HCG test is a pregnancy associated hormone commonly used to confirm pregnancy and ectopic

pregnancy. It is also used as a tumor marker for the management of gestational trophoblastic pathology.
The HCG test kit is a rapid chromatographic immunoassay for the qualitative detection of HCG in urine,
serum, plasma to aid an early detection of pregnancy. In this assay we use serum from a pregnant women
and serum from the non-pregnant women.
First, we test each specimen to different test kits by adding 3 drops of serum in each test kit and
wait for 5 minutes. The serum of pregnant women yields positive reaction by having a pink/red line in both
C and T line in the kit. While the serum from the non-pregnant women results negative by exhibiting only
one line in C line on the kit.
Then do a serial dilution from the serum of the pregnant women labeling each tube as 1:10, 1:100,
and 1:1000. Adding 900uL of diluent in each tube, then add 100uL of sample to 1:10, then get 100uL from
1:10 tube and transfer to 1:100, then get another 100uL from 1:100 tube to be transferred in 1:1000 tube.
Open 3 test kits and label each as 1:10, 1:100, and 1:1000. Then add 3 drops of specimen in each test kit
corresponding from what is labeled in the kit and wait for 5 minutes for the results. The 1:10 test kit yields
positive since there is a pink/red line in both C and T line, while for the 1:100 it is considered positive even
though the test kit have red/pink line in C line and faint pink line in T line, and lastly for 1:1000 results for
negative for having only one line in C line in the kit. If the line does not appear in C line the test is considered
invalid.

Calcium plays an important role in many cell functions intracellularly in muscle contraction, and glycogen
metabolism, extracellularly - bone medialization, blood coagulation, and in transmission of nerve impulses.
Decreased total calcium level can be associated in different disease of the bone apparatus especially
osteoporosis, kidney disease under dialysis, defective intestinal absorption, and hypothyroidism. Increased
total calcium can be measured in hyperparathyroidism, malignant disease with metastasis and sarcoidosis.
This measurement can also help in monitoring of calcium supplementation mainly in prevention of
osteoporosis. To start the test add 10uL of sample and add 1000uL of reagent then cover with parafilm and
invert to mix and incubate for 5 minutes. The result is 4mg/dL.

Test Calcium Measurement

Amount of sample 10uL
Amount of reagent 1000uL
Blank Reagent
Incubation time 5 minutes
Color of reagent blank Reddish purple
Color of sample Purple
Wavelength 623nm
Temperature 37C
Method 3
Standard 10
Old standard 11
Measured volume 450uL
Result 4mg/dL
Reference Range 8.6 -10.3 mg/dL
Calcium: Precautions:

Serum Calcium level is maintained at a constant 1. Only use disposable materials.

level for the optimal excitability of neural and
muscular tissue and the coordinated 2. Traces of chelating agent such as EDTA
functioning of various organ systems in the can prevent the formation of the
human body colored complex.

Serum/Plasma: 8.6-10.3 mg/dL (2.15 –2.57 HYPERCALCEMIA

mmol/L)
1. Disorders of Parathyroid Glands and
Urine: Women <250 mg/24h (6.24 mmol/24h) CSR’s

Men <300 mg/24h (7.49 mmol/24h) 2. Cancer-mediated hypercalcemia

Method: Photometric test using arsenazo III 3. Granulomatous Diseases

Principle: Calcium with arsenazo III at neutral 4. Medications

pH yields a blue colored complex, whose
5. Miscellaneous
intensity is proportional to the calcium
concentration. Interference by HYPOCALCEMIA
magnesium is eliminated by addition of 8-
hydroxyquinoline-5-sulfonic acid. 1. Endocrine Causes

Wavelength: 623 nm 2. Deficiency of Vitamin D

3. Hypomagnesemia
 LABORATORY ACTIVITY 8
Calcium

I. Desired learning outcomes

Calcium plays an essential role in many cell functions: intercellularly in muscle contraction and glycogen
metabolism, extracellularly in bone mineralization, in blood coagulation and in transmission of nerve
impulses. Calcium is present in plasma in three forms: free, bound to proteins, or complexed with ions
such as phosphate, citrate, and bicarbonate. Decreased calcium levels is associated with diseases of
bone apparatus, kidney diseases, defective intestinal absorption, and hypothyroidism. Increased calcium
levels can be measured in hyperthyroidism, malignant diseases with metastasis, and sarcoidosis.
After performing this activity, students should be able to:
1. correlate the clinical laboratory data with the possible pathologic conditions associated with
thyroid;
2. discuss factors that contribute to the alteration of calcium.

II. Materials
Serum/Heparinized plasma Calcium Reagent
Calcium Standard 10mg/dL Photometer

Test Principle:
Calcium with arsenazo III at neutral pH yields a blue colored complex whose intensity is proportional to
the calcium concentration. Interference by magnesium is eliminated by addition of 8-hydroxyquinoline-
5-sulfonic acid.
1. Label test tubes as STANDARD, TEST, and BLANK.
2. Pipette 1000 µL of calcium reagent to all tubes.
3. Add 10 µL of serum to respective tubes and mix.
4. Cover with parafim. Invert to mix.
5. Incubate for 5 minutes.
6. Read at 623 nm against reagent blank.
7. Record and interpret result using the reference values:

Serum/Plasma: 8.6 -10.3 mg/dL

Human Chorionic Gonadotropin (HCG) Urine HCG False Positive

□ Is produced by the trophoblast cells of the

placenta during pregnancy
□ It serves to maintain progesterone
production by the corpus luteum in the
early pregnancy
□ It can be detected 2 – 3 days after
ovulation

Qualitative test for urine samples has detection

limits 50 mIU/mL

Indicative of pregnancy/trophoblastic disease:

Urine HCG False Negative
>5 mIU/mL

Serum level during the first trimester of

pregnancy: >100,000 mIU/mL
 LABORATORY ACTIVITY 7
Human Chorionic Gonadotropin

I. Desired learning outcomes

Human chorionic gonadotropin (hCG) is a pregnancy-associated
hormone commonly used to confirm pregnancy and diagnosis of
ectopic pregnancy. It is also used as a tumor marker for the
management of gestational trophoblastic pathologies.
After performing this activity, students should be able to:
1. describe how hCG test be useful in confirming pregnancy,
diagnosing abnormal pregnancy, and pathology of
pregnancy;
2. state the principle of the method used; and
3. determine the laboratory protocol on HCG testing in order
to produce a qualitylaboratory result.

II. Materials

Freshly collected urine specimen of pregnant Test kit Test tube and rack
woman
Blood sample of the same pregnant woman Timer
Freshly collected Urine specimen from non- centrifuge
pregnant woman

Test Principle:

The One Step hCG Single Test Card is a rapid chromatographic immunoassay
for the qualitative detection of human chorionic gonadotropin (hCG) in
urine/serum/plasma 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 immersing the strip of the test card
in a urine/serum/plasma specimen 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 at the control line region if the test has been performed properly.
1. Collect fresh urine sample and blood sample from a woman who is pregnant. Ask data like
age, last menstrual period, and history of ectopic pregnancy. Do the same from a non-
pregnant woman.
2. Do serial dilution of the 2 samples (1:10; 1:100 and 1:1000) but
first do a qualitativetest.

3. Remove the testing device from the foil pouch by tearing at the notch and place in on a
flatsurface (Remember to open only the pouch when you are ready to perform the
procedure).
4. Holding a sample dropper vertically, add exactly four drops of the urine specimen to the
samplewell.
5. Read results in 5 minutes. Do not read result after more than 5 minutes.
6. Follow the same procedure to the diluted samples, one at a time.
7. Interpret the result
Negative Pink/Red band in the C area
Positive Pink/Red bands in both C and T areas
Invalid No Pink/Red band in the C area
 COLEGIO SAN AGUSTIN-BACOLOD
College of Health and Allied Professions MLS 111B LABORATORY ACTIVITY SHEET
Medical Technology Program

Test SGPT SGOT

Volume of Sample 100uL 100uL
Volume of working reagent 1000u/L 1000u/L
Working solution 4 parts of reagent 1 4 parts of reagent 1
1 part of reagent 2 1 part of reagent 2
Reagent 1 TRIS-pH 7.8, L-alanine, MDH, & TRIS-pH 7.8, L-alanine, MDH, &
LDH LDH
Reagent 2 2-oxaloglutarate and NADH 2-oxaloglutarate and NADH
Blank Air Blank Air Blank
Temperature 37C 37C
Unit u/L u/L
Wavelength 340 nm 340nm
Method 28 29
Factor 1745 1746
Delay 0 0
Delta 3 3
Time delta 60 seconds 60 seconds
Measured volume 450u/L 450 u/L
Delta 1 0.202 0.4016
Delta 2 -0.008 -0.008
Delta 3 0.013 0.017
Result 19u/L 23u/L

In preparing SGPT and SGOT we need to prepare the working solution. Then label each tube according
to its test, add 100u/L of sample. Add the working solution when the sample is read in the photometer
in 340nm against air blank. The result for SGPT is 19u/L and for SGOT 23u/L.

Enzymatic measurement reflects the condition of the organ producing the enzyme.
Transaminases are associated with the heart, liver and several other organs.

As liver specific enzyme, ALT(SGPT) is only significantly elevated in hepatobiliary

diseases. Increased AST(SGOT) levels, however, can occur in connection with damages of
heart or skeletal muscle as well as parenchyma. Parallel measurement of AST and ALT is
therefore applied to distinguish liver from heart or skeletal damages. The AST/ALT ration is
used for differential diagnosis in liver diseases.

After performing this activity, students should be able to:

1. identify the importance of the enzymatic reaction in the experiment conducted;

For CSA-B students use only. Prepared by Gemma B. de los Reyes & Mark P. Garcellan
 COLEGIO SAN AGUSTIN-BACOLOD
College of Health and Allied Professions MLS 111B LABORATORY ACTIVITY SHEET
Medical Technology Program
2. determine the clinical association of the results to the condition of the organ secreting
transaminases; and
3. explain why enzymes are reported in Katal or U/L.

I. Materials

Freshly collected serum sample Photometer Test tubes and rack

ALT reagent Pipettes

For CSA-B students use only. Prepared by Gemma B. de los Reyes & Mark P. Garcellan
 COLEGIO SAN AGUSTIN-BACOLOD
College of Health and Allied Professions MLS 111B LABORATORY ACTIVITY SHEET
Medical Technology Program

A. AST (SGOT)
Test Principle:
GOT
L-Aspartate + 2-Oxyglutarate Oxaloacetate + L-Glutamate

MDH
Oxaloacetate + NADH + H+ L-Malate + NAD+

*NADH is oxidized to NAD, the resulting decrease in absorbance at 340nm is directly proportional to the activity
of GOT in the sample.

Assay Procedure:
1. Prepare working reagent by combining 4 parts of reagent 1 (TRIS-pH 7.8, L-alanine, MDH, & LDH) and 1 part
of reagent 2(2-oxaloglutarate and NADH).
2. Label test tube as TEST.
3. Pipet 100 µL specimen to this test tube.
4. Add 1000 µL of working reagent 1 to this tube.
5. Invert to mix and read immediately against air blank at 340 nm.
6. Add only working reagent if it is your turn to read.
7. Record end color and result.
8. Reference ranges:
Men <35 U/L
Women <31 U/L

B. ALT (SGPT)
Test Principle:
GPT
L-Alanine + 2-Oxyglutarate Pyruvate + L-Glutamate

LDH
Pyruvate + NADH + H+ L-Lactate + NAD+

*NADH is oxidized to NAD, the resulting decrease in absorbance at 340nm is directly proportional to the activity
of GPT in the sample.

Assay Procedure:
1. Prepare working reagent by combining 4 parts of reagent 1 (TRIS-pH 7.5, L-alanine, LDH) and 1 part of
reagent 2(2-oxaloglutarate and NADH).
2. Label test tube as TEST.
3. Pipet 100 µL specimen to this test tube.
4. Add 1000 µL of working reagent 1 to this tube.
5. Invert to mix and read immediately against air blank at 340 nm.
6. Add only working reagent if it is your turn to read.
7. Record end color and result.
For CSA-B students use only. Prepared by Gemma B. de los Reyes & Mark P. Garcellan
 COLEGIO SAN AGUSTIN-BACOLOD
College of Health and Allied Professions MLS 111B LABORATORY ACTIVITY SHEET
Medical Technology Program
8. Reference ranges:
Men <45 U/L
Women <34 U/L

For CSA-B students use only. Prepared by Gemma B. de los Reyes & Mark P. Garcellan