BACHELOR OF SCIENCE IN MEDICAL

LABORATORY SCIENCE:
CLINICAL CHEMISTRY 2
COURSE MODULE LABORATORY ACTIVITY SESSION

5 4 5

CALCIUM DETERMINATION

Group Members: Agraviador, Suzanne Romero, Charles Baldwin

Centeno, Conrado Saldivar, John Rafael
Hernandez, Ivan Salvador, Andreana Khielle
Laforga, James John Vanguardia, Angelic
Section: MEDTECH 3-YC-IRR2 Group: 2 Date: July 25, 2021
CALCIUM DETERMINATION
1. What are the main functions of calcium in the body?
The following are the functions and importance of calcium in the body:

• Calcium is the 5th most common element and the most prevalent cation in the body
• Calcium comes in 3 forms, the highest form (50%), protein bound (40%) which is bound in plasma
protein and the complex form with anion (10%)
• Calcium is important in skeletal mineralization
• Calcium preserves the cell membrane's integrity and permeability particularly in terms of sodium and
potassium exchange
• Calcium is involved in glandular synthesis and regulation of exocrine and endocrine glands
- In addition, calcium plays a vital role in the following:

• Blood coagulation
• Neural transmission
• Enzyme activity
• Maintenance of normal tone
 • Maintenance of normal bone
• Excitability of skeletal and cardiac muscle

2. State the principle involved in the manual procedure of calcium determination.

To generate a compound with Calcium (Ca2+), the two most often used techniques for total Ca2+
analysis use either ortho-cresolphthalein complexone (CPC) or arsenazo III dye. Ca2+ is freed from its
protein carrier and complexes prior to the dye-binding process by acidifying the sample. To avoid Mg2+
interference, the CPC technique employs 8-hydroxyquinoline. Although it is rarely used in clinical settings,
Atomic Absorption Spectrophotometry (AAS) remains the gold standard for total Ca2+ measurement.
ISEs are currently used in commercial analyzers that measure ionized/free Ca2+. Membranes
impregnated with specific compounds that selectively but reversibly bind Ca2+ ions could be used in these
systems. As Ca2+ binds to these membranes, an electric potential develops across the membrane that is
proportional to the ionized Ca2+ concentration.

3. Give the clinical significance of serum calcium values.

Total calcium reference values vary slightly with age, with younger individuals having higher
concentrations due to bone growth. These values generally decrease as they grow older.

Reference Ranges for Calcium:

Total Calcium Ionized Calcium
Serum Plasma Urine Serum Plasma Whole Blood
Adult 2.15-2.50 2.15-2.50 2.50-7.50 1.16-1.32 1.03-1.23 1.15-1.27
mmol/L (8.6- mmol/L (8.6- mmol/L mmol/L (4.6- mmol/L (4.1- mmol/L (4.6-
10.0 mg/dL) 10.0 mg/dL) (100-300 5.3 mg/dL) 4.9 mg/dL) 5.1 mg/dL)
Child 2.20-2.70 2.20-2.70 mg/d) 1.20-1.38 - -
(<12y) mmol/L (8.8- mmol/L (8.8- mmol/L (4.8-
10.8 mg/dL) 10.8 mg/dL) Note: Varies 5.5 mg/dL)
with diet

When calcium levels are decreased, it is referred to as hypocalcemia. There are numerous causes for
hypocalcemia such as: Primary hypoparathyroidism, hypomagnesemia, hypermagnesemia, hypoalbuminemia,
acute pancreatitis, vitamin D deficiency, renal disease, rhabdomyolysis, and pseudohypoparathyroidism.
Neuromuscular irritability and cardiac irregularities are common symptoms indicating hypocalcemia. Symptoms
occur usually when there is severe hypocalcemia, where total Ca2+ values are below 1.88 mmol/L (7.5 mg/dL).

On the contrary, high levels of calcium indicate hypercalcemia, which the causes include: Primary
hyperthyroidism, hyperthyroidism, malignancy, multiple myeloma, increased vitamin D, Thiazide diuretics, and
prolonged immobilization. Mild hypercalcemia (2.62-3.00 mmol/L [10.5-12 mg/dL]) is often asymptomatic but
more than that may result in renal, neurologic, and GI symptoms. Neurologic symptoms may include mild
drowsiness, weakness, depression, lethargy, and coma. GI symptoms may include constipation, nausea,
vomiting, anorexia, and peptic ulcer disease. Lastly, renal symptoms may cause nephrolithiasis and
nephrocalcinosis.
 Causes of Hypercalcemia Causes of Hypocalcemia
Primary hyperparathyroidism—adenoma or Primary hypoparathyroidism—glandular aplasia,
glandular hyperplasia destruction, or removal
Hyperthyroidism Hypomagnesemia
Benign familial hypocalciuria Hypermagnesemia
Malignancy Hypoalbuminemia (total only, ionized calcium not
Multiple myeloma affected by)—chronic liver disease, nephrotic syndrome,
Increased vitamin D malnutrition
Thiazide diuretics Acute pancreatitis
Prolonged immobilization Vitamin D deficiency
Renal disease
Rhabdomyolysis
Pseudohypoparathyroidism

4. Explain the reciprocal relationship between calcium and phosphate.

• The amount of phosphate in the blood affects the level of calcium in the blood. Calcium and phosphate in
the body react in opposite ways: as blood calcium levels rise, phosphate levels fall. A hormone called
parathyroid hormone (PTH) regulates the levels of calcium and phosphorus in your blood. When the
phosphorus level is measured, a vitamin D level, and sometimes a PTH level, is measured at the same time.
Vitamin D is needed for your body to take in phosphate.
The relation between calcium and phosphate may be disrupted by some diseases or infections. For this
reason, phosphate and calcium levels are usually measured at the same time.
• Calcium - The most common mineral in the body. Calcium is essential for healthy bones and teeth. It is
also important for controlling heart function, blood clotting, and muscle functioning, such as contraction
and relaxation. Calcium levels are often abnormal in people with kidney disease. Raised calcium levels may
cause headaches, nausea, sore eyes, aching teeth, itchy skin, mood changes, and confusion.
• Phosphate - A mineral that, together with calcium, keeps your bones strong and healthy. Too much
phosphate causes itching and pain in the joints, such as the knees, elbows and ankles. When the kidneys
are not functioning properly, high levels of phosphate accumulate in your blood.
 COURSE MODULE LABORATORY ACTIVITY SESSION

5 5.1 5

HCG DETERMINATION

HCG DETERMINATION

1. Give the principle of urine pregnancy test.

The hCG Card Pregnancy Test is a rapid chromatographic
immunoassay for the qualitative detection of human chorionic
gonadotropin in urine to aid in the early detection of pregnancy. The
test utilizes a combination of antibodies including a monoclonal hCG
antibody to selectively detect elevated levels of hCG. The assay is
conducted by adding a urine specimen to the specimen well of the
test device and observing the formation of colored lines. The
specimen migrates via capillary action along the membrane to react
with the colored conjugate.
Positive specimens react with the specific antibody-hCG-colored conjugate to form a colored line at
the test line region of the membrane. Absence of this colored line suggests a negative result. To serve as a
procedural control, a colored line will always appear in the control line region indicating that proper volume of
specimen has been added and membrane wicking has occurred.

2. Enumerate other possible specimen for pregnancy testing aside from urine.
Aside from urine, blood may also be a specimen for pregnancy testing. Using blood as a sample offers
a 99% accuracy in detecting pregnancy. It can detect lower amounts of HCG than urine pregnancy test and can
detect even before you missed a period.
In addition, there are 2 types of blood pregnancy test:
• Quantitative blood pregnancy test that can measure exact amount of HCG in the blood
• Qualitative blood pregnancy test that checks presence of HCG, however, it can’t measure exact HCG

Blood should be collected aseptically into a clean tube without anticoagulants. Separate the serum from
the blood as soon as possible to avoid hemolysis. Use clear non-hemolyzed specimens when possible.

Serum or urine specimens may be stored at 36-46°F (2-8°C) for up to 48 hours prior to testing. For
prolonged storage, specimens may be frozen and stored below -4°F (-20°C). Frozen specimens should be
thawed and mixed before testing.
3. What are the possible cases aside form pregnancy which produces a positive result in urine pregnancy
testing?
Aside from pregnancy, the following are the possible cases of positive result in urine pregnancy testing:
• Recent miscarriage or abortion
During pregnancy, hCG levels continue to rise as the placenta grows, doubling every few days
and peaking at around 10 weeks. When a pregnancy ends, hCG levels begin to recede, but it’s a slow
process. The hormone can remain in your blood and urine for up to six weeks following the end of the
pregnancy. It’s possible to have a false-positive test until your hCG levels return to their prepregnancy
state.
• User error
Even with these safeguards, user error can occur. One of the most common mistakes is taking the
test too early during your cycle. This can cause either a false negative or a false positive. It’s also important
to use the test when your urine isn’t diluted excessively with water. Use the test when your urine is very
concentrated, like when you first wake up in the morning
• Evaporation lines
Sometimes an evaporation line can be mistaken for a positive pregnancy test. Some at-home tests
show two lines when hCG is detected and one line when hCG isn’t detected. Evaporation lines may show
up on a test you view after your urine has evaporated completely. Sometimes they’re caused by hormonal
levels that don’t represent pregnancy.
• Medications
Other medications can also cause false-positive pregnancy tests. They include but aren’t limited
to:
• anti-anxiety medications, like diazepam (Valium) or alprazolam(Xanax)
• antipsychotics, such as clozapine or chlorpromazine
• anticonvulsants, like phenobarbital or other barbiturates
• Parkinson’s disease medications, including bromocriptine (Parlodel)
• diuretics, like furosemide (Lasix, Diuscreen)
• antihistamines, including promethazine
• methadone (Dolophine)
• Certain Medical Conditions
Rarely, certain medical conditions can cause a home pregnancy test to give a false positive. These
include:

• urinary tract infection

• kidney disease that causes blood or white blood cells in the urine
• ovarian cysts, particularly corpus luteum cysts
• more serious diseases, such as ovarian cancer
• pituitary problems (very rarely)
• Ovarian and testicular cancer
• Gestational trophoblastic diseases
 COURSE MODULE LABORATORY ACTIVITY SESSION

5 5.2 5

ALCOHOL DETERMINATION

ALCOHOL DETERMINATION
1. The Saliva alcohol test strip only provides a preliminary result for the relative Blood Alcohol
concentration (BAC). What should be performed to provide a confirmatory result for BAC?
Gas chromatography should be performed to provide a confirmatory result for BAC.
Gas chromatography is used to separate mixtures of volatile or potentially volatile substances. GC can be
either gas–solid chromatography (GSC) or gas–liquid chromatography (GLC) with a nonvolatile liquid stationary
phase. In clinical laboratories, GLC is often utilized. The setup is similar to HPLC, with the exception that the
mobile phase is a gas and samples are divided into a gaseous mobile phase and a liquid stationary phase. Nitrogen,
helium, or argon can be used as the carrier gas. The detector utilized in the device determines the carrier gas to
use. If a sample contains components with varying volatilities, the instrument can be set to run at a constant
temperature or designed to run at different temperatures. This is analogous to the HPLC gradient elution method.
The sample must be injected as a gas or the injection port temperature must be above the boiling point of
the components so that they vaporize upon injection. The sample vapor is swept through the column in two
phases: partially as a gas and partially as a liquid. Volatile chemicals that are mostly present in the gas phase have
a low partition coefficient and travel fast across the column. Higher boiling point compounds will travel more
slowly through the column. The effluent flows through a detector, which generates an electric signal proportional
to the volatile component concentration. The chromatogram, like HPLC, is used to identify chemicals based on
their retention time and to quantify their concentration based on the area under the peak.
2. Interpreting the results for rapid tests such as the saliva alcohol test strip has subjective variability.
Enumerate the factors which influence the interpretation of results. What measures should be taken to
avoid misinterpretation of results?

The following are the factors that influence the interpretation of the results for rapid saliva
alcohol test strip:
• Color perception
• Presence or absence of inhibitory factors
• Lighting
Measures to avoid misinterpretation:

• Atlas Saliva Alcohol Test Strip (Saliva) provides only a preliminary result for the relative BAC. A
secondary analytical method must be used to obtain a confirmed result. Gas chromatography (GC) is
the preferred confirmatory method.
• Failure to wait 15 minutes after smoking or placing food, drink, or other non‐alcoholic
materials in the mouth before performing the test can produce erroneous results due to
possible contamination of the saliva by interfering substances.
• Interpretation of visual results is dependent on several factors: the variability of color
perception, the presence or absence of inhibitory factors, and the lighting conditions when the
 strip is read. Caution should be taken when interpreting test results due to the subjective nature
of the test.
• Atlas test strip should not be used to determine the presence of alcohol in beverages, in
undiluted alcohol, or in other liquid solutions.
• Atlas test strip is highly sensitive to the presence of alcohol. Alcohol vapors in the air are
sometimes detected by the test strip. Alcohol vapors are present in many institutions and
homes. Alcohol is a component in many household products such as disinfectant, deodorizers,
perfumes, and glass cleaners. If the presence of alcohol vapors is suspected, the test should be
performed in an area known to be free of vapors.
• Ingestion or general use of over‐the‐counter medications and products containing alcohol
such as cold medicines, breath sprays and mouthwashes can produce positive results. Wait at
least 20 minutes after ingesting any such products before using the test strip.

3. Give 5 examples of substances which could interfere with the Saliva Alcohol Test Strip
The following are the examples of substances which could interfere with the Saliva Alcohol Test Strip:

• Peroxidases
• Ascorbic Acid
• Uric acid
• Bilirubin
• L-dopa

References:

• Bishop, M. L. (2020). Clinical Chemistry: Principles, Techniques, and Correlations, Enhanced Edition: Principles, Techniques,
and Correlations, Enhanced Edition (8th ed.). Jones & Bartlett Learning.

• Gallieni, M., & Cozzolino, M. (2008). Calcium and phosphate: serum levels versus body balance. Kidney
International, 74(2), 246. https://doi.org/10.1038/ki.2008.176

• Phosphate in Blood Test | Michigan Medicine. (2021, March 31). University of Michigan Health.
https://www.uofmhealth.org/health-
library/hw202265#:%7E:text=The%20amount%20of%20phosphate%20in%20the%20blood%20affects%20
the%20level,and%20phosphorus%20in%20your%20blood

• PREGNANCY TESTING URINE AND SERUM | Student Health Center Manuals. (2007, November 1).
Student Health Center Manual. http://shs-manual.ucsc.edu/policy/pregnancy-testing-urine-and-serum

• Volpicellim, Joseph R., M.D., Ph.D.: Alcohol Dependence: Diagnosis, Clinical Aspects and Biopsychosocial
Causes, Substance Abuse Library, University of Pennsylvania, 1997

• Whelan, C. (2017, May 11). 7 Causes for a False-Positive Pregnancy Test. Healthline.
https://www.healthline.com/health/pregnancy/false-positive-pregnancy-test#medical-conditions
 EXERCISE: DETERMINATION OF ALCOHOL

Group Members: Agraviador, Suzanne Romero, Charles Baldwin

Centeno, Conrado Saldivar, John Rafael
Hernandez, Ivan Salvador, Andreana Khielle
Laforga, James John Vanguardia, Angelic
Section: MEDTECH 3-YC-IRR2 Group: 2 Date: July 25, 2021

or

PATIENT’S NAME: James John Laforga SEX: Male

AGE: 21
1. Color change on reaction pad of Saliva Alcohol Test Strip:
% O - 08%
-

EB •oEE8
2. Interpretation of Result:

POSITIVE ; 0.08%

NEGATIVE: No color change appears on the reaction pad. The color should
match the color block on the pouch corresponding to a negative (-) result. This
indicates that alcohol has not been detected.
POSITIVE: A color change appears on the reaction pad. The BAC will range
from 0.02% to 0.30%, with the color on the reaction pad varying from a light blue
to dark blue, falling on or between the corresponding color blocks on the pouch.
INVALID: The outer edges of the reaction pad produces a slight color but
the majority of the reaction pad remains colorless. Repeat the test using a new
test strip, ensuring complete saturation of the reaction pad with saliva.