Technical Procedure

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Table of Contents
Types of Blood Collection Tubes 1
General Procedure 2
Patient Identification 2
Skin Puncture 2
Venipuncture Method 3
Venipuncture Procedure 4
Capillary Blood Collection or Skin Puncture 6
HEMATOLOGY
Complete Blood Count 7
DYMIND DH36 Auto Hematology Analyzer 7
Hematocrit Determination 10
Hemoglobin Determination 10
Hemoglobin Determination Table 1 10.1
Differential Count 11
Blood Smear Preparation (Slide/Wedge Method) 11
Staining the Smear 12
Hemacytometry 12.1
Clotting Time and Bleeding Time 13
SEROLOGY
Pregnancy Test 14
Blood Typing 14
Hepatitis B Surface Antigen 19
Dengue NS1 Antigen 22
CLINICAL CHEMISTRY
Precise Determination of Glucose 24
Precise Determination of Serum Creatinine from Venous Blood and/or 24-Hour Urine 33
Precise Determination of Uric Acid from Venous Blood 40
Precise Determination of SGPT/ALT 47
Precise Determination of Urea from Venous Blood and/or 24-Hour Urine 53
Precise Determination of Total Cholesterol 61
Precise Determination of Triglycerides from Venous Blood 68
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Precise Determination of HDL-Cholesterol Using Venous Blood 75

Calculation of LDL-Cholesterol Using Previously Determined TAG, Total Cholesterol and HDL-C 82
Precise Determination of Sodium 85
Precise Determination of Potassium 91
Glycosylated/Glycated Hemoglobin (HbA1C) 97
OGTT103
OGCT 104
2-Hour PPBS 104
CLINICAL MICROSCOPY
Introduction to Urinalysis 106

Physical Examination of Urine110
Chemical Examination of Urine 112
Microscopic Examination of Urine 114
Introduction to Fecalysis 124
Physical/Macroscopic Examination 134
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GENERAL PROCEDURE
Patient Identification
The phlebotomist’s role requires a professional, courteous, and understanding manner in
all contacts with the patient. Greet the patient and identify yourself and indicate the procedure
that will take place. Effective communication, both verbal and non-verbal is essential.
Proper patient identification is mandatory. An out-patient must provide identification
other than the verbal statement of a name. Using the requisition for reference, ask a patient to
provide additional information such as surname or birthdate.
If possible, speak with the patient during process. The patient who is at ease will be
focused on the procedure. Always thank the patient and excuse yourself courteously when
finished.
Skin Puncture
Used when only small quantities of blood are required.
Sites of Puncture
1. Margin of earlobe
2. Palmar surface of the 3rd or 4th finger
3. Plantar surface of the heel and big toe.
Sites to Avoid
1. Inflamed and pallor areas
2. Cold and cyanotic areas
3. Congested and edematous areas

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4. Scarred and heavily calloused areas
EQUIPMENT
The following are needed for routine venipuncture:
1. Syringes
2. Needles – the gauge number indicates the bore size: the larger the gauge number, the
smaller the needle bore.
3. Tourniquet – wipe off with alcohol and replace frequently.
4. Gauze/sponges/cotton – for application on the site from which the needle is withdrawn.
5. Adhesive tape – protects the venipuncture site after collection
6. Needle disposal unit – needles should be removed, bent or recapped. Needles should be
placed in a proper waste disposal immediately after use.
7. Gloves – worn to protect the phlebotomist
8. Evacuated collection tubes – the tubes are designed to fill with a predetermine volume of
blood. The rubber stoppers are color coded according to the additive that tube contains.
SELECT VENIPUNCTURE METHOD
1. Evacuated tube system: Evacuated tubes have color-coded tops and fill by vacuum. The
tube is used with a collection tube holder and a safety needle to collect blood directly into
the tube. The collection tube holder helps in handling the needle and collection tube and
allows for changing tubes to obtain multiple samples with one stick. When the collection
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tube is disengaged from the needle, a small rubber sleeve covers the needle and prevents
loss of blood while the tube is changed.
2. Syringe method: Uses a 10-mL syringe and a needle to puncture the vein and aspirate
blood. The needle is then inserted into the colored tube top for transportation to the lab.
3. Butterfly method: Uses a small butterfly needle and a syringe to obtain the venous
sample.
Venipuncture Procedure
It is a complex, requiring both knowledge and skill to perform. Each phlebotomist
generally establishes a routine that is comfortable for him her. Several essential steps are
required for every successful collection procedure.
1. Observe PPE
2. Approach the patient in a friendly, calm, manner. Make sure that he/she is in supine and
comfortable position.
3. Identify patient correctly by letting him/her state his/her name.
4. Properly fill out the appropriate requisition forms, indicating the test period.
5. Verify the patient’s condition. Fasting, dietary constrictions, medications, timing and
medical treatment are all of concern and should be noted on the laboratory requisition.
6. Place the patient in a supine or semi-Fowler’s position with ventral surfaces of both arms
up.
7. Examine both arms for suitability.

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1. Never draw blood from the arm if it is on the side of a mastectomy, is edematous, has
an IV below the venipuncture site, has a vascular access or fistula, or has a
hematoma.
8. Tie a tourniquet approximately 3-4 inches above the intended venipuncture site. Be sure
that it is tight but not uncomfortable.
9. Ask the patient to make a fist.
10. Assess the antecubital area of the arm. The basilic, cephalic, and median cubital veins are
all usually near the skin surface.
11. Palpate the vein. It should rebound (bounce). If the antecubital veins of both arms are not
suitable, assess the forearms first and then the hands and wrist.
2. lf a suitable vein cannot be found, DO NOT ATTEMPT BLOOD DRAW. Remove
the tourniquet and inform the nurse in charge or call the physician.
12. Observe Standard Precautions.
13. Don gloves.
14. Open an alcohol pad and rub the site in concentric circles, working outward from the
center. To avoid patient discomfort, allow the area to air dry for 30-60 sec (or wipe with a
sterile, dry gauze pad). Povidone-iodine preps may be used. Check if patient is allergic.
15. With your non-dominant hand, stabilize the vein by holding the vein between the index
finger and thumb.
16. With your dominant hand grasp the collection tube holder and turn so that the bevel is
UP. Holding the needle at a 15 to 30-degree angle, enter the skin directly above the vein
and in the same I direction as the blood flow. Advance needle into vein. Entry should be
smooth and quick.

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17. When the blood is enough, remove the tourniquet.
18. Remove the needle from the patient’s arm using a swift backward motion.
19. Apply gauze/cotton once the needle is out of the arm. Ask the patient to apply pressure or
press down the cotton for 3-5minutes to avoid trauma or hematoma.
20. Dispose of contaminated materials/supplies in designated containers.
21. Label all tubes accordingly.
Capillary blood collection or Skin Puncture Procedure
5. Position the patient.
6. The best locations for finger puncture are the 3 rd (middle) or 4th (ring) fingers of the non-
dominant hand. Do not use the tip or the center of the finger. Avoid the side of the finger
where there is less soft tissue, where vessels and nerves are located, where the bone is
closer to the surface. The 2nd (index) finger tends to have thicker, callused skin. The 5 th
finger tends to have less soft tissue overlying the bone. Avoid puncturing a finger that is
cold, cyanotic, swollen, scarred, or covered with rash.

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7. Obtain a sterile lancet, make a skin puncture just of the center of the finger pad. The
puncture should be made perpendicular to the ridges of the fingerprint so that the drop of
blood does not run down the ridges.
8. Wipe away the first drop of blood, which tends to have excess tissue fluids.
9. Collect drops of blood into the heparinized capillary tube by gently massaging the finger.
10. Have the patient hold a small gauze pad over the puncture site for a couple of minutes to
stop the bleeding.
11. Dispose contaminated materials/supplies in designated containers.
12. Label all specimens properly and accordingly.
COMPLETE BLOOD COUNT
6 Determinations
1. Red blood cell count
2. White blood cell count
3. Hemoglobin determination
4. Hematocrit determination
5. Differential count
6. Stained Red blood cell count
Methods Performed
1. Automated (DYMIND DH36 Auto Hematology Analyzer)
2. Manual method
DYMIND DH36 Auto Hematology Analyzer

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This Auto Hematology Analyzer is a quantitative, automated hematology analyzer and 3-
part differential counter used in clinical laboratories.
Purpose and principle of the test
The measurement methods used in this analyzer are the Electrical Impedance method for
determining the WBC, RBC and PLT and their volume distribution; the colorimetric method for
determining the HGB. During analysis cycle, the sample is aspirated, diluted and mixed before
the determination for each parameter is performed.
WBCs/RBCs/PLTs are counted and sized by the Electrical Impedance Method. This
method is based on the measurement of changes in electrical resistance produced by a particle,
which in this case is a blood cell, suspended in a conductive diluent as it passes through an
aperture of known dimensions. An electrode is submerged in the liquid on both sides of the
aperture to create an electrical pathway. As each particle passes through the aperture, a transitory
change in the resistance between the electrodes is produced. This changed produces a measurable
electrical pulse. The number of pulses thus generated is equal to the number of particles that
passed through the aperture. The amplitude of each pulse is proportional to the volume of each
particle.
Each pulse is amplified and compared to the internal reference voltage channel, which
only accepts the pulses of certain amplitude. If the pulse generated is above the WBC/RBC/PLT
lower threshold value, it is counted as WBC/RBC/PLT. The cell volume distribution is
determined by the cell counting within each channel classified by the pulse amplitude.
The analyzer presents the WBC/RBC/PLT histogram, where the x-coordinate represents
the cell volume and the y-coordinate represents the number of cells.
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Procedure
Venous Whole Blood Sample/Venipuncture
1. Turn on the power switch at the back of the analyzer.
2. Click [Home] and select [Sample Analysis]
3. Collect venous blood with a K2EDTA anticoagulant collection tube.
4. Input patient’s name and age
5. Mix the blood sample very well and present the sample tube to the probe so that the
probe tip is so well into the sample.
6. Press the aspirate key to start analysis.
Note: Refer to the analyzer’s Operator’s Manual for maintenance and troubleshooting
Capillary Whole Blood Samples/Finger Puncture
1. Turn on the power switch at the back of the analyzer.
2. Click [Home] and select [Sample Analysis]
3. Click the Add Diluent icon.
4. Take a clean centrifugal tube, uncap it and present it to the sample probe in which the
sample probe tip is vertically in contact with the bottom of the tube so as to avoid
bubbles, liquid attached to the inner wall or spatter.
5. Press the aspirate key and add the diluent (180 µL at a time)
6. After the diluent is added and you hear a beep, you can remove the centrifugal tube.
7. Click Cancel to exit dispensing the diluent.
8. If more portions of diluent are needed, repeat steps 2-7.
9. Click [Sample Analysis]

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10. Click Mode & ID, a dialog box will pop up
11. Select blood sample mode Pre-dilute (PD).
12. Collect capillary whole blood sample/s with heparinized microhematocrit tube.
13. Add 20 µL of blood to the diluent, close the tube cap and shake the tube to mix sample
14. Click the Pre-entry icon
15. Input patient’s name and age and Press OK
16. Mix the blood sample very well and present the sample tube to the probe so that the
probe tip is so well into the sample.
17. Press the aspirate key to start analysis.
Note: Don’t forget to change the Mode & ID back to Venous Whole Blood (VWD) when
running non-diluted samples.
RBC STUDIES
Hematocrit Determination
1. Collect blood with heparinized tube/s. Fill heparinized tubes until 2/3 to ¾ full
without air spaces from the 3rd or 4th finger palmar surface of the fingertip.
2. Invert tube at least four times to mix blood and anticoagulant.
3. Seal one end of the tube with sealing clay.
4. Centrifuge at 10,000 to 15, 000 rpm for 5 minutes in a microhematocrit centrifuge
provided with special head for capillary tubes or with rubber adapter.
5. Read hematocrit using a microhematocrit reader.
6. Record result.
Hemoglobin Determination
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*see table at the next page
RBC count: Refer to Microscopic hematocytometry
WBC STUDIES
Corrected WBC count
This should be done when a high WBC count is obtained and there are more than
10 nucleated RBCs per 100 WBC in the blood smear.
100
Corrected WBC count = X Uncorrected WBC count
100 + no. Nucleated RBCs
WBC count: Refer to Microscopic hematocytometry
Differential count
Express in % (percent) the relative number of the various types of leucocytes
present in the peripheral (venous) blood.
4 General Steps
1. Prepare blood smear
2. Stain the smear
3. Count the cells
4. Report
BLOOD SMEAR PREPARATION (TWO-GLASS SLIDE/SWEDGE METHOD)

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1. Collect blood through skin puncture or venous blood.
2. Label the slide with patient’s name, age, and date smeared.
3. Put a small drop of blood near one end of the slide.
4. Using another clean slide as a “spreader”, touch the blood with the spreader and
allow the blood to run along its edge. Firmly and rapidly push the spreader along
the slide, keeping the spreader at an angle of 33-45 degrees. Make sure the
spreader is in even contact with surface of the slide all the time the blood being
spread.
5. Allow to dry in room temperature.
STAINING THE SMEAR
1. Air dry smear before staining.
2. Dip air-dried smear 5 times for about 1 second each dip into Solution 1.
5. Rinse the slide into the Solution 4.
6. Allow to dry.
7. Examine under oil immersion lens.
COUNTING THE CELLS
Only 1 method should be used in scanning one smear to avoid counting the same
cells twice or more.
REPORTING
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Report in relative count. Normal values in an average adult as follows:
Neutrophil 55-70 %
Lymphocyte 20-35 %
Basophil 0-1 %
Eosinophil 1-4 %
CLOTTING TIME (Slide or Drop Method)

Monocyte 1-6%
1. Disinfect the site of puncture of 70% alcohol sponge and dry.
2. Puncture to a depth of 3 mm.
3. Wipe off 1st drop of blood.
4. Start the timer as soon as the 2nd drop of blood appears.
5. Transfer the 2nd drop of blood onto the center of the glass slide.
6. Pass the tip of the needle through the drop of blood every 30 seconds and note for the
formation of fibrin strands.
7. Stop the timer as soon as fibrin strands are seen clinging at the tip of the needle.
NORMAL VALUES = 5-8 MINUTES
BLEEDING TIME (Duke’s Method)
1. Disinfect the site of puncture of 70% alcohol sponge and dry.
2. Puncture to a depth of 3-4 mm with sterile disposable lancet.
3. Immediately start the timer as soon as the 1st drop of blood appears.
4. Blot the drop of blood with filter paper every 30 seconds, making sure that the filter
paper does not touch the wound.

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5. Stop the timer as soon as the last drop of blood disappears or as soon as bleeding
stops.
NORMAL VALUES = 2-5 MINUTES
PREGNANCY TEST
INTENDED USE
This is a rapid chromatographic immunoassay for the qualitative detection of human
chorionic gonadotropin in urine to aid in the early detection of pregnancy.
PRINCIPLE
This test uses two lines to indicate results. The test line utilizes a combination of
antibodies including a monoclonal hCG antibody to selectively detect elevated levels of hCG.
The control line is composed of goat polyclonal antibodies and colloidal gold particles. 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
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colored line will always appear in the control line region indicating the appear volume specimen
has been added and membrane wicking has occurred.
SPECIMEN COLLECTION
1. Specimens may be collected in any clean, dry, plastic container and labeled with patient
identifiers.
2. First morning specimen is recommended for it contains the highest concentration of hCG.
3. If the test is to be analyzed within 24 hours after collection, the specimen should be
stored in the refrigerator. Stored specimens should be brought to room temperature before
analysis.
PROCEDURE
1. Follow hand hygiene protocol and put on gloves.
2. Remove the test device from the protective pouch and place it on a flat, dry surface.
3. Label test device with two patient identifiers.
4. Dispense 3 to 4 drops of urine into the sample well. Wait for the red lines to appear.
5. Read results after 3 minutes and no later than 5 minutes. READ UNDER DIRECT
LIGHT TO AVOID OVERSHADOWING IN THE T (TEST) AND C (CONTROL)
LINES.
6. Document patient result.
INTERPRETATION OF RESULTS
 Negative: The presence of only control band (C) within the results window
indicates a negative result.

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 Positive: The presence of the test line (T) and the control line (C) within the
results window, regardless of which band appears first, indicates positive result.
 Invalid: If the control band (C) is not visible within the result window after
performing the test, the result is considered invalid. Instructions may not have
been followed correctly or the test may have deteriorated beyond the expiration
date. It is recommended that the specimen be re-tested using a new test kit.
BLOOD TYPING
(FORWARD TYPING/SLIDE METHOD)
SUPPLIES
Blood typing reagents (ABO and Rh typing)
2-3 toothpicks
1 clean glass slide
Receptacle to hold supplies
Waste receptacles
Handwashing materials
Blood collection lancet
Alcohol swab
Cotton
PROCEDURE
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5. Position the patient.
6. The best locations for finger puncture are the 3 rd (middle) or 4th (ring) fingers of the non-
dominant hand. Do not use the tip or the center of the finger. Avoid the side of the finger
where there is less soft tissue, where vessels and nerves are located, where the bone is
closer to the surface. The 2nd (index) finger tends to have thicker, callused skin. The 5 th
finger tends to have less soft tissue overlying the bone. Avoid puncturing a finger that is
cold, cyanotic, swollen, scarred, or covered with rash.
7. Obtain a sterile lancet, make a skin puncture just of the center of the finger pad. The
puncture should be made perpendicular to the ridges of the fingerprint so that the drop of
blood does not run down the ridges.
8. Wipe away the first drop of blood, which tends to have excess tissue fluids.
9. Place 3 drops of blood on the slide (at the center and right and left side of the slide).
10. Stop the bleeding on the finger of the patient by putting a clean, dry cotton on the wound
and let the patient hold it in place for at least 5 minutes. Let patient remove the cotton
from finger after 5 minutes (if bleeding has stopped). Discard cotton.
11. Place a drop of anti-B typing reagent on a drop of blood at one end of the slide and drop
anti-A typing reagent on a drop of blood at other end of the slide.

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12. Place a drop of anti-D typing reagent on a drop of blood at the center of the slide.
13. Thoroughly mix the reagent and blood using a toothpick.
14. Observe for clumping or agglutination of blood.
15. Report result. Thank the client for the cooperation and give post-procedure instructions.
16. Do after care, remove gloves and do handwashing.

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HEPATITIS B SURFACE ANTIGEN

(SD HBsAg)
INTENDED USE
The HBsAg test is an in-vitro immunochromatographic, one-step assay designed for
qualitative determination for HBsAg in human serum or plasma. This is intended for professional
use, only for an initial screening test and reactive samples should be confirmed by a
supplemental assay. This test is suitable for pregnant women, but in case of neonates, test
haven’t performed.
PRINCIPLE
This test device contains a membrane strip, which is pre-coated with mouse monoclonal
anti-HBs antibody on test band region. The mouse monoclonal anti-HBs antibody and the sample
move along the membrane chromatographically to the test region (T) and forms a visible line as
the antibody-antigen gold particle complex forms.
KIT STORAGE AND STABILITY
1. The test kit should be stored between 1°C and 30°C. Do not freeze kit.
2. The test device is sensitive to both heat and humidity.
3. Check the humidity indicator on the desiccant for color change and throw the pouch if
the color indicates saturation (yellow to green).
4. Perform the test immediately after removing the test device from the foil pouch.
5. Do not use the test kit beyond its expiration date.
6. The shelf-life of the kits is indicated on the outer package.
7. Do not use the test kit if the pouch is damaged or the seal is broken.
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SPECIMEN COLLECTION AND STORAGE
1. For plasma, collect the whole blood into the collection tube (containing
anticoagulants such as heparin, EDTA, and sodium citrate) by venipuncture and then
centrifuge blood to get plasma specimen. Anticoagulants such as heparin, EDTA,
sodium citrate do not affect the test result.
2. For serum, collect the whole blood into the collection tube (not containing
anticoagulants such as heparin, EDTA, and sodium citrate) by venipuncture, leave to
settle for 30 minutes for blood coagulation and then centrifuge blood to get serum
specimen of supernatant.
3. If plasma or serum specimens are not tested immediately, they should be refrigerated
at 2-8°C. For storage period longer than 3 days, freezing (below -20°C) is
recommended. They should be brought to 15°C to 30°C prior to use.
4. Plasma or serum specimens containing a precipitate may yield inconsistent test
results. Such specimens must be clarified prior to assaying.
MATERIALS
Micropipette
HBsAg test kit
Disinfectant
Tissue
Waste container
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PROCEDURE
1. Allow kit components and specimen to room temperature between 15°C to 30°C prior to
testing.
2. Remove the test device from the foil pouch and place it on a flat, dry surface.
3. Pipette 100 µL of plasma or serum.
4. Dispense the 100 µL plasma or serum into the sample well.
5. As the test begins to work, you will see purple color to move across the result window in
the center of the test device.
6. Interpret test results after 20 minutes.
1. A purple color band will appear in the left section of the results window to show that test
is working properly.
2. The right section of the results window indicates the test results. If another band appears
in the right section of the results window, this is the test band.
 Negative: The presence of only control band (C) within the results window
indicates a negative result.
 Positive: The presence of the test line (T) and the control line (C) within the
results window, regardless of which band appears first, indicates positive result.
 Invalid: If the control band (C) is not visible within the result window after
performing the test, the result is considered invalid. Instructions may not have
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been followed correctly or the test may have deteriorated beyond the expiration
DENGUE NS1 ANTIGEN

(LABiTDX DENGUE NS1 Ag)
INTENDED USE
One step kit for the detection of NS1 against dengue virus using whole blood.
PRINCIPLE
The Dengue NS1 Ag test is a chromatographic immunoassay kit for rapid detection of
dengue virus NS1 antigen using human blood. It is used as a screening test. When dengue
antigen-positive specimen is loaded into the sample injection point, the antigen is captured by the
immobilized anti-dengue NS1 antibodies to make a visible band in the test line.
TEST PROCEDURE
 Place all specimens, materials and test kits on a clean, flat table. Allow these materials to
cool to room temperature prior to using them.
Specimen Collection and Assay
1. Twist and pull the cover of the lancet to open it.
2. Clean the tip of the finger with alcohol swab. The middle or ring finger are good sites
for specimen collection.
3. Place the colored end of the lancet on the fingertip.
4. Push the lancet against the fingertip to activate the lancet mechanism.
5. Squeeze the tip of the finger to form a drop of blood.

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6. Drop blood directly from the fingertip into the sample well (S), load at least 6 drops.
7. Interpret results between 15-20 minutes. Do not read the results after 20 minutes.
1. A color band will appear in the upper section (C) of the window to show that the test is
working properly. This band is the control band.
2. A line below the control band is the test band.
Negative: The presence of only control band (C) within the results window indicates a
negative result.
Positive: The presence of the control band (C) and the test band (T) within the results
window, indicates positive result.
Invalid: If the control band (C) is not visible or only (T) band visible within the result
window after performing the test, the result is considered invalid. Instructions may not
have been followed correctly or the test may have deteriorated beyond the expiration
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PRECISE DETERMINATION OF GLUCOSE
CLINICAL SIGNIFICANCE
Glucose is the main source of energy for the human body. Glucose is converted either
into glycogen to be stocked in the liver or into triglycerides to be stocked in fatty tissues.
Glucose concentration in blood is regulated by several hormones, including two antagonists:
insulin and glucagon. Quantification of glucose in blood is used to diagnose metabolic
carbohydrates disorders such as diabetes, neonatal glycaemia, idiopathic hypoglycemia and
pancreatic disease. The main physiological troubles are linked to hyperglycemia (type I Diabetes
mellitus and type II Diabetes mellitus). Type I diabetes mellitus is insulin-dependent, and
appears mainly before 30 years old. Type II diabetes mellitus is non-insulin-dependent, and
usually appears after 40 years old, but can occur earlier for obese people. Other diabetes has
secondary origin, and appear after endocrinal or hepatic diseases.
METHOD
Enzymatic colorimetric.
Trinder - Kinetic.
PRINCIPLE
Enzymatic determination of glucose according to the following reactions:
Glucose oxidase
Glucose + O2 Gluconic acid + H2O2
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Peroxidase
2H2O2 + Phenol + 4-Aminoantipyrine Quinoneimine + 4H2O
REAGENTS COMPOSITION
Reagent R
Phosphate buffer, pH 7.4 13.8 mmol/L
Phenol 10 mmol/L
4-Aminoantipyrine 0.3 mmol/L
Glucose oxidase ≥10 000 U/L
Peroxidase ≥700 U/L
Sodium azide <0.1%
Standard: Std
D-Glucose 100 mg/dL
5.55 mmol/L
PRECAUTIONS
 This reagent and this standard are professional in vitro diagnostic use only.
 The reagent contains less than 0.1 % sodium azide. Sodium azide can react with copper
and lead plumbing to form explosive metal azides. When disposing of these reagents
always flush with copious amounts of water to prevent azide buildup.

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 Take normal precautions and adhere to good laboratory practice.
 Use clean or single use laboratory equipment only to avoid contaminations.
 The standard should be immediately and tightly capped to prevent contamination and
evaporation.
 For more information, Material Safety Data Sheet (MSDS) is available on request for
professional user.
STABILITY OF REAGENTS
Store at 2-8°C and protected from light. Do not freeze.
Do not use after expiration dates indicated on the vial labels.
On board stability:
The stability is specific for each analyzer
REAGENT DETERIORATION
 This reagent and standard solution should be clear. Cloudiness would indicate
deterioration.
 Do not use the product if there is visible evidence of biological, chemical or physical
deterioration.
PREPARATION
The reagent and the standard are ready to use.

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SAMPLES
 Fasting for 8-12 hours is recommended.
 Serum free of hemolysis to avoid false decrease results.
 Plasma collected on sodium fluoride/potassium oxalate or any inhibitors of glycolysis or
lithium heparin.
 Do not use other specimens.
STORAGE
 Serum and plasma collected without sodium fluoride are stable for 8 hours at room
temperature and up to 3 days at 2-8°C.
 Serum and plasma collected with sodium fluoride are stable for 2 days at room
temperature and up to 7 days at 2-8°C.
MATERIALS:
 Pipettes (1 mL, 0.5 mL)
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
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PROCEDURE
A. Preparation of Spectrophotometer
1. Before starting anything, turn on the spectrophotometer according to the machine's
instructions.
2. Set the machine for Glucose to read the absorbance.
3. Set the wavelength to 500 nm at 37°C.
B. Preparation of working reagent
 Reagents are liquid, ready to use.
1. Pipette 500 µL of Glucose reagent to 5mL test tube/s.
C. Preparation of blank
1. Label clean and dry test tube/s according to protocol.
2. Pipette 5 µL of distilled water to working reagent test tube.
3. Mix thoroughly by aspirating or cover with parafilm and mix by inversion.
4. Incubate for 5 minutes at 37°C.
5. Aspirate or read absorbance to the spectrophotometer at 500 nm at 37°C.
D. Preparation of Standard
2. Pipette 5 µL of Standard (Std) to working reagent test tube.

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E. Preparation of Control
2. Pipette 5 µL of Control to working reagent test tube.
F. Preparation of Unknown Using Serum
2. Pipette 5 µL of Unknown to working reagent test tube.
5. Record results before releasing.
Note: The preparation of the control, blank, standard and unknowns can be done
simultaneously. Recalibrate when lots change, when quality control results fall outside the
established range and after maintenance check.
REFERENCE VALUES (3)
Serum, plasma:
33
Fasting Blood Glucose 75 – 115 mg/dl
Random Blood Glucose <60 years old: 45 mg/dL – 130 mg/dL

> 60 years old: 70 mg/dL – 160 mg/dL
CONVERSION
 To convert the result from mg/dL to mmol/L, multiply value in mg/dl by 0.0555.
CALIBRATION:
Concentration value of Glucose Standard 100 mg/dL is traceable to the Standard
Reference Method ID-MS (Isotope Dilution – Mass Spectrometry).
The calibration stability is specific for each analyzer.
INTERFERENCES
According to SFBC recommendations, studies have been performed to determine
the level of interference from different compounds:
Turbidity: No significant interference up to 600 mg/dL Triglyceride equivalent (6
g/L, 6.9 mmol/L).
Unconjugated bilirubin: Negative bias from 6 mg/dL (60 mg/L, 100 μmol/L) on
normal serum. Negative bias from 11 mg/dL (110 mg/L, 190μmol/L) on pathological
serum.
Conjugated bilirubin: Negative bias from 2.1 mg/dL (21 mg/L, 35 μmol/L) on
normal serum. No significant interference up to 25 mg/dL (250 mg/L, 427μmol/L) on
pathological serum.
Hemoglobin: No significant interference up to 500 mg/dL (5 g/L).

34
Ascorbic acid: No significant interference up to 7 mg/dL (70 mg/L, 0.40 mmol/L).
QUALITY CONTROL:
These controls must be performed and validated before the patient samples are assayed.
The control frequency must be at least once a day, after each calibration and should be adapted to
quality control procedures of each laboratory and the regulatory requirements. Results should be
within the defined ranges. If values fall outside of the defined ranges, each laboratory should
take corrective measures. Quality control materials should be used in accordance with local
guidelines.
CALCULATION:
A
Sample
xn n = concentration of standard
A
Standard
WASTE MANAGEMENT
Disposal of all waste material should accordance with local and legal requirements.
35
REFERENCES
1. Bishop, M. L., Duben-Engelkirk, J. L., & Fody, E. P. (2000). Clinical Chemistry:
Principles, procedures, correlations (4th ed.). Philadelphia, PA: Lippincott Williams &
Wilkins.
2. Rodriguez, M. T. T., (Revised 2014). Clinical Chemistry: Review Handbook for Medical
Technologist. Copyright Ed. Cattleya Star Copy Center and Book binding, 1205 Asturias
St., Sampaloc, Manila.

36
PRECISE DETERMINATION OF SERUM CREATININE FROM VENOUS BLOOD
AND/OR 24 HOUR URINE
Creatinine is a waste product formed in muscle from the high energy storage compound,
creatinine phosphate. The amount of creatinine produced is fairly constant (unlike Urea) and is
primarily a function of muscle mass. It is not greatly affected by diet, age, sex or exercise.
Creatinine is removed from plasma by glomerular filtration and then excreted in urine without
any appreciable resorption by the tubules.
Creatinine is used to assess renal function, however, serum creatinine levels do not start to rise
until renal function has decreased by at least 50%.
PRINCIPLE
Creatinine reacts with alkaline picrate to produce an orange-yellow color (the Jaffe’s reaction).
Specificity of the assay has been improved by the introduction of an initial rate method.
However, Cephalosporin antibiotics are still major interference. The absorbance of the orange-
yellow color formed is directly proportional to creatinine concentration and is measured
photometrically at 490-510 nm.
REAGENT COMPOSITION
R1: Sodium Hydroxide = 394 mmoI/I
R2: Picric Acid = 11 mmol/l
R3 standard: see bottle label

37
STABILITY AND STORAGE
 The unopened reagents are stable till the expiry date stated on the bottle and kit label
when stored at 2-25°C.
SPECIMEN COLLECTION AND HANDLING
 Unhemolytic serum, plasma (heparin, EDTA) or urine.
 It is recommended to follow NCCLS procedures (or similar standardized conditions).
Stability
In serum / plasma:
7 days at 4-25°C
at least 3 months at -20°C
In urine:
2 days at 20-25°C
6 days at 443°C
6 months at -20°C
For the determination in urine creatinine, use 24-hour urine specimen. It is important to
exactly measure the volume of collected urine. Dilute urine samples in 1:20 ratio with distilled
water and multiply results by 20. Discard contaminated specimens.

38
INTERFERENCES
 Following substances do not interfere:
Hemoglobin up to 10g/L
Bilirubin up to 10 mg/dL
Triglycerides up to 1700 mg/dL
NORMAL RANGE:
Male = 0.9 mg/dl – 1.3 mg/dl
Female = 0.6 mg/dl – 1.1 mg/dl
CONVERSION
 To convert the result from mg/dL to µmol/L, multiply value in mg/dl by 88.4.
MATERIALS:
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
39
PROCEDURE
instructions.
2. Set the machine for Creatinine to read the absorbance.
3. Set the wavelength to 492 nm (490 nm - 510 nm).
 The Working Reagent is stable for 1 month at 2-8°C.
1. Mix 250 µL of Reagent 1 and 250 µL of Reagent 2 to 5mL test tube/s. Wait for 15
minutes before use.
C. Preparation of blank
 The absorbance of the reagent blank should be approximately <0.15 at 505 nm when read
against distilled water.
4. Aspirate or read absorbance immediately to the spectrophotometer at 492 nm (490 nm -
510 nm).
40
 Label clean and dry test tube/s according to protocol.
 Pipette 50uL working standard to working reagent test tube.
 Mix thoroughly by aspirating or cover with parafilm and mix by inversion.
 Read absorbance immediately against blank to the spectrophotometer at 492 nm (490 nm
- 510 nm.
4. Pipette 50uL control to working reagent test tube.
6. Read absorbance immediately against blank to the spectrophotometer at 492 nm (490 nm
- 510 nm).
F. Preparation of Unknown Using Serum or Diluted Urine as Specimen
1. Label clean and dry test tube/s either for urine or serum in accordance to protocol.
2. Pipette 50uL of unknown to working reagent test tube.
4. Read absorbance immediately against blank to the spectrophotometer at 492 nm (490 nm
- 510 nm).
Note: The preparation of the control, blank, standard and unknowns can be done simultaneously.
Recalibrate when lots change, when quality control results fall outside the established range and
after maintenance check.

41
F. Computation of Results
1. Get the absorbance of control, standard and unknown.
2. Compute the values for the control and unknown using Beer’s Law.
3. Using a semi-log paper, make a standard concentration curve.
4. Plot the absorbance of your control and unknown and determine their respective values.
5. Compare plotted values with the computed values.
6. To compute for the value of creatinine in your unknown using urine as sample, use the
formula:
Unknown Abs x n x Sample Dilution Factor n = concentration of standard

Standard Abs
QUALITY CONTROL:
guidelines.
WASTE MANAGEMENT
42
Sources:
Wilkins.
St., Sampaloc Manila.
3. Erba Mannheim Package Insert (Revised Ed., 2015, July 28th). www.
erbamannheim.com. Erba Lachema s.r.o., Karásek 1d, 621 00 Brno, CZ

43
PRECISE DETERMINATION OF URIC ACID FROM VENOUS BLOOD
Uric acid is the major product of the catabolism of endogenous and exogenous (dietary)
purine nucleosides (adenosine and guanosine). This transformation mainly occurs in the liver.
Approximatively 75% of uric acid is eliminated by kidneys; the remainder is secreted into the
gastrointestinal tract, where it is degraded by bacterial enzymes. Uric acid is not very soluble in
water; urate crystals can occur in urines when the concentration is abnormally high. it can also
happen in plasma, crystals then deposit essentially in joints, which induce intense inflammatory
responses (gout). Some causes for increasing uric acid rate in serum are: increasing of purines
synthesis, metabolic disorders (Lesch-Nyhan syndrome for example), nutritional troubles,
increasing of nucleic acid tum-over in case of proliferation of tumor cells, leukemia, psoriasis,
cytotoxic drugs, renal failures... Decreasing of uric acid rate in serum is more uncommon. it can
occur in different cases: failure in renal elimination of uric acid (Fanconi syndrome), Hodgkin's
disease for example.
The quantitation of urinary uric acid is used to define the cause of hyperuricemia (excess
purines or renal retention) and define appropriate treatment.
METHOD
Uricase-Peroxidase Method (Colorimetric-Enzymatic Method)
PRINCIPLE
Enzymatic determination of uric acid according to the following reactions:
Ascorbic acid + O2 ASCORBATE OXIDASE

Dehydroascorbic acid + H2O
44
URICASE
Uric acid + H2O + O2 Allantoin + CO2 + H2O2
TOOS + 4-AAP + 2H2O2 + H+ PEROXIDASE

Quinoneimine + 4H2O
By using ascorbic oxidase to eliminate the interference of ascorbic acid, uric acid is
catalyzed to produce H2O2 which oxidize the 4-AAP to yield a colored dye quinoneimine. The
absorbency decrease is directly proportional to the concentration of uric acid.
SPECIMEN COLLECTION AND PREPARATION
 Serum, lithium heparinized plasma or EDTA plasma, and urine are suitable samples
 24-hour urine should be diluted 1:15 ratio with saline solution NaCL 9 g/L.
 Whole blood or hemolyzed samples are not recommended for use of samples.
 Freshly drawn specimen is the preferred specimen.
 Assay urinary uric acid as soon as possible. Do not refrigerate.
Storage
 Serum or heparinized plasma samples are stable 3 to 5 days if stored at 2-8°C, 6 months
at -20°C.
 Urine are stable 3 to 4 days upon addition of NaOH at 20-25°C.

45
LIMITATIONS AND INTERFERENCES
 Do not use visibly turbid or hemolyzed samples
 Do not report results outside of the usable range.
The following substances in their respective concentrations have no significant interference
(NSI) on the assayed value:
Ascorbic acid = 15 mg/dL
Bilirubin = 20 mg/dL
Lipemia = 500 mg/dL
Hemoglobin = 250 mg/Dl
REAGENT COMPOSITION
R: Phosphate buffer, peroxidase, Uricase, 4 - aminoantitipryine, EHSPT, ferrocyanide, sodium
azide
Standard (Std): Uric acid = 6mg/dL
NORMAL RANGE
Male = 3.4 mg/dl – 7.0 mg/dl
Female = 3.0. – 5.7 mg/dl

46
MATERIALS:
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
PROCEDURE
instructions.
2. Set the machine for uric acid to read the absorbance.
1. Pipette 600 µL to 5mL test tube/s.
C. Preparation of Blank

47
3. Mix thoroughly by aspirating or cover with parafilm and mix by inversion
4. Incubate for 5 minutes at 37°C
2. Pipette 15 µL working standard to working reagent test tube.
5. Aspirate or read absorbance to the spectrophotometer against blank at 546 nm at 37°C
2. Pipette 15 µL control to working reagent test tube.
5. Aspirate or read absorbance to the spectrophotometer against blank at 546 nm at 37°C
E. Preparation of Unknown Using Serum
2. Pipette 15 µL of unknown to working reagent test tube.

48
5. Read absorbance immediately against blank at 546 nm at 37°C.
6. Record results to the log book.
QUALITY CONTROL:
guidelines.
WASTE MANAGEMENT
49
REFERENCES
Wilkins.

50
PRECISE DETERMINATION OF SGPT/ALT
Alanine aminotransferase (ALT), also known as glutamate pyruvate transaminase (GPT).
is a transaminase. ALT catalyzes the transfer of the amino group of L-alanine to a-ketoglutarate
to give L-glutamate. The highest levels are found in the liver and the kidneys, and in smaller
amounts in heart and skeletal muscle. ALT concentration is increased when hepatic cells are
damaged (liver cell necrosis or injury of any cause). Indeed, viral and toxic hepatitis induce a
marked elevation of ALT activity in serum. Intake of alcohol, delirium tremens. and
administration of various drug induce slight or moderate elevation of ALT. ALT concentration in
serum is also slightly increased in various conditions such as: muscular dystrophy, hemolytic
disease, myocardial infarction.
ALT is more liver specific than AST (Aspartate aminotransferase). Measurement of both
AST and ALT has some value in distinguishing hepatitis from other parenchymal lesions. ALT
serum level can be decreased in case of vitamin B6 deficiency.
METHOD
IFCC Method without pyridoxal phosphate (P-5'-P)
Kinetic, UV
Coupled Enzymatic Reaction

51
PRINCIPLE
Kinetic determination of the alanine aminotransferase (ALT) activity:
ALT
L-Alanine + α-Ketoglutarate Pyruvate + L-Glutamate
Pyruvate + NADH + H+ LDH

L-Lactate + NAD+
Specimen Collection and Preparation
1. Serum free hemolysis
2. Lithium heparinized plasma free of hemolysis
3. ALT is stable in sample for 3 days at room temperature or 7 days at 2-8°C. ALT stability
is better maintained at -70°C.
Note: Hemolyzed samples should not be used since significant hemolysis may increase
ALT concentration because of high levels of ALT in erythrocytes.
REAGENT COMPOSITION
Reagent 1 (R1):
Tris buffer, pH 7.5 (30°C)
L-alanine
LDH
Reagent 2 (R2):
α-ketoglutarate
52
NADH
STORAGE AND STABILITY
1. The reagents are stable until the expiry date stated on the label.
2. Store at 2-8°C and protect from light.
Glucose = up to 500 mg/dL
Ascorbic acid = up to 40 mg/dL
Lipemia = up to 600 mg/dL
Bilirubin = up to 20 mg/Dl
MATERIALS:
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
PROCEDURE
53
instructions.
2. Set the machine for ALT/SGPT to read the absorbance.
1. Mix 400 µL of Reagent 1 (R1) and 100 µL Reagent 2 (R2) to 5mL test tube/s.
2. Read against distilled water at 340 nm at 37°C.
C. Preparation of Standard
4. Aspirate or read absorbance immediately to the spectrophotometer against distilled water
at 340 nm at 37°C
D. Preparation of Control
at 340 nm at 37°C

54
at 340 nm at 37°C.
5. Record results to the log book before releasing.
QUALITY CONTROL:
guidelines.
WASTE MANAGEMENT
55
REFERENCES
Wilkins.
3. ALT/GPT 4+1 SL. Elitech: Clinical Systems. SAS - Zone Industrielle – 61500 SEES
France.
56
PRECISE DETERMINATION OF UREA FROM VENOUS BLOOD
AND/OR 24-HOUR URINE
Uremia is also increased by high-protein diet, state of dehydration, muscle wasting (as in
starvation). The determination of urea rate is used together with the determination of creatinine
rate to discriminate between pre-renal (normal creatinine) and renal! Post-renal (increased
creatinine) disorders.
Urine urea may be used as an indicator of overall nitrogen balance and as a guide to total
amino acid requirements for patients with parenteral nutrition.
METHOD
UV Enzymatic Method - Kinetic.
PRINCIPLE
Enzymatic determination according to the following reactions:
UREASE
Urea + H2O 2NH4+ + CO2
GLUTAMATE DEHYDROGENASE
NH4 + 2-oxoglutarate + NADH Glutamate + NAD + H2O
Urea in the sample interacts with enzyme urease found in the reagent. This leads to the
production of ammonium ion and carbon dioxide. In a secondary reaction the ammonium ion,
together with salicylate and sodium perchlorate, is acted upon by nitroprusside to produce
indophenol inducing a color change.

57
SPECIMEN COLLECTION AND PREPARATION
 Serum, or lithium heparinized plasma.
 Urine (24-hour urine) diluted with normal saline or distilled water. Make sure to measure
the total volume of your 24° urine specimen prior to taking out aliquot and diluting.
 Heparin is recommended as anticoagulant.
 Urea is stable at room temperature if microbial growth is prevented.
STORAGE AND STABILITY OF SPECIMEN
 Serum and plasma are stable up to 24hrs at room temperature, 1 week at 2-8°C and at
least 3 months when frozen (-20°C).
 Urine samples are stable up to 4 days if stored at 2-8°C. Urines can be preserved by
maintaining the pH below 4. Addition of thymol (if available) as preservative is not
recommended as it inhibits urease activity.
REAGENT COMPOSITION
R1: Tris buffer, pH 7.6 (37°C), ADP, α-ketoglutarate, urease, GIDH, sodium azide
R2: NADH, sodium azide
Standard (Std): Urea = 50 mg/dL
REAGENT STABILITY AND STORAGE
 Reagents and standards are stable until expiry date stated on the bottle label when stored
tightly closed and if contaminations are prevented during their use.

58
 Store at 2-8°C and protect from light. Do not freeze.
Indications of deterioration:
Reagents: Presence of particulate material, turbidity
Absorbance of blank above 0.250 at 340 nm.
Standard: Presence of particulate material, turbidity
The following substances in their respective concentrations may interfere with the assayed
values:
 Hemolysis (Hgb) = up to 500mg/Dl
 Elevated ammonia
The following substances in their respective concentrations have no significant interference
(NSI) on assayed value:
 Bilirubin = up to 30mg/dL
 Lipemia (triglycerides) = up to 614 mg/dL
NORMAL RANGE
5 mg/dl – 23 mg/dl
59
MATERIALS:
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
PROCEDURE
Preparation of Spectrophotometer
instructions.
2. Set the machine for Creatinine to read the absorbance.
3. Set the wavelength to 492 nm (490 nm - 510 nm).
A. Preparation of working reagent
1. Mix 400 µL of Reagent 1 and 100 µL of Reagent 2 to 5mL test tube/s.
B. Preparation of Blank

60
5. Aspirate or read absorbance immediately at 340 nm at 37°C.
4. Read absorbance immediately against blank at 340 nm at 37°C
3. Cover with parafilm and mix by inversion.
4. Read absorbance immediately against blank at 340 nm at 37°C
E. Preparation of Unknown Using Serum or Diluted Urine as Specimen
4. Read absorbance immediately against blank at 340 nm at 37°C.

61
F. Computation of Results
1. Get the absorbance of control, standard and unknown.
2. Compute the values for the control and unknown using Beer’s Law.
3. Using a semi-log paper, make a standard concentration curve.
4. Plot the absorbance of your control and unknown and determine their respective values.
5. Compare plotted values with the computed values.
 To compute for the value of creatinine in your unknown using serum or urine as sample,
use the formula:
A
Unknown x n x Sample Dilution Factor n = concentration of standard
A
Standard
G. Computation of Clearance
1. Using the formula for creatinine clearance, substitute the appropriate
variables with the obtained values for serum and 24-hour urine creatinine to compute for
clearance.
Urine Creatinine (mg/dl) x Urine Volume (ml/24h) x 1.73

Creatinine Clearance = A
Serum Creatinine
1.73
= normalization factor for body surface area
A
62
QUALITY CONTROL:
guidelines.
WASTE MANAGEMENT
63
REFERENCES
Wilkins.
3. Urea UV SL Package Insert (2016, March). www. elitechgroup.com. Elitech Clinical
Systems SAS - Zone Industrielle - 61500 SEES FRANCE.

64
PRECISE DETERMINATION OF TOTAL CHOLESTEROL
Measurement of serum cholesterol levels can serve as an indicator of liver function,
biliary function, intestinal absorption. propensity towards coronary artery disease, thyrond
function and adrenal disease. Cholesterol levels are important in the diagnosis and clasification
of hyperlipoproteinaemias. Stress, age. gender, hormonal balance and pregnancy affect normal
cholesterol levels.
METHOD
Cholesterol Oxidase - Peroxidase Method
PRINCIPLE
The determination of cholesterol after enzymatic hydrolysis and oxidation. The colorimetric
indicator is quinoneimine which is generated from 4-aminoantypirine and phenol by hydrogen
peroxide under the catalytic action of peroxidase (Trinder's reaction). The intensity of the
pink/red color is proportional to cholesterol concentration in the sample.
CHOLESTEROL ESTERASE
1. Cholesterol ester + H2O Cholesterol + Fatty acids
CHOLESTEROL OXIDASE
2. Cholesterol + O2 Cholest-4-en-3-one +H2O2
PEROXIDASE
3. 2H2O2 + 4-aminoantipyrine Quinoneimine dye + 4H2O
65
REAGENT COMPOSITION
R1: Good‘s Buffer, Phenol, A-aminoantipyrine, Cholesterol esterase, Cholesterol
oxidase, Peroxidase
R2: standard = See bottle label
SPECIMEN COLLECTION
1. Fasting for 8-10 hours is recommended.
2. Use serum, plasma (heparin, EDTA)
3. It is recommended to follow NCCLS procedures (or similar standardized conditions).
Stability: at 20-25°C for 7 days
at 4-8°C for 7 days
at -20°C for 3 months
REAGENT STABILITY AND STORAGE
 Protect reagent from light.
 Close the reagent bottles immediately after use.
 Avoid contamination of opened reagent.
 Do not freeze the reagent.
 Reagents are stable up to expiration date stated on the bottle and kit label when stored at
2-8°C.
NORMAL RANGE
66
< 200 mg/dl
INTERFERENCES
Following substances do not interfere:
1. Hemoglobin up to 5 g/L
2. Bilirubin up to 20 mg/Dl
3. Triglycerides up to 2000 mg/dL
Blood withdrawal should be performed prior to administration of drugs.
MATERIALS:
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
PROCEDURE
instructions.
2. Set the machine for cholesterol to read the absorbance.
3. Set the wavelength to 500 nm (546 nm) at 37°C.

67
5. Aspirate or read absorbance to the spectrophotometer against blank at 500 nm (546 nm)
at 37°C.
at 37°C

68
at 37°C.
at 37°C.
Note: All preparations must be read spectrophotometerically within 60 minutes after the final
incubation. Preparations that have stood for more than an hour are considered invalid and
should no longer be read.
The preparation of the control, blank, standard and unknowns can be done
CONVERSION
 To convert the result from mg/dL to mmol/L, multiply value in mg/dL by 0.026.
69
QUALITY CONTROL:
These controls must be performed and validated before the patient samples are assayed. The
control frequency must be at least once a day, after each calibration and should be adapted to
guidelines.
WASTE MANAGEMENT
70
REFERENCES
1. Rifai, N., Bachorik, P..,S &Albers J..J (1999). Lipids, lipoproteins and apolipoproteins.
In: C. A. Burtis & E. R. Ashwood (Eds..) Tietz textbook of clinical chemIstry (3rd ed. )
(pp. 809 861). Philadelphia: W. B. Saunders Company.
2. Recommendation of the second joint task force of European and other societies on
coronary prevention: Prevention of coronary heart disease in clinical practice. (1998). In
Eur Heart J, 19 1434-1503.
3. Artis, J. D., & Zak, B. Measurement of cholesterol concentration. (1997). ln N.Rifai, G.
R. Warnick, G. R. & M. H. Dominiczak (Eds.). Handbook of Iipoprotein testing (pp.94-
114). Washington: AACC Press.
4. Cholesterol Package Insert. (2017, January 5th). Erba Lachema s.r.o,. Karásek 2219/1d,
62100 Brno, CZ.
5. Bishop, M. L., Duben-Engelkirk, J. L., & Fody, E. P. (2000). Clinical chemistry:
Principles, procedures, correlations (4th ed.). Philadelphia, PA: Lippincott Williams and
Wilkins.
6. Rodriguez, M. T., RMT, MAEd, MSMT. (Revised 2014). Clinical chemistry: Review
Handbook for Medical Technologists (pp 66-69). Copyright ed. Cattleya Star Copy
Center and Book Binding, Asturias St., Sampaloc Manila

71
PRECISE DETERMINATION OF TRIGLYCERIDES FROM VENOUS BLOOD
Triglycerides are a family of lipids absorbed from the diet and produced endogenously
from carbohydrates. Measurement of triglycerides is important in the diagnosis and management
of hyperlipidemias. These diseases can be genetic or secondary to other disorders including
nephritis, diabetes mellitus and endocrine disturbances. Elevation of triglycerides has been
identified as a risk factor for atherosclerotic disease.
METHOD
Glycerokinase Oxidase - Peroxidase Method
PRINCIPLE
The series of reactions involved in the assay system is as follows:
LIPASE
Triglycerides + 3H2O Glycerol + Free fatty acids
GLYCEROKINASE
Glycerol + ATP Glycerol-3-Phosphate + ADP
GLYCEROL-3-PHOSPHATE
Glycerol-3-Phosphate + O2 Dihydroxyacetone phosphate + H2O2
PEROXIDASE
H2O2 + 3-aminoantipyrine + 4-chlorophenol Quinoneimine + HCl + 2H2O
Triglycerides are enzymatically hydrolyzed by lipase to free acids and glycerol. The
glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to
produce glycerol-3-phosphate and adenosine diphosphate (ADP). Glyerol-3-phosphate is

72
oxidized to dihydroxy-acetone phosphate (ADP) by glycerol phosphate oxidase producing
hydrogen peroxide (H2O2).
In a Trinder type colour reaction catalyzed by peroxidase the reacts with 4-
aminoantipyrine (4AAP) and 4-chlorophenol to produce a red colored dye. The increase in
absorbance is directly proportional to the concentration of triglycerides.
SPECIMEN COLLECTION
1. Serum, heparin or EDTA plasma is suitable for samples.
2. Whole blood and hemolyzed specimen are not recommended for use as samples.
3. Freshly drawn serum is the preferred specimen.
4. Use the suitable tubes or collection containers and follow the instruction of the
manufacturer.
5. Centrifuge samples containing precipitate before performing assay.
Stability
5 - 7 days at 2-8°C
7 days at 4-8°C
at least a year at -20°C
REAGENTS
R1: Phosphate buffer, 4-chlorophenol, ATP, Mg2+, Glycerolkinase, Peroxidase,
Lipoprotein lipase, 4-aminoantipyrine, Glycerol-3-phosphate-oxidase
Standard (Std): See bottle label
STORAGE AND STABILITY

73
 The unopened reagents are stable up to expiration indicated on the label when stored
unopened at 2-8°C and protected from light.
 Contamination of reagents must be avoided.
 Do not freeze reagents.
 Hemoglobin = 500 mg/dL
NORMAL VALUES
< 200 mg/dl
MATERIALS:
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
PROCEDURE
instructions.
2. Set the machine for cholesterol to read the absorbance.

74
at 37°C.
at 37°C
75
at 37°C.
at 37°C.
The preparation of the control, blank, standard and unknowns can be done simultaneously.
CONVERSION
 To convert the result from mg/dL to mmol/L, multiply value in mg/dL by 0.0113
76
QUALITY CONTROL
guidelines.
WASTE MANAGEMENT
77
REFERENCES
1. Rifai, N., Bachorik, P..,S &Albers J..J (1999). Lipids, lipoproteins and apolipoproteins.
In: C. A. Burtis & E. R. Ashwood (Eds..) Tietz textbook of clinical chemIstry (3rd ed. )
(pp. 809 861). Philadelphia: W. B. Saunders Company.
2. Artis, J. D., & Zak, B. Measurement of cholesterol concentration. (1997). ln N.Rifai, G.
R. Warnick, G. R. & M. H. Dominiczak (Eds.). Handbook of Iipoprotein testing (pp.94-
114). Washington: AACC Press.
3. Triglycerides. (2017, January 5th). Erba Lachema s.r.o,. Karásek 2219/1d, 62100 Brno,
CZ.
Wilkins.

78
PRECISE DETERMINATION OF HDL-CHOLESTEROL USING VENOUS BLOOD
High-density lipoproteins (HDL) compose one of the major classes of plasma
Lipoproteins. They are synthesized in liver as complexes of apolipoprotein and phospholipid and
are capable of picking up cholesterol and carrying it from arteries to the liver, where the
cholesterol is converted to bile acids and excreted into the intestine.
An inverse relationship between HDL Cholesterol (HDL-C) levels in serum and the
incidence/prevalence of coronary heart disease (CHD) has been demonstrated in a number of
epidemiological studies. The importance of HDL-C as a risk factor for CHD is now recognized.
Accurate measurement of HDL-C is of vital importance when assessing patient’s risk for
CHD.
METHOD
Colorimetric, Endpoint, Increasing reaction, Precipitation
PRINCIPLE
Phosphotungstate + Magnesium
Serum/Plasma HDL + (LDL + VLDL + CHYLOMICRONS)
(Supernatant) (Precipitate)
Chylomicrons, LDL and VLDL (low and very low density lipoproteins) are precipitated
from serum by phosphotungstate in the presence of divalent cations such as magnesium. The
HDL cholesterol remains unaffected in the supernatant and is estimated using ERBA Cholesterol
reagent.
79
SPECIMEN COLLECTION AND HANDLING
 Use serum, plasma (EDTA)
 Serum should be separated from blood clot as rapidly as possible.
Stability:
For 24 hours at 20-25°C
7 days at 4-8°C
12 weeks at -20°C
REAGENT COMPOSITION
R1: Phosphotungstic Acid, Magnesium Chloride
R2: Standard: See bottle label
STABILITY AND STORAGE
 The unopened reagents are stable till the expiry date stated on the bottle and kit label
when stored at 2~8°C.
 Close reagent bottles immediately after use.
 Avoid contamination of opened reagent.
NORMAL RANGE
≥ 35 mg/dl
UNIT CONVERSION
80
 To convert the result from mg/dL to mmol/L, multiply value in mg/dL by 0.026.
MATERIALS:
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
PROCEDURE
instructions.
2. Set the machine for HDL-C to read the absorbance.
 Reagent is liquid, ready to use.
1. Pipette 250 L of HDL-PREC to 5mL test tube/s.
C. Precipitation Reaction
A. Preparation of Control
a) Label clean and dry test tube/s according to protocol.

81
b) Pipette 125 µL of Control to the working reagent test tube.
c) Aspirate or cover with parafilm and mix by inversion.
d) Stand for 10 minutes at room temperature.
e) Centrifuge at 4000 rpm (1800 x g) for 10 minutes to obtain a clear supernatant
B. Preparation of Standard
b) Pipette 125 µL of Standard to the working reagent test tube.
e) Centrifuge at 4000 rpm (1800 x g) for 10 minutes to obtain a clear supernatant
C. Preparation of Unknown
b) Pipette 125 µL of Unknown to the working reagent test tube.
e) Centrifuge at 4000 rpm (1800 x g) for 10 minutes to obtain a clear
supernatant.
Note: The precipitation reaction for the unknown, control, standard and blank can be done
simultaneously.
D. Preparation of Blank
1. Label clean and dry test tube according to protocol.
2. Pipette 500 µL Cholesterol reagent to test tube.

82
3. Pipette 30 µL distilled water to the Cholesterol reagent test tube.
6. Aspirate or read absorbance spectrophotometerically at 500 nm (546 nm) at 37°C.
E. Preparation of Standard
2. Pipette 500 µL Cholesterol reagent to a test tube.
3. Pipette 30 µL supernatant from Standard precipitation to the Cholesterol reagent test
tube.
F. Preparation of Control
3. Pipette 30 µL supernatant from Control precipitation to the Cholesterol reagent test tube.
G. Preparation of the Unknown

83
3. Pipette 30 µL supernatant from Unknown precipitation to the Cholesterol reagent test
tube.
The preparation of the control, blank, standard and unknowns can be done simultaneously.
QUALITY CONTROL
guidelines.
84
WASTE MANAGEMENT
REFERENCES
Wilkins.
3. HDL-C PREC. (2017, January 5th). Erba Lachema s.r.o,. Karásek 2219/1d, 62100 Brno,
CZ.
85
CALCULATION OF CHOLESTEROL USING PREVIOUSLY DETERMINED
TRIGLYCERIDE, TOTAL CHOLESTEROL AND HDL-CHOLESTEROL VALUES
Because of the effect of lipids to cardiovascular health, initial screening is recommended
for individuals who are aged 20 and older. Doctors often order a lipid panel test to screen
individuals for any risk for cardiovascular diseases such as coronary heart disease (CHD). The
lipid panel test commonly includes tests for cholesterol, HDL-cholesterol, LDL-cholesterol and
triglycerides. While measure of total cholesterol, HDL- cholesterol, LDL-cholesterol and
triglycerides are commonly offered in most laboratories, the method of LDL-cholesterol
measurement is too complicated to be performed for routine purposes. One method of LDL-
cholesterol measurement involves beta quantification where plasma is ultracentrifuge for 18
hours at 105K x gravity. The protracted length of the procedure makes it impractical to be used
for simple screening purposes.
In order to improve the speed with which LDL-cholesterol levels can be re reported, the
routine method is the indirect method of quantitation LDL-cholesterol through its calculation.
Cholesterol-LDL levels are derived by subtracting HDL and VLDL levels from total cholesterol
using the Friedewald formula.
LDL-Cholesterol = Total Cholesterol – HDL – VLDL
If VLDL is not measured, then the formula can be modified by using triglyceride
measurement instead of VLDL. The VLDL is divided by 2.175 or 2.2. For plasma triglyceride
levels which are in mmol/L, and by 5.0 for those in mg/dl.

86
TAG in mmol/L
LDL-Cholesterol = Total Cholesterol – HDL –
2.175
TAG in mg/dl
LDL-Cholesterol = Total Cholesterol – HDL –
5
LDL, or also known as the “bad cholesterol”, is synthesized in the liver and it is a product
of the catabolism of VLDL. In plasma, 50% of lipoproteins are in this form. The main function
of LDL is the transport of cholesterol to peripheral tissues where cholesterol is then endocytosed
by LDL-receptors. LDL contains the most cholesterol of all lipoproteins which makes it
atherogenic or capable of causing atherosclerosis. Increased level of LDL-cholesterol is a cause
for concern and lowering it is the main target of cholesterol therapy. LDL is a risk factor CHD.
After the initial screening, lipid panel testing should be done once every five years
thereafter. Lipid profile testing can be done at least 3 weeks after any trauma, bacterial or viral
infection and at least 3-4 months after giving birth.
METHOD
Indirect Determination of LDL
PRINCIPLE
LDL cholesterol values can be calculated using Friedewald formula which is reliable if
chylomicrons are absent in the sample, the triglycerides concentration is < 400 mg/dl and the
samples are not derived from patients with type III hyperlipoproteinemia.
NORMAL RANGE
 Desirable = ≤130 mg/dl
 Bordeline high risk = 130-160 mg/dl

87
 High risk = > 160 mg/dl
REFERENCES
1. Friedewald, W. T. (1971). Clinical chemistry, 18, 499
Wilkins.
88
PRECISE DETERMINATION OF SODIUM
Sodium measurements are used in the diagnosis and treatment for disturbances of fluid
and electrolyte balance, e.g. due to loss of water or salt, and other serum electrolytes deviating
from their reference interval by polyuric-polydypsic syndromes and impaired thirst, renal
diseases, hypertension, disorders of the acid-base balance, some endocrine diseases, edema,
excessive sodium intake.
METHOD
Mg-Uranylacetate Method, Colorimetric
PRINCIPLE
Sodium is precipitated with Mg-uranyl acetate; the uranyl ions remaining in suspension
form a yellow-brown complex with thioglycolic acid. The difference between reagent blank
(without precipitation of sodium) and analysis is proportional to the sodium concentration.
SPECIMEN COLLECTION
 Unhemolyzed serum
REAGENTS
PREC: Uranyl acetate, Magnesium acetate
RGT (colour reagent): Ammonium thioglycolate
STD: Sodium = 150 mmol/L

89
REAGENT STABILITY
 Stored unopened at room temperature and in the dark, the contents are usable until
the expiry date printed on the label.
NORMAL RANGE
Male = 135-148 mg/dl
Female = 135-155 mg/dl
MATERIALS:
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
PROCEDURE
instructions.
2. Set the machine for Sodium (Na+) to read the absorbance.
3. Set the wavelength to Hg 365 nm, or Hg 405 nm, or 410 nm (360-410 nm) at 20...25°C.
90
Note: The photometer is zeroed against distilled water. Optical density results must be corrected
for the reagent blank (RB). Only one RB per series is required.
1. Pipette 500 µL PREC reagent to 5mL test tube/s.
2. Pipette 500 µL RGT to 5mL test tube/s.
C. Precipitation Reaction
1) Label clean and dry test tube according to protocol.
2) Pipette 10 µL Control to the PREC reagent test tube.
3) Mix thoroughly by aspirating several times.
4) Cover with Parafilm
5) Allow it to stand for 5 minutes at room temperature.
6) Shake intensively for at least 30 seconds.
7) Allow to stand for 30 minutes at room temperature.
8) Centrifuge at high speed for 10 minutes to obtain clear supernatant.
B. Preparation of Standard
2) Pipette 10 µL Standard to the PREC reagent test tube.
4) Cover with parafilm.

91
C. Preparation of Unknown
2) Pipette 10 µL Unknown to the PREC reagent test tube.
4) Cover with Parafilm
Note: The precipitation reaction for the unknown, control, standard and blank can be done
simultaneously.
C. Preparation of Reagent Blank
1) Label a clean and dry test tube according to protocol.
2) Pipette 500 µL RGT to a 5 mL test tube.
3) Pipette 20 µL PREC reagent to the RGT test tube.
4) Cover with parafilm.
5) Allow to stand for 10 minutes.
6) Aspirate or read absorbance spectrophotometerically at 360-410 nm in 20...25°C.

92
1) Label a clean and dry test tube according to to protocol.
2) Pipette 20 µL supernatant from Standard precipitation to the RGT test tube.
4) Cover with parafilm
5) Allow to stand for 10 minutes
6) Aspirate or read absorbance spectrophotometerically at 360-410 nm at 20...25°C.
1) Label a clean and dry test tube according to to protocol.
2) Pipette 20 µL supernatant from Control precipitation to the RGT test tube.
6) Aspirate or read absorbance spectrophotometerically at 360-410 nm at 20...25°C.
F. Preparation of the Unknown
1) Label a clean and dry test tube according to protocol.
2) Pipette 20 uL supernatant from the Unknown precipitation to the RGT test tube.

93
6) Aspirate or read absorbance spectrophotometerically at 360-410 nm at 20-25°C.
QUALITY CONTROL
guidelines.
WASTE MANAGEMENT
REFERENCES
1. Henry R.J. et al., Clinical Chemistry., Harper & Row New York, Sec. edit. 643 (1974).
2. Human (2016, April 16th). Sodium rapid: Package Insert.

94
PRECISE DETERMINATION OF POTASSIUM
METHOD
Photometric Turbidimetric Test
PRINCIPLE
Potassium ions in a protein-free alkaline medium react with sodium tetraphenylboron to
produce a finely dispersed turbid suspension of potassium tetraphenylboron. The Turbidity is
proportional to the potassium concentration and read photometrically.
REAGENT COMPOSITION
PREC 50 ml Precipitant (white cap)

Trichloroacetic acid (TCA)
TPB 50 ml TPB-Na-Reagent (black cap)

Sodium tetraphenylboron
NaOH 50 ml NaOH Reagent (red cap)

Sodium hydroxide
STD 5 ml Standard
Potassium (K+)
REAGENT STABILITY
 The reagents are stable up to given expiry date when stored at 2-25°C
SPECIMEN
 Serum and lithium heparin plasma

95
NORMAL RANGE
Potassium = 3.5 – 5.3 mmol/L
MATERIALS:
 5mL test tubes
 Parafilm
 Pipette tips
 Tissue paper
 Waste container
PROCEDURE
A. Precipitation Reaction
2. Pipette 250 µL of PREC reagent to 5 ml test tube/s.
3. Pipette 25 µL Control to the PREC reagent test tube.
4. Mix thoroughly by aspirating several times.
5. Cover with Parafilm
6. Allow it to stand for 10 minutes at room temperature.
7. Centrifuge at high speed for 5-10 minutes to obtain clear supernatant.
B. Preparation of Unknown

96
2. Pipette 250 µL of PREC reagent to 5 ml test tube/s.
3. Pipette 25 µL Unknown to the PREC reagent test tube.
6. Allow it to stand for 10 minutes at room temperature.
7. Centrifuge at high speed for 5-10 minutes to obtain clear supernatant.
Note: The precipitation reaction for the unknown, and control can be done simultaneously.
C. Preparation of Reagent Blank
1. Label a clean and dry test tube according to protocol.
2. Mix 250 µL of TPB reagent and 250 µL of NaOH to 5 ml test tube/s.
 Allow to stand for 15-30minutes prior to use
3. Pipette 50 µL of PREC reagent to TPB and NaOH test tube.
5. Cover with parafilm.
6. Allow to stand for 10 minutes at temperature.
7. Aspirate or read absorbance spectrophotometerically at 578 nm at 20-25°C.
1. Label a clean and dry test tube according to to protocol.
2. Mix 250 µL of TPB reagent and 250 µL of NaOH to 5 ml test tube/s.
3. Pipette 50 µL Standard to the TPB and NaOH test tube.

97
5. Cover with parafilm
6. Allow to stand for 10 minutes at room temperature
1. Label a clean and dry test tube according to to protocol.
2. Mix 250 µL of TPB reagent and 250 µL of NaOH to 5 ml test tube/s
3. Pipette 50 µL supernatant from Control precipitation to the working reagent test tube.
6. Allow to stand for 10 minutes at room temperature.
F. Preparation of the Unknown
1. Label a clean and dry test tube according to protocol.
2. Mix 250 µL of TPB reagent and 250 µL of NaOH to 5 ml test tube/s
3. Pipette 50 µL supernatant from Control precipitation to the working reagent test tube.
6. Allow to stand for 10 minutes at room temperature.

98
Note: The unknown, control, blank and standard can be prepared simultaneously.
QUALITY CONTROL
guidelines.
WASTE MANAGEMENT
Notes
1. Use non-hemolytic serum or heparin plasma as specimen.
2. As red blood cells contain 25X the amount of potassium, they have to be separated
from the serum within one hour after blood collection. Otherwise, falsely elevated
potassium concentrations will be found.
3. Traces of detergents produce turbidity which leads to falsely elevated potassium
concentrations. They therefore have to be avoided

99
4. In blood collection, prolong application of tourniquet can cause falsely elevated
potassium. Application of tourniquet must be less than 1 minute.
REFERENCES
 Potassium Liquirapid (2008, June 16th). Human: Package Insert.

100
GLYCOSYLATED/GLYCATED HEMOGLOBIN (HbA1c)

CLOVER A1c® Self: Test Analyzer
INTENDED USE
The CLOVER A1c® Self Analyzer is an IVD (In Vitro Diagnostic Device) device for
measuring Hemoglobin A1c by the well-established method of boronate affinity. It is able to
analyze both capillary whole blood and venous blood. Anticoagulants may be used, such as
EDTA (ethylenediaminetetracetic acid) and heparin etc. This CLOVER A1c® Self system is
designed to help controlling diabetes and it is intended to be used by patients and professionals at
home, laboratories, clinics and hospitals. Same as all diagnostic tests, do not make a definitive
diagnosis of diabetes based on the result of single test. A physician should make a diagnosis after
all clinical and laboratory findings are evaluated.
PRINCIPLE
The CLOVER A1c® Self system uses reflectance spectrophotometer in a fully automated
boronate affinity assay for the determination of the percentage of Hemoglobin A1c (HbA1c) in
whole blood. The reagent pack is pre-filled with reagent and rinsing solution. The reagent
solution contains agents that lyse erythrocytes and boronate bead that binds cis-diols of glycated
hemoglobin.
SAMPLE COLLECTION AND HANDLING
 Capillary whole blood
 Venous whole blood with K2EDTA or K3EDTA, lithium heparin, sodium citrate or
sodium fluoride/oxalate as anticoagulants

101
 Venous whole blood can be stored at 2-8°C for 7 days with unbroken seal (3 days if
broken) and at 20-25°C for 3 days.
 Allow blood sample to reach room temperature. Anticoagulated blood should be mixed
well prior to testing.
NORMAL VALUES
HbA1c = 4 – 5.4%
 5.5%-6.8%: Pre diabetic
 6.8%-7.6%: Fair control
 7.6% above: Poor control
MATERIALS
Daily Check Cartridge
Monthly Check Cartridge
Test Cartridge/s
Lancet (for capillary whole blood processing)
Cotton
Waste receptacle
Tissue
102
PREPARATION OF THE SPECTROPHOTOMETER
 When the power is connected, the display will show “Warming up” for 5 minutes
depending on the ambient temperature. This
PROCEDURE
1. Open the lid of the CLOVER A1c® Self Analyzer, when the analyzer is in “Stand-by”
mode, displaying the “Open the Lid” icon.
2. Open the Test Cartridge pouch by tearing the pouch on the side with serrated edge. Do
not use scissors to open to avoid damaging the reagent pack.
3. Gently insert the cartridge into the cartridge compartment when “Insert Test Cartridge”
is shown. Hold the cartridge with barcode facing left. Ensure a gentle snap is either heard
or felt to confirm proper placement.
4. The display will show “Apply Sample to sample area” and “Insert Reagent Pack”
icon.
5. Gently mix the reagent pack 5-6 times before applying blood sample.
6. Apply the blood sample by gently touching the drop of the blood either from the fingertip
or from the venous blood with the tip of the sampling area. Ensure the sampling area is
completely filled.
Capillary Whole Blood Process
Prick the finger tip of the patient to get a minimum of 4µL of capillary blood sample.
Touch softly the blood sample with capillary tip of the reagent pack. The blood is
automatically drawn up.
Make sure that the sample area is completely filled

103
Venous Whole Blood Process
Venous whole blood collected in tubes with K 2EDTA or K3EDTA, lithium heparin,
sodium citrate or sodium fluoride/oxalate as anticoagulants can be used.
Allow blood sample to reach room temperature. Anticoagulated blood should be
mixed well prior to testing.
Remove the stopper from the holder and take out a drop of blood sample on clean
container.
Softly touch the sampling area of the reagent pack on the blood sample, and wait until
the sampling area is completely filled.
Note: Do not wipe off excess blood outside the sampling area. Do not touch the open
end of the sampling area.
7. Insert the reagent pack into the cartridge in the analyzer. The “Close the lid” icon will be
displayed.
8. The test starts automatically when the lid is closed.
9. Record results after 5 minutes.
10. After the test is completed, open the lid. “Remove catridge” from the analyzer by gently
pushing it to the left and pulling it out.
11. Dispose all waste in accordance with applicable national and/or local regulations.
QUALITY CONTROL
The CLOVER A1c® Self Check Cartridge checks the optical and operating systems of
the analyzer.
104
Types of Check Cartridges
1. CLOVER A1c® Self Daily Check Cartridge
 Use before samples are tested
a) Open the lid CLOVER A1c® Self analyzer
b) Press “Down” button for 3 seconds to enter into the “Daily Check” mode.
c) Insert the Daily Check Cartridge while Daily and Check are blinking.
d) Close the lid. The test starts automatically.
e) After 1 minute “OK” or error message will be displayed.
f) Press the “Mode” button and remove the Daily Check Cartridge when the test is
completed.
2. CLOVER A1c® Self Monthly Check Cartridge
This is use when there is a concern that the test result may be incorrect, after an
error message (refer to “Instructions for Use” manual for troubleshooting) and once a
month before samples are tested.
a) Open the lid CLOVER A1c® Self analyzer
b) Press “Up” button for 3 seconds to enter into the “Monthly Check” mode.
c) Insert the Monthly Check Cartridge while Daily and Check are blinking.
d) Gently mix the Check Reagent Pack before use.
e) Insert the Check Reagent Pack, when the “Insert Check Reagent Pack” icon is
displayed. Insert it respectively.
f) Close the lid. The test starts automatically.

105
g) After 5 minutes “OK” or error message will be displayed.
h) Press the “Mode” button and remove the Monthly Check Cartridge when the
test is completed.
CALIBRATION
The CLOVER A1c® Self analyzer is factory calibrated. The analyzer automatically self-
adjusts during each power-up and during assay. An error message will appear if the analyzer is
not able to make appropriate internal adjustments.
REFERENCES
 Clover A1c Self: Test analyzer (Revise Ed. 2017/05/01). Instruction for use for
measuring Hemoglobin A1c. Diabetes Management Technology.

106
OTHER GLUCOSE TESTS
1. ORAL GLUCOSE TOLERANCE TEST (OGTT)
Requirements for OGTT:
 Patient should be ambulatory.
 Unrestricted diet of 150 g carbohydrate/day for 3 days prior to testing.
 The patient should not smoke and drink alcohol prior testing.
 Fasting for 8-14 hours or 8-12 hours
 The patient should avoid exercise, eating, drinking (except water) and smoking during the
test.
 For non-pregnant women and adults, only fasting and the 2-hour sample may be
measured.
GLUCOSE LOAD:
75 g - WHO standard glucose load
100 g - pregnant women
1.75 g glucose/kg body weight – for children (to a maximum of 75 g)
PROCEDURE
1. Collect the fasting blood sample. (Urine may also be collected)
2. Instruct the patient to drink the glucose load within 5 minutes.
3. Collect blood sample after 1-hour, 2-hour or 3-hour, respectively.
NORMAL VALUES
107
FBS = 75-115 mg/dl 1-hour = 95-165 mg/dl 2-hour = 80-130
mg/dl
2. ORAL GLUCOSE CHALLENGE TEST
 Fasting is not required.
 It can be done any time of the day, not on an empty stomach.
GLUCOSE LOAD: 50 g glucose load
PROCEDURE
1. Instruct the patient to drink the glucose load within 5 minutes.
2. Collect sample after 1-hour
NORMAL VALUES = 95-165 mg/dl
3. 2-HOUR POST PRANDIAL BLOOD SUGAR
 Two-Hour Post Prandial or Post Cibum Glucose Levels are used to screen for
Diabetes Mellitus, to diagnose diabetes, and to monitor glucose control in the
known diabetic. Usually the maximum increase in blood glucose following a meal
occurs at 60 to 90 minutes, and by two hour levels are similar to fasting levels.
PATIENT PREPARATION
1. Unrestricted diet of 150 g carbohydrate/day
2. Fasting for 8-14 hours or 8-12 hours
3. The patient should avoid exercise, eating, drinking (except water) and
smoking during the test.

108
PROCEDURE
1. Extract fasting blood sugar only if ordered by the physician. It is common for
both fasting and 2-hour samples to be requested for a diabetic screen.
2. The patient consumes a breakfast or lunch containing 50-100 grams of
carbohydrate. Often patients have instructions from their physician’s office for a
carbohydrate-rich meal which they need to follow. An acceptable breakfast meal
consists of orange juice, cereal with sugar, toast and milk. The patient should
remain at rest and avoid medications, smoking, caffeine and alcohol during the
test. Water is permitted.
3. Draw patient blood sample for glucose determination 2 hours after completion of
the meal.
NORMAL VALUES: 70-150 mg/dl

109
CLINICAL LABORATORY DIAGNOSIS USING URINE AND STOOL
Clinical Examination Using Urine (URINALYSIS)
INTRODUCTION
Urine is produced by the kidneys to maintain constant plasma osmotic concentration; to
regulate pH, electrolyte, and fluid balances, and to excrete some 50 grams of waste solids
(mostly urea and sodium chloride). Examination of urine or urinalysis helps evacuate the normal
functioning of the kidneys as well as provide valuable information for the detection of diseases
related to the kidney and urinary tract. There are several types of urine specimens. Nonetheless,
for routine urinalysis, the most commonly examined is random, midstream, clean catch urine.
This urine is placed in the container. However, the ideal specimen for urinalysis is the early
morning, midstream catch, volume is uniform and sediments are still intact. Also, it is
recommended that genitalia have to be washed properly prior collection. Approximately 10-15
ml of urine has to be submitted to the laboratory for examination. Routine urine screening tests
are done to get valuable information for the detection of disease related to the kidney and urinary
tract.
SPECIMEN COLLECTION
1. Random Urine Collection
 For routine and qualitative urinalysis
 If time permits, collect the first voided specimen of the morning.
 Collect the specimen in a nonsterile container with a screw top lid.

110
 If the specimen cannot be sent to the lab immediately, refrigerate until taken to the lab.
Specimens can be refrigerated for 24 hr. They should be received in the laboratory within
1 hour of removal from refrigeration.
2. Clean Catch Midstream Urine Collection
 For routine screening and bacterial culture
For Female Patients
Tell the patient to:
1. Wash her hands well and remove the lid from the collection cup, and open the towelette
package.
2. Separate the labia and keep the labia separated until the urine is collected.
3. Cleanse the meatus with one towelette using a front to back wipe.
4. Repeat with next towelette.
5. Keep the labia separated and begin to urinate into the toilet.
6. Move the collection cup into the urinary stream making sure the cup does not come in
contact with clothing, legs, or genitals.
7. Fill the container halfway, remove the cup from the urinary stream, and finish voiding
into the toilet.
8. Wash her hands, replace the lid on the container, and dry the outside of the container.
111
For Male Patients
Tell the patient to:
1. Wash his hands well, remove the lid from the collection cup, and open the towelette
package.
2. Pull back the foreskin, if present, to expose the urinary meatus.
3. Wash the meatal opening with the towelette using a circular motion.
4. Repeat with the remaining towelettes.
5. Begin voiding into the toilet.
6. Move the collection cup into the urinary stream making sure the cup does not come i
contact with clothing, legs, or genitals.
7. Fill the container halfway, remove the cup from the urinary stream, and finish voiding
into the toilet
8. Wash his hands, replace the lid on the container, and dry the outside of the container.
3. First Morning Urine
Ideal specimen for routine Urinalysis and pregnancy test (hCG). It is the most
concentrated, most acidic (for well preservation of cells and casts). And it is for
evaluation of orthostatic proteinuria.
4. Catheterized
 For bacterial culture
5. Pediatric specimen
112
 Use soft, clear plastic bag with adhesive
6. Timed specimen
 24-hour:
 Day 1: 7am, Patient voids and discard specimen; collects all urine for the next 24
hours.
 Day 2: 7am, patient voids and adds this urine to previously collected urine.
 12-hour: For Addis count
 4-hour: For Nitrite determination
 Afternoon (2pm - 4pm): For Urobilinogen determination
SPECIMEN INTEGRITY
 Following collection, urine specimens should be delivered to the laboratory promptly
and tested within 2 hours.
SPECIMEN PRESERVATION
 Refrigeration - most routine method of preservation at 2°C to 8°C.
IDEAL SPECIMEN CONTAINERS
 Clean, dry, leak-proof containers
 Wide mouth, tight fitting lid

113
SPECIMEN REJECTION
 Unlabeled specimens
 Nonmatching labels and requisition forms
 Specimens contaminated with feces or toilet paper
 Containers with contaminated exteriors
 Insufficient quantity
 Improper transport
PHYSICAL EXAMINATION OF URINE
VOLUME
 Average volume required for submission is 12 mL (10 mL - 15 mL).
COLOR
Normal = colorless to deep yellow (straw - amber)
Major pigments
1. Urochrome (major pigment) – yellow
2. Uroerythrin - amorphous urates and uric acid; pink
3. Urobilin - Imparts an orange-brown color to a urine that is not fresh; dark-yellow/orange;
Indicator of hydration
114
Abnormal color
1. Dark yellow/Amber/Orange - presence of the abnormal pigment bilirubin (yellow
foam); increase concentration of protein (white foam); photo-oxidation of large amounts
of urobilinogen to urobilin; phenazopyridine administration
2. Yellow-green - presence of biliverdin
3. Red/Pink/Brown - RBCs (Cloudy/smokey red); Hemoglobin (Clear red)
4. Brown/Black - methemoglobin (acidic urine); homogentisic acid (alkaline urine);
Melanin (upon air exposure)
5. Blue/Green - caused by indican in urine or bacterial infection (Klebsiella or Providencia)
CLARITY/TRANSPARENCY
Normal = Clear
Abnormal
1. Hazy: Few particulates, transparent
2. Cloudy: Many particulates, print blurred through urine
3. Turbid: Print cannot be seen through urine
4. Milky: May precipitate or be clotted

115
URINE ODOR
Normal = Aromatic
Abnormal
1. Foul, ammoniacal
2. Fruity, sweet
3. Maple syrup
4. Mousy
5. Rancid
6. Sweaty feet
7. Cabbage
8. Bleach
CHEMICAL EXAMINATION OF URINE
Reagent Strip:
 Provide a simple, rapid means for performing medically significant chemical analysis
of urine.
 Contains chemical impregnated absorbent pads attached to a plastic strip: pH, protein,
glucose, ketones, blood, bilirubin, urobilinogen, nitrite, leukocytes and specific
gravity.
 Must be checked with both negative and positive controls a minimum of once every
24 hours
116
Reagent Strip Technique
a. Dip the reagent strip into a well-mixed uncentrifuged urine specimen at room
temperature.
b. Remove excess urine by touching the edge of the strip to the container as the
strip is withdrawn.
c. Blot the edge of the strip on a disposable absorbent pad
d. Wait the specified amount of time for the reaction to occur
e. Compare the color reaction of the strip pads to the manufacturer's color chart in
good lighting.
Care of Reagent Strips
a. Store with desiccant in an opaque, tightly closed container.
b. Store below 30°C; do not freeze
c. Do not expose to volatile fumes.
d. Do not use past the expiration date
e. Do not use if chemical pads become discolored
f. Remove strips immediately prior to use.

117
Reading Urine
Principle Positive
Time Parameter
Glucose Double sequential enzyme reaction Gree-brown

30 secs
tan or pink to
Bilirubin Diazo reaction
violet
Sodium nitroprusside reaction
40 secs Ketones Purple
(Legal's Test
45 secs Specific Gravity pKa change of polyelectrolyte Blue to yellow
Protein (Sorensen's) error of
Protein Blue
indicator
pH Double indicator system Orange
Uniform
60 secs green/blue (Hgb or
Pseudoperoxidase activity of
Blood Mb)
hemoglobin
Speckled/spotted
(intact RBCs)
Urobilinogen Ehrlich reaction Red
Nitrite Greiss reaction Uniform pink
120 secs Leukocytes Leukocyte Esterase Purple
MICROSCOPIC EXAMINATION OF URINE
 To detect and to identify insoluble materials present in urine
Volume: 10 - 15 mL urine (Average: 12 mL)
Centrifugation: 400 RCF for 5 minutes
MICROSCOPIC TECHNIQUES
1. Bright-field microscopy - for routine urinalysis
2. Phase - contrast microscopy - enhances visualization of elements with low
refractive indices (ex: hyaline cast)
3. Polarizing microscopy - Identification of cholesterol in oval fat bodied, fatty
casts, and crystals.
4. Dark-field microscopy - Identification of Treponema pallidum

118
5. Fluorescence microscopy - Visualization of fluorescent microorganisms or
those stained by fluorescent dye.
6. Interference microscopy - 3-D microscopy image & layer-by-layer imaging
of a specimen.
a. Nomarski (Differential)
b. Hoffman (Modulation)
SEDIMENT CONSTITUENTS
A. CELLS
RBCs (Hematuria)
NV = 0-2 or 0-3/HPF
o Smooth, non-nucleated, biconcave disks
o Hypertonic urine = crenate/shrink
o Hypotonic urine = swell/hemolyze; “ghost cell”
o Glomerular membrane damage: dysmorphic with projections, fragmented
o Sources of error: Yeasts, oil droplets, air bubbles, calcium oxalate crystals
Remedy: Add 2% acetic acid to lyse RBCs but not the others
WBCs (Pyuria or Leukocyturia)
NV = 0-5/HPF or 0-8/HPF
119
o Increased number indicates the presence of an infection or inflammation.
a. Neutrophils (predominant) – granulated and multilobed. In hypotonic,
granules swell an undergo BROWNIAN MOVEMENT producing a
sparkling appearance (GLITTER CELLS).
b. Eosinophils – NV = <1%; Increased amount may have associated with
drug-induced interstitial nephritis.
c. Mononuclear cells – small numbers
EPITHELIAL CELLS
A. Squamous epithelial cell – largest cells with abundant, irregular cytoplasm
and prominent nucleus. This is the point of reference. Sometimes, it is
covered or studded with bacteria or CLUE CELL (Gardnerella vaginalis).
REPORTING: RARE, FEW, MODERATE OR MANY/LPF
B. Transitional epithelial (Urothelial) cell – spherical, polyhedral or caudate
with centrally located nucleus. ↑ numbers are seen in catheterization.
REPORTING: RARE, FEW, MODERATE OR MANY/HPF
C. Renal tubular epithelial cells – most clinically significant epithelial cell;
originally from nephron. It is rectangular, polyhedral, cuboidal, or columnar
with an eccentric nucleus. >2 RTE/HPF indicates TUBULAR INJURY.
REPORTING: AVERAGE NUMBER/HPF

120
BACTERIA – UTI = Bacteria + WBCs; Enterobacteriaceae for example E. coli
(most common cause of UTI); Staphylococcus, Enterococcus
YEASTS – Yeasts + WBCs (true yeasts infections); small retractile oval structures
that may or may not bud. Candida albicans = seen in DM & vaginal moniliasis
(candidiasis).
PARASITES
a. Trichomonas vaginalis – most frequent parasite seen the urine;
pear-shaped flagellate with jerky motility; agent of “PING-PONG
DISEASE”
b. Schistosoma haematobium ova – blood fluke with terminal spine;
causes hematuria; associated with blood cancer
Specimen: 24-hour unpreserved urine
c. Enterobius vermicularis ova – most common fecal contaminant
SPERMATOZOA – after sexual intercourse
REPORTING: PRESENT, BASED ON LABORATORY PROTOCOL
MUCUS THREADS – Major constituents = Tamm-Horsfall protein (uromodulin)
REPORTING: RARE, FEW, MODERATE OR MANY/LPF

121
B. CASTS (Cylinduria)
 Unique to the kidney
 Formed in the DCT and collecting duct
 Major constituent = Tamm-Horsfall protein; produced by RTE cells
 Performed along to the edges of the coverslip with subdued light
Cast Information Clinical Significance

Physiologic: Stress, Strenous
exercise
Prototype cast; Beginning of all types of casts Pathologic: Glomerulonephritis,
1. Hyaline cast
NV = 0-2/LPF Pyelonephritis, Congestive Heart
Failure
Glomerulonephritis, Strenuous
2. RBC cast Bleeding within nephron exercise
Pyelonephritis; Acute interstitial
3. WBC cast Inflammation within nephron nephritis
4. Epithelial (RTE) Advance tubular detsruction; Renal
cell cast tubular damage
5. Bacterial Cast Identified by performing gram stain Pyelonephritis
Granules are derived from lysosomes of RTE Glomerulonephritis, Strenuous
6. Granular cast cells during normal metabolism (non- exercise; stress; pyelonephritis
pathologic)
Nephrotic syndrome (lipiduria)
Not stained with Sternheimer-Malbin stain
Identification:
TAG &Neutral fats = stain with Oil Red
7. Fatty cast
O & Sudan III
Cholesterol = Polarizing microscope
(MALTESE CROSS)
Final degenerative form of all types of casts; Stasis of urine flow; Chronic renal
8. Waxy cast failure
brittle, highly refractile with jagged ends
Also known as Renal Failure Casts; indicates Extreme urine stasis; Renal failure
9. Broad cast destrruction (widening) of the tubular walls;
any type of casts can be broad
122
C. CRYSTALS
 Precipitation of urine solutes (salts, organic compounds, medication)
 Factors that contribute to crystal formation:
o Temperature
o Solute concentration
o pH
REPORTING: RARE, FEW, MODERATE OR MANY / HPF
REPORTING OF ABNORMAL CRYSTALS: AVERAGE / LPF
NORMAL URINARY CRYSTALS

pH Crystals Information Significance
Brick dust / yellow brown
granules; Pink sediment
(uroerythrin)
Amorphous urates (acidic urine)
Product of purine metabolism; Lesch-Nyhan
Rhombic, wedge, hexagonal, syndrome (Orange-
four-sided flat tape Sand diaper);
(whetstone), lemon-shaped Chemotherapy
(Increased
metabolism of
nuclei); Gout
Uric acid (acidic urine)
ACIDIC
Forms: ↑ Food high in

1. Dihydrate ascorbic acid
(Wheddelite): (tomatoe /
envelope/pyramidal asparagus);
2. Monohydrate (Whewellite): Ethylene Glycol
oval/dumbell poisoning (anti-
freeze agent): ↑
Monohydrate
CaOX
Calcium oxalate
(acidic/neutral/rarely alkaline urine)
"Cigarette-butt" in appearance
Calcium sulfate (acidic urine)
Yellow-brown or colorless
Hippuric acid (acidic urine) elongated prisms
123
NORMAL URINARY CRYSTALS

Amorphous Phosphates Granular appearance; white
(alkaline/neutral) precipitate
Yellow-brown "thorny apples; Presence of Urea-
Ammonium biurate (alkaline) Seen in old specimens splitting bacteria
Colorless, prism shape, or

Triple phosphate/Magnesium "coffin-lid", feathery
ammonium phosphate/ Struvite appearance when they Presence of Urea-
(alkaline) disintegrate, Fern leaf splitting bacteria
ALKALINE
Colorless, flat plates, thin

prisms in rosette form; Rosettes
may resemble sulfonamide
crystals
Forms:
a. Hydroxyapatite (basic
calcium phosphate)
Calcium phosphate/Apatite b. Brushite
(alkaline/neutral) (calcium hydrogen phosphate)
Small, colorless, dumbbell or

spherical-shaped; Formation of
gas (effervescence) after
Calcium carbonate (alkaline) adding acetic acid
ABNORMAL URINARY CRYSTALS
Colorless, hexagonal plates; Cystinuria;
Cystine (acid) mistaken as uric acid crystals cystinosis
Rectangular plates with a notch

in one or more corners
(staircase pattern); Resemble Nephrotic syndrome
Cholesterol (acid) crystals of radiographic dye (lipiduria)
Colorless to yellow needles in Liver disease (more
Tyrosine (acid) clumps or rosettes common)
ACIDIC
Yellow-brown spheres with

Leucine (acid/neutral) concentric circles and striations Liver disease
Clumped needles or granules
Bilirubin (acid) with bright yellow color Liver disease
Colorless to yellow-brown
needles, sheaves of wheat,
rosettes, arrowhead, petals, and Possible tubular
round forms; Mistaken as damage (may
Sulfonamide (acid/neutral) Calcium phosphate crystals deposit in nephrons)
Colorless needles that tend to
form bundles followign Massive dose of
Ampicilin (acid/neutral) refrigeration penicillin
124
Urinary Sediment Artifacts
1. Starch granules
 Spheres with dimples center
 “Maltese cross” formation on polarizing microscope
2. Oil droplets
3. Air bubbles
4. Pollen grains (spheres with a cell wall and concentric circles
5. Hair & Fibers (mistaken as casts)
6. Fecal contamination
SUPPLIES AND EQUIPMENT
1. Clean test tubes
2. Test tube rack
3. Slides and coverslip
4. Reagent strip
5. Waste receptacle
6. Microscope
PROCEDURE
1. Wash hands and put on PPE
2. Obtain urine specimen from patient with proper label
3. Mix the urine specimen thoroughly and fill a clean test tube with urine until ½ to 2/3 full.
4. Place on the test tube rack.

125
5. Examine the physical characteristics – color and degree of turbidity or transparency, but
placing the test tube at eye level, with adequate light. Note and record the color and
transparency.
6. Place the test tube back to the test tube rack.
7. Open the reagent strip (dipstick) bottle, get one strip. Place strip in a clean paper
receptacle, DON’T PUT IT ANYWHERE TO AVOID BEING CONTAMINATED.
Close the lid of the bottle immediately after getting the strip. DON’T ALLOW THE
REAGENT BOTTLE TO STAY OPEN FOR A LONG TIME because moisture can
cause chemical changes in the strips and this will give false results.
8. Dip the strip into a well-mixed urine sample. DON’T KEEP THE STERILE STRIP IN
THE URINE FOR A LONG TIME because impregnated chemicals might be dissolved
and produce inaccurate results.
9. Remove the excess sample by passing the underside of the strip to pass the mouth of the
test tube or blot of with clean tissue paper. The readings will be inaccurate if the reagent
chemicals are mixed.
10. Observe the required time for the reaction to occur (precise timing makes reaction to
occur completely, thus, invalid or false results) and compare the color or depth of color of
each reagent area to the color chart at the bottle label under adequate light.
11. Fill up your test tube with urine at least 2/3 full.
12. Centrifuge at 400 RCF for 5 minutes.
13. Remove supernatant by decantation. Leave about 1 ml and mix.
14. Put a small amount of urine sediment on a slide and place the cover slip.
15. Examine using the LPO and HPO of the microscope.

126
16. Record results.
17. Do aftercare and hand washing.
REFERENCES
1. Strasinger, S. K. & Di Lorenzo, M. S. (2014). Urinalysis and Body Fluids (6 th ed.). F. A.
Davis Company, Philadelphia, Pennsylvania.

127
STOOL EXAMINATION
Rationale
Specimens submitted to the laboratory for examination for parasites must be collected
properly and transported to the laboratory without delay. Upon receipt, specimens should be
processed immediately, or preserved to maintain the quality of the specimen. Improperly
collected specimens may lead to the inability to identify parasites or incorrect interpretation of
results. Processing of specimens involves a number of standardized procedures used in the
routine analysis for ova and parasites (e.g. stool specimen for ova and parasites or 0 & P), as well
as specialized procedures performed only upon request.
Collection and Handling of Fecal Specimens
Fecal specimens should be collected in clean, wide-mouth containers with tight-fitting
lids, and sealed in plastic bags for transport. All specimens should be collected prior to
radiological studies using barium or the administration of bismuth, mineral oil or certain
antibiotics and antidiarrheal medications that may interfere with the detection and identification
of intestinal parasites. If therapy has already begun, stool samples should not be collected until 5-
7 days after completion of treatment. Care should be taken to avoid contamination with urine or
water, which might harm existing organisms or introduce free-living organisms from the
environment.
General Considerations in Stool Examination
1. Stools after enemas are suitable for examination of worms, eggs and protozoan
parasites."
128
2. Stool obtained after taking in oil laxatives, barium or bismuth salts are not suitable for
examination.
3. Urine kills protozoan trophozoites rapidly, so never accept stool mixed with urine.
4. Stool should not be contaminated with water for it will speed up growth of nonpathogenic
organisms making examination difficult.
5. Trophozoites and cysts appear in stool at intervals. A series of several specimens should
be examined to rule out a negative fecalysis result.
6. Formed stools may contain protozoan cysts while watery or diarrheic stools contain
trophozoites.
7. Prompt examination of specimen must be observed to prevent disintegration of protozoan
trophozoites.
8. Never leave stool specimen exposed to the air without lids.
9. Never freeze stool samples nor keep them in Incubators.
10. Various structures may be mistaken for protozoan cysts or worms by beginners.
a. Epithelial cells and macrophages can be confused with amoebic trophozoites,
especially macrophages that show slight amoeboid movement and may contain
red blood cells. The nuclei which can be seen in BMB (Buffered Methylene Blue)
stained mounts, appear much larger than nuclei of amoeba and usually contain
several granules or particles of chromatin.

129
b. Pus cells can be confused with amoebic cysts. The nuclei appear as 3 or 4 rings
and usually stain heavily. The cytoplasm is ragged and the cell membrane is often
not seen. Amoebic cysts have a distinct cell wall.
c. Hair and fibers may be confused with larvae. However, hairs and fibers do not
have the same internal structure as larvae.
d. Plant cells (e.g. molds or yeast) can be confused with cyst or eggs. Plant cells
usually have a thick wall; cyst has a thin wall. Yeast and mold are usually smaller
than amoebic cysts and do not have nuclei such as are seen in amoebic cysts.
e. Eggs of arthropods and plant nematodes may be mistaken as parasites.
Amount and Timing of Collection
Collection of sufficient quantity of stool is necessary in order to permit detection of
parasites even if in low concentration and to prevent drying of the specimen. Acceptable
specimens should contain a sufficient amount of material to perform the examination procedures.
For formed stool sample, approximately 5-7 grams of feces or about the size of a marble or
thumb size is required. For watery or diarrheic stool, 10 ml or: 10 cc is required. Intestinal
parasites may not be shed into the feces in consistent numbers on a daily basis to facilitate
adequate recovery and identification. For this reason, as series of three specimens should be
submitted for routine examination, on alternate days if possible, or within no more than 10-day
interval. When physician suspects intestinal amoebiasis, a total of six specimens may be ordered.
Collection should be completed within no more than 14 days. If any stool specimen tests positive
for parasites, the remaining specimens in the series may be unnecessary.

130
Patients who have been treated for protozoan infections are typically checked 3 to 4
weeks after therapy. A patient treated for helminth infection may be checked 1 to 2 weeks post
therapy and checks for Tenia may be delayed for 5 to 6 weeks post therapy.
Container for stool samples should be clean a dry, wide-mouthed & made of plastic or glass
(leak-proof). Preferably, container should have tight-fitting lid to avoid spillage to avoid
contamination & for retention of moisture.
Specimens should always be properly labeled, including the patient’s name, and the date
and time that the specimen was collected & initials of the examiner. Specimen should be
processed and examined as soon as possible after arrival at the laboratory. Examination must be
done within 1 hour after collection but not more than 2 hours. If a number of specimens are
received at the same time, pick out liquid or watery stool and those with mucus or blood because
parasites such as protozoan trophozoites will deteriorate rapidly.
Preservation of Stool Specimens
In cases when examination is delayed, appropriate preservative may be used to preserve
protozoan morphological features and prevent possible destruction of helminth eggs and larvae.
Stool samples must be adequately mixed with selected preservative in a proportion of 1part stool
to 3 parts preservative. Any of the following stool preservatives can be used:
1. Refrigeration at 4° to 8°C preserves protozoan trophozoites for several days and cysts
for several weeks.
2. Formalin is an all-purpose fixative. A 5% concentration is recommended for protozoan
cyst while a 10% concentration is recommended for helminth eggs and larvae. However,
131
protozoan trophozoites are destroyed. Preserved stool can be concentrated using
Formalin-Ether or Formalin-Ethyl Acetate Concentration Technique (FECT):
3. Schaudinn's solution is frequently used for fixation of fresh fecal material. It is used for
preparing permanent-stained smears to demonstrate intestinal protozoa. Although it is
less effective than PVA, it is still very useful when PVA is unavailable. The greatest
problem in this fixative is that it contains mercuric chloride which is highly toxic to
humans. Problems of mercury disposal may therefore arise.
4. Polyvinyl alcohol (PVA) is as an excellent fixative for preservation of morphologic
features of intestinal protozoa, in particular trophozoite stages, as well as helminth eggs
and larvae. PVA technique utilizes a plastic resin mixed with stool in a ratio of 3 parts
PVA to 1part stool. Samples fixed with PVA can best be stained either immediately or
weeks or months later. Well-fixed specimens will remain stable for a year or longer.
However, it may distort slightly their cysts.
5. Merthiolate-Iodine-Formaldehyde (MIF) is a combination of preservative and stain for
fecal specimens. It is especially useful in early surveys. It preserves all stages of
helminthes and protozoa as well as stains protozoans for better identification. However,
care must be exercised in removing fecal material from the MIF sample to avoid taking
up too much liquid. Furthermore, iodine component of the fixative is unstable and not
applicable for concentration procedures.
6. Sodium-Acetate Formaldehyde SAF) is an alternative to Schaudinn’s because of the
concern surrounding mercuric chloride. SAF contains 10% formalin as a fixative plus
acetate which acts buffer. It is easy to prepare and store, eliminates the use of mercuric
132
compounds, can be used for concentration and permanent smears and does not interfere
with monoclonal antibody studies. It also aids in the recovery of protozoan cysts and
trophozoites, helminth eggs and larvae and intestinal coccidian.
Dispatching Mailing of Stool Specimens
Stools may be sent to specialized laboratory for identification of rare parasites that are difficult to
recognize. Below are preservatives that can be used.
1. 10% formalin (for wet mount)-prepare a mixture containing about 1 part of stool to 3
parts of formalin solution. Place in a vial; crush the stool thoroughly. It preserves
specimen indefinitely if the bottle is tightly closed.
2. MIF (for wet mount) - Just before dispatching, mix in a tube or vial 4.7 ml of MIF
solution and 0.3 ml of Lugol's iodine-solution. Add a portion of stool, approximately 2
ml. Crush with a glass rod or stick. It preserves specimen indefinitely.
3. PVA (for permanent staining) - in a bottle or vial, pour about 30 ml of PVA fixative or 3
quarters full; add enough Wesh stool to fill the container. Break the stool with a stick and
cover. It preserves all forms of parasites indefinitely.

133
FECALYSIS
Feces end product of body's metabolism which provide a valuable diagnostic
information. It takes 18-24 hours for food to be converted as feces.
Importance
1. Detect parasitism
2. Detect malfunction of the GIT, liver and pancreas.
3. Detect GIT bleeding
4. Detect steatorrhea, amylorrhea and inflammation
Terminologies
Hematofezia. presence of blood in stool Amylorrhea. increased starch in stool
Steatorrhea. increased fats in stool
Two Types of Fecalysis
1. Routine Fecalysis is composed of macroscopic and microscopic parts.
2. Chemical Analysis is a special tests done to stool samples
Composition (2/3 liquid and 1/3 solids)
1. Undigested foodstuff (cellulose)
2. Sloughed intestinal epithelium

134
3. Intestinal bacteria (normal flora)
4. GIT secretions (digestive enzymes)
a. Trypsin
b. Chymotrypsin
c. Aminopeptidase
d. Lipase
e. Elastase
5. Bile pigments - digestion of fats
6. Electrolytes
7. Water (60-80%)
Specimen Collection
Patient Education
1. Instruct the patient on proper collection of stool sample.
2. Importance of Proper Collection
 Improper collected specimens may lead to the inability to identify parasites or
incorrect interpretation of results
TYPES OF SPECIMEN
135
1. Random specimen is suitable for qualitative testing for blood and microscopic exam
such as leukocytes, muscle fibers, and fecal fats
2. Timed Specimen is for quantitative examination.
* 3-day stool (most representative sample) and collected in paint cans
Ideal Container for Stool
1. Clean and dry
2. Wide-mouth
3. Leak-proof (made of glass or plastic)
4. With tight-fitting lids or cover.
 To prevent spillage of sample
 To retain moisture within the container
 To avoid outside contamination (from the environment)
Amount to be submitted
 Acceptable specimen - sufficient amount of material to perform the examination
 Amount would depend on the methods to apply
For routine stool exam (Fecalysis)
 Ideal amount - 5-7 grams of feces or about the size of a marble or thumb size (if formed)
136
 If watery about 10 ml or cc
For chemical examination
 3-day stool sample for Quantitative Fecal Fat determination
Specimens to be Rejected
1. Specimen contaminated with urine, tissue paper toilet water soil & water
2. Specimens not properly labeled
3. Old specimens
Proper Labeling
1. Patient’s name
2. Age and Gender
3. Identification number (if any)
4. Physician’s name
5. Date & time collected or time submitted in the laboratory

137
ROUTINE FECALYSIS
2 PARTS OF FECALYSIS:
PHYSICAL/MACROSCOPIC EXAM
COLOR (by visual assessment)
Normal Color: light brown-dark brown Pigments responsible:
1. Stercobilin - major pigment
2. Urobilin
3. Mesobilin
Abnormal Color
1. Yellow
 Non-pathologic: milk diet, corn meal, santonin, starchy food
 Pathologic: amylorrhea & unchanged bilirubin
2. Green
 Non-pathologic: spinach, calomel, cooked green vegetables
 Pathologic unchanged biliverdin

138
3. Black or Tarry
 Non-pathologic: iron, bismuth, charcoal, blackberries, huckleberries
 Pathologic: upper GIT bleeding (esophagus, stomach or gastric ulcer) > hemoglobin is
converted to hematin when combined with HCl
4. Red
 Non-pathologic: beets, tomatoes *Pathologic: lower GlT bleeding, hemorrhoids,
carcinoma
5. Clay/Tan/Putty (acholic)
 Pathologic: absence of bile in obstructive jaundice
6. Dark red or chocolate brown
 Non-pathologic: coffee, cocoa, chocolate, too much red meat diet
CONSISTENCY
Normal: soft to formed
Abnormal
1. Soft, watery - diarrhea, irritation of GlT
a. "rice - watery" - characteristics of cholera b. “pea-soup” - characteristics of typhoid fever
2. Extremely hard (scybalous) -also known as “goat droppings”, prolonged constipation;
seen in spastic colitis, atony of the colon

139
3. Ribbon-like or flattened stool also known as “pipe stem”
 Seen in obstruction of lower colon, syphilis, ulcer and tumor
4. Mucoid - amoebiasis, dysentery, inflammation of large intestines
5. Small Caliber - seen in spasms due to hemorrhoids, CA & ulcer
6. Large Caliber - constipation seen among children with Hirschsprung’s disease
7. Gaseous or fermentative stool - soft & mushy appearance; seen in increase
carbohydrate fermentation
ODOR:
Normal Odor: peculiar offensive but not extremely foul
Substances responsible: butyric acid, indole & skatol
Abnormal odor
 Extremely foul - amoebic dysentery CA of intestine, putrefaction
 Putrid -necrosis, hemorrhage, malignancy
 Sour & Rancid - hyperacidity; gas formation & unabsorbed fats

140
Wet Mounts
Rationale
The preparation of direct wet mounts is a simple and efficient procedure for the
examination of feces. The mixing action of the intestinal tract usually results in an even
distribution of parasitic organisms in stool specimens. Wet mounts should be examined
immediately after preparation to avoid evaporation of liquid. Several techniques can be applied
to preserve the smears for several hours such as ringing the coverslip with petroleum jelly,
paraffin wax, nail polish & Eukitt.
Wet mounts are recommended for the detection of motile trophozoites when liquid or diarrheic
specimens are received.
DIRECT FECAL SMEAR
Principle
The direct wet mount is performed to detect motile protozoan trophozoites and to
determine cellular morphology.
Materials & Reagents
Stool sample
Microscope
141
Clean glass slides
Applicator stick
Coverslips (22X22 mm)
Marking pens
NSS or 0.9 % NaCl Solution
Procedure:
1. On a clean glass slide, place a drop of saline (NSS or 0.9% NaCl).
2. Using an applicator stick, pick about 2 mg feces (enough to form a low cone at the end of
the wooden applicator stick) and mix with NSS. The feces should be thoroughly
comminuted in a drop so that a uniform suspension will spread evenly to all edges of the
coverslip.
3. Cover with coverslip and exclude bubbles.
4. Examine the entire preparation systematically and thoroughly under the microscope using
the Low Power objective. Confirmation of parasites can be made by switching to High
Power objectives.
Report results as:
Protozoa
Positive for (name of parasite) stage of parasite if parasites are present.
None seen if negative for protozoa
Helminths
142
Number of eggs parasite same per coverslip
No ova seen if negative for helminths
Note: A properly prepared DFS will allow ordinary newspaper print to be barely legible
through the preparation. Too thick smear will make microscopic observation difficult; while too
thin a smear will make examination unsatisfactory.

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1

Precise Determination of HDL-Cholesterol Using Venous Blood 75

Introduction to Urinalysis 106

The phlebotomist’s role requires a professional, courteous, and understanding manner in

Proper patient identification is mandatory. An out-patient must provide identification

provide additional information such as surname or birthdate.

Used when only small quantities of blood are required.

2. Palmar surface of the 3rd or 4th finger

3. Plantar surface of the heel and big toe.

1. Inflamed and pallor areas

2. Cold and cyanotic areas

3. Congested and edematous areas

4. Scarred and heavily calloused areas

The following are needed for routine venipuncture:

smaller the needle bore.

3. Tourniquet – wipe off with alcohol and replace frequently.

5. Adhesive tape – protects the venipuncture site after collection

placed in a proper waste disposal immediately after use.

7. Gloves – worn to protect the phlebotomist

SELECT VENIPUNCTURE METHOD

loss of blood while the tube is changed.

It is a complex, requiring both knowledge and skill to perform. Each phlebotomist

required for every successful collection procedure.

3. Identify patient correctly by letting him/her state his/her name.

7. Examine both arms for suitability.

an IV below the venipuncture site, has a vascular access or fistula, or has a

that it is tight but not uncomfortable.

9. Ask the patient to make a fist.

all usually near the skin surface.

2. lf a suitable vein cannot be found, DO NOT ATTEMPT BLOOD DRAW. Remove

12. Observe Standard Precautions.

13. Don gloves.

finger and thumb.

smooth and quick.

17. When the blood is enough, remove the tourniquet.

press down the cotton for 3-5minutes to avoid trauma or hematoma.

20. Dispose of contaminated materials/supplies in designated containers.

21. Label all tubes accordingly.

Capillary blood collection or Skin Puncture Procedure

2. Identify patient correctly by letting him/her state his/her name.

5. Position the patient.

cold, cyanotic, swollen, scarred, or covered with rash.

blood does not run down the ridges.

stop the bleeding.

11. Dispose contaminated materials/supplies in designated containers.

12. Label all specimens properly and accordingly.

COMPLETE BLOOD COUNT

1. Red blood cell count

2. White blood cell count

6. Stained Red blood cell count

1. Automated (DYMIND DH36 Auto Hematology Analyzer)

DYMIND DH36 Auto Hematology Analyzer

This Auto Hematology Analyzer is a quantitative, automated hematology analyzer and 3-

part differential counter used in clinical laboratories.

Purpose and principle of the test

the determination for each parameter is performed.

method is based on the measurement of changes in electrical resistance produced by a particle,

lower threshold value, it is counted as WBC/RBC/PLT. The cell volume distribution is

Venous Whole Blood Sample/Venipuncture

1. Turn on the power switch at the back of the analyzer.

2. Click [Home] and select [Sample Analysis]

3. Collect venous blood with a K2EDTA anticoagulant collection tube.

4. Input patient’s name and age

probe tip is so well into the sample.