2. OUTLINE
• Introduction
• Indications for Calcium analysis
• Patient preparation & specimen
collection
• Methodologies
• Brief history of methods
• Routine / Field methods
• Definitive / Reference methods
• Conclusion
• References
2
3. INTRODUCTON
• Calcium, an alkaline earth metal in the periodic table, is a
divalent cation needed as an essential element in the
human body; where it is found 99% as hydroxyapatite
complex in bone and 1% in soft tissues and blood.
• Intracellularly, calcium functions in muscle contraction,
hormone secretion, coenzymes in intermediary metabolism
and cell division; while extracellular calcium is essential for
blood coagulation and cell membrane excitability.
3
4. …hypocalcemia Symptoms of
hypocalcemia
Nausea and
vomiting
Frequent
urination
Increased thirst
Constipation
Abdominal pain
Loss of appetite
• Hypoparathyroidism,
Malabsorption,
Osteomalacia, Pancreatitis,
Renal failure, Rickets and
vitamin D deficiency, Liver
disease (decreased albumin
production), Low serum
magnesium.
INDICATIONS FOR CALCIUM ESTIMATION 4
5. …indications in hypercalcemia
Symptoms of
hypercalcemia
Nausea and
vomiting
Frequent
urination
Increased thirst
Constipation
Abdominal pain
Loss of appetite
• Hyperparathyroidism,
Hyperthyroidism, Metastatic
bone tumor, Milk-alkali
syndrome, Vitamin D/Lithium
toxicity, Excessive calcium
intake, Prolonged
immobilization, Multiple
myeloma, Thiazide diuretics,
Tumors producing a PTH-like
substance, Paget's disease,
Sarcoidosis.
5
6. PATIENT PREPARATION AND SPECIMEN
COLLECTION
• Sample: heparinized plasma (preferable), serum or
whole blood. Whole blood specimen should be
analyzed within 30 mins – 1 hour of collection at room
temperature, or within 4hrs when stored at 4ºC.
• Acid – washed test tubes should be used for calcium
analysis, to eradicate the effect of domestic soaps
forming insoluble complexes with the calcium in
sample.
6
7. • Ideally, no tourniquet should be use for
venipuncture for Calcium analysis, as it increases
total calcium by 012 – 0.25 mmol/L. If in use for
localization of vein, tourniquet should be released
after gaining venous access by needle/canula.
• This is because venous occlusion by tourniquet
dams the vessels, causing efflux of water from
intravascular compartment increase in protein –
bound Ca2+ during stasis.
7
8. …Patient prep & sample collection
• Changes in posture from sitting to standing causes
fluid shift within 10 mins, decreases intravascular
water, increases albumin conc. and thus total Ca2+
by up to 0.2 mmol/L.
• Fist clenching, should be avoided during phlebotomy
as it causes lactic acidosis and elevated free
Ca2+.
8
9. • Hemolysis: cause dilution by cell contents and can
also cause spectral interferences, hence, emptying of
collected sample with small bore needles into
vacuum tubes should be avoided.
• Prolonged immobilization and bed rest increase bone
resorption increased total and free calcium.
• Exclude drug use: prolonged Antacids, Vitamin D,
Lithium, Thiazide diuretics and Thyroxine can cause
increased plasma calcium.
9
10. • Early in the historical pathway of calcium methodology,
gravimetry and visual titrimetry were used as main
analytical techniques for the determination of calcium in
biological fluids.
• Advancement to modern era began with the development
of colorimetric complexing indicators to determine the
endpoints of those titrations, and the use of photometric
devices to read-off optical density of the complexes at
endpoints.
METHODOLOGIES 10
11. …history
• Historical trends in determination of calcium in body fluids
were: Gravimetry Titrimetry Molecular absorption
spectrophotometry & Fluorometry Atomic absorption
spectrophotometry Ion selective electrode
Automation techniques.
• The quantitative determination of serum calcium began with
gravimetry. As early as 1871, Pribham precipitated calcium
directly from serum with ammonium oxalate and then weighed
the washed precipitate. This was unrefined and wroth with a
lot of errors, as impurities easily affected the results.
11
12. …history
• Kramer and Tisdall (1884) discovered a then advanced
system in which the washed calcium oxalate was
dissolved with acid, and the oxalate moiety was titrated
at an unstated warm temperature with standardized
potassium permanganate to a self indicated
endpoint…read-of visually.
• Diehl Labs (1912) developed the metallochromic
indicators. This was followed by the introduction of
photometric devices into calcium analysis.
12
13. ..history
• Because of the subjective nature of the visual method, this
led to the substitution of the spectrophotometer for the
human eye for analyzing the complex, in the titration of
serum calcium.
• Then came other spectral analysis such as Fluorometry,
but these has been less sensitive than the photometric
methods with metallochromic indicators used recently in
routine calcium analysis.
13
14. Routine Laboratory /
Field methods
Reference / Definitive
methods
Enzymatic: o-
Cresolphthalein
Complexone (o-CPC),
Arsenazo III
Ion Selective Electrode
(ISE)
Reference: Atomic
absorption
spectrophotometry; AAS
(CLSI approved)
Definitive: Isotope dilution
– mass spectrometry; ID-
MS (NIST developed)
14
15. Enzymatic: o-Cresolphthalein Complexone method
• In alkaline medium, o-Cresolphthalein
Complexone (CPC) forms a red chromophore
with Calcium…measured spectrophotometrically
between 570 and 580 nm against a reagent
blank.
15
16. …CPC
• Because calcium exists in complexes with other
physiologic anions such as proteins, phosphate and
sulphate, sample is diluted with acid to release
protein-bound and complexed calcium, for total
calcium to be assayed.
• Organic base, most often diethylamine or
ethylaminoethanol is added to buffer the reaction and
produce an alkaline pH for the CPC reaction to take
place.
16
17. …CPC
• Interference by magnesium can be removed either
by:
adding 8-hydroxyquinoline,
buffering the reaction mixture to near pH 12, or
measuring the optical density near 580 nm
• Urea may be added to reduce turbidity of lipemic
specimens.
17
18. Enzymatic: Arsenazo III method
• The compound Arsenazo III, at mildly acidic pH, has
more affinity for calcium than magnesium, and binds
it to produce an intense purple complex, measured at
650 nm against reagent blank.
18
20. …Arsenazo III
• Operating at a pH of about 6.0, Arsenazo III much
more stable as a single reagent than CPC, and has
an accuracy of 97+/-2% when compared to the
definitive method: Isotope dilution – mass
spectrometry.
• Ethylene diamine tetracetic acid (EDTA) and Citrate
have been seen to be a negative interference.
20
21. ION SELECTIVE ELECTRODE (ISE)
• Membrane potentials are caused by the permeability of
certain types of membranes to selected anions or
cations.
• Calcium ISEs contain a calcium-selective membrane
which encloses an inner reference solution of calcium
chloride often containing saturated AgCl and
physiologic concentrations of NaCl/KCl, and an internal
reference electrode (usually Ag/AgCl).
21
22. …ISE
• The electrochemical
cell is completed by
the external
reference electrode:
an Ag/AgCl or
calomel electrode,
which is in contact
with the specimen by
a liquid/liquid junction
or a salt bridge.
22
23. …ISE
• The potential difference produced at the membrane –
sample interface is proportional to the logarithm of the ionic
activity, and related to the concentration of the ion in
question (Ca2+), via Nernst equation:
23
24. …ISE
• Modern calcium ISEs use liquid membranes containing
organophosphate sensors, i.e. ion-selective calcium
sensor dissolved in an organic liquid trapped in a
polymeric matrix.
• Within the ISE, measurement of Total & Free calcium takes
two steps: the available free calcium is first measured, then
the specimen is acidified to convert protein – bound and
complexed calcium to free calcium, for total calcium to be
measured by the ISE.
24
25. …ISE
• ISE machine uses 75 – 150
uL of sample for
analysis…an average of 25
uL for each analyte.
• Results are displayed as
ready-to-use values on a
read-out screen, or may be
printed out for use directly.
25
27. • The Clinical Laboratory & Standards Institute
(CLSI) approved atomic absorption
spectrophotometry as the reference method for
measuring total calcium.
• Principles: the ground state calcium atom absorbs
light energy at 422.7 nm as it enters the excited
state.
…AAS 27
28. …AAS
• As the number of calcium atoms in the light path
increases, the amount of light absorbed also
increases.
• By measuring the amount of light absorbed, a
quantitative determination of the amount of calcium
present can be made.
28
29. …AAS
• 0.5 % Lanthanum solution is added to each
standard, control and sample to prevent chemical
and ionization interference.
• This method has been compared with ID – MS
(the definitive method), and seen to have a very
high accuracy.
29
30. ISOTOPE DILUTION – MASS SPECTROMETRY
• Basic principle of isotope dilution: the addition of an
isotopically altered standard to the sample changes the natural
isotopic composition of the analyte.
30
31. This altered /
labelled sample is
then coupled to a
mass spectrometer
for high accuracy
measurement.
By measuring the
resulting isotopic
composition, it is
possible to
calculate the
amount of the
analyte present in
the sample.
31
32. …ID – MS
• A mass spectrometer generates multiple ions from the sample
under investigation, separates them according to their specific
mass-to-charge ratio (m/z), and then records the relative
abundance of each ion type.
Schematic representation of mass spectrometry
32
33. …ID – MS
• The method of isotope dilution coupled to mass
spectrometry is regarded in clinical chemistry
measurement methods as having the highest
metrological standing, and is used as the definitive
method for analyzing almost all analytes in clinical
chemistry.
33
35. Conclusion
• Measurement of calcium is important in the management of certain
abnormality states with alteration in calcium homeostasis.
• Calcium status in a patient is more accurately determined by
measuring free calcium, which is the tightly regulated,
biologically active form, although total calcium is also
important when adjusted with albumin concentration.
• A knowledge of procedures and methodology for assaying
calcium is imperative in ensuring proper management of
patients by clinicians.
35
36. REFERENCES
• Tietz Clinical Chemistry and Molecular Diagnostics, 5th Ed
• Bishops Clinical Chemistry – Principles, Techniques, Correlations, 7th Ed.
• Cowley, D.M. et al. Improved linearity of calcium – cresolphthalein
complexone reaction with sodium acetate. Clin. Chem., 32: 894-5, 1986.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612271/
• Dito, W.R. Microdetermination of serum calcium by parallel fast analyzer. Am.
J. Clin. Pathol., 65: 1016-21, 1976.
https://emedicine.medscape.com/article/874690-overview
36
….to prevent lactic acidosis formation by anaerobic glycolysis
1.
Of the metallochromic indicators (dyes) that form change colors on selectively binding with Ca2+, CPC & Arsenazo III are the most widely ued.
CLSI – Clinical Lab & Standards Institute
NIST – Nat’l Institute of Standards & Technology
2. As in patients with recent citrated blood transfusion.
Isotopes are variants of a particular chemical element which differ in neutron number.
