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Clinical- Chemistry-2- Laboratory- Finals (1) 230522 2007 12

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Clinical Chemistry 2 (MDT 3122L)

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CLINICAL CHEMISTRY 2 (Laboratory)

College Of Medical Laboratory Science Our Lady of Fatima University – Pampanga FINALS LESSON 1: DETERMINATION OF HUMAN CHORIONIC GONADOTROPIN (HCG)

ADVANCED QUALITY ONE STEP PREGNANCY TEST

  • This test is a rapid qualitative immunochromatographic assay. Rapid because you will simply need to drop sample of a urine in the test cassette and is able to determine the presence of early pregnancy.

DETERMINATION OF HUMAN CHORIONIC

GONADOTROPIN (HCG)

  • Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by the developing placenta shortly after fetilization. o It is produced by syncytiotrophoblast of placenta (syncytiotrophoblastic cells of the placenta)
  • In normal pregnancy, hCG is detected in urine and serum soon after conception and levels of 5 - 50 mIU/mL are seen within 1 week of implantation. At the time of first missed menstrual period, hCG concentration in urine and serum are about 100mIU/mL, peak levels of 100,000-200,000 mIU/mL reached at the end of first semester.
  • Elevated urine and serum hCG are observed not only in pregnancy but also in some malignant conditions.
  • Elevated urine and serum hCG are observed also in trophoblastic or non-trophoblastic neoplasm, like hydatidiform mole, and choriocarcinoma. o Hydatidiform mole, and choriocarcinoma are examples of Gestational trophoblastic disease (GTD) o This type of neoplasm is actually a tumor that derives from the syncytiotrophoblast.
  • Lower levels of hCG hormone may be associated with placental insufficiency, threatened spontaneous abortion, and ectopic pregnancy. o Placental Insufficiency – in this condition, oxygen and nutrients are not sufficiently transported to the growing fetus via the placenta. o Threatened Spontaneous Abortion – when we say spontaneous abortion, that’s the pregnancy loss before 20 weeks of gestation. o Ectopic Pregnancy – the fertilized egg was implanted outside the uterus.

PRINCIPLE

  • Advanced Quality Pregnancy Test is a sandwich immunoassay. o It involves the interaction of the antibody and antigen.
  • The test has a sensitivity of 25 mIU/mL hCG for urine, which is sufficient to detect pregnancy the first day of the missed period. o In first day of missed period the concentration of hCG can reach up to 100mIU/mL. o The test can also detect lower than 100.
  • The sample is applied to the card and reacts initially with the specific, anti-beta hCG monoclonal antibody/colloidal gold conjugate on the test membrane. The mixture moves along the membrane, by capillary action, and reacts with a specific anti-hCG in the test region.
  • If hCG is present in the sample, the result is the formation of a colored band in thet test region. If there is no hCG in the sample, the area will remain white. o There are two regions in the test casettes: ▪ Test Region (indicator that hCG is present in the sample) ▪ Control Region (for quality control to determine if the cassette is really working)
  • The sample continues to flow to the control region and forms a pink to purple color, indicating the test is working and the result is valid.

MATERIALS:

  • Urine specimen cups
  • Pregnancy test kit

STORAGE AND STABILITY:

  • The test device can be stored under refrigeration and room temperature ( 2 - 30ºC) and will be stable until the expiration date. o You cannot use the test kit beyond the expiration date.

SAMPLE COLLECTION:

  • Collect the urine in a clean, dry glass or plastic container. o Urine specimens can be collected at any time of the day. o It is not necessary to obtain the first-morning specimen However, the concentration of the hCG may be higher in this specimen.
  • Sample can be refrigerated up to 72 hours prior to testing. o Refrigerated samples must be allowed to warm to room temperature and mix before testing

PROCEDURE:

  1. Bring all materials and specimen to room temperature
  2. Remove test card from the sealed foil pouch.
  3. Place the test card on a flat dry surface.

UNIT OUTLINE

I. Determination of Human Chorionic Gonadotropin (hCG) II. Determination of Alcohol III. Drug Test Specimen Collection and Accessioning IV. Determination of Methamphetamine and Tetrahydrocannabinol V. Determination of Barbiturates VI. Determination of Cocaine VII. Determination of Morphine VIII. Determination of Methadone IX. Principle for Drugs and Abuse Determinations

  1. Using provided plastic dropper, dispense 100uL of urine sample (3 drops) to the sample well of the test card. Start timing.
  2. Read result between 3 - 10 minutes after adding the sample.

INTERPRETATION OF RESULTS:

NEGATIVE

  • Only one colored band appears on the control region “C”

POSITIVE

  • In the addition to the control band, a distinct pink/ purple colored band also appears on the test region “T” o 2 lines. One in the control region and one on the test region. o There are conditions that may also give a positive result with the pregnancy test kit:

▪ trophoblastic or non-trophoblastic

▪ if the urine is diluted (diluted urine may contain or may not contain an sufficient level of the hCG)

INVALID

  • If there are no pink/purple bands in the control region. o If it’s invalid, you need to retest in a new device/test kit

LESSON 2: DETERMINATION OF ALCOHOL

BLUE SCREEN ONE STEP SALIVA ALCOHOL TEST

STRIP

  • It is a rapid saliva test for the determination of relative Blood Alcohol Concentration (BAC)

ALCOHOL

  • Alcohol is consumed by two-thirds of adults. Especially in the form of ethanol by drinking alcoholic beverages. It has been well established that the concentration of alcohol in saliva is comparable to that in the blood. o Saliva is a filtered blood. It is an ultrafiltrate of the blood. It also reflects the level of alcohol in the blood.
  • It is metabolized by alcohol dehydrogenase in the liver and produces acetaldehyde, then acetic acid by aldehyde dehydrogenase as its by- product. It affects the central nervous system and is among the leading causes of intoxication-related road accidents. (Alcohol (Aldehyde Dehydrogenase) Dehydrogenase)

Ethanol ------------------> Acetaldehyde ----------------->

Acetic Acid

  • Acetaldehyde is the one responsible for hangover syndrome

PRINCIPLE

  • It is a chemical assay based on an alcohol sensitive enzymatic reaction. Alcohol, if present in the saliva specimen, reacts with chemicals on the reaction pad and causes a color change. o We use a test strip and at the end of a test strip, it has a reaction pad which is the one that we will saturate with saliva. o Specimen: Saliva
  • It is consists of a plastic strip with a reaction pad attached at the tip. The reaction pad employs a solid phase chemistry system which uses a highly specific enzyme reaction. On contact with solutions of alcohol, the reaction pad will rapidly change colors depending on the concentration of alcohol present. This color change is proportional to the concentration of alcohol in the saliva. By comparing with the color blocks on the color chart printed on the pouch, an approximate Blood Alcohol Concentration (BAC) can be determined.

Control Region

Test Region

  • Some over ‐the ‐counter medications and products containing alcohol such as cold medicines, breath sprays and mouthwashes can produce false positive results. Wait at least 20 minutes after ingesting any such products before using the test strip
  • Assay Specificity: o The Blue Screen One Step Saliva Alcohol Test Strip (Saliva) will react with methyl, ethyl, and allyl alcohols. LESSON 3: DRUG TEST SPECIMEN COLLECTION AND ACCESSIONING

DRUG TESTING

  • The best practices for drug testing in the Philippines are based on the general principles that have been established internationally. They are designed to ensure that the entire drug testing process is conducted to give accurate and reliable information about client/donor/subject’s drug use, as mandated by Republic Act 9165 otherwise known as “Comprehensive Dangerous Drugs Act of 2002”.
  • The East Avenue Medical Center is designated as the National Reference Laboratory (NRL) for environmental and Occupational Health, Toxicology, and Micronutrient Assay by virtue of Department Order No. 393 - E s. 2000.

TYPES OF SPECIMEN FOR COLLECTION

SPECIMEN REASON FOR

COLLECTION

MINIMUM

QUANTITY

URINE Pre-employment, random, reasonable suspicion/cause, mandatory 60 mL SALIVA (ORAL FLUID) Pre-employment, random, reasonable suspicion/cause 2 mL SCALP HAIR Pre-employment, random, return to duty, follow up 100 mg or equivalent number of strands

CHAIN OF CUSTODY

  • A laboratory shall use documented chain of custody procedures to maintain control and accountability of specimens. The date and purpose shall be recorded on an appropriate Custody and Control Form each time a specimen is handled or transferred and every individual in the chain shall be identified.

ACCESSIONING

  1. Provide a unique accession number upon entry of the specimen to the laboratory;
  2. Inspect the specimen submitted and the CCF to verify the integrity and identity of the specimen.
  3. Examine the packaging for evidence of tampering in transit; 4. Compare the information on the sample bottles within the package; and 5. Document all discrepancies.

DRUG TEST SPECIMEN COLLECTION

  • As mandated by Republic Act 9165, specimens which, will be tested for the presence of substances of abuse should be collected following the standard set forth by the National Reference Laboratory under the Department of Health.

MATERIALS:

  • Zip-lock plastic containers
  • Wide mouth plastic urine containers
  • Analytical balance

PROCEDURE:

1. URINE

a. Collect 60ml into a clean, dry wide-mouth plastic container. b. Label as follows: o Name: o Age: o Sex: o Volume: o Date Collected: o Time Collected: o Signature of authorized specimen collector: o Signature of client: o Storage: Prolonged storage - 20 ̊C

2. SCALP HAIR PROLONGED STORAGE

a. Weigh 100 mg hair or its equivalent in number of strands centimeters above the scalp o Name: o Age: o Sex: o Weight: o Date Collected: o Time Collected: o Signature of authorized specimen collector: o Signature of client: o Storage: Cool, dry place

3. SALIVA (ORAL FLUID)

a. Collect 2 ml in a dry, clean plastic container avoiding inclusion of food debris. o Name: o Age: o Sex: o Volume: o Date Collected: o Time Collected: o Signature of authorized specimen collector: o Signature of client: o Storage: Frozen at - 8 to - 10 ̊C

DRUG TESTING ACCESSIONING

1. DRUG TESTING CONSENT FORM

  • Fill up the drug testing consent form. It will be filled up by the client or donor of the sample
  • Counseling is included here before doing the test. Explaining the purpose of the procedure o Positive results o The things to do after having a positive result

2. CUSTODY AND CONTROL FORM

  • This part will be completed by the collector or employer representative. NOTES:
  • Drug test to be performed: o THC – Tetrahydrocannabinol (marijuana) o COC – Cocaine o PCP – Phencyclidine o OPI – Opiates o AMP – Ampethamine

3. DRUG TEST REPORT

4. RECEIVING/ACCESSIONING/RELEASING

LOGBOOK

PROCEDURE:

  1. Answer the following forms to be utilized in drug testing if you were the client: a. Drug testing consent form b. Custody and control form (Copy for the screening laboratory) c. Custody and control form (Copy for the donor) d. Custody and control form (Copy for the collection site) e. Custody and control form (Copy for the confirmatory)
  2. Answer the following forms to be utilized in drug testing if you were the analyst: a. Custody and control form (Copy for the screening laboratory) b. Custody and control form (Copy for the donor)| c. Custody and control form (Copy for the collection site) d. Custody and control form (Copy for the confirmatory e. Drug test result
  3. Answer the following forms to be utilized in drug testing if you were the accessioner. a. Custody and control form (Copy for the screening laboratory) b. Custody and control form (Copy for the donor) c. Custody and control form (Copy for the collection site)

NOTE:

  • In the test kit, there are two T regions. o T1: MET o T2: THC LESSON 5: DETERMINATION OF BARBITURATES TAKE NOTE:
  • All procedures in the following lessons are qualitative tests. It means that the result will only be positive or negative.
  • The principles of all the methods in the following lessons are lateral flow chromatographic immunoassay. In this principle, the urine sample is simply dropped in the sample well of the kit test. In the kit test, there is a membrane where the urine will flow. Impregnated to that membrane are the antibodies that will react with the dangerous drugs that are possibly present in the urine.

BLUE SCREEN ONE STEP BAR TEST DEVICE

  • Upon the receipt of laboratory specimen in the laboratory, the medical technology should check the label (name, age, sex, date and time of collection) attached to the specimen container together with the requisition slip. o Compare the label and the requisition slip if it has the same content with the request form. o Make sure that the client filled up the consent form because in every drug testing, there must be a consent form.
  • He should verify the proper patient preparation, collection procedure and transportation of specimen to the patient. Sample received should be noted to appropriate logbooks in the laboratory for documentation purposes. o It is part of chain custody. From the time that you collect the specimen up to the time that you released the result, it must be documented in the laboratory log book.
  • Barbiturates are sedatives, hypnotics, anti-convulsants, which are also CNS depressants. o Sedatives are “pampakalma”. o Hypnotics cause sleepiness or “pampaantok o pampatulog”.
  • Chronic use of Barbiturates leads to tolerance to physical dependence. o When we say tolerance, the old dose of barbiturates you use, just like an anti- convulsants, it increases the dosage that you need for it be effective. If it is only the usual dosage, it may not work. That’s why it increases the dosage up to the point that you’re already dependent in that drugs. o When we say “drug dependence”, it means there are physical activities that you can’t do without taking the barbiturates. And when you stop taking the drug, it may cause withdrawal syndrome.
  • Barbiturates taken at 400 mg/day for 2 - 3 months produce significant dependence. The withdrawal symptoms of drug abstinence can be severe enough to cause death. o For example, you are already dependent in barbiturates and all of a sudden you stop, tendency because of the withdrawal syndrome, it can cause death.
  • Barbiturates are unaltered in the urine and could be detected for 4 - 7 days.

REAGENTS FOR BARBITURATES DETERMINATIONS:

  • The test device contains mouse monoclonal anti- barbiturates antibody coupled particles and Barbiturates-protein conjugate. A goat antibody is employed in the control line system. o Mouse monoclonal anti-barbiturates antibody coupled particles is the one that will react on the test region of the kit test. It is the one responsible for the presence of color reaction in the test region which means the absence of barbiturates in the urine specimen. o Barbiturates-protein conjugate is present in the test region and also in the control region. When Barbiturates are absent, basically these antibodies will bind to the protein conjugate for the barbiturates producing a colored line. But if the barbiturates is present in the urine, tendency these barbiturates will saturate all the binding sites into the antibodies. Because it is saturated, lahat ng pwedeng gamiting antibodies para makadikit sa protein conjugate ay saturated na, therefore it will not bind the protein conjugate. Therefore, there is no colored line that will produce. o In drug test, positive result is the absence of colored line. o A goat antibody is the one that will bind to the barbiturates-protein conjugate to the control region. And there must always be a color reaction or colored line in the control region. If there is no line in the control region means the kit is not working. It means the results are invalid and you need to repeat the test using a new test kit. LESSON 6: DETERMINATION OF COCAINE

BLUE SCREEN ONE STEP COC TEST DEVICE

  • Cocaine (COC), is a potent CNS stimulant and a local anesthesia. o Anesthesia blocks all the sensation including the vibrations, pain, and pressure. Unlike in pain reliever, it targets only the preceptor for the pain.
  • Initially, it brings about extreme energy and restlessness while gradually resulting in tremors, over- sensitivity and spasms. In large amounts, Cocaine causes fever, unresponsiveness, difficulty in breathing and unconsciuosness.

DETERMINATION OF COCAINE

  • Cocaine is self-administered through nasal inhalation, intravenous injection and free base smoking. It is excreted in the urine in a short time primarily as Benzoylecgonine. Benzoylecgonine, a major metabolites of Cocaine, has a longer half life ( 5 - 8 hours) than Cocaine (0.5-1), and can generally be detected for 24 - 48 hours after Cocaine exposure.

REAGENTS FOR COCAINE DETERMINATIONS:

  • The test device contains mouse monoclonal anti- Benzoylecgonine antibody coupled particles and Benzoylecgonine-protein conjugate. A goat antibody is employed in the control line system. o Mouse monoclonal anti-Benzoylecgonine antibody is the one that will react with the Benzoylecgonine-protein conjugate. It is responsible for the color lined production. o But once is the cocaine is present higher than the cut off level for the cocaine, it will saturate the binding sites. So, because of that, wala na siyang binding sites na pwedeng kumapit sa protein conjugate, so there is no a colored formation in the test region if the urine is positive for cocaine. o The control region must have a colored line in able for us to say that the test kit is properly working and test is not invalid. LESSOM 7: DETERMINATION OF MORPHINE

ONE STEP MOP TEST DEVICE

  • Opioid analgesics comprise a large group of substances which control pain by depressing the CNS. Large doses of Morphine (MOP) can produce higher tolerance levels and physiologic dependency in users, and may lead to substance abuse. o Before, they use morphine as pain reliever.
  • It is excreted unmetabolized in urine. Morphine is also the major metabolic product of codeine and heroin. It is detectable in the urine for several days after an opiate dose.

REAGENTS FOR MOP DETERMINATIONS:

  • the test device contains mouse monoclonal anti- morphine antibody coupled particles and Morphine- protein conjugate. A goat antibody is employed in the control line system. o Same purpose like in other test determinations. LESSON 8: DETERMINATION OF METHADONE

ONE STEP MOP TEST DEVICE

  • Methadone (MTD) is a narcotic pain relieve for medium to severe pain. It is also used in the treatment for Heroin (Opiate dependence: Vicodin, Percocet, Morphine, etc.) addiction.
  • MTD is a long acting pain reliever producing effects that last between 12 - 48 hours. Ideally, MTD frees the client from the pressures of obtaining illegal heroin, from the dangers of injection, and from the emotional roller coaster that most opiates produce. MTD if taken for long periods and at a large doses can lead to a very long withdrawal period. The withdrawals from MTD are more prolonged and troublesome than those provoked by heroin cessation, yet the substitution and phased removal of MTD is an acceptable method of detoxification for patients and therapists.

REAGENTS FOR MTD DETERMINATIONS:

  • The test device contains mouse monoclonal anti- methadone antibody coupled particles and Methadone-protein conjugate. A goat antibody is employed in the control line system. LESSON 9: PRINCIPLE FOR DRUGS OF ABUSE DETERMINATIONS

PRINCIPLE FOR DRUGS OF ABUSE DETERMINATIONS

  • Blue Screen BAR/COC/MOP/MTD One Step Test Device is an immunoassay based on the principle of competitive binding. Drugs that may be present in the urine specimen compete against the drug conjugate for binding sites on the antibody. o Positive result is the absence of colored line in the test region. o Cut-off level: 300 ng/mL ▪ Below the cut-off level, it can’t saturate all the binding sites in the antibodies. Therefore, those antibodies will stick in the protein conjugate producing a colored line.
  • During testing, a urine specimen migrates upward by capillary action. BAR/COC/MOP/MTD if present in the urine specimen below the cut off level, will not saturate the binding sites of the antibody in the test. The antibody coated particles will then be captured by immobilized BAR/COC/MOP/MTD-protein conjugate and a visible colored line will show up in the test line region. The colored line will not form in the test region if the BAR/COC/MOP/MTD level exceeds the cut-off level, because it will saturate all the binding sites of anti- BAR/COC/MOP/MTD antibodies.

RESULTS FOR BAR/COC/MOP/MTD DETERMINATIONS

  • a drug positive urine specimen will not generate a colored line in the test line region because of drug competition, while a drug-negative urine specimen or a specimen containing a drug concentration less than the cut-off will generate a line in the test line region.
  • To serve as a procedural control, a colored line will always appear at the control line region indicating that proper volume of specimen has been added and membrane wicking has occured.

MATERIALS:

  1. Test devices
  2. Specimen collection container
  3. Timer
  4. Droppers NOTE:
  • The urine specimen must be collected in a clean and dry container. Urine collected at any time of the day may be used. Urine specimens exhibiting visible particles should be centrifuged, filtered, or allowed to settle to obtain a clear specimen for testing
  • Urine specimens may be stored at 2 - 8°C for up to 48 hours prior to testing. For long-term storage, specimens may be frozen and stored below - 20°C.
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Clinical- Chemistry-2- Laboratory- Finals (1) 230522 2007 12

Course: Clinical Chemistry 2 (MDT 3122L)

165 Documents
Students shared 165 documents in this course

University: Our Lady of Fatima University

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CLINICAL CHEMISTRY 2 (Laboratory)
College Of Medical Laboratory Science
Our Lady of Fatima University – Pampanga
OCAMPO, MARY GRACE O. [SECOND TERM, 3-Y2-1]
1
FINALS
LESSON 1: DETERMINATION OF HUMAN CHORIONIC
GONADOTROPIN (HCG)
ADVANCED QUALITY ONE STEP PREGNANCY TEST
• This test is a rapid qualitative immunochromatographic
assay. Rapid because you will simply need to drop
sample of a urine in the test cassette and is able to
determine the presence of early pregnancy.
DETERMINATION OF HUMAN CHORIONIC
GONADOTROPIN (HCG)
• Human chorionic gonadotropin (hCG) is a
glycoprotein hormone secreted by the developing
placenta shortly after fetilization.
o It is produced by syncytiotrophoblast of
placenta (syncytiotrophoblastic cells of the
placenta)
• In normal pregnancy, hCG is detected in urine and
serum soon after conception and levels of 5-50 mIU/mL
are seen within 1 week of implantation. At the time of
first missed menstrual period, hCG concentration in urine
and serum are about 100mIU/mL, peak levels of
100,000-200,000 mIU/mL reached at the end of first
semester.
• Elevated urine and serum hCG are observed not only in
pregnancy but also in some malignant conditions.
• Elevated urine and serum hCG are observed also in
trophoblastic or non-trophoblastic neoplasm, like
hydatidiform mole, and choriocarcinoma.
o Hydatidiform mole, and choriocarcinoma are
examples of Gestational trophoblastic
disease (GTD)
o This type of neoplasm is actually a tumor that
derives from the syncytiotrophoblast.
• Lower levels of hCG hormone may be associated with
placental insufficiency, threatened spontaneous
abortion, and ectopic pregnancy.
o Placental Insufficiency – in this condition,
oxygen and nutrients are not sufficiently
transported to the growing fetus via the
placenta.
o Threatened Spontaneous Abortion – when
we say spontaneous abortion, that’s the
pregnancy loss before 20 weeks of gestation.
o Ectopic Pregnancy – the fertilized egg was
implanted outside the uterus.
PRINCIPLE
• Advanced Quality Pregnancy Test is a sandwich
immunoassay.
o It involves the interaction of the antibody and
antigen.
• The test has a sensitivity of 25 mIU/mL hCG for urine,
which is sufficient to detect pregnancy the first day of the
missed period.
o In first day of missed period the concentration
of hCG can reach up to 100mIU/mL.
o The test can also detect lower than 100.
• The sample is applied to the card and reacts initially with
the specific, anti-beta hCG monoclonal antibody/colloidal
gold conjugate on the test membrane. The mixture
moves along the membrane, by capillary action, and
reacts with a specific anti-hCG in the test region.
• If hCG is present in the sample, the result is the
formation of a colored band in thet test region. If there
is no hCG in the sample, the area will remain white.
o There are two regions in the test casettes:
▪ Test Region (indicator that hCG is
present in the sample)
▪ Control Region (for quality control
to determine if the cassette is really
working)
• The sample continues to flow to the control region and
forms a pink to purple color, indicating the test is
working and the result is valid.
MATERIALS:
• Urine specimen cups
• Pregnancy test kit
STORAGE AND STABILITY:
• The test device can be stored under refrigeration and
room temperature (2-30ºC) and will be stable until the
expiration date.
o You cannot use the test kit beyond the
expiration date.
SAMPLE COLLECTION:
• Collect the urine in a clean, dry glass or plastic
container.
o Urine specimens can be collected at any time
of the day.
o It is not necessary to obtain the first-morning
specimen However, the concentration of the
hCG may be higher in this specimen.
• Sample can be refrigerated up to 72 hours prior to
testing.
o Refrigerated samples must be allowed to warm
to room temperature and mix before testing
PROCEDURE:
1. Bring all materials and specimen to room temperature
2. Remove test card from the sealed foil pouch.
3. Place the test card on a flat dry surface.
UNIT OUTLINE
I. Determination of Human Chorionic Gonadotropin
(hCG)
II. Determination of Alcohol
III. Drug Test Specimen Collection and Accessioning
IV. Determination of Methamphetamine and
Tetrahydrocannabinol
V. Determination of Barbiturates
VI. Determination of Cocaine
VII. Determination of Morphine
VIII. Determination of Methadone
IX. Principle for Drugs and Abuse Determinations

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