unit: ph and blood gases

MLAB 2401- Clinical Chemistry Lab Manual CD 87

UNIT: pH and Blood Gases 11pH.wpd

Purpose

To become acquainted with theory and methods of measuring pH and blood gases.

Objectives

Upon completion of this exercise, the student will be able to:

1. Review classroom notes on pH, Henderson-Hasselbach equation, normal values andexpected ratios, and pH electrodes.

2. 2 2Discuss the basic principles involved in pH, pO and pCO determinations on whole blood.

3.Interpret the basic clinical significance of blood gas values.

4.Discuss the basic theory behind operation of the pH electrode.

5.Discuss the operation of the pH meter and the blood gas machine.

Principle I

Determination of blood gases enables the evaluation of a patient's acid-base balance. Blood gasinstruments in the laboratory are designed to measure the partial pressures of carbon dioxide

2 2(pCO ) and of oxygen (pO ) as well as blood pH. Specialized electrodes designed for each gasdetermination are placed within the instrument so that one small blood sample suffices for allmeasurements.

2 2After measuring pH and pCO directly, it is possible to obtain bicarbonate and CO content values

2by calculation or with the use of a nomograph. From a direct measurement of pH and pO , bloodoxygen % saturation can be determined.

Principle II

The carbonic acid-bicarbonate buffer system is the most important buffer system in the regulationof hydrogen ion balance in the body. In plasma, the relationship between pH and the bicarbonate-carbonic acid buffer system is expressed by the Henderson-Hasselbach equation:

With this mathematical expression of the relationship between the bicarbonate ion and carbonicacid, it is possible to calculate pH.

Principle III

3 2 3The ratio of HCO (salt) to H CO (acid) is normally 20:1. With this ratio, the blood pH is 7.40.–

The pH falls (acidosis) as bicarbonate decreases in relation to carbonic acid. The pH rises(alkalosis) as bicarbonate increases in relation to carbonic acid.

Principle IV

Four categories of acid-base imbalance may be encountered: metabolic acidosis; metabolicalkalosis; respiratory acidosis; and respiratory alkalosis. In this context, “metabolic” refers to the

UNIT: pH and Blood Gases (continued)

D 88 C MLAB 2401 - Clinical Chemistry Lab Manual

bicarbonate concentration in the Henderson-Hasselbach equation and the “respiratory” to thecarbonic acid.

Principle V

Blood gases and pH are generally performed on arterial blood to provide acid-base and respiratoryinformation on a sample that is a mixture of blood from all parts of the body to tell how well thelungs are oxygenating the body.

1. pH is the only way to determine if the body is too acid or too alkaline.

a. Acidemia and alkalemia refer to a condition of the blood.

b. Acidosis and alkalosis refer to the process occurring in the patient which caused thecondition.

2 22. The respiratory parameter is the pCO , or pressure/ tension exerted by dissolved CO gas

2in the blood. pCO is influenced only by the function of the lungs.

2a. CO gas should be considered an acid substance because in water carbonic acid

2 3(H CO ) is formed.

2b. Transport forms of CO

21) dissolved CO gas

2 2 3 32) combined with H O 6 H CO 6 HCO + H– +

3) as carboxyhemoglobin

2 2 23. Under normal conditions, dissolved CO gas has a pCO of approximately 40 mmHg. pCO

3 2of 40 mm = 1.2 mEq/L. Normal HCO = 24 mEq/L. Total CO content = 25.2 mEq/L (or–

mmol/L).

2 3 34. The H produced by the breakdown of H CO 6 HCO + H+ – +

a. is loosely carried and thus buffered with plasma proteins

4b. H is excreted by the kidney as NH+ +

25. CO is removed by the lungs

2 2High PCO – hypoventilation – respiratory acidosis by decreased elimination of CO bylungs.

2 2Low PCO – hyperventilation – respiratory alkalosis by increased elimination of CO bylungs.

3Non-Respiratory Parameters: HCO and Base Excess (BE)–



Bicarbonate ion and BE are influenced by metabolic causes. Metabolic acid-base is under thecontrol of the kidneys.

31. HCO is an alkaline (base) substance excreted or conserved by the kidneys.–

32. A high HCO or positive BE indicates metabolic alkalosis caused by loss of non-volatile acid–

3or gain of HCO –

a. excess vomitingb. excess diuretic therapyc. excess bicarbonate intake

3 33. A low HCO or negative BE indicates metabolic acidosis caused by loss of HCO or its– –

neutralization by a non-volatile acid.

a. seen in conditions causing the accumulation of organic acids as in diabeticketoacidosis, lactic acidosis, renal failure, etc.

b. deficit of bicarbonate due to diarrhea, renal tubular acidosis, ammonium chloridetreatment.

Oxygen

2 2 2The relationship between pO and hemoglobin O (HbO ) is an s-shaped curve, the hemoglobin-oxygen dissociation curve.

To fulfill its function as respiratory pigment, hemoglobin must specifically bind with high affinity tolarge quantities of oxygen, transport them, and release them in appropriate tissues. It is thetetrameric structure of hemoglobin that provides its unique oxygen-binding capacity and makesit superior.

Each molecule of hemoglobin contains four (ferrous) iron-containing heme groups, each capable

2 2 2 4 2 2of binding one molecule of O (� Hb + 4 O º Hb(O ) ), each O added depends on the (pO )).

The position of the oxygen-dissociation curve isdetermined by a number offactors in addition to thepreviously mentionedpartial pressure of oxygen;body temperature, red cell2, 3, DPG concentration,and pH (Bohr effect).

T o p l e f t , o x y g e ndissociation curves forhuman blood with different

2pH(S) but constant pCO(40 mm Hg). DPGc o n c e n t r a t i o n i n



erythrocytes (4.8 mmol/L), and temperature (37°C).Principle VI

Review of Potentiometric Methods

1. Potentiometric methods are based on the measurement of a potential (voltage) differencebetween two electrodes immersed in a solution under the condition of zero current. Theelectrodes and the solution constitute an electrochemical cell. Each electrode ischaracterized by a half-cell reaction with a corresponding half-cell potential. The potentialdifference between the two electrodes is usually measured using a pH/milli-volt meter.

2. Indicator/measuring electrodes' half-cell potential responds to changes in the activity orconcentration of the substance/species in the solution being measured.

3. Reference electrodes serve as an internal standard as their half-cell potential does notchange. They consist of a metal and its salt in contact with a solution of the same ion (beingmeasured) of known concentration. Reference electrodes are usually a calomel or silver-silver chloride type.

4. The salt bridge or liquid junction is a device that allows ionic movement betweencompartments of an electrochemical cell to maintain electrical contact and at the same time,prevents mixing of the separate solutions of the half-cells.

Principle VII

pH Electrodes

– The first ion-selective electrode to be widely used was the pH electrode designed to measurehydrogen ion activity. This was made possible by the development of a special pH-sensitive glass.When a thin membrane of this glass separates two solutions of differing hydrogen ionconcentrations, a hydrogen ion exchange takes place in the outer hydrated layers of the glass,causing a potential to develop across the glass membrane. If a calomel half-cell (referenceelectrode) is also immersed in the solution and the two are connected through a pH meter, themeter can measure the potential difference (in millivolts) between the electrodes and convert thisto pH units.



1. Calomel or silver-silver chloride electrode2. Buffer-chloride solution3. Saturated (3.5M) chloride solution4. pH sensitive glass membrane5. Liquid junction

Principle VIII

2 2 2The pCO Electrode – The pCO electrode consists of a pH electrode with a CO permeablemembrane covering the glass membrane surface. Between the two is a thin layer of dilute

2bicarbonate buffer. The aspirated blood sample is in contact with the CO -permeable membrane,

2and as CO diffuses from the blood into the buffer, the pH of the buffer is lowered. The change

2of pH is proportional to the concentration of dissolved CO in the blood. The glass electrode

2responds to the buffer pH change, and the meter is calibrated to read the pCO in mm of mercury.

2This type of pCO electrode is known as the Severinghaus electrode.

2In a patient's blood the three values, pH, pCO and bicarbonate, are all inter-related according tothe Henderson-Hasselbach equation. If any two of the values are known, the third can be

2calculated. Since blood gas instruments designed today measure the pH and pCO , the

2 2bicarbonate value may be calculated. Often a total CO , (which includes dissolved CO , carbonicacid, and bicarbonate) is ordered.

Principle IX

2The pO Electrode – The Clark electrode for measuring the partial pressure of oxygen in the bloodis based on a different principle from that of pH measurement. While the pH electrode measures

2a voltage difference when no current is flowing; the pO electrode measures the current that flowswhen a constant voltage is applied to the system. The current is the stream of electrons that flowas the oxygen molecules are reduced at the cathode:

2 2½ O + H O = 2e- + 2OH-



The source of the electrons is the silver-silver chloride anode where the silver molecules areoxidized:

Ag 6 Ag + e-+

The amount of current that flows through the system is a direct measure of the number ofelectrons released to the oxygen and is consequently a measure of the number of oxygen

2molecules available for reduction. The current is directly linear with O concentration as long asthe constant voltage is maintained.

A platinum wire forms the cathode; the anode is a silver wire in AgCl. The contact between thepoles is an electrolyte solution that is separated from the test sample (blood) by a membrane

2 2permeable to O molecules. Dissolved O diffuses from the blood through the membrane and isreduced at the cathode. The rate-limiting factor in the system is the diffusion of oxygen molecules

2through the membrane. The diffusion rate depends directly upon the pO of the sample, so the

2change in current flow offers a direct measurement of the pO .

Principle X

Blood Gas Instruments Calibration and Maintenance – Blood gas instruments must be monitored

2 2constantly and calibrated frequently. All three electrodes (pH, pO , and pCO ) are calibrated bysetting with two standard concentrations. Two buffers in the physiologic range are used for pH

2 2calibration, and two gases (high and low concentrations of O and CO ) are used for the gaselectrodes. The gases are bubbled through water in the instrument to saturate them with watervapor. Gases dissolved in the blood would be comparably saturated. Corrections must be madefor water vapor pressure and for the barometric pressure, which must be checked regularlythroughout the day.

Since the pressure of gases and pH is dependent upon temperature, the temperature of the bathsurrounding the electrodes must be carefully monitored and closely controlled. Usually the bathis maintained at 37°C ± 0.1°C.

2 2Both the pO and pCO electrodes require regular maintenance to keep the membranes intact,taut, and clean. Obstruction to diffusion, such as protein build-up on the membrane, slows downthe response and may give low results.

Principle XI

Blood Gas Measurement

Normally arterial blood is drawn for blood gas studies since it will provide the best informationconcerning overall acid-base balance as well as lung efficiency. Arterialized capillary blood isacceptable and preferred when working with infants and small children.

The heparinized sample must be drawn, handled, and measured anaerobically, as any exposureto atmospheric gases will change the patient's sample.

The sample is transported in crushed ice to slow cell metabolism. Testing should begin ASAP.

The sample should be evaluated for the presence of clots or air bubbles, then well-mixed beforeintroducing it into the blood gas instrument. The blood gas instrument is maintained at 37°C to



determine the pH of the blood as it existed in the patient's body. Temperature control is veryimportant because pH is temperature dependent and decreases about 0.015 units for each degreerise in temperature.

Principle XII

Siggard-Andersen Nomograph

2 2In routine blood gas measurement, three parameters (pH, pCO , and pO ) are measured. The

2 3total CO or bicarbonate ion (HCO ) and another parameter known as base excess (BE) can be–

obtained by calculation or through the use of the Siggard-Andersen nomograph.

2To obtain total CO (or bicarbonate ion), and base excess values from the Siggard-Anderson

2 2nomograph, use a straight edge to mark a line that crosses pCO , pH, BE grid, and CO scales.



Siggaard-Andersen nomograph.



Table 1Primary Blood Gas Classifications

Base

2 3Primary Ventilatory pCO pH HCO Excess

1. Acute ventilatory failure I D N N

2. Chronic ventilatory failure I N I I

3. Acute ventilatory insufficiency D I N N

4. Chronic ventilatory insufficiency D N D D

Primary Acid-Base

1. Uncompensated acidosis N D D D

2. Uncompensated alkalosis N I I I

3. Partly compensated acidosis D D D D

4. Partly compensated alkalosis I I I I

5. Compensated acidosis/alkalosis I or D N I or D I or D

I = increased; D = decreased; N = normal

Normal Blood Gas Values

Arterial Blood Mixed Venous Blood

pH

2pO

2O saturation

2pCO

3HCO –

2Total CO

Base Excess

7.35 - 7.45

80 - 100 mmHg

$ 95%

35 - 45 mmHg

22 - 26 mEq/L

23 - 27 mmol/L

-2 to +2

7.31 - 7.41

35 - 40 mmHg

70 - 75%

41 - 51 mmHg

22 - 26 mEq/L

23-27 mmol/L

-2 to +2



Name

Date

Study Questions

Instructions: Unless otherwise indicated, each question is worth one point. Indicate youranswers in an appropriate manner or in the space provided.

1. pH is defined as theA. H concentration+

B. log of the H concentration+

C. negative log of the H concentration+

D. log of the reciprocal of the OH concentrationE. sum of the OH and H– +

2. The pH meter actually measuresA. currentB. pHC. voltageD. ion numbers

23. Most of the CO present in blood is in the form ofA. carbonic acid

2B. dissolved COC. calcium carbonateD. a protein complexE. bicarbonate ion

4. Which of the following are compensatory mechanisms used by the body in acid-basedisturbances?A. blood buffer systemsB. respiratory mechanismsC. renal mechanismsD. all of these

5. In uncompensated metabolic acidosis, which of the following would be observed?

3A. pH decreased, HCO increased–

3B. pH decreased, HCO decreased–

3C. pH increased, HCO decreased–

3D. pH increased, HCO increased–

6. Making an electrode selective for an ion other than hydrogen is made possible byA. increasing the buffer pHB. changing the reference electrodeC. using stronger buffer solutionsD. decreasing the buffer temperatureE. changing the chemical composition of the glass membrane

7. What is the difference between an indicator electrode and a reference electrode? (2 points)



8. Using classroom and laboratory notes, define/describe a “salt bridge.” Be sure to includeits purpose.

9. State the Henderson-Hasselbach equation. What is it? (2 points)

10. What is the normal bicarbonate ion to carbonic acid ratio in the body?

11. What is the most important single factor or system responsible for keeping the pH of theblood within normal range?

12. Which compensatory mechanism will respond the quickest to a sudden change in the acid-base status of a patient?

213. Use the Siggard-Anderson nomograph to determine the total CO ; bicarbonate level and

2base excess (BE) of a patient with 7.47 pH and a PCO of 37 mmHg.

2Total CO bicarbonate base excess

The Severinghaus electrode measures

14.

while the Clark electrode measures .

15. State the normal body pH range.

16. Normally, the body is slightly (acidic / alkaline). Circle the correct response.

Case Study Questions17. (5 points) A medical laboratory technician obtained the following blood-gas results from a

2 3well-iced arterial specimen: pH = 7.14, pCO = 51 mmHg, and HCO = 10 mmol/L.–

1. Are each of the individual results consistent with acidosis? alkalosis?pH ___________________

2pCO _________________

3HCO _________________

2. Which of the results indicate respiratory acid / base?

3. Which of the results indicate metabolic acid / base?,



4. Are these results consistent with a metabolic or respiratory problem?

The specimen is re-examined, and small clots are noted. A repeat blood-gas specimen is

2 2obtained and analysis yields the following data: pH - 7.23, PCO = 22 mmHg, PO = 55

3mmHg, and HCO = 10 mmol/L.

1. What type of acid-base disorder do these results indicate, if any?

2. What other laboratory results do you think may be abnormal in a patient with theseblood-gas results?

A half hour later a request for a stat lactic acid is received. The result is 21 mmol/L (21mEq/L). (NV = 0.5 - 1.9 mmol/L)

1. Is this result consistent with the above results?

CASE STUDY: Acid-base Balance 18. (5 points) A 27 year old man was comatose with depressed respiration upon arrival at the

ER. A friend stated that the man had overdosed on “sleeping pills.” The following blood gasresults were obtained:

pH 7.29

2pCO 58 mmHg

3HCO 25 mmol/L

2pO 72 mmHg

1. Evaluate these ABGs and determine the patient’s acid-base balance. Is there anycompensation occurring?

2. What has caused the acid-base state of this man?

23. Using the Siggard - Anderson nomograph, calculate the total CO and BE.

4. What parameter would change if complete compensation occurs?

unit: ph and blood gases

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