct is by measuring the conductivity of the whole blood sample using a pair of calibrated electrodes as disclosed in U.S. Pat. No. 4,686,479. The Hct sensor may be factory calibrated or calibrated using aqueous standards. 
     TCO 2  plasma obtained from analyzing a whole blood sample following the method of the invention is equivalent to the TCO 2  determined after isolating the plasma from the whole blood sample by centrifugation. Table I lists TCO 2  data of patients that was measured in whole blood and in the separated plasma. The plasma samples acquired from the corresponding blood samples were subsequently analyzed under identical conditions to obtain the directly measured TCO 2  plasma. Hct of the whole blood sample was measured by the method disclosed in U.S. Pat. No. 4,686,479. Finally, the TCO 2  whole blood was adjusted according to equations (2) and (3). The data in Table I indicate that TCO 2  plasma measured using the preferred method correlates with TCO 2  plasma measured directly in plasma samples. 
     
                       TABLE I                                                     
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       Measured             TCO.sub.2 blood                               
                                    Measured                              
Patient                                                                   
       TCO.sub.2 blood      [1-Hct/100]                                   
                                    TCO.sub.2 plasma                      
ID     (mmol/L)   Hct (%)   (mmol/L)                                      
                                    (mmol/L)                              
______________________________________                                    
 1     15         45        27      27                                    
 2     16         38        26      25                                    
 3     16         41        27      27                                    
 4     16         39        26      24                                    
 5     16         41        27      24                                    
 6     14         44        25      27                                    
 7     14         42        24      25                                    
 8     15         45        27      25                                    
 9     19         38        31      30                                    
10     14         45        25      25                                    
11     13         52        27      26                                    
12     12         42        21      22                                    
13     16         38        26      25                                    
14     16         39        26      23                                    
15     14         44        25      27                                    
16     13         45        24      22                                    
17     15         43        26      28                                    
18     16         41        27      26                                    
19     15         43        26      28                                    
20     16         41        27      31                                    
21      8         43        14      13                                    
22     16         41        27      26                                    
23     29         37        46      47                                    
24     11         43        19      17                                    
25     27         37        43      44                                    
26      7         40        12      11                                    
27     26         37        41      42                                    
28      9         42        16      14                                    
29     15         44        27      29                                    
30     15         42        26      26                                    
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     The method of the invention can be used in most typical blood chemistry analyzers. The features of a preferred blood chemistry analyzer are presented in the figure, which shows the preferred arrangement of pumps and analysis chambers in the analyzer. The analyzer includes two peristaltic pumps and a series of analysis chambers. Pump 1 draws the sample through tube 10 into analysis chamber 12 where the Hct of the sample is determined if the sample is a whole blood sample. The sample then flows through a set of analysis chambers 14 where other sample chemistry is probed. The sample and acidifying agent are then combined in mixing chamber 16. The acidifying agent is delivered to chamber 16 by pump 2. The mixture flows to chamber 18 which houses a TCO 2  gas electrode. The liberated CO 2  gas is measured in chamber 18 using a TCO 2  gas electrode, and related to TCO 2  in the sample. The TCO 2  in the whole blood sample is then converted to TCO 2  plasma using the data collected for that sample. 
     The TCO 2  plasma measurement described above can be incorporated into a NOVA CRT12 or CRT14 Chemistry Analyzer, which is available from Nova Biomedical Corp., with appropriate modification. The analyzer allows most of the blood chemistry to be determined in a single run of a whole blood sample, decreasing sample analysis time which is critical in emergency situations. 
     Other embodiments are within the claims.