ACID BASE

BALANCE
DEFINITIONS: ACID,
BASE, BUFFER
 WHAT IS ACID?
ARRHENIUS’DEFINITION:

 An acid is a substance that increases the concentration of

hydrogen ion (H+) when dissolved in water

B R O N S T E D A N D L O W RY ’ S D E F I N I T I O N :

 An acid is a substance that donates a proton in a reaction

LEWIS’ DEFINITION:

 An acid is a molecule or ion that accepts a pair of

electrons to form a covalent bond

 WHAT IS A BASE?
ARRHENIUS’ DEFINITION:

 A base is a substance that increases the concentration of hydroxyl ion (OH-) when
dissoved in water

BRONSTED AND LOWRY’S DEFINITION:

 A base is a substance that accepts a proton in a reaction

LEWIS’ DEFINITION:

 A base is a molecule that donates a pair of electrons for a covalent bond.

 WHAT IS BUFFER?
 A buffer consist of a weak acid and a salt of its conjugate base and it resists the

change in pH upon adding acod or base.

 The effectiveness of a buffer depends on the pKa of the buffering system and

the pHof the environment in which it is placed. In plasma, the bicarbonat-

carbonic acid system, is one of the principal buffers: H2CO3 ↔ HCO3- + H+

 Buffers consist of a weak acid, such as a carbonic acid (H 2CO3) and a salt of its

conjugated base such as bicarbonate (HCO3-) which forms bicarbonate-carbonic

acid buffer system.

ACID BASE
BALANCE
 MAINTENANCE OF H+ (hydrogen ion)

 The normal concentration of H+ in the extracellular body fluid ranges

from 36 to 44 nmol/L (pH 7.34 to 7.44)

 Lungs and kidneys are involve in maintaining pH homeostasis

 Any H+ value within the outside range will cause alterations in the

chemical reaction rate and can also lead to alterations in

consciousness, neuromuscular irritability, tetany, coma, and death.

The logarithmic pH scale expresses H+ concentration:
 The arterial pH is controlled by systems that regulate the production and retention of

acids and bases {buffers, respiratory center/lungs, and kidneys}

The reference value for arterial blood pH is 7.40 and is equivalent to an H+

concentration of 40 nmol/L

 An increase in H+ concentration decreases the pH whereas a decrease in H+

concentration increases the pH.

 A pH below the reference range is reffered to as acidosis and a pH above the

reference range is referred to as alkalosis.

 BUFFER SYSTEM: REGULATION OF H+ AND THE
HENDERSON-HASSELBALCH EQUATION
BUFFER
BUFFER PAIR:
-WEAK ACID + CONJUGATE BASE
- WEAK BASE+ CONJUGATE ACID

The buffer solution resist the change in pH

In plasma, the bicarbonate-carbonic acid system, is one of the principle
buffer:
H2CO3 HCO3- + H+
 BUFFER SYSTEMS

1. BICARBONATE (ECF)- the most important buffer

A. H2C03 dissociates into CO2 and H2O, allowing CO2 to be

eliminated by the lungs and H+ as water.

B. changes in CO2 modify the ventilation (respiratory) rate

C. HCO3- concentration can be change by the kidneys

CO2 + H2O H2CO3 H+ HCO3-

2. PHOSPHATE (URINARY AND ICF)

Two types: INORGANIC AND ORGANIC BUFFER

3. PROTEIN (ICF) : e.g., HEMOGLOBIN

 Regulation of Acid-Base Balance: Lungs and
Kidneys
 Carbon dioxide, the end product of most aerobic metabolic processes, easily
diffuses out of the tissue where it is produced and into the plasma and red celss
in the surrounding capillaries.
 In plasma, a small amount of CO2 is physically dissolved or combined with
proteins to form carbamino compounds.
 The reaction is accelerated by the enzyme carbonic anhydrase found in the red
cell membrane.
 The dissociation of H2CO3 causes the HCO3- concentration to increase in the red
cells and diffuse into the plasma. To maintain electroneutrality (the same number
of positively and negatively charged ions on each side of the red cell membrane),
chloride diffuses into the cell. This is known as the chloride shift.
 LUNGS
 In the lungs, the process is reversed. Inspired O2 diffuses from the alveoli into
the blood and is bound to hemoglobin, forming oxyhemoglobin (O 2Hb).

 The H+ that was carried on the (reduced) hemoglobin in the venous blood is
released to recombine with HCO3- to form H2CO3, which dissociates into H2O
and CO2. The CO2 diffuses into the alveoli and is eliminated through ventilation.

 When the lungs do not remove CO2 at the rate of its production (as a result of
decreased ventilation or disease), it accumulates in the blood, causing the
increase in H+ concentration. If, however, CO2 removal is faster than production
(hyperventilation), the H+ concentration will be decreased.
 A change in the H+ concentration of blood that results from
nonrespiratory disturbances causes the respiratory center to respond by
altering the rate of ventilation in an effort to restore the blood pH to
normal.

 The lungs, by responding within seconds, together with the buffer system,

provide the first line of defense to changes in acid-base status.

 KIDNEYS
 The kidney’s main role in maintaining acid-base homeostasis is to reclaim
HCO3- from the glomerular filtrate. Without this reclamation, the loss of HCO 3-
in the urine would result in the excessive acid gain in the blood.
 The main site for HCO3- reclamation is the proximal tubules. The glomerular
filtrate contains essentially the same HCO3- levels as plasma.

 The process is not a direct transport of HCO3- across the tubule membrane
into the blood. Instead, soduim (Na+) in the glomerular filtrate is exchanged for
H+ in the tubular cell.
 The H+ combines with HCO3- in the filtrate to form H2CO3, which is converted
into H2O to CO2 by carbonic anhydrase.
 The CO2 easily diffuses into the tubule and reacts with H2O to reform
H2CO3 and then HCO3-, which is reabsorbed into the blood along with
sodium. With alkalotic conditions, the kidney excretes HCO3- to
compensate for the eleveated blood pH.

 Reabsorption or reclamation - refers to the process of re-entering the

blood.

 Secretion or excretion - the tubule cells concentrate or remove

substances form the filtrate. These reactions determine the pH of the urine,
as well as the pH of the blood.
 Under normal conditions, the body produces a net excess (50 to 100 mmol/L) of
acid (H+) each day that must be excreted by the kidney. Because the minimum
urine pH is approximately 4.5, the kidney excretes little nonbuffered H +.
 The remainder of the urinary H+ combines with monohydrogen phosphate
(HPO42-) and ammonia (NH3) and is excreted as dihydrogen phosphate (H2PO 4-)
and ammonium (NH4+).

 The amount of HPO42- available form combining with H + is fairly constant;

therefore, the daily excretion of H + in urine is largely depends on the amount of
NH4+ formed.

 The renal tubular cells are able to generate NH 3 from glutamine and other amino
acids, the concentration of NH3 will increase in response to a decreased blood pH.
 Various factors affect the reabsorption of HCO3-. When the blood or plasma
HCO3- level is higher that 26 to 30 mmol/L, HCO3- will be excreted. It is unlikely
that the plasma will exceed an HCO3- value of 30 mmol/L unless these excretory
capabilities fail (e.g., kidney failure occurs). However, a frequent exception to
this is compensatory retention of HCO3- in chronic hypercarbia as seen with
chronic lung disease.

 Several factors may result in decreased HCO3- levels. Most diuretics, regardless
of the mechanism of action, favor the excretion of HCO 3-. Reduced HCO3-
reabsorption also occurs in conditions in which there is an excessive loss of
cations. In kidney dysfunction (such as chronic nephritis or infections), HCO3-
reabsorption may be impaired.
ASSESSMENT OF
ACID-BASE
HOMEOSTASIS
 BICARBONATE BUFFERING SYSTEM

 Are measured and calculated in assessing acid-base homeostasis.

 The dissolve CO2(dCO2) is in equilibrium with CO2 gas, which can be expelled

by the lungs where in it is referred to as an open system and the dCO 2 which is

controlled by the lungs is the respiratory component.

 HENDERSON-HASSELBANCH EQUATION

States the relationship between lungs, kidney and pH.

 It indicates that pH depends on the ration of HCO3/pCO2.

 When the kidneys and the lungs are functioning properly, a 20:1 ratio of HCO3 to

H2CO3 will be maintained, and it is expressed by the Henderson-Hasselbalch

equation.
 ACID-BASE DISORDERS
 ACIDEMIA- blood pH is less than the reference range, which reflects excess
acid or H+ concentration.
 ALKALEMIA- or excess base, when pH is greater than the reference range.
 PRIMARY RESPIRATORY ACIDOSIS OR ALKALOSIS- a disorder caused by
ventilatory dysfunction (a change in the PCO2, the respiratory component).
 ACIDOSIS- refers to a decrease in blood pH.
 ALKALOSIS- refers to an increase in blood pH.
 NON-RESPIRATORY DISORDER- a disorder resulting from a change in the
bicarbonate(HCO3) level (a renal or metabolic function)
 MIXED ACID-BASE DISORDERS- refers to a clinical condition in which two or
more primary acid-base disorders coexist.
• The body's cellular and metabolic activities are pH dependent, thus during

compensation, the body tries to return the pH toward normal whenever an

imbalance occur.

• After full compensation, pH would returned to its normal range

• After partial compensation, the pH would be near normal.

OXYGEN AND GAS
EXCHANGE
 OXYGEN AND GAS EXCHANGE

 The role of oxygen in metabolism is crucial to life. In cell mitochondria,

electron pairs from the oxidation of NadH and FadH2 are transferred to

molecular oxygen.

----> that causes release of the energy used to synthesize ATP from the

phosphorylation of ADP.
 7 conditions that is adequate in tissue oxygenation:

1. available atmospheric oxygen

2. adequate ventilation

3. gas exchange between lungs and arterial blood

4. loading of oxygen onto hemoglobin

5. adequate Hgb

6. adequate transport (cardiac output)

7. release of oxygen to the tissue

 Dalton’s Law
 The total atmospheric pressure is the sum of the individual gas pressures
atmosphere -------- = 760 mmHg (sea level)
composed of: O2 = (20.93%)
CO2 = (0.03%)
Nitrogen = (78.1%)
Inert gas = (1%)

pO2 = (atmosphere - vapor pressure of water) X 20.93%

pCO2 = (atmosphere - vapor pressure of water) X 0.03%
 DEAD SPACE AIR - dilutes the air being inspired

 INSPIRED AIR - air that move into the lungs

 EXPIRED AIR - air that move unto the lungs/airways

4 FACTORS AFFECTING THE DECREASE IN pO2:

1. warmed to 37oC and fully saturated with water vapor

2. the percentage of O2 in inspired air

3. the amount of pCO2 in the expired air

4. ratio of the volume of inspired air to the volume of the dead space air.
 Influence the amount of pCO2:

1. patient with increase metabolism

2. increase of pCO2 in blood and expired gas and greater dilution of inspired air.

Influence the amount of oxygen that moves to the tissue:

3. destruction of the alveoli

4. pulmonary edema
5. airway blockage
6. inadequate blood supply
7. diffusion of CO2 and O2
 How oxygen transport to the tissue

Factors affecting the amount of O2 loaded unto Hgb:

1. availability of O2

2. presence of interfering substances

3. pH

4. temperature of blood

5. levels of pCO2, 2,3 - DPG

 How Hgb works in 4 conditions:
1. O2Hb describes reversibly bound to Hgb

2. Deoxyhemoglobin is Hgb not bound to O2 but capable of froming a

bond when O2 is available

3. Carboxyhemoglobin is Hgb bound to CO2. Bond between CO and Hgb is

reversible but is 200 times as strong as the bond between O2 and Hgb

4. Methemolgobin is Hgb unable to bind O2 because iron is in an oxidized

rather that reduce state The FE3+ can be reduced by the enzyme
Methemoglobin reductase (which is found in RBC).
 Quantities Associated with Assesing a
Patient’s oxygen status:

1. oxygen saturation (SO2)

2. fractional oxyhemoglobin (FO2Hgb)

3. trends in oxygen saturation assessed by transcutaneous (TC), pulse

oximetry (SpO2) assessments

4. amount of O2 dissolve in plasma (pO2)

 Oxygen Saturation (SO2)

 Represents the ratio of O2 that is bound to the carrier protein, Hgb,

compared with the total amount of Hgb capable of binding O2
 Fractional Oxyhemoglobin (FO2Hgb)

 the ratio of the concentration of oxyhemoglobin to the concentration of

total Hgb (c + Hgb)
 Pulse Oxymeter (SpO2)
 Devices that pass light of two or more wavelengths trough the tissue in

the capillary bed of the toe, finger, and ear.

Amount of the O2 dissolve in plasma (pO2)

 From every mmHg of pO2 0.00314 mL of O2 will be dissolved in 100 mL of

plasma at 370C
MEASUREMENT
 Spectrophotometric (Co-oximeter)

Determination of Oxygen Saturation

The actual percent oxyhemoglobin can be determined spectrophotometrically using a

co-oximeter designed to directly measure the various hemoglobin species.

Instruments, at a minimum, should have four wavelengths for measurements of HHb,

O2Hb, and the two most common dyshemoglobins, COHb, and MetHb.

Some newer co-oximeters employ hundreds of wavelengths, which has greatly reduced
measurement interferences.

 Microprocessors control the sequencing of multiple wavelengths of light
through the sample and apply the necessary matrix equations after absorbance
readings are made to calculate the percentage of the individual hemoglobin
species:

O2HB = a1A1 + a2A2 + . . . + anAn

HHb = b1A1 + . . . + bnAn

COHB = c1A1 + c2A2 . . . + cnAn

MetHb = d1A1 + d2A2 + dnAn

 The primary purpose of determining O2Hb is to assess oxygen transport from

the lungs, it is best to stabilize the patient’s ventilation status before blood
sample collection.
 Blood Gas Analyzers: pH, pCO2 and pO2
 Blood gas analyzers use electrodes (macroelectrochemical or
microelectrochemical sensors) as sensing devices to measure pO2, pCO2, and
pH.
 The cathode can be defined in at least three ways:
a. negative electrode
b. a site to which cations tend to travel
c. a site at which reduction occurs
 Anode:
a. positive electrode
b. the site which anions migrate or site at which oxidation occurs.
 The blood gas analyzer can calculate several additional parameters: bicarbonate,
total CO2, base excess, and SO2.
A. Measurement of pO2:

 Also called as Clarke electrode

 It measure the amount of current flow in a circuit that is related to the

amount of oxygen being reduced at the cathode.

 The primary source of error for pO2 measurement is associated with the

buildup of protein material on the surface of the membrane.

 Contamination of the sample with room air (pO2 150 mm Hg) can result in
B. Measurement of pH and pCO2:
 To measure pH, a glass membrane sensitive to H is placed around an internal
Ag–AgCl electrode to form a measuring electrode.
 pCO2 is determined with a modified pH electrode, called a Severinghaus
electrode.
Type of Electrochemical Sensors:
 Macroelectrode sensors have been used in blood gas instruments since the
beginning of the clinical measurement of blood gases.

 Microelectrodes basically are miniaturized macroelectrodes.

 Thick and thin film technology is a further modification of electrochemical

sensors.
 Optical Sensors

 A technology for blood gas measurements is based on the fact that certain

fluorescent dyes will react predictably with specific chemicals.

Optical technology has been applied to indwelling blood gas systems.

Fiberoptic bundles - carry light to sensors positioned in the tip of

catheters.
 Calibration
 Temperature is an important factor in the measurement of pH and blood
gases.

 Nernst Equation - specifies the expected voltage output of an electrochemical

cell at a given temperature.

 The solubility of gases in a liquid medium also depends on the temperature:

 The pH electrode is usually calibrated with two buffer solutions TRACEABLE to

standards prepared by the National Institute of Standards and Technology
(NIST).

- One calibrator is near 6.8 and the other is near 7.38.

 Two gas mixtures are used for pCO2 and pO2

A. No O2 to set the zero point of the O2 electrode. The same gas has
approximately 5% CO2 because this is the null point for the CO2 electrode.

B. The other gas sets the gain.

 Most instruments are self-calibrating and are programmed to indicate a

calibration error if the electronic signal from the electrode is inconsistent with
the programmed expected value.
 Calculated parameters
● Several acid- base parameters can be calculated from measured pH and pCO2
values

-The calculation of HCO3 is based on the HendersonHasselbalch

equation

●Some clinicians use base excess to assess the nonrespiratory (metabolic)

component of a patient’s acid-base disorder.

-Positive value (base excess)

-Negative value (base deficit)

 Correction of Temperature

• Values for pH, pCO2, and pO2 are temperature dependent - 37 degree

Celsius
 QUALITY
ASSURANCE
 Preanalytic Considerations
 Blood gas measurements, like all laboratory measurements, are subject to
preanalytic, analytic and postanalytic errors.
 The steps included in the analytic area are under the direct control of the
laboratory.
 Start with proper patient identification

 Once collected the specimen must be correctly labeled and accompanied by

accurate information

Note: Only personnel who have experience with the drawing equipment and
technique and have knowledge of the possible sources of error should draw
samples for pH and blood gas analyses.
 Site of Choice in Drawing Blood Gas
Analysis
 Radial

 Brachial

 Femoral

— While arterial samples for pH and blood gas studies are recommended,
peripheral venous samples can be used if pulmonary function is not being
assessed.
 Sources of error in thecollection and handling of blood gas
specimens include:

 Collection device

 Form and concentration of heparin used for anticoagulation

 Speed of syringe filling

 Maintenance of the anaerobic environment

 Mixing of the sample to ensure dissolution and distribution of the heparin

 Transport and storage time before analysis

 Proper interpretation of blood gas results:

 The patient's status-ventilation and body temperature at the time of sample

collection must be documented.
 The ideal collection device for arterial blood sampling is a 1- to3-ml.
 Once drawn, the blood in the syringe must be mixed thoroughly with the
heparin to prevent microclots from forming.
 Adequate mixing is also important immediately before analysis to resuspend
the settled cells.
 Slow filling of the syringe may be caused by a mismatch of syringe and needle
sizes.
 Maintenance of an anaerobic environment is critical to correct results.

 Any air trapped in the syringe during the draw should be immediately
expelled at the completion of the draw.

Note: Evacuated collection tubes are not appropriate for blood gases.While
both dry and liquid heparin are acceptable anticoagulants
 Avoiding preanalytic errors
 The best practice in avoiding many of the preanalytic errors is to analyze the
sample as quickly as possible.
 Oxygen and carbon dioxide levels in blood kept at cool room temperatures
for 20 to 30 minutes or less are minimally affected except in the presence of
an elevated leukocyte or platelet count.
 The CLSI guidelines advocate samples be kept at room temperature and
analyzed in less than 30 minutes.
 Consideration should be given to the additional sources of preanalytic
errors for samples that are to be analyzed on multi-analyte instruments.
—For example, prolonged ice water slurry storage can result in falsely
elevated potassium in whole blood samples. Consult manufacturer
manuals for preanalytic considerations.
MEASUREMENT OF
SIGNIFICANT
PARAMETERS
 Parameters in the Assessment of Acid-Base
Balance
1. Evaluate the pH

Normal pH: 7.35 - 7.45

< 7.35 : ACIDOSIS

> 7.45 : ALKALOSIS

2. Evaluate the ventilation (LUNGS) : pCO2

Normal pCO2 : 35 - 45 mmHg

< 35 : Respiratory Alkalosis

> 45 : Respiratory Acidosis

3. Evaluate the metabolic process (KIDNEYS) : HCO3

Normal HCO3 : 21 - 28 mEq/L

< 21 : Metabolic Acidosis

> 28 : Metabolic Alkalosis

4. Evaluate the degree of oxygenation : pO2

Normal pO2 : 81 - 100 mmHg

Levels of Hypoxemia

▲ MILD : 61 - 80

▲ MODERATE : 41 - 60

▲ SEVERE : 40 or less
4. Evaluate the degree of oxygenation : pO2

 If the patient is with oxygen support, degree of oxygenation is

interpreted as corrected, uncorrected, or overcorrected
oxygention.
--> (81 - 100 mmHg)

 If the patient is without oxygen support, degree of oxygenation is

interpreted as normoxemia, or normal oxygenation ; mild,
moderate, severe hypoxemia.
Determine the degree of compensation

 non-compensatory; partial ; or complete/fully compensated

COMPENSATION pH pCO2 HCO3

Non- A N
A
compensatory N A
Partial A A A

Complete Nearly N A A
 SPECIMEN
COLLECTION AND
CONSIDERATIONS
 Safety and Disposal Considerations in
Specimen Collection
In all settings in which specimens are collected and prepared for testing,
laboratory and health care personnel should follow current recommended sterile
techniques, including precautions regarding the use of needles and other sterile
equipment. Treat all biological material as material that is potentially hazardous
as well as contaminated specimen collection supplies. For all those who are
involved in specimen collection and preparation, the responsibility to adhere to
current recommendations designed to maintain the safety of both patients and
health care workers does not end when the patient is dismissed.

There are four steps involved in obtaining a good quality specimen for
testing: (1) preparation of the patient, (2) collection of the specimen, (3)
processing the specimen, and (4) storing and/or transporting the specimen
 Preparation
Prior to each collection, review the appropriate test description, including
the specimen type indicated, the volume, the procedure, the collection materials,
patient preparation, and storage and handling instructions.
 Preparing the Patient. Provide the patient, in advance, with appropriate
collection instructions and information on fasting, diet, and medication
restrictions when indicated for the specific test.
 Preparing the Specimen. Verify the patient's identification. Proper identification
of specimens is extremely important. All primary specimen containers must be
labeled with at least two identifiers at the time of collection. Submitted slides
may be labeled with a single identifier, but two identifiers are preferred. patient's
name (patient's first and last name exactly as they appear on the test request
form), date of birth, hospital number, test request form number, accession
number, or unique random number.
 Avoiding Common Problems

Careful attention to routine procedures can eliminate most of the

potential problems related to specimen collection. Materials provided by the
laboratory for specimen collection can maintain the quality of the specimen
only when they are used in strict accordance with the instructions provided.
 Considerations In Specimen Collection:

• Avoid patient identification errors.

• Draw the tubes in the proper sequence.
• Use proper containers for collection.
• Mix all tubes ten times by gentle inversion immediately after
collection.
• Do not decant specimens from one type of container into
another.