ASTRUP BLOOD GAS ANALYZER

Procedure

• Heparinized blood ( capillary /arterial )

• Wax sealing

• Remix before use

• Poor peripheral circulation –caution

• eg ABL /Roche blood gas analyzer

Laboratory determination of blood gas analysis –Micro method 1.

•Procedure :By using heparinized capillaries ,blood can be collected from fingertips , a lobe of ear or a heel. After filing with the blood capillaries are sealed by using modelling wax. Immediately before use blood is remixed & by cutting the sealed ends the blood is introduced into each equilibration chamber of apparatus. After determining blood pH & p CO₂ ,the bicarbonate & base deficit ( or excess ) are determined by using a Nomograms.

Laboratory determination of blood gas analysis –Micro method 2.

•Although under ordinary conditions capillary blood is similar to arterial , it can be assumed to so in patients with poor peripheral circulations . For reliable results a good flow of capillary blood is essential. If this cannot be obtained arterial blood must be used.

• In hypothermia following corrections has to be made. The measurements are made at 38 ⁰ C.

pH at T ⁰ C = p H at 38 ⁰ C+ 0.0146 ( 38 –T )

PCO₂ at T ⁰ C = Antilog ( log PCO₂ at 38 ⁰ C – 0.021 (38-T )

• Blood must not be exposed to air in any circumstances before its analysis .

Blood sample collection for blood gas analysis

•

PRINCIPLE OF BLOOD GAS ANALYSER

BLOOD GAS ANALYSER works with three in-built electrodes1. pco₂ electrode2. pO ₂ electrode3. pH sensitive glass electrode

pco₂ electrode

1. A. Glass electrode-sensitize to p H

B.silver /silver chloride reference electrode

2.Bicarbonate Buffer

3. Plastic membrane selectivity permeable to gaseous CO₂ but not dissolved ions

4.pH α p CO₂, inverse relationship betweenp H & log p CO₂

pO₂ Measurement1.Polari graphic method

A. platinum electrode

B.silver /silver chloride reference electrode

2.NaCl / phosphate buffer

3. Membrane permeable to gaseous O₂

4.A .O₂ + H₂O +4 e⁻ 4 OH⁻

B. 4 Ag + 4 Cl⁻ 4AgCl +4 e⁻

Important components of Blood gas Analyzer -1. pH sensitive glass electrode

Reference electrode ( silver –silver chloride ) immersed in bicarbonate buffer

• Principle :The pCO₂ Electrode : Test solution –Arterial blood separated by plastic membrane permeable to gaseous CO₂ but not permeable to dissolved ions .

• CO ₂ of blood diffuses through plastic membrane & react with the buffer system to change pH.

• The pCO ₂ electrode takes advantage of linear correlation between p H & log PCO₂ over the range 11-90 mm Hg .

• The hydrogen ion concentration change due to the dissolution of CO₂ is detected by the pH sensitive glass electrode .

• A potential difference exist between glass electrode & Reference electrode This is measured on the meter. The meter’s scale is usually calibrated for in semi logarithmic fashion, since pH is inversely proportional to the log of pCO ₂.

Important components of Blood gas Analyzer 2 Principle : pO ₂ electrode –Polarographic method

Reference platinum & ( silver –silver chloride ) electrode is immersed in a buffer containing ( phosphate & sodium chloride ) .These electrodes are separated from test solution.

• Test solution –Arterial blood separated by plastic membrane permeable to gaseous O₂ but not permeable to dissolved ions .

• O₂ of blood diffuses through plastic membrane & react with the buffer Following reactions take place –

• At platinum electrode ( cathode ) electro reaction occurs .

O₂ + 2H ₂ O+ 4 e⁻ 4 OH ⁻

• The electrons necessary for electro reduction are produced at reference electrode (anode ) as follows-

4 Ag⁺ + 4 Cl ⁻ 4 AgCl + 4 e⁻

• A potential difference exist between glass electrode & Reference electrode This is measured on the meter. The meter’s scale is usually calibrated for in semi logarithmic fashion. The current through the system is directly proportional to PO ₂ & can be recorded directly after amplification into PO ₂.

pH electrode

Principle of p H electrode :

The measurement of pH is called potentiometric analysis. It has been reported that a difference in electrical potential could be measured between two solutions of different pH separated by a thin glass membrane. The potential thus produced varies with the hydrogen ion concentration of two solutions .The glass membrane is sensitive for H ⁺ions. It is on this principle that the glass electrode is constructed for pH measurement.

Combined pH electrode

Integral Parts of combined p H electrode :

1. Simple glass electrode

2. Calomel reference electrode

Combined pH electrode Integral Parts of combined p H electrode :

1. A simple glass electrode:

Modern glass electrodes are constructed from glass containing Lithium oxide which is soft ,hygroscopic imparting low resistance. The inner surface of glass membrane is in contact with a buffer pH. Into this buffer dips a silver /silver chloride electrode ,the internal glass electrode .The glass electrode functions like semi permeable membrane selectively permeable only to H⁺ ions.

2.Calomel reference electrode : consist of Mercury (Hg ⁺²) in contact with a solution of potassium chloride saturated with Calomel (HgCl ).It is surrounded by a outer vessel holding saturated Potassium chloride which acts as a salt bridge between reference and test solution. This electrode is not sensitive to pH .

Combined pH electrode Only when glass electrode is coupled with a Calomel reference electrode the

potentiometric measurements are possible. The potential difference ( or electrical voltage ) between two electrodes depends upon the hydrogen ion concentration of test or standard solution .

It is the logarithmic response measured in millivolts on which pH meter is calibrated in both millivolts and pH.

pH measurements vary with temperature and all measurements should be made at a temperature of 25° C . With increasing temperature there will be fall in of pH. It is therefore important to record the temperature of liquid before measuring pH by adjusting dial on p H meter and then record p H .

Some pH meters have facilities whereby a temperature thermometer can be incorporated into circuit and variation above and below 25 °C are automatically corrected by pH meter itself.

Combined pH electrode Scehatic representation of combined electrode

Ag /AgCl /HCl/glass membrane Test solution KCl/HgCl/Hg

Glass electrode Reference electrode

is the boundary between the two immiscible solutions.

Combined electrode of p H meter

Combined electrode of pH meter

Modern Combined glass electrodefor p H determination.

Single blood sample is in contact With pCO2, pO₂ & pH electrodes for their Simultaneousmeasurement .

Typical reference ranges in blood gas analysis-1 Parameter Normal range

p H 7.35 -7.45

H⁺ ions ( 35-45mmol/ lt )

Arterial pO₂ 95- 100 mm of Hg

Arterial pCO₂ 4.7 -6.0 Kpa ( 1.02 -1.35 mmol/ lt or 35-45 mm Hg )

Total CO₂ 25-39 mmol /lt

Carbon dioxide combining power 53-75 ml per 100ml of plasma

HCO₃⁻ 21- 28 mmol /lt ( mequ/lt )

Base excess of blood 2.3 to + 2.3 mequ /lt

T O₂ Sum of O₂ dissolved in plasma & chemically bound to Hb

H₂ O + CO ₂ ↔ H₂CO₃ ↔ H⁺ + HCO₃ ⁻ uncompensated phase – CONC CO₂ ↑ pCO₂ ↑, Total CO₂ ↑(RESPIRATORY ACIDOSIS )

Typical reference ranges in blood gas analysis-2Parameter Normal range Acidosis Alkalosis

p H 7.35 -7.45 < 7.35 > 7.45

H⁺ ions ( 35-45mmol/ lt ) > 45 mmol/ lt < 35 mmol/ lt

Arterial pO₂* 95- 100 mm of Hg9.3-13.3 kPa

Arterial pCO₂ ◊ 4.7 -6.0 Kpa ( 1.02 -1.35 mmol/lt at 35-45 mm Hg )

High ( respiratory acidosis –under ventilation ) uncompensated phase

low ( respiratory alkalosis –Hyper/over ventilation )-uncompensated phase

T O₂ Sum of O₂ dissolved in plasma & chemically bound to Hb

H₂ O + CO ₂ ↔ H₂CO₃ ↔ H⁺ + HCO₃⁻ * PO ₂ Low =Hypoxemic,< 60 mm Hg- supplemental O ₂ needed ,< 26 mm Hg-Risk of death requires immediate O ₂ supplementation

Typical reference ranges in blood gas analysis-3 Parameter Normal range Acidosis Alkalosis

Total CO₂ ( HCO₃⁻ + PCO₂)Conc HCO₃⁻ : m MPCO₂ :Kpa

25-30 mmol /lt ( Total CO₂= (HCO₃⁻ ) +α PCO₂) α = 0.0226mmol /KpamM /Kpa

Carbon dioxide combining power

53-75 ml per 100ml of plasma

HCO₃⁻ 21- 28 mmol /lt( mequ/lt )

Low( metabolic acidosis )

High ( metabolic alkalosis )-kidney function normal compensating for respiratory issues

Base excess of blood 2.3 to + 2.3 mEq /lt

Typical reference ranges in blood gas analysis-3

• ◊ Arterial pCO₂ ◊- can become abnormal when respiratory system is working to compensate for metabolic issue so as to normalize the blood p H & elevated pCO₂ level is desired in some disorders associated with respiratory failure ,this is known as ‘Permissive hyper apnea ’. (↑ CO₂ )

• HCO₃⁻ : levels can become abnormal when kidneys are working to compensate for respiratory issues so as to normalize blood pH.

Laboratory determination of blood gas analysis (1) The pH of blood : heparinized whole arterial blood ( or heparinized capillary blood ) is used. The pH determination is performed immediately after collection of blood .The blood can be stored at 0-4 ⁰ C up to 2-3 hrs,without significant change in pH .(2) PCO₂ : ( The respiratory parameter ) plasma carbonic acid can not to be found directly .But it is determined by measuring PCO₂ . The PCO₂ of arterial blood is usually directly proportional to the amount of carbon dioxide which is being produced in the body & inversely proportional to the rate of alveolar ventilation in the lungs .(3) TCO₂ : Total CO₂ is mainly bicarbonate & also includes dissolved carbon dioxide. Measurement of CO₂ ,carbonic acid & bicarbonate of plasma derived from blood plasma ,collected under liquid paraffin gives measure of CO₂ content . It is reported as CO₂ per 100ml at standard conditions of temperature & pressure . If the plasma is equilibrated with normal alveolar air ( 40 mm Hg ) before it is measured ,the CO₂ combining power is obtained.Ordinarily the CO₂ content & CO₂ combining power are practically identical.

Laboratory determination of blood gas analysis (4)The plasma bicarbonate : ( non respiratory parameter )can be determined by finding out the actual bicarbonate concentration of plasma separated from blood taken anaerobically & expressed as mill equivalents per liter .This can be calculated from the p H & PCO₂ by using the Henderson's –Hassel Balch equation or from total carbon dioxide & the PCO₂ .

(5) Standard bicarbonate : is expressed as milliequivalents per liter, is theconcentration of bicarbonate in plasma separated from whole blood taken anaerobically which has ben equilibrated at 37⁰ C a PCO₂ of 40 mm Hg with oxygen to give full saturation to the hemoglobin.The plasma concentration bicarbonate is influenced by change in PCO₂ & degree of oxygen saturation . Alkali reserve is plasma bicarbonate . It is this fraction of plasma which is used to neutralize all the acidic compounds entering the blood & tissue. Plasma bicarbonate can also be calculated ( in terms of mequ /lt ) by dividing CO₂ combing power by 2.24.

Laboratory determination of blood gas analysis (5)PO₂ : determination is carried out to assess the oxygen carrying capacity of blood hemoglobin . The increased oxygen affinity of hemoglobin is indicated by elevated PO₂ values. The measurement of arterial PO₂ is also used in conjunction with that of PCO₂ in assessment of respiratory disorders .A low PO₂ is a measure of anoxia. It may also occur with a high PCO₂ when there is alveolar hypo ventilation due depression or obstruction of respiration . A low PO₂ with low PCO₂ may also be observed in pulmonary edema .

( 6 ) Base excess : is the amount of acid required to titrate blood to p H 7.4 at 37⁰ C & PCO₂ at 40 mm Hg . Base deficit is the reverse concept.

Henderson’s Hassel Balch equation :

Henderson’s Hassel Balch equation for pH determination

BLOOD GAS ANALYSIS

The interrelation of TCO₂ , Bicarbonate , Carbonic acid , PCO ₂, & p H in the Henderson’s Hassel Balch equation :

p H = p ka + log (congugate base= HCO₃⁻)

(non ionized acid = H₂CO₃)

At 38⁰ C p ka = 6.1 therefore pH=6.1 + log (HCO ₃⁻ )/ H₂CO₃H₂CO₃ ( mequ /lt ) =(0.0301 X pCO ₂)

pCO ₂= partial pressure of CO ₂ in mmHg equilibrated with plasma at 38⁰ C.

For normal plasma at 38⁰ C at pCO ₂ of 40mm Hg

H₂CO₃ = 0.0301 X 40 (pCO ₂)

H₂CO₃ = 1.204 mequ /lt

p H of plasma at 38⁰ C=

6.1 + log ( total CO ₂ )- (0.0301 X PCO ₂)

( 0.0301 )X 40 (pCO ₂)

Laboratory determination of blood gas analysis • Standardization : by measuring the p H or the blood at its actual PCO₂ & at

accurately known PCO₂ values ,one higher & one lower than the normal PCO₂. This can be achieved by equilibrating two portions of the blood with carbon dioxide & oxygen mixture with PCO₂ values between 30 -60 mmHg respectively .This reduces experimental error since the actual PCO₂ values are between 30-60 mmHg.

• 2. A Nomogram is constructed by plotting log PCO₂ against p H .It is possible to calculate standard bicarbonate & base excess ( or deficit ) .

Laboratory findings in compensated acidosis & alkalosis

Clinical conditions pH PCO₂ Bicarbonate PO₂

Respiratory acidosis Fall Rise Rise Rise

Respiratory alkalosis Rise Fall Fall Fall

Metabolic acidosis Fall Fall Fall Fall

Metabolic alkalosis Rise Rise Rise Rise

pH determination for urine , saliva and low volume body fluids

Pulse oximetry