ELECTROLYTE TESTING

Supernatant - 20 uL 20 uL
SODIUM from Step 1
• Most abundant CATION in EXTRAcellular R1 20 uL - -
fluid (ECF) [cation – positive charge] Mix well and allow it to stand at room temperature for 5
• SPECIMEN: Serum, Plasma (lithium minutes. Measure the absorbance of standard and sample
heparin, ammonium heparin, and lithium against reagent blank.
oxalate), Sweat and 24hr Urine [Hemolysis TEST PARAMETER
does not cause significant change, bcoz Mode of Reaction End Point
sodium is the most abundant cation in ECF, decrease Slope of Reaction Increasing
levels intracellular. However, if MARKED HEMOLYSIS Wavelength 546nm
sodium levels may be decrease as a result of dilutional Temperature 30°c
effect] [Whole blood sample may be used in other Standard Concentration 150 mmol/L
analyzer only depends manual of acceptability of Linearity 200 mmol/L
machine] [Sweat – Sweat Electrolyte Test (also called Blank Reagent
Iontophoretic sweat test/ Chloride sweat test)– detects Incubation Time 5 min
the amount of sodium & chloride in sweat, primarily used Sample Volume 10 uL
in px. w/ symptoms of CF/Cystic Fibrosis]
Reagent Volume (R1)1mL + (R2)1mL
• METHODS:
Cuvette 1 cm light path
1. Flame Emission Spectrophotometry (FES):
REFERENCE RANGE
yellow Serum/Plasma: 128-160 mmol/L
[Measure light emitted by excited of atoms, widely used to
determine concentration of Sodium, Potassium and POTASSIUM
Lithium; no longer used in CCLab] • Major INTRAcellular CATION
2. Ion Selective Electrode (ISE) • COLLECTION of SAMPLES:
[used different membranes/ electrodes; Not directly 1. Serum K may be 0.1-0.7 mmol/L higher
concentration is being measure but the Activity of Ion] than plasma K concentration [if sample is
o Most routinely used clotted, the coagulation process is activated
o Glass Aluminum Silicate and releases potassium from platelets; Serum –
o Source of error: protein buildup on the membrane
coagulated; px w/Thrombocytosis/ elevated platelets
through continuous use [protein-coated membranes
count serum potassium may be further elevated]
cause poor selectivity, which results in poor
reproducibility of results] 2. Tourniquet is left on the arm too long or if patients
o 2 Types of ISE Based on Sample Preparation excessively clench their fists or otherwise exercise
▪ Direct – provides undiluted sample to interact w/ their forearms before venipuncture [Tourniquet - to avoid
ISE membranes which is the glass aluminum used of heparinized tube to prevent clotting]
silicate; more accurate to use 3. Whole blood samples for K determinations should be
▪ Indirect – uses diluted sample stored at room temperature (never iced) and analyzed
[no significant difference between direct & indirect promptly or centrifuged
result except if the sample is Hyperlipidemic/ 4. If hemolysis occurs after the blood is drawn, K may be
Hyperproteinemic can displace plasma water falsely elevated - the most common cause of artifactual
causing false decrease] hyperkalemia
3. Atomic Absorption Spectrophotometry (AAS)
• SPECIMEN:
o Serum, Plasma, and 24hr Urine [24hr – to
REAGENT COMPOSITION
eliminates the diurnal variation of K]
Component Description
o Hemolysis must be avoided
Sodium R1 Uranyl acetate
o Anticoagulant of choice: Heparin
Magnesium acetate
• METHODS:
Sodium R2 Ammonium thioglycolate
1. Flame Emission Spectrophotometry (FES): violet
Ammonia
2. Ion Selective Electrode (ISE)
Sodium Standard Sodium standard
concentration o Common method
o Valinomycin Gel
PROCEDURE
3. Atomic Absorption Spectrophotometry (AAS)
Step I-Precipitation Laboratory Procedure for
Semi Auto Analyzer REAGENT COMPOSITION
Sodium R1 (Precipitating Standard Sample Component Description
Reagent) 1000 uL 1000 uL Potassium Reagent Sodium tetraphenylboron
Sodium Standard 10 uL - Potassium Standard Potassium Standard
Sample - 10 uL PROCEDURE
Shake vigorously and incubate at room temperature for 5 Laboratory Procedure for Semi Auto Analyzer
minutes. Then centrifuge at 2000-3000 RPM for 2 minutes
Standard Sample
to obtain a clear supernatant. Transfer the supernatant
Potassium Reagent 1000 uL 1000 uL
immediately after centrifugation for standard and test.
Step II- Sodium Estimation Standard 25 uL -
Sodium R2 Blank Standard Sample Sample - 25 uL
(Colour Mix well and allow it to stand at room temperature for 5
1000 uL 1000 uL 1000 uL minutes. Measure the absorbance of standard and sample
Reagent)
against distilled water at 578nm within 10 mins.

MONTEMOR, DJ 7
 TEST PARAMETER 2. Sample (lonized Ca2+)
Mode of Reaction End Point o samples must be collected anaerobically [loss of CO2
Slope of Reaction Increasing will increase the pH of sample]
Wavelength 578nm o Preferred sample: heparinized whole blood [can’t
Temperature 30°c use liquid heparin such as sodium heparin and
Standard Concentration 5 mmol/L lithium heparin, will partially bind calcium and will
Linearity 14 mmol/L lower the ionized calcium concentration]
Blank DI/Distilled water o serum from sealed evacuated blood-collection tubes
Incubation Time 5 min may be used if clotting and centrifugation are done
Sample Volume 25 uL quickly (<30 minutes) and at room temperature
3. Ca2 in urine
Reagent Volume 1000 uL
Cuvette 1 cm light path o Acidified with 6mol/L HCI, with approximately 1mL of
the acid added for each 100 mL of urine
REFERENCE RANGE
Serum/Plasma: 3.6-5.5 mmol/L • METHODS:
1. Total Ca2+ Analysis
o Ortho-cresolphthalein complexone (CPC) or
CHLORIDE Arsenzo III dye
• Major EXTRAcellular ANION [Prior to the dye-binding reaction, Ca 2+ is released
• SPECIMEN: from its protein carrier and complexes by
1. Serum, Plasma, Sweat and 24hr Urine acidification of the sample]
2. Anticoagulant of choice: Lithium Heparin ▪ form a complex with Ca2
▪ CPC method: uses 8-hydroxyquinoline to
prevent Mg2 interference
[Chloride is the negative charge ionic form of Chlorine] o Atomic Absorption Spectrophotometry (AAS):
• METHODS: reference method [rarely used]
1. Mercurimetric Titration: Schales & Schales 2. Ionized/ Free Ca2+
CI + Hg2 + → HgCl2 o lon Selective Electrode (ISE)
Excess Hg2+ + Diphenylcarbazone = Blue [use membranes impregnated with special molecules that
2. Ion Selective Electrode (ISE) selectively, but reversibly, bind Ca 2+ ions. As Ca 2+
o Common method binds to these membranes, an electric potential develops
o Ion Exchange Membrane across the membrane that is proportional to the ionized
REAGENT COMPOSITION Ca 2+ concentration.]
Component Description REAGENT COMPOSITION
Chloride Reagent Mercuric (II) thiocyanate Component Description
Nitric Acid R1 Calcium Base Reagent O-Cresolphthalein complex
Ferric Nitrate 8-Hydroxyquinoline
PROCEDURE R2 Calcium Dye Reagent Diethylamine
Laboratory Procedure for Semi Auto Analyzer PROCEDURE
Blank Standard Sample Laboratory Procedure for Semi Auto Analyzer
Working Reagent 1000 uL 1000 uL 1000 uL Blank Standard Sample
Standard - 10 uL - Working 1000 uL 1000 uL 1000 uL
Sample - - 10 uL Reagent
Mix and incubate for 1 min. Measure the absorbance of Standard - 10 uL -
standard and sample against reagent blank Sample - - 10 uL
TEST PARAMETER Mix and incubate for 5 min. at room temperature. Read the
Mode of Reaction End Point absorbance of standard and sample against reagent blank.
Slope of Reaction Increasing TEST PARAMETER
Wavelength 505nm Mode of Reaction End Point
Temperature RT Slope of Reaction Increasing
Standard Concentration 100 mEq/L Wavelength 578nm
Linearity 130 mEq/L Temperature 30°c
Blank Reagent Standard Concentration 10 mg/dL
Incubation Time 1 min Linearity 15 mg/dL
Sample Volume 10 uL Blank Reagent
Reagent Volume 1000 uL Incubation Time 5 min
Cuvette 1 cm light path Sample Volume 10 uL
REFERENCE RANGE Reagent Volume 1000 uL
Serum: 97-108 mEq/L Cuvette 1 cm light path
Urine: 120-240 mEq/L/24 hr REFERENCE RANGE
Serum: 8.8 – 10.2 mg/dL
CALCIUM Urine: 100-400 mg/24 hrs
• MOST ABUNDANT ION in the whole body
• SPECIMEN:
1. Preferred specimen (Total Ca2+): Serum
or Plasma (lithium heparin) [EDTA & oxalate
anticoagulant can bind the calcium and
interfere with the measurement]

MONTEMOR, DJ 8
 ACID - BASE
o removal of carbon is low compared of rate of
• TERMINOLOGY production = lungs should exhale the carbon dioxide;
o Acid if dse state increased of rate of production increased
▪ Chemical compound capable of donating H+ ▪  CO2 in blood = hydrogen=  pH – acidic
o Base o removal of carbon is high compared of rate of
▪ Chemical compound capable of accepting H+ production
o Buffer ▪ CO2= hydrogen= pH - Alkaline
▪ A weak acid/base and its conjugate salt • Regulation of Acid Base Balance
▪ Minimizes pH changes • Rule of thumb: H+ HCO3 <-> H2CO3 <-> CO2 + H2O
▪ HCO3 (bicarbonate) and H2CO3(Carbonic o Increased either side, the buffer system increased
acid): serve as buffers in the blood – considered also other side to maintain equilibrium
as the most important buffer pair in the blood
- decreased pH = acidic - high hydrogen concentration
- increased pH- low hydrogen proportion
• Buffer Systems: Regulation of H+
o Bicarbonate-Carbonic acid System
▪ first line of defense against the changes in
hydrogen concentration
▪ low buffering capacity

• Regulation of Acid Base Balance: Kidneys

o proximal tube – site of bicarbonate reclamation
o if kidney is defective the lungs will compensate and
vice versa

(1) H2CO3 dissociates into CO2 and H2O, allowing

CO2 to be eliminated by the lungs and H as water
(2) Changes in CO2 modify the ventilation (respiratory)
rate
(3) HCO3 concentration can be altered by the kidneys. • Henderson Hasselbalch Equation

• Regulation of Acid Base Balance

o pKa=constant (6.1)
o TCO2 - H2CO3
o pCO2 x 0.03
o TCO2 - (pCO2 x 0.03) [expanded formula]
• Arterial Blood Gas Reference Range
o pH: 7.35-7.45
▪ <7.35= acidosis
▪ >7.45= alkalosis
o pCO2: 35-45 mmHg
▪ lungs
▪ <35= respiratory alkalosis
▪ >45= respiratory acidosis
o HCO3: 22-26 mEq/L
• Regulation of Acid Base Balance: Lungs ▪ kidneys
▪ <22= metabolic acidosis
▪ >26= metabolic alkalosis
o pO2: 81-100 mmHg

• ACID BASE IMBALANCES [mnemonic = RO-ME

(Respiratory Opposite, Metabolic Equal)]
Acid-Base Imbalance pH pCO2 H2CO3 HCO3
Respiratory Acidosis    N
Respiratory Alkalosis    N
Metabolic Acidosis  N N 
Metabolic Alkalosis  N N 

MONTEMOR, DJ 9
• PRIMARY COMPENSATION The potential that
develops at the glass
o Respiratory membrane as a
▪ Acidosis: Kidneys retain HCO3 and excrete H+ result of H from the
▪ Alkalosis: Kidneys excrete HCO3 and retain H+ unknown solution
o Metabolic diffusing into the
membrane’s surface
▪ Acidosis: Hyperventilate (blow off CO2) is proportional to the
▪ Alkalosis: Hypoventilate (retain CO2) difference in H
o Partially Compensated Acidosis/ Alkalosis between the
unknown sample
▪ When compensatory mechanism/s are evident and the buffer
but normal normal pH is not yet restored. solution inside the
o Fully Compensated Acidosis/ Alkalosis electrode.
▪ When compensatory mechanism/s have pCO PCO2 An outer
2 electrode semipermeable
succeeded in restoring normal pH but other (Severinghaus) membrane that
parameters are not at their normal level. allows CO2 to diffuse
• EXAMPLE into a layer of
electrolyte, usually a
Acid-Base Imbalance pH pCO2 H2CO3 HCO3 bicarbonate buffer,
Metabolic Alkalosis  N N  covers the glass pH
Partially Compensated     electrode. The CO2
that diffuses across
Fully Compensated N    the membrane reacts
with the buffer,
Acid-Base Imbalance pH pCO2 H2CO3 HCO3 forming carbonic
acid, which then
Respiratory Acidosis    N dissociates into
Partially Compensated     bicarbonate plus H.
Fully Compensated N    The change in
activity of the H is
measured by the pH
BLOOD GAS ANALYSIS electrode and related
to PCO2
• SPECIMEN COLLECTION/ CONSIDERATIONS
o Specimen: arterial blood or arterialized capillary
blood
o Anticoagulant
▪ Heparin (0.05 mg/mL blood) liquid- used
▪ never use EDTA/ citrate/ other anticoagulants
[alter pH]
o Transportation
▪ Within 30 minutes
▪ Ice water [prevent oxygen consumption by the
RBC as well as release of acidic metabolites]
• INTERFERENCES
ERROR EFFECT REMEDY
Specimen exposed to air False Tightly cap
ALKALOSIS
Unprocessed specimen False Ice water
>30 minutes at RT ACIDOSIS
Carry-Over VARIABLE Wash machine
Uncalibrated machine VARIABLE Calibrate
Excess protein build-up VARIABLE Clean
at electrode
Presence of air bubble False Expel
ALKALOSIS
Too much heparin False Blood
ACIDOSIS anticoagulant
ratio

INSTRUMENTATION: PO2, PCO2 AND PH

ELECTRODE/ Measur Principle
Instrument es
pO2 pO2 electrode Change AMPERO- A gas permeable
(Clarke) in METRIC membrane covering
current the tip of the
electrode selectively
allows the O2 to
diffuse into an
electrolyte and
contact the cathode.
Electrons are drawn
from the anode
surface to the
cathode surface to
reduce the O2.
pH pH electrode POTENTI- A glass membrane
Reference: Voltage OMETRIC sensitive to H is
1. Calomel change placed around an
[Hg-HgCl] due to internal Ag-AgCl
2. Ag-AgCl H+ electrode to form a
half-cell measuring electrode.

MONTEMOR, DJ 10