National Autonomous University of Mexico Delivery date

09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

Summary
The qualitative determination of the human chorionic gonadotropin hormone is the
test known worldwide as a pregnancy test. The results of these tests contribute
significantly to diagnostic decision-making. Chorionic gonadotropin is a
glycoprotein hormone, with a molecular weight of approximately 37,000 daltons,
produced by trophoblastic cells of the placenta during pregnancy. Two units of the
glycoprotein called alpha and beta are distinguished, the alpha unit being identical
to that of the luteinizing hormone LH and the beta subunit differing from LH by the
presence of 30 amino acids in the carboxy terminus that do not exist in LH. HCG is
secreted 6 to 8 days after conception, increasing rapidly to a peak of 50,000 to
200,000 mIU/ml in the 6th to 8th week. For this qualitative determination of HCG, a
urine sample was required from an anonymous patient who was in the 1st trimester
of pregnancy with a confirmed diagnosis of pregnancy. Two immunoassays were
carried out, the first test was carried out using a commercial reagent strip by
placing a drop of urine at the application point and after 10 minutes two blue bands
were observed, indicating a positive test for the presence of chorionic
gonadotropin. human above the sensitivity of the test strip. The second
immunoassay was performed by the direct agglutination method with latex particles
which contained specific antibodies to the human chorionic gonadotropin antigen.
A drop of urine from the donor and a drop of urine from a healthy male donor were
placed as a negative test. In different parts of the observation plate, a drop of the
latex reagent with antibody was placed and left to react for 10 minutes. After this
time, the precipitation of the latex particles on the observation plate was observed

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 1
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

considerably. In the urine sample of the anonymous patient, no agglutination was

observed in the observation plate of the male donor. This result confirms the
presence of human chorionic gonadotropin in the sample of the anonymous donor,
above the sensitivity levels of the method used.

Theoretical framework:

hCG measurements have many other clinical applications. They are used to
screen prenatal maternal serum for Down syndrome and in the treatment of
pregnancy-related complications, including miscarriage, ectopic pregnancy, benign
gestational trophoblastic disease (hydatidiform mole), and 3 molar pregnancy. 4.
hCG is also an important serum tumor marker, particularly for testicular and other
germ cell cancers 1, 2, 3, 4. Additionally, hCG measurement is important in sports
medicine. The hormone is on the World Anti-Doping Agency (WADA) list of
prohibited substances (http://www.wada-ama.org/Documents/World_Anti-
Doping_Program/WADP-Prohibited-list/2014/WADA-prohibited-list -2014-EN.pdf)
because it can be abused by male athletes, either to mask the effect of anabolic
steroids, or to increase testosterone production.

hCG measurements have generally been useful in meeting the needs of physicians
and patients in a wide spectrum of applications, as summarized in Table 1.
However, there is a need for more accurate measurement, description, and
reporting of hCG results to minimize the risk of analytical error or clinical
Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 2
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

misinterpretation leading to an adverse patient outcome. Continued causes of

confusion include lack of clarity in nomenclature, particularly the continued use of
the term “βhCG.” This terminology was originally used to differentiate hCG-specific
radioimmunoassays (RIA) from early hCG RIA that cross-reacted with luteinizing
hormone (LH), but can be misinterpreted to mean that only the free hCGβ subunit
is being measured. Clinically, lack of clarity about which isoforms should be
measured under certain clinical conditions can also cause confusion. Analytically, a
lack of knowledge of clinically relevant interferences (e.g., high-dose binding in the
presence of a marked excess of antigen or the presence of heterophilic
antibodies), and a lack of knowledge about how best to measure the most clinically
relevant isoforms of hCG, can also be problematic. Addressing these issues
requires universal adoption of unambiguous nomenclature, evidence-based
guidance on which hCG variants should be measured in different clinical
applications, and knowledge of how to select antibodies and optimize methods to
meet these requirements. In this review, which focuses on the measurement of
hCG in pregnancy but also addresses its use in oncology, we describe how the
outcome of three major international workshops. Addressing these issues requires
the universal adoption of unambiguous nomenclature, evidence-based guidance
on which hCG variants should be measured in different clinical applications, and
knowledge of how to select antibodies and optimize methods to meet these
requirements. In this review, which focuses on the measurement of hCG in
pregnancy but also addresses its use in oncology, we describe how the outcome of
three major international workshops. Addressing these issues requires the

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 3
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

universal adoption of unambiguous nomenclature, evidence-based guidance on

which hCG variants should be measured in different clinical applications, and
knowledge of how to select antibodies and optimize methods to meet these
requirements. In this review, which focuses on the measurement of hCG in
pregnancy but also addresses its use in oncology, we describe how the result of
three major international workshops 6, 7, 8, 9, 10 will help reduce the future risk of
damage to the patient due to erroneous or misinterpreted hCG results.

Structure of hCG and related variants

What is measured as 'hCG' is actually a complex mixture of closely related

heterogeneous glycoprotein variants. Bioactive intact hCG itself is a heterodimer
consisting of non-covalently linked α and β subunits (hCGα and hCGβ). The α
subunit, encoded by a single gene, is common to all glycoprotein hormones. The
hCGβ genes have evolved from an ancestral human LH β subunit (hLHβ) gene
through gene duplications and mutations. hCGβ confers molecular specificity,
differentiating hCG from LH, follicle-stimulating hormone (FSH), and thyroid-
stimulating hormone (TSH) 12, 13, 14. Genomics, biochemically, and
immunologically, these four hormones are members of a family of glycoproteins
that share structural homology with the cystine knot growth factor superfamily. Two
twisted β-sheet loops (Ł1 + 3) protrude from one side of the central cystine knot
(ck) of each subunit and a single second loop (Ł2) protrude from the opposite side
(box 1, Figure I, Figure II). hCGβ differs from hLHβ as it incorporates an extended
carboxyl-terminal peptide (hCGβCTP, amino acid residues 112 – 145) [12].
Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 4
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

Metabolic variants include nicked hCG (hCGn), nicked hCGβ (hCGβn), hCGβ core
fragment (hCGβcf), and truncated hCGβCTP variants (-CTPhCG and -CTPhCGβ)

PRINCIPLE OF THE METHOD (test strip)

The hCG present in the sample connects to the monoclonal antibody-dye

conjugate making an antibody-antigen complex. It flows through the absorbent
area

of the test strip/test plate being connected to the anti-hCG antibody in the area of
the positive reaction (T), determining the emergence of a light pink-colored band if
the concentration of hCG in the sample is greater than 25 mIU /ml.

In the absence of hCG there will be no appearance of the colorful band in the T
area. The reaction mixture continues to flow reaching the control area (C). The
conjugate not connected to the antigen binds to the reagents in this area producing
a light pink-colored band, showing that the reagents are working correctly.

Qualitative determination of the hormone human chorionic gonadotropin (hCG)

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 5
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

PRINCIPLE OF THE METHOD (latex particle agglutination)

The hCG-Latex test is a slide agglutination technique for the direct qualitative
detection of hCG in urine. Latex particles coated with anti-hCG monoclonal
antibody (Note 1) are agglutinated by hCG molecules present in the patient
sample.

CLINICAL SIGNIFICANCE

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 6
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

hCG is a hormone secreted by the placenta of the pregnant woman that appears
relatively soon in the blood and urine after implantation of the fertilized embryo. It
can be detected in urine from the third day after the loss of the menstrual period
and its concentration continues to increase until reaching very high levels after 10
weeks of gestation.
PROCEDURE

1. Temper the reagents and samples to room temperature. The sensitivity of the
assay decreases at low temperatures.

2. Place 100 µL of the sample to be tested and one drop of each of the Positive
and Negative controls on different circles on a slide.

3. Mix the hCGlatex reagent vigorously or vortex before use. Place a drop (50 µL)
next to each of the previous drops.

4. Mix the drops with a toothpick, trying to spread the mixture over the entire inner
surface of the circle. Use different toothpicks for each sample.

5. Place the slide on a rotary shaker at 80–100 rpm for 2 minutes.

READING AND INTERPRETATION

Examine macroscopically for the presence or absence of agglutination immediately

after removing the slide from the shaker. The presence of agglutination indicates
an hCG concentration equal to or greater than 200 IU/L.

LIMITATIONS OF THE PROCEDURE

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 7
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

- Pituitary hormones such as FSH and LH cross-react with hCG when their
concentration levels are very high.

- A negative result does not exclude the existence of a pregnancy. It is

recommended to repeat the test a few days later.

- Urine from patients with trophoblastic diseases such as carcinoma or hydatidiform

cyst could cause false positive results. [3]

Goals.

1. Determine the importance of hormonal changes in pregnancy.

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 8
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

2. Know the agglutination technique for pregnancy tests.

3. Using the agglutination technique, check the presence of hCG

Material, reagents and equipment :

-Stereoscope
-Pregnancy test
-Reagent kit for hCG determination
-Urine sample (first trimester of pregnancy)
-
Results:

Fig 1. Test: Inhibition of agglutination of latex particles.

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 9
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

Fig 2. Test: Chromatographic immunoassay.

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 10
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

Analysis of results

In practice, two simple tests were carried out for the determination of hCG in a
pregnancy test, the first the inhibition of the agglutination of latex particles and the
second the immunochromatographic assay. The presence of gonadotropin in
maternal plasma and its excretion in urine are the basis of pregnancy studies
because this hormone reaches its maximum level in the first trimester of
pregnancy.

In the agglutination inhibition, the urine was mixed with the antibodies against hCG
and for the positive test, no agglutination occurred when adding the hCG-coated
latex particles since the hormone reacted with the antibody, while in the negative
test the Antibody reacts with latex particles producing agglutination.

In the case of the chromatographic immunoassay, it was carried out using a

reagent strip (which consists of 3 antibodies Ac1, Ac2 and Ac3) that when
immersed in the urine, it migrates by capillary action to react with Ac1 forming a
complex which was dragged away. to Ac2 forming a new junction, a colored line
appearing, and the antibody Ac1 that did not bind to hCG migrated to Ac3 forming

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 11
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

the second line. It is important to note that hCG is not the only hormone present in
pregnancy, there are others that are equally important.

Other types of hormones can be found during pregnancy, secreted by the placenta:
placental lactogen (hLP) or human chorionic somatomammotropin (hCS), high
concentration from the 5th week of gestation until the end of pregnancy, its main
function is to alter availability of energy such as maternal lipolysis and fatty acid
metabolism to save glucose, insulin resistance in the mother to save glucose for
the fetus and stimulation of breast growth and development. Progesterone is also
found having inhibition of the smooth muscle of the uterus to prevent premature
expulsion and estrogens having functions in the metabolism and growth of the
mammary gland, growth of the uterus and high blood flow.

Conclusion

The tests carried out are important, quick and simple, since you are certain of a
positive or negative result if you are pregnant. These tests are carried out between
three and six months of pregnancy since this is where the hCG hormone can be
found. , which is what is present in pregnancy. It is carried out between this time
since after three months it reaches its maximum level and after six months its
concentration is low.

Two methods were performed for the qualitative determination of chorionic

gonadotropin, agglutination inhibition and the chromatographic immunoassay. In
the first test, the physical difference in the urine of a 6-month pregnant woman and
a non-pregnant woman was observed, by adding a drop of antibody solution and
latex particles that in the case of Non-pregnancy as there was no hCG in the urine,
the antibody reacts with the latex particles covered with hCG which will produce
agglutination and in the case of Pregnancy is when we can find hCG in the urine,

Members: Belmar Portillo Gibran, Mendoza Mendez Gabriel, Reyes Padilla Ivan Jahaziel, Weir
Reyes Melissa Page 12
National Autonomous University of Mexico Delivery date
09/06/2018
Faculty of Higher Studies Zaragoza
Activity: Endocrinology of pregnancy Responsible
Advisor: Carolina
Group: 1503 BLOCK 1 TEAM 5 PRACTICE 5 Sauer Ramirez
Lab: L-404 Module: Cellular and tissue
biology II

since it will react with the antibody and agglutination will not occur when adding the
coated latex particles with hCG.

In the second process, a test strip is used for the early diagnosis of pregnancy.
Two test lines are used, one of which is a combination of the hCG monoclonal
antibody for its detection and the control line, which is composed of polyclonal
antibodies and colloidal particles. By having both marked it is

Conclusion:

References.

1.Sources Arderiu 51 / 261 Clinical Laboratory Codex: Indications and

Interpretation of Laboratory Tests. Madrid: Elsevier; 2003.

2.González de Buitrago J. Technology and methods of the clinical laboratory.

Barcelona: Salvat; 1992.

3. Silverthorn, DE. Human physiology. A Pan-American Medical Officer; 2008.

4. Costanzo LS. Physiology. Barcelona, Spain: Wolters Kluwer/Lippincott Williams

& Wilkins; 2007.

5. Cunningham FG, Williams Obstetrics. 21st Edition. Spain: Editorial Médica

panamericana, 202.

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