MT6328 - AUBF LAB (Comprehensive)

MT
UNIT 1: Urine Specimen Collection Mr. Joemarie Malana | Dr. John Henrick Uy LAB
2022 MT6328: Analysis of Urine and Body Fluids 1st Shifting | Medical Technology | University of Santo Tomas
OUTLINE ● Information on label:

URINE SPECIMEN COLLECTION ○ Patient’s name (complete name)
Specimen Collection 1 ■ First name, middle name (not initial), last name
Specimen Labeling ○ ID number
Specimen Rejection
Specimen Integrity ■ Hospital ID number or medical records number (MRN)
Specimen Preservation 2 ○ Date
Types of Specimen ○ Time
- Random
- First Morning ● Additional information:
- Fasting ○ Age
- 2-hour Postprandial ■ Correlation purposes
- Glucose Tolerance
- 24-hour (Timed) ○ Location
- Catheterized 3 ■ Correlate to specimen handling
- Midstream Clean Catch ■ What floor or which hospital it came from
- Supra-pubic aspiration
- Prostatitis
○ Physician
- Pediatric 4 ■ For consultation or inquiries regarding abnormal results
Drug Specimen Collection ● Time of collection
LEGEND ● Long-standing urine may be compromised and affect the
BLACK TEXT COLORED TEXT integrity of the results
Based from ppt Based from lecture proper
● Always available especially for unpreserved specimen
UNIT 1: URINE SPECIMEN COLLECTION ● Place label on container, not lid
● The analysis of urine requires a specific type of specimen collection ○ Placing on lid - mortal sin
or an optimal specimen collection procedure ● Requisition form: must accompany specimen
○ If not followed, results will be compromised ○ Information must match label
○ Compromised results = compromised clinical diagnosis ○ Time of receipt is stamped on requisition
■ in order to know where the process was delayed
SPECIMEN COLLECTION
● Urine is a biohazardous substance ○ Other info: type of specimen, interfering meds, type of test
○ May contain infectious materials that can harm any person ○ Date and time of collection is optional in the requisition form
exposed to it ○ Must be comprehensive so that MTs are properly instructed on
● Disposable, wide-mouthed, and flat-bottom containers with screw what to do with the specimen
caps are recommended ○ Must be written clearly; clarify before performing analysis
○ Used in the routine setting where patient is ambulatory
● Clear containers / at least 50 mL capacity (optimal) SPECIMEN REJECTION
○ Actually accommodates 60mL of urine ● Unlabeled containers
● Adhesive bags for pediatrics and large plastic containers for 24-hour ● Non-matching labels and requisition
specimens ○ Should create suspicion that there was a significant event that
○ Adhesive bags (wee bag) are attached to the genitalia of the resulted to the mislabelling the container, or submitting the
infant, and it allows urine to be collected any time the patient wrong requisition form
urinates ● Contaminated specimens - feces, paper
○ Large plastic containers can accommodate 1.5 L of urine ○ Depends on the hospital’s protocol
■ usually an amber bottle -> to protect the urine from light ○ Usually occurs when patient is not properly instructed on
as some analytes are photosensitive; resistant to specimen collection
corrosion ● Contaminated containers
● Wear gloves when working with urine ● Insufficient quantity
○ to protect oneself from urine spillage/ chemicals around the ○ Defined as the insufficient amount to perform a test
container ○ Depends on the hospital’s protocol
○ Routine Urinalysis: bottle should be at least half filled
○ Minimum amount - up to the first line
● Delayed or improper transport
○ Ice, refrigeration
○ Usually placed in a thermal bag with dry ice pack if specimen
is transported from hospital to hospital
■ Sometimes done even in hospital premises
■ Because urine analytes are sensitive
○ Quality is compromised if improperly transported
● Labs have written policies for rejection
○ Vary from lab to lab
SPECIMEN INTEGRITY
Figure. 1 Clear screw-cap container with flat bottom ● Test within 2 hours of collection
- Label should always be attached to the body and not on the ○ some of the analytes, sediments, and properties of the urine
cap -> prone to misidentification of the specimen if the cap is
will be lost when you let it stand for a long time
labeled
● Refrigerate if testing is delayed
○ there is a dedicated urine refrigerator in the laboratory
SPECIMEN LABELING
○ 2 to 8 degrees celsius, but it is normally set at 4 degrees
● Required information in Joint Commision International (JCI)
celsius (the range of the ref temperature)
accredited hospitals for standard safety:
● Most problems are caused by bacterial multiplication
○ at least two identifiers
● Delayed testing of urine will result to:
○ Complete patient's name & birthday
○ Increased:
○ Complete patient's name & MRN or ID number
IRINEO | TAN, K. | TENG | 3I-MT Hustle kahit Hassle 💯 1
MT UNIT 1: Urine Specimen Collection Mr. Joemarie Malana | Dr. John Henrick Uy LAB
■ Color Random Specimen

■ Odor ● Most common type received
■ pH ● Routine screening for obvious abnormalities
■ Nitrite ● May be collected at any time
■ Bacteria ● Dietary intake and activity may alter results
■ Turbidity ○ eg. Orthostatic proteinuria because of the posture of the patient
○ Decreased: prior to the collection of the urine
■ Glucose ■ Upright position: abnormally large level of protein
■ Ketones ■ Supine position (nakahiga; face up): normal level of
■ Bilirubin protein
■ Urobilinogen ● Patients may have to collect a follow-up specimen
■ RBCs ○ Requested when abnormal results are obtained
■ WBCs First Morning Specimen
■ Casts ● Ideal screening specimen
● More concentrated than a random specimen
CHANGES IN UNPRESERVED URINE ○ Epithelial cells and metabolites are present
ANALYTE CHANGE CAUSE ● Patient is in a basal state
Color Modified / Darkened Oxidation or reduction of ○ Approx 12 hours after last ingestion of food or nutrition
metabolites ● Use for orthostatic protein confirmation and urine pregnancy
Odor Increased Multiplication of bacteria tests
or bacterial breakdown ○ Orthostatic proteinuria: if this is observed in the random
of urea to ammonia specimen, first-morning will be requested kasi last position mo
pH Increased Breakdown of urea to bago gumising is nakahiga, so the protein level will decrease if
ammonia by you have this disorder
urease-producing
bacteria/loss of CO2 ● Patient collects immediately on arising, delivers to lab within 2
hours
Nitrite Increased Multiplication of
nitrite-reducing bacteria Fasting Specimen
Commonly seen in ● Actually is the second specimen voided - collected after the first
cases of UTI morning specimen
Bacteria Increased Due to multiplication ● Does not contain metabolites from the evening meal
Clarity Decreased Bacterial growth and ● Recommended for glucose monitoring
precipitation of
amorphous material 2-hour Postprandial Specimen
1. Patient voids before eating routine meal
Glucose Decreased Glycolysis and bacterial
utilization 2. Eats meal
3. Collects next specimen 2 hours after finishing meal
Ketones Decreased Volatilization and
bacterial metabolism ● Monitors insulin therapy
Bilirubin Decreased Exposure to light / photo ● Results can be compared with fasting urine specimen and blood test
oxidation to biliverdin results
Urobilinogen Decreased Oxidation to urobilin Glucose Tolerance Specimen
Blood cells and Decreased Disintegration in dilute ● Institutional option for collection with blood glucose tolerance test
casts alkaline urine (OGTT) - not frequently done
● Specimens are collected at the same time intervals as the blood
SPECIMEN PRESERVATION samples
● Ideal is bactericidal: inhibits urease and p reserves formed ○ Patient fasts for 8 hours, then you extract the blood, then you
elements let them consume a glucose load (e.g. 75g), after 1 hour you
● Routine is refrigeration; this is a must for culture specimens extract blood, then after 2 hours your extract blood again
○ Causes the precipitation of amorphous crystals (OGTT); if 100g: requires another blood extraction; Iba iba
■ Causes an increase in turbidity yung protocol depending on the load
○ Must return to room temperature for chemical testing ● Used to correlate renal threshold with the patient’s ability to
■ Stand the urine in room temp until it equilibrates metabolize glucose
■ 18 to 24 degrees celsius (room temp) PRESERVATION
● Commercial transport tubes are available but they must be 24-hour (Timed) Specimen
compatible with tests ● Required for quantitative results
○ In routine urinalysis: routine transport tubes are not used often ● Needed for measuring substances:
● HCL / Hydrochloric acid ○ with diurnal variation (results differ and pm) &
○ commonly used preservative especially for chemical tests in the ○ that vary with meals, activity, and body metabolism
CC section in determining urine metabolites ● Usually used for creatinine clearance
■ eg. Catecholamines, Vanylyl Mandelic acid (VMA) ● The midpoint or endpoint serum creatinine is also collected in
conjunction with urine specimen
TYPE OF SPECIMENS ○ Midpoint: 12 hours in between or within the collection of
● The composition of urine depends on the patient’s metabolic state urine specimen
and the timing and procedure used for collection ○ Endpoint: Performed after urine collection
● The type of urine specimen is determined by the type of test and ● Can also be used for: total protein, catecholamines, serotonin, VMA
the method of collection ● Shorter timed specimens can be used for substances with
● Patient must be instructed when special collection techniques are consistent levels
required ● Accurate timing is critical for accurate results

Timing Schedule Example (Timed Specimens)

● 7AM - patient voids and discards urine Midstream Clean-Catch Specimen
● Patient begins collecting urine ● Alternative to catheterized specimen
● 7AM second day - patient voids and adds this urine to the specimen ● Less contaminated than routine collection
container ● Provide patient with mild cleansing material and container and
● Principle: collection must begin and end with an empty bladder instructions:
● Calculation for units per 24 hours includes the volume in milliliters ○ Wash hands
of urine c ollected ○ Clean genitalia with supplied cleanser
● The patient is to refrigerate the urine at home and should submit or ■ More problems arise when collecting specimens from
be transported in ice female patients than male patients
■ Epithelial cells from the genitalia of the female obscure the
Handling of Timed Specimens
microscopic findings
● Thoroughly mix specimen and measure
■ Providing the cleanser is not really done
● Save a large enough aliquot to test, and repeat test if necessary
○ Void into toilet, then into container, and finish into toilet
○ A large graduated cylinder is used to measure the total volume
● Do not touch or contaminate inside of container
of urine collected, then an aliquot is collected
● Keep specimen on ice or refrigerated d uring collection
Supra-Pubic Aspiration
● Use appropriate and nontoxic preservatives ● Completely free of contamination for culture and cytology
● Review instructions with patient ○ Possible contamination from the lower urinary tract is avoided
○ Patients should be well educated about the procedure ○ When proper aseptic technique is performed, bacteriologic
○ Ex.: HCl (preservative) is corrosive findings from this specimen can confirm the presence of a
■ Usually 10 mL, but it has exact proportions and rules (di bacterial infection
raw matandaan ni sir; review daw niya) ● External needle aspiration from the bladder
■ Collection should be done in a way that the skin is not ○ Invasive procedure because you use a needle
exposed to HCL / preservative ● Possible pediatric specimen
→ Huwag hahawakan ang loob
→ Huwag idederetso yung genitalia sa container, kasi
HCL emits fumes and the patient will be burnt
■ Don’t dispose the preservatives without gloves
Catheterized Specimen
● Sterile specimen collected from bladder with a catheter
● Most common test is culture and sensitivity
● Culture first before performing routine urinalysis
● Specimen sharing: Clinical Microbiology -> Clinical Microscopy ->
Clinical Chemistry
○ 1st: Clinical Microbiology/ Bacteriology
■ culture always first
○ 2nd: Clinical Microscopy/ AUBF
■ for physical, microscopic, and chemical analysis of urine
using the urine strip Figure. Suprapubic Aspiration
○ 3rd: Clinical Chemistry
■ if there are other metabolites to be determined Prostatitis Specimen
○ This sequence applies to shared specimens and is only done if ● Collection similar to midstream clean-catch
the patient cannot provide separate specimens for the lab ● 3-glass collection:
● Midstream clean catch ○ Container 1 - first urine passed
● Foley catheter is used but this is not cultured and not submitted to ○ Container 2 - midstream urine
the laboratory; only the urine should be submitted ○ Massage prostate to obtain prostatic
● Urine should NOT be collected from the bag ○ Container 3 - remaining urine and fluid
○ urine in the bag has been standing for a couple of hours (>2 ○ Quantitative cultures on all 3 specimens,
hours) and could have undergone microbial proliferation ■ examine 1 and 3 microscopically for WBCs
○ urine catheter should be separated from the urine bag Interpretation of Results
○ urine should be collected from the CATHETER SECURING ● Prostatic Infection
DEVICE 1. Higher WBC/hpf count in specimen 3 than specimen 1
■ WBCs are related to infection
■ If the count is higher in specimen 3 than specimen 1, the
prostatic fluid in specimen 3 caused the increase in
WBC count -> prostatic infection
2. Bacterial count in specimen 3 is 10 times higher than
specimen 1
● Specimen 2 is a control for bladder or kidney infection
○ Positive culture in specimen 2 invalidates positive culture in
specimen 3 (cannot differentiate urinary tract infection from
prostate infection)
○ used to determine if there is no interfering infection from the
bladder
○ If specimen 2 is (+), di na malalaman if galing ba sa bladder or
Figure. Foley Catheter sa prostate yung infection
● Basically, if the first two specimens collected (S1 and S2) are
negative, but S3 is positive this is indicative of prostatitis, because
the first two specimens do not contain any prostatic fluid, since the
prostatic massage will only be performed after S1 and S2
Prostate Specimen Variation
1. Stamey-Meares 4 - glass collection
○ VB1: Initial voided
○ VB2: Midstream
■ VB: voided bladder
○ EPS: Massaged prostate excretions
■ EPS is Expressed Prostate Secretions
Figure. Wee Bag
○ VB3: Post-massage urine
● Cultures on all specimens
DRUG SPECIMEN COLLECTION
○ VB1 and VB2 (+) = urinary infection
● Guided by RA 9165
■ Urinary tract infection; coming from the bladder
● Proper collection, labeling, handling must be documented
○ EPS examined for WBCs >10- 20 / hpf = abnormal
○ 60mL bottle must be filled -- because challenge tests may be
○ VB1 and VB2 (-) and EPS and VB3 (+) = prostatitis
requested (another urine specimen cannot be collected since
2. Pre and Post-massage test:
time is a very significant factor)
● Specimen 1: midstream clean-catch specimen
○ The screening test sample will also be used for the
● Specimen 2: post-massage specimen
confirmatory testing for drug analysis
● Prostatitis is indicated by a quantitative culture result in the second
○ The temperature should be checked (fresh voided urine is
glass that is 10 times higher than specimen 1
warm), does not really require a thermometer
● The massaging of the prostate in the anterior portion of the rectum
○ Prone to adulteration by guilty patients
will induce the release of fluid from the prostate
○ Labelling is very important (needs to be complete)
○ If di mo nimassage di mo malalaman or madidifferentiate which
● Chain of Custody
is positive and yung may infection (urinary bladder vs prostate)
○ documentation from the time of specimen collection until the
kasi they flow on the same urethra
time of receipt of laboratory results; standardized form
always accompanies specimen
○ Uses a carbon paper
○ First part -> specimen collection and the signature of the
specimen collectors
○ You cannot analyze the specimen without this form
● Specimen must withstand legal scrutiny
○ IDTOMIS - only those who have access to this are allowed to
perform the specimen analysis; platform where results are
uploaded
○ If chinallege ng patient ung result, you cannot recollect the
sample and so the urine should enough for additional tests
● Points to consider:
○ Photo ID of urine donor or ID by employer
○ No unauthorized access to specimen
■ authorized persons can be any laboratory personnel who
is trained by a certified laboratory test analyst
Figure. Prostatitis Specimen Collection ○ No adulteration, substitution, or dilution of specimen
Prostatic massage is usually performed by a urologist, without this ■ To avoid dilution, there is usually no water in the bowl
massage prostatic fluid will not be collected ■ Bluing agent is used in the toilet bowl to know if the
specimen is already adulterated
Pediatric Specimens
→ Adulterated urine will turn green when reacted
● Soft, clear plastic bags, with hypoallergenic tape applied to genital
with the bluing agent (but not always, as some urine
area (Wee bag)
are straw colored)
○ Wee bag is submitted to the lab when optimum amount is
● Witnessed vs. unwitnessed collection
reached
○ Determined by test orderer
● Monitor bag frequently
○ Both specimens must be handed immediately to collector
● Clean-catch method with sterile bag can be used
● Adulteration Tests:
○ Especially if urine is for culture
● Bags with tubes to a larger container are available for timed ○ Temperature taken within 4 min must be 32.5-37.7oC
specimens ○ Report temperatures outside of range immediately
● At least 40 mL (depending on the protocol) ○ Collect another specimen ASAP
○ Inspect urine color for anything unusual
● Follow laboratory instruction for labelling, packaging, and transport
● The refrigerator used for this is locked, and not mixed with routine
specimens
○ Only the certified drug analyst has access to this
○ It may be stored indefinitely as long as it is frozen

Mr. Joemarie Malana
MT UNIT 2: Methods and Procedures in Performing Physical Examination of Urine Dr. John Henrick Uy LAB
OUTLINE ● Turbid urine may indicate infection, but a clear urine specimen
PHYSICAL EXAMINATION OF URINE does not immediately mean that the patient does not have an
Urine Color 1 infection
Urine Clarity
○ Infection - presence of bacteria, making the urine turbid
Urine Specific Gravity
- Refractometer ○ Clear specimen does not rule out infection, as it has to be
- Urinometer 2 correlated to other parameters of urinalysis
- Harmonic Oscillation Densitometry 3 ○ Gold standard (for UTI): performing culture
- Reagent Strip Specific Gravity
● Can all turbid urine specimens be assumed as pathologic?
LEGEND
○ No
BLACK TEXT COLORED TEXT
Clarity Term
PHYSICAL EXAMINATION OF URINE Clear No visible particulates, transparent; see through, print
● The physical examination of urine should be correlated with other seen easily
parameters in the urinalysis and the clinical condition of the patient. Hazy Few particulates, print easily seen through the urine
URINE COLOR Cloudy Many particulates, print blurred through urine
● Examine the specimen under: Turbid Print cannot be seen through the urine
○ Good light source Milky May precipitate or be clotted
○ Looking down through the container ● If the patient has chyluria or a damaged lymphatic system, the
■ Sometimes the container is not standardized patient’s urine will appear milky
■ Mix urine specimen by swirling
→ Then transfer it to a labelled wassermann test tube
■ In a wassermann test tube, accompanied by a good light
source, we look at the color and clarity against a white
background
○ Against a white background
● The color of urine may be used as an indicator for different
diseases or disorders, but should not be the only parameter used
as a basis
○ Colorless or straw colored
■ Possible recent fluid consumption Figure 1. Urine Clarity Determination
○ Pale yellow
■ Patient with diabetes Urine Color and Clarity Procedure
● Evaluate an adequate volume of the specimen
→ Experiencing polyuria - excreting glucose in the urine
● Use a well mixed specimen
○ Dark yellow
● View the urine through a urine container
■ May be due to:
● View the urine against a white background using adequate room
→ dehydration
→ medications: lighting
⇒ Vitamin B ● Maintain adequate room lighting
⇒ Vitamin C - yellow to orange urine ● Evaluate a consistent volume of urine
⇒ Bilirubin (may be accompanied by a yellow ○ Determine the urine color
foam after shaking) ○ Determine the urine clarity
○ Dark or orange
URINE SPECIFIC GRAVITY
■ Liver malfunction
● Definition: comparing the sg of the urine to the sg of the water
■ Presence of bilirubin
○ Red colored urine (Blood) Current Urine Specific Gravity Measurements
■ Stones Method Principles
■ Menstrual bleeding leading to contamination Refractometry Refractive Index
■ Trauma Osmolality Changes in colligative properties by
→ patient may have been involved in a vehicular particle number
accident which led to myoglobinuria Reagent Strip pKa changes of a polyelectrolyte by
Additional information: ions present
○ Bubbles
■ Increased protein concentration Refractometer
● Determines the concentration of dissolved particles in a
■ Foam shake test - very crude (not that reliable) method
specimen by measuring refractive index
of determining presence of protein in urine
● Temperature correction is NOT necessary but requires corrections
→ ( + ) presence of foam/bubbles upon shaking
for glucose and protein
■ Yellow bubbles if bilirubin is present
● Calibrator
○ NOTE: See Strasinger for different colors of urine and its
○ standardized solutions used to determine if the refractometer is
possible causes
working properly:
■ Potassium sulfate - 1.015
URINE CLARITY
■ Distilled water - 1.00
● Done by visually examining the mixed specimen while holding it in
■ 5% NaCl - 1.022 +/- 0.001
front of a light source
■ 9% sucrose - 1.034 +/- 0.001
○ Usually read against a newspaper print
○ If the refractometer is deviating, you can always use the
■ White background with print
calibration screw to recalibrate the refractometer to the
● The specimen should be in a clear container.
specific gravity value that you are expecting of the calibrator
● Color and clarity are routinely determined at the same time
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Mr. Joemarie Malana
○ When you drop the calibrator on the prism, the refractometer STEPS ON USING THE REFRACTOMETER:
should show the known specific gravity of the solution 1. Put one or two drops of sample on the prism
■ eg. when you drop potassium sulfate, the refractometer 2. Close the daylight plate gently
should show you 1.015 as its specific gravity 3. The sample must spread all over the prism surface
■ If that is not the case, you can recalibrate the ○ The drop should be enough to fill the daylight plate
refractometer using the calibration screw to the specific ○ The urine should not have bubbles so that no holes will be
gravity of the solution; adjust the calibration screw to show present when the specimen is dropped on the plate
you 1.015 4. Look at the scale through the eyepiece
5. Read the scale where the boundary line intercepts it
Refractometer ○ May light kang makikita, and kung saan nag touch yung light,
Advantages Disadvantages ‘yun ‘yong specific gravity ng solution na nilagay mo sa daylight
Small volume of specimen Corrections for glucose and protein plate
must be calculated
○ Sample image: 1.030 reading
Temperature corrections are not 6. Wipe the sample from the prism clean with a tissue paper and water
necessary Requires a light source
Easy to use and calibrate
Handheld refractometer
Figure 5. Digital refractometer
Historical Significance: Urinometer

● Consists of a weighted float attached to a scale that has been
calibrated in terms of urine specific gravity
Figure 2. Parts of a handheld refractometer ● Historical significance na lang siya: because it is not as accurate
● Daylight plate compared to other methods & it requires large volumes of urine
○ Where the urine specimen is placed
○ Should be completely filled; no more, no less
● Calibration screw
○ This is where you calibrate the refractometer, in order for it to
tell the accurate specific gravity
○ Used against a standardized solution
Figure 6. Urinometer set-up; graduated cylinder (left) weighted float

(right)
Figure 3. View of the scale ( Specific Gravity: 1.040); since the principle
of the refractometer is the refractive index, kung saan nag touch yung
light (color blue) yun yung specific gravity of the solution
Figure 7. Parts of a Urinometer
Procedures
1. Fill a measuring cylinder with 5
0mL of urine
2. Lower urinometer gently into the urine and let it float freely
○ Dapat wala siyang nababangga, in order to read the specific
gravity
Figure 4. Steps in using a handheld refractometer (See typed text) 3. Let urinometer settle; it should NOT touch the sides or bottom of
the cylinder
Mr. Joemarie Malana
4. Take the reading of SG on the scale (lowest point of meniscus) at Osmolality vs Osmolarity
the surface of the urine Osmolality: number of particles Osmolarity: both size and number of
5. Take out the urinometer and immediately note the temperature of only particles
urine with a thermometer
○ Aside from correcting for protein and glucose, you also have to ● Solute dissolved in solvent causes the following changes in
correct it for temperature colligative properties:
6. Compute for temperature correction: ○ lower freezing point, higher boiling point, increased osmotic
For every 3ºC increase/decrease add/subtract 0.001 pressure, and lower vapor pressure
7. Compute for protein/glucose correction:
SUBTRACT: Particle Changes to Colligative Properties
-- 0.003 for each gram% of protein present in urine Property Normal Pure Water Effect of 1 Mole of
Point Solute
-- 0.004 for each gram% of glucose present in urine
Freezing Point 0ºC Lowered 1.86ºC
Example Boiling Point 100ºC Raised 0.52ºC
● given: Temp of urine is 32ºC; calibration temp is 20ºC Vapor Pressure 2.38 mm/Hg at 25ºC Lowered 0.3 mm/Hg at
○ 32ºC is above the calibration temperature (20ºC) by 12ºC 25ºC
Osmotic Pressure 0 mm/Hg Increased 1.7 x 10 9
TAKE NOTE: mm/Hg
○ If the temp is ABOVE the calibration temperature, ADD
● If you add salt, the longer the time it maintains the cool temperature
○ If the temp is BELOW the calibration temperature, SUBTRACT
● Adding solute to the water raises the boiling temperature that is why
○ Divide by 3 because for every 3ºC ang pag-correct it takes longer to boil
○ The answer from this is what will be added to the measured
RATIONALE:
specific gravity to obtain the corrected specific gravity
● Nilalagyan ng mga sorbetero ng salt: m ore salt = lower freezing
● given: Sp gravity of the urine is measured at 1.011
point; mas matagal siyang malamig, mas matagal before mag-melt
● Corrected specific gravity:
● Water + Salt = mas matagal bago kumulo, because a dding solute
y 0.52ºC
raises the boiling point b
Reagent Strip
● Most commonly used method in determining specific gravity in the
Urinometer
laboratory
Advantages Disadvantages
Requires large volume ● Principle: The reagent strip reaction is based on the change in pKa
Easy to observe May take time for the result (dissociation constant) of a polyelectrolyte in an alkaline medium
Less accurate than the other ○ The more solute present in urine, the more hydrogen ions
methods currently available are released by the polyelectrolyte = ALKALINE
Corrections for glucose and protein ○ Less solute in the urine; less hydrogen released = ACIDIC
must be calculated ● As it increases
Corrections for temperature ○ The indicator changes from blue (1.00 [alkaline]), through
must be calculated shades of green, to yellow (1.030 [acid])
○ If there is not much solute in the urine, polyelectrolyte will not
Historical Significance: Harmonic Oscillation Densitometry release hydrogen so it will not be acidic: bromothymol blue will
read it as blue or as alkaline
○ So as the solute in the urine increases, the release of hydrogen
ions is also increased
■ pH of bromothymol blue: decreases - read from green
going to yellow (1.030 sp. gravity; most acidic)
● Readings can be made in 0.005 intervals by careful comparison
with the color chart
○ In some laboratories, it is read through an automated machine
● Bromothymol blue as indicator (pH indicator)
Figure 8. Harmonic Oscillation Densitometry

● Harmonic oscillation densitometry is based on the principle that
the frequency of a sound wave entering a solution changes in
proportion to the density of the solution
● Osmolality is affected only by the number of particles present
○ Size of molecule does not matter, only how many of that
particle is present
● Molecules contribute equally to the osmolarity of the specimen thus
a more representative measure of renal concentrating ability can
be obtained by measuring osmolarity
○ Does not only account for the number of particles, but also
accounts for the size of particles ● Time: time at which it could be read
○ Specific gravity - 45 seconds after it is dipped in urine
specimen
MT UNIT 3 - 11: Chemical Examination of Urine Mr. Joemarie Malana LAB
OUTLINE UNIT 3: QUALITATIVE TESTS FOR ALBUMIN

UNIT 3: QUALITATIVE TEST FOR ALBUMIN 1
Heat and Acetic Acid Test
- Reagents, Materials and Equipment ● Albumin is a protein
- Procedure
- Video ● These are conventional methods. The reagent strip is commonly
Sulfosalicylic Acid Test (SSA) 2 used to test for the different analytes found in the urine
- Reagents, Materials, and Equipment ● The tests may be used for confirmatory testing
- Procedure
- Video 1 ● Main principle: protein would precipitate in the presence of heat or a
- Video 2 reagent.
Heller’s Test 3
- Reagents, Materials, and Equipment Heat and Acetic Acid Test
- Procedure ● Denaturation of protein by HEAT
- Video 1 ● Precipitation of protein by adding 5% or 10% ACETIC ACID
Robert’s Test 4
- Reagents, Materials, and Equipment ● Buffering with acetate - Prevents the following:
- Procedure ○ Formation of soluble acid
○ Albumination of alkali precipitates
UNIT 4: QUANTITATIVE TEST FOR ALBUMIN
Kwilecki’s Modification of Esbach Method ○ Precipitation of phosphates
- Reagent, Materials, and Equipment
- Procedure Reagents, Materials, and Equipment
Kingsbury and Clark Method
- Reagents, Materials, and Equipment ● Urine
- Procedure ○ First morning specimen is preferred
○ Random specimen can be used
UNIT 5: TESTS FOR GLUCOSE 5
Qualitative Tests for Glucose ● Test Tube
- Benedict’s Test ○ Where the reaction would take place
- Fehling’s Test ● 5%-10% Acetic Acid
- Nylander’s Test 6
- Moore-Heller’s Test ● Centrifuge
Quantitative Tests for Glucose ● Alcohol Lamp
- Benedict’s Quantitative Method ○ Or a bunsen burner
- Fehling’s Quantitative Method 7
UNIT 6: TESTS FOR OTHER SUGARS Procedure

Fructose 1. Fill a test tube three-fourths full of clear or centrifuged urine
- Seliwanoff’s Test
- Borchardt’s Test
2. Gently heat the upper inch to boiling. Leave the lower portion
Lactose 8 unheated for contrast
- Rubner’s Method 3. Add 3 to 5 drops of 5% acetic acid and heat again
Pentose
4. If albumin is present, the urine becomes cloudy
- Bial Orcinol Method
○ The cloudier it is, the larger the amount of albumin is
UNIT 7: TESTS FOR KETONE BODIES present.
‒ LEGAL'S TEST ○ Due to the denaturation of albumin
‒ GUNNING'S TEST 9
‒ GERCHARDT'S TEST ○ The amount of albumin is proportional to the cloudiness of the
‒ 2,4-DINI5ROPHENYL HYDRAZINE TEST sample
‒ SODIUM BISULPHITE TEST 5. Record the results
‒ SODIUM NITROPRUSSIDE TEST 10
UNIT 8: TESTS FOR BILE PIGMENTS Video

BILE PIGMENTS ● Clinical use: for bedside detection of albumin in urine in patient with
SMITH'S TEST 11
HARRISON SPOT TEST Nephrotic syndrome, Glomerulonephritis, etc
PETTENKOFER'S TEST ● Principle: on application of heat, albumin gets denatured and
coagulum forms at the upper heated part of the test tube
UNIT 9: TEST FOR INDICAN
Indican ● Methods by which proteins get precipitated:
- Obermayer’s Test ○ Denaturation
- Jaffe’s Test 12 ○ Dehydration of outer shell
UNIT 10: TEST FOR URINE CALCIUM
○ At its isoelectric point
Calcium ○ Neutralization of charge
- Sulkowitch’s Test ● Denaturation - loss of secondary, tertiary, and quaternary structure
Supplementary Video
Preliminary Test for Calcium Ion
of proteins, only primary structure remains intact
- Dry Heating Test ● Coagulum: thick viscous precipitate
- Charcoal Cavity Test
Analysis of Calcium Ion 13
Procedure
Confirmatory Tests for Calcium Ion
- Ammonium Oxalate Test ● Fill ⅔ of the test tube with urine
- Flame Test ● Heat the upper ⅓ part of the test tube
○ Coagulum forms in upper ⅓ part
UNIT 11: TEST FOR URINE CHLORIDE 14
Chloride ○ Lower part serves as a control for comparison
- Precipitation ● Add 2 drops of acetic acid into the test tube to adjust the pH at
LEGEND isoelectric point of albumin (pH 4.7)
Interpretation
● Albumin (+): If turbidity (coagulation) forms, appears, intensifies or
remains as it is after the addition of acetic acid
● Phosphates or carbonate: If turbidity disappears
IRINEO, Ritzl, TAN, Kashlee, TENG, Pamela | 3I-MT Hustle kahit Hassle 💯 1
2. Observe and record your results according to the table on the next
slide
Reporting SSA Turbidity

Grade Turbidity Protein Range (mg/dL)
Negative No increase in turbidity Less than 6
Trace Noticeable turbidity 6 - 30
1+ Distinct turbidity, 30 - 100
no granulation
2+ Turbidity, granulation, no 100 - 200
flocculation
3+ Turbidity, granulation flocculation 200 - 400
4+ Clumps of protein Greater than 400
Table 1. SSA Precipitation Grading Table
Video 1 (kasama nito yung Heller’s Test)

● Materials
○ Urine sample in a test tube
○ 30% sulfosalicylic acid
○ Dropper
● Procedure (see Figure 3)
● Conclusion
○ When sulfosalicylic acid reacts with albumin it causes
denaturation of protein in the sample that appears in the form
of precipitate
Fig 1. Heat and Acetic Acid Test. (1-4 from top to bottom)
(1) How the test tube should be heated
(2) How the specimen looks like when boiling
(3) Dropping of acetic acid
(4) Coagulum formation indicating positive (+) result
Sulfosalicylic Acid (SSA) Test

● Other name: Exton’s Test
● Cold precipitation technique
● Sulfosalicylic acid precipitates proteins irreversibly.
Reagents, Materials, and Equipment

Fig 4. Procedure for SSA.
● Urine As the sulfosalicylic acid is dropped into the urine. A cloudy turbid
○ Random specimen can be used solution or precipitate appearing in the solution indicates the presence
● Test Tube of albumin in the urine sample.
● Exton’s Reagent
○ Sulfosalicylic acid Video 2
○ Sodium sulfate Introduction
● Detects the presence of protein in urine
Procedure ● Proteins such as albumin, globulin, glycoprotein, and Bence Jones
1. Mix equal volumes of urine and reagent in a test tube Proteins coagulate on addition of sulphosalicylic acid
Samples Only black lines are visible

● Freshly voided clean midstream morning urine
● Can be stored at 2-8C for 24 hours if analysis cannot be performed Indicative of 40-100mg/dl of
immediately protein in urine
● Grossly contaminated and unlabelled sample is rejected
(2+) = Only lines seen,
newsprint not seen
Reagent
● 20% w/v aqueous solution of sulphosalicylic acid
● Methyl Alcohol
● Preparation of working reagent
○ Mix 100ml of 20% SSA + 100mL methanol = 10% SSA Black lines are not visible
○ Mix 30ml of 10% SSA + 100mL methanol = 3% SSA
● Store the reagent at room temperature in a glass bottle (labelled) Indicative of 145-400mg/dL of
● Reagent is stable for 1 year after date of preparation protein in urine
Equipment (3+) = Both newsprint and

● Test tubes lines not seen
● Disposable droppers
Procedure
● Take 5mL of urine and place it in a test tube Indicative of more than
● Centrifuge this for 10 minutes at 1500rpm 500mg/dL of protein
● Transfer the supernatant in a clear test tube
(4+) = Dense flocculation in
● Add an equal amount of 3% SSA
the test tube
● Invert to mix
● Let stand for exactly 10 minutes
● Invert again twice
● Using ordinary room light, observe the degree of turbidity and
precipitation and grade it according to the following description
○ Normal total protein excretion
■ < 150 mg/24 hours
Quality Control
■ < 10 mg/100 mL
● Internal Quality Control is maintained by correlation with urine
○ Proteinuria
rapid strip test
■ > 150 mg/day
● Proficiency Testing is done by split sample testing once a month
○ Clear solution
by two technicians
■ Declared as negative
● Inter Lab Comparisons is done per feasibility
Heller’s Test
● Ring test
● Protein precipitation by addition of nitric acid.
● Precautionary measure: perform under the fume hood
Reagents, Materials, and Equipments

● Urine
● Test Tube
Fig 4. Comparison between a clear test tube and test tube with trace amounts of ● Heller’s Reagent
protein
○ Concentrated Nitric Acid
Interpretation
A newspaper print can be read
through the test tube Procedure
1. Place a small quantity of concentrated nitric acid in a test tube.
Indicative of approximately 2. Stratify a small amount of urine on the nitric acid according to the
30mg/dl of protein in urine
accompanying table for ring test. (See table below)
(1+)= both newsprint and ● Precautionary measure: perform the Heller’s test under a fume hood
lines are seen
Video for SSA and Heller’s Test
● Albumin is a protein produced by the liver that is mainly found in the
blood
● A trace of protein is found in normal urine daily
● In the pathological conditions like albuminuria the level of albumin
will be way above the normal level
● In kidney disturbance and in high blood pressure, albumin in urine is
significantly high

● Urine
○ First morning specimen is preferred
● Test Tube
● Robert’s Reagent
○ Magnesium sulfate
○ Nitric Acid
Procedure
● If urine is cloudy, filter or centrifuge it first.
Fig 2. Albumin levels in healthy vs damaged kidney ● If it is clear, proceed to the next step.
● Place 3.0 mL of Robert’s reagent in a test tube.
Heller’s Test ● Stratify 3.0 mL of urine on the reagent.
● Materials required ● A white ring at the point of contact indicates albumin
○ Concentrated Nitric Acid in a test tube ○ The larger the ring, the greater the quantity of the albumin
○ Urine sample ● Observe and record your results according to the accompanying
○ Dropper table for ring test.(Same table used for Heller’s)
● Procedure (see Fig. 4)
● Conclusion
Results for Ring Tests
○ Nitric acid causes denaturation of proteins with the formation of
a white precipitate. Grade Presence / Absence of Ring
Negative (-) No ring at point of contact
Barely perceptible ring against black background
Trace (+/-)
(+) Ring is distinct against black background. Seen
when held up to the light
Positive (++) Ring is definitive against light. Faintly visible when
viewed from above
(+++) Ring is heavy against light. Distinct cloudiness
when viewed from above
(++++) Ring is thick and dense against light. Opaque when
viewed from above
While inclining the test tube containing the conc. Nitric acid, slowly drop Table 2. Results for Ring Test
the urine sample along the inner side of the test tube
UNIT 4: QUANTITATIVE TEST FOR ALBUMIN
KWILECKI’S MODIFICATION OF ESBACH’S METHOD

● Principle is precipitation by heating

● 24 hour (timed) urine specimen
● Esbach’s tube
● 10% Ferric chloride
● Esbach’s reagent
As the urine sample comes in contact with the nitric acid, a white ring is
○ Picric acid
formed at the point of contact
○ Citric acid
● Water bath (Set to 72C)
● Thermometer
Procedure
● Fill the Esbach’s Tube to the mark “U” with urine
● Add 10 drops of 10% ferric chloride and mix
● Fill the tube to the mark “R” with the Esbach’s reagent
● Place in a water bath at 72C for five minutes
● Immediately after heating, read the height of the coagulum formed
The presence of a white ring at the junction of two layers indicates the and report in grams %
presence of albumin in the sample ● Height of coagulum
○ Grams albumin present in 1000mL of urine
Fig. 4. Heller’s Test Procedure ○ To obtain grams% -> divide the height of the coagulum by 10
Robert’s Test KINGSBURY AND CLARK METHOD

● Ring Test Reagents, Materials, and Equipment
● Protein precipitation by addition of magnesium sulfate
● 24 hour (timed) urine specimen
● Test tube
● 3% Sulfosalicylic acid
● Centrifuge
Procedure ● Fehling’s Solution B

1. Using a pipette, transfer 2.5 mL of centrifuged urine in a test tube ○ Rochelle’s Salt (Potassium sodium tartrate)
graduated at 10 mL and add 3% sulfosalicylic acid up to the 10 mL ○ KOH (Potassium hydroxide)
mark.
2. Invert the tube to mix and allow it to stand for 10 minutes. Procedure
3. Record according to the table on the next slide and report in 1. Mix equal parts of Fehling’s solution A and B in a test tube
grams%. 2. Dilute the mixture with 2 or 3 times the amount of water and boil for
a few seconds
Results for Kingsbury Clark Method 3. If the mixture remains clear after boiling, the solution may be used
Grade Flocculation Protein Range (mg/dL) 4. Continue boiling. Add the urine drop by drop until about 5 mL
+ No definite flocculation. Cloud is 0.01-0.03 gram% albumin 5. Interpret results. Refer to the table
distinguishable but not granular
++ No definite flocculation. Cloud is 0.04-0.1 gram% albumin Table for Benedict’s and Fehlings
distinct and granular Grade Turbidity
+++ Marked flocculation with dense 0.2-0.3 gram% albumin (-) Negative No change in color
cloud (+/-) Trace Green opacity, no precipitate
++++ Heavy thick precipitate almost to 0.5 gram% albumin or (+) Positive Green solution, with yellow precipitate
boiling solid higher; 3 grams% boils
solid (++) Green to yellow solution, with yellow precipitate
TABLE 3. Results for Kingsbury Clark Method (+++) Muddy orange solution with yellow precipitate
● Flocculation is similar to precipitation, but in serology there is a (++++) Orange to brick red precipitate
different or very distinct definition. Flocculation in serology,
when you are talking about antigen-antibody reactions you are Video for Benedict’s and Fehling’s Test
to verify or view it under the microscope. ● Sugar is ordinarily not present in normal urine
● When the sugar level in blood rises above the normal level, the
kidney eliminates the extra sugar through the urine. Sugar then
UNIT 5: TESTS FOR GLUCOSE starts to appear
● Benedict’s and Fehling’s test are used to detect the presence of
QUALITATIVE TESTS FOR GLUCOSE sugar in urine. These tests give colored precipitate
Benedict’s Test
● PRINCIPLE: In hot alkaline solution, glucose reduces the copper
salts to copper oxide
● In the reaction, precipitation of copper hydroxide should be
prevented by adding potassium sodium tartrate

● Urine
○ Freshly voided or random specimen
○ In long standing urine, glucose can be decreased because of
the utilization of bacteria of the sample or by glycolysis
Fig 5. Colored precipitates depending on the concentration of sugar
○ Sample should alway be fresh to be able to correctly identify
the presence of glucose in the sample
● Test Tube Benedict’s Test
● Benedict’s Reagent ● Materials required
○ Copper sulfate ○ Urine Sample
○ Sodium citrate ○ Benedict’s Reagent
○ Sodium carbonate ○ Burner
○ Test tube holder
Procedure ○ Dropper
1. Place 5.0 mL of Benedict’s qualitative solution in a test tube. ● Procedure (see Fig. 6)
2. Add 8 to 10 drops of urine and mix. ● Conclusion
3. Place the test tube in a boiling water bath for 2 to 3 minutes. ○ On boiling the urine sample with the Benedict’s reagent, the
4. Let stand and cool. cupric ion present in the Benedict’s reagent is reduced by the
5. Interpret results. Refer to the table on the next slides. reducing agent (sugar), to form a brick red colored precipitate
of cuprous oxide
Fehling’s Test
● In hot alkaline solution, glucose reduces the copper salts to copper
oxide
○ Formation of copper hydroxide prevented by the addition of
potassium sodium tartrate

● Urine
○ Freshly voided or random
● Test tube
● Fehling’s Solution A
○ Cupric Sulfate Add the Benedict's reagent to the urine sample, then boil the sample
over a burner for two minutes (done in intervals. Keep shaking the test Procedure
tube as it is being heated. 1. Place 5 mL of urine in a test tube.
2. Add 0.5 mL of Nylander’s reagent.
3. Heat for 3 to 5 minutes using an alcohol lamp.
4. Allow to stand for a few minutes before reading.
5. Interpret the results. Use the interpretation on the next slide.
Interpretation For Nylander’s Test

Color Interpretation
Black Presence of Sugar
Brown Presence of Sugar (TRACE only)
A brick red precipitate may appear, indicating the presence of sugar No change in color Negative
Fig. 6. Benedict’s Test Procedure Solution turns black Presence of other substances other than sugar
upon cooling
Fehling’s Test
● Materials Needed Moore-Heller’s Test
● Caramelization of glucose by strong alkali with the aid of heat
○ Urine sample in test tube
○ Test tube holder Reagents, Materials, and Equipment
○ Fehlings’s solution A ● Urine
○ Fehling’s solution B ○ Freshly voided or random
○ Dropper ● Test tube
○ Burner ● Moore Heller’s reagent
● Procedure (Fig 7) ○ 10% KOH
● Conclusion ● Alcohol lamp
○ The cupric ion present in the Fehling’s solution is reduced
when boiling by the reducing substance (sugar), to form the Procedure
brick red ion precipitate of cuprous oxide. 1. Place in a test tube 2 parts urine with 1 part 10% KOH
2. Boil the upper portion for 2 to 3 minutes
3. Interpret the results. Use the table:
Moore-Heller’s Test
Color Percentage of Sugar
Canary yellow Less than or equal to 1% sugar
Wine yellow 1-2% sugar
Cherry color 2-3% sugar
Rum color 3-4% sugar
Dark brown to black More than 4% sugar
Add Fehling’s solution A to the urine sample (left), followed by the
addition of Fehling’s solution B (right). Boil the sample over a burner
for two minutes Keep shaking the test tube while heating. QUANTITATIVE TESTS FOR GLUCOSE
● Benedict’s Quantitative Method
● Fehling’s Quantitative Method
Benedict’s Quantitative Method

● Determined through the reaction with copper
○ The reducing property of glucose is utilized to reduce cupric
ions to cuprous ions through titration

A brick red precipitate appears, indicating the presence of sugar.
Fig 7. Fehling’s Test Procedure ● 24 hour or timed urine
● Serological pipettes
● Benedict’s volumetric solution
● Powdered pumice
NYLANDER’S TEST ● Sodium carbonate powder
● In hot alkaline solution, glucose reduces the bismuth salts to metallic ● Erlenmeyer flask
bismuth ● Alcohol lamp

Procedure
● Urine
1. Place in a 250-mL Erlenmeyer flask the following:
○ Freshly voided or random
○ 25.0 mL Benedict’s volumetric solution
● Test tube ○ 5 to 10 grams sodium carbonate
● Nylande’s reagent ○ Pinch of powdered pumice
○ Rochelle salt (potassium sodium tartrate) 2. Heat the mixture to boiling.
○ 10% NaOH or KOH 3. Slowly add urine drop by drop by using a serological pipette.Stir the
○ Bismuth subnitrate mixture constantly while maintaining the boil. Continue adding the
● Alcohol lamp
urine until the last trace of blue color disappears.This is the Seliwanoff’s Test Procedure Vid
endpoint. ● for ketose sugar
4. Read the volume of mL of urine used in the reaction. ○ fructose
5. Calculate the sugar present: ○ sucrose
● Principle: ketose sugar (fructose) reacts immediately with mild acid
(HCl) to give furfural derivatives. These furfural derivatives combine
with resorcinol to give cherry red colored complex
6. Multiply the answer above by 100. ● procedures:
○ Mg glucose in 100 mL urine 1. Take a test tube and hold it with a test tube holder
7. Divide the answer (in number 6) by 1000. 1. Add 5mL of Seliwanoff reagent
○ Grams glucose in 100 mL urine (% sugar present in the 2. Add 0.5mL to 1 mL of sample
specimen) 3. mix the test tube and boil for 30 seconds
4. Shake the test tube while boiling it
Fehling’s Quantitative Method 5. Cool the test tube under tap water
● Determined through the reaction with copper
6. Observation - cherry red colored complex (+)
○ The reducing property of glucose is utilized to reduce cupric
○ Ketoses dehydrated early with HCl.
ions to cuprous ions through titration
○ time for boiling is important (30 seconds)

● 24-hour or timed urine
● Test tube
● Alcohol lamp
● Fehling’s reagent (A and B)
● Serological pipette
Procedure
1. In a test tube, dilute 2 mL Fehling’s solution with 4 mL distilled
2. water. Heat to boiling.
3. Using a 1 mL serological pipette, add urine drop by drop until all
Borchardt’s Test
blue color disappears from the Fehling’s reagent. Each mL used in ● Heated with concentrated hydrochloric acid
the disappearance of the color contains 0.005 g glucose. ● Formation of oxymethylfurforol
4. Calculate the number of grams glucose per 100 mL urine. ● Positive color: Red (due to resorcin)
● The same as Seliwanoff’s Test but uses the Borchardt’s reagent
UNIT 6: TESTS FOR OTHER SUGARS
● First morning urine
● Fructose
● Borchardt’s reagent
○ Seliwanoff’s Test
○ 25% hydrochloric acid
○ Borchardt’s Test
○ Resorcinol Crystals
● Lactose
○ Rubner’s Method
● Pentose
○ Bial Orcinol Method
FRUCTOSE
Seliwanoff’s Test
● Heated with concentrated hydrochloric acid
● Formation of oxymethylfurforol
● Positive color: Red (due to resorcin)

● Test tube ● Solid sodium hydroxide or potassium hydroxide
● Seliwanoff’s reagent ● 1:1 Acetic acid-ether mixture
○ Resorcinol ● Test tube
○ Concentrated hydrochloric acid ● Alcohol lamp - heating process
● Alcohol lamp - for heating the sample and reagent Procedure
1. In a test tube, place 5mL of urine and 5
mL 25% hydrochloric acid
Procedure
2. Add a few crystals of resorcinol
1. In a test tube, place 5mL of urine and 2mL Seliwanoff’s reagent.
Heat the solution
2. If fructose is present, the solution turns red and precipitates a dark
sediment. The sediment is soluble in alcohol giving a bright red
color.
3. Report as positive or negative.
● (+): red color with dark sediment precipitate
● (-): no color change
3. Boil for less than 30 seconds. PENTOSES

4. A red color represents fructose. Bial Orcinol Method
● preliminary confirm the presence of fructose in the ● Urine is heated with concentrated hydrochloric acid
solution ● Pentoses lose a molecule of water
● *in vid demo after cooling pa para makita yung red ○ Conversion to furfurol
color ● Positive color: GREEN (with orcinol)
● Bial Orcinol Reagent
○ Orcinol
○ Hydrochloric acid
○ Ferric chloride
● Test tube
● Alcohol lamp
5. Cool the mixture and alkalinize using solid NaOH or KOH Procedure
6. Add a few drops of 10% KOH/ potassium hydroxide to alkalinize the 1. In a test tube, boil 5mL of Bial Orcinol reagent
solution (used in the vid demo) 2. Remove from the flame and add urine drop by drop until no more
7. Add 3mL 1:1 acetic acid-ether mixture and shake than 1mL has been used.
3. If pentoses are present, the solution turns green almost
immediately.
4. Report as positive or negative
● (+): green
● (-): no green coloration
8. Yellow ethereal layer will confirm the presence of fructose.

9. Report as positive or negative. If fructose is present, the solution
turns red and precipitates a dark sediment. The sediment is soluble UNIT 7: TESTS FOR KETONE BODIES
in alcohol giving a bright red color.
● (+): yellow ethereal layer after the addition of 1:1 acetic
acid-ether mixture ● Legal’s Test - Acetone and diacetic (acetoacetic) acid
○ does not only stop with the red color for determining the ● Gunning’s Test - Acetone
presence of fructose ● Gerhardt’s Test - Diacetic (acetoacetic) acid
○ but also needs the addition of acetic acid-ether mixture to KETONE BODIES
confirm the presence of fructose through the formation of ● Ketones are organic compounds containing ketonic functional group,
the yellow ethereal layer in which the carbonyl carbon is attached to two R groups. The R
groups may be alkyl or aryl groups (vid demo)
LACTOSE
Rubner’s Method
● Urine is treated in lead acetate
● Heated in the presence of ammonia
● Positive color: RED

● Rubner’s reagent
○ Lead acetate powder ● Three intermediate products of fat metabolism
○ Concentrated ammonium hydroxide ○ Acetone
● Alcohol lamp ○ Acetoacetic acid
● Beta-hydroxybutyric acid
Procedure ● Normal urine: k etone bodies are NOT detected
1. In a test tube, place 10 mL of urine and 3 g lead acetate ● Diminished source of carbohydrates
2. Filter off precipitate. Heat the filtrate for a few minutes. Upon ○ Fats are utilized and ketone bodies are produced
appearance of yellowish-brown color, add ammonium hydroxide ○ High amounts of ketone bodies in body → can be detected in
and continue heating urine
3. If lactose is present, a brick red color solution with cherry red or
copper-colored precipitate will appear. The supernatant becomes Legal’s Test
colorless after formation of the precipitate ● detect for Acetone and diacetic (acetoacetic) acid
4. Report as positive or negative ● Reaction with nirate (-NO) group
● (+) : brick red color with cherry red or copper-colored ● Sodium nitroprusside, acetone, and acetoacetic acid form
precipitate isonitroacetone
○ Remains trapped in the complex anion
Reagents, Materials, and Equipment 3. Observe the result and report as positive or negative
● Urine ○ ORDEAUX RED
diacetic acid is present - urine will become B
○ Preferred: First morning urine ○ diacetic acid is due to drugs
○ Freshly voided or random can be used ■ does not disappear upon heating
■ random specimen can be used but it should be freshly ■ does not reappear upon cooling
voided
■ as ketone bodies are volatile in long standing urine which * 2,4-Dinitrophenyl Hydrazine Test
can cause a false decrease or cause inappropriate test Reagents, Materials, and Equipment
results ● Organic compound
● Sodium hydroxide or potassium hydroxide ● Rectified spirit
● Sodium nitroprusside ● 2,4-DInitrophenyl hydrazine solution
● Concentrated acetic acid ● Test tube
● Droppers
Procedure
1. In a test tube, add few drops of urine.
2. Add enough NaOH or KOH solution to render the solution
slightly alkaline.
3. Add a few drops of sodium nitroprusside solution.
4. Add a few drops of concentrated nitric acid.
5. Observe the result and record as positive or negative
○ purple or violet red color: Acetone
○ red: Diacetic acid / Alcohol / Acetic aldehyde
Procedure
Gunning’s Test
● for Acetone 1. Take a small quantity of organic compound in a test tube
2. To this add a small amount of rectified spirit using a dropper
● Reaction of acetone bodies and alcoholic iodine
3. Shake the test tube well to dissolve the compound in rectified
● Production of iodoform crystals
spirit
● Sodium nitroprusside, acetone, and acetoacetic acid form
4. Using another dropper, add a small amount of
isonitroacetone
2,4-Dinitrophenyl hydrazine solution into the test tube
○ Remains trapped in the complex anion
● Urine
○ Preferred: First morning urine
○ Freshly voided or random can be used
● Concentrated ammonium hydroxide
● Lugol’s solution
● Test tube
● Microscope
2,4-Dinitrophenyl hydrazine reacts with the carbonyl group present in
Procedure ketone to form a yellow or orange precipitate of 2,4-Dinitrophenyl
1. In a test tube, place 5mL urine hydrazone
2. Add 5 drops of concentrated ammonium hydroxide
3. Add Lugol’s solution enough to produce a black cloud, which does * Sodium Bisulphite Test
not disappear immediately Reagents, Materials, and Equipment
4. Let stand for a few minutes
● Organic compound
5. Take a small quantity of the sediment and examine microscopically
● Saturated solution of sodium bisulphite
○ Iodoform crystals - yellowish six-pointed stars or six-sided
● Boiling tube
plates
● Dropper
6. Report as positive or negative
● Cork
● (+) yellow six-pointed stars or plates = iodoform crystals
Procedure
Gerhardt’s Test
● Diacetic (acetoacetic) acid 1. Take a small quantity of saturated solution of sodium bisulphite in a
boiling tube
Reagents, Materials, and Equipment 2. To this, add a small quantity of organic compound using a dropper
3. Cork the test tube with the cork and shake it well and leave it for
● Urine
some time
● 10% ferric chloride
● Test tube
● Pasteur pipette
Procedure
1. In a test tube, add few drops of urine. Ketone gives an addition product with sodium bisulphite which is white
2. Add 10% ferric chloride solution drop by drop until no further crystalline in nature
precipitation occurs
* Meta-dinitrobenzene Test
● Meta-dinitrobenzene
● Dilute sodium hydroxide
● Test tube
● Spatula
● Dropper
Procedure
1. Take a small quantity of organic compound in a test tube
2. To this, add a small quantity meta-dinitrobenzene using a 3. Shake the test tube well to dissolve the crystals in water
spatula 4. To this solution, add a small amount of organic compound
using a dropper (dropper daw tapos isinalin :”) )
3. Using a dropper, add a small amount of dilute sodium

hydroxide solution to the test tube
5. Now, add a small amount of dilute sodium hydroxide solution

using another dropper
4. Shake the test tube well

5. Ketones, on reaction with meta-dinitrobenzene and sodium
hydroxide, give a violet coloration that slowly fades away
* Sodium Nitroprusside Test

The anion of the ketone formed by sodium hydroxide, reacts with
Reagents, Materials, and Equipment nitroprusside ion to form a red colored complex
● Sodium nitroprusside crystal
● Dilute sodium hydroxide
● Distilled water
● Test tubes
● Dropper
● Spatula
Procedure
1. Using a spatula take few crystals of sodium nitroprusside in a
test tube
UNIT 8: TESTS FOR BILE PIGMENTS
● Smith’s Test
● Harrison’s Spot Test
● Pttenkofer's Test
BILE PIGMENTS
● Bile – composed of bile acids or salts
○ help digests the fats that are ingested in the diet
○ hepatic causes of hyperbilirubinemia
■ bile pigments are found in urine
● Bile pigments – can be extracted from blood
○ Bilirubin – principal pigment; degradation product of
2. Add a small amount of distilled water to the test tube hemoglobin
■ Two major forms of bilirubin:
→ B1 - indirect bilirubin; bound to albumin so it cannot ● Test tube

be filtered by the glomerulus ● Filter paper
→ B2 - direct bilirubin; found in urine bc it is water ● Pasteur pipette
soluble
⇒ water soluble = B2 Procedure
● Tests for bile depends upon the oxidation of bile pigments by 1. In a test tube, place 5 mL urine and 5 mL barium chloride. Mix and
acids with the formation of a series of colored derivatives of stand for a few minutes.
bilirubin 2. Filter the solution. Place the precipitate in another piece of dry filter
○ GREEN – biliverdin paper.
■ Verde - green 3. Place 3 drops of Fouchet’s reagent on the precipitate.
○ BLUE – bilicyanine 4. Observe the result and report as negative or positive.
■ Cyan - blue ○ Positive result – BLUE TO GREEN COLOR
○ YELLOW – choletellin → presence of bile pigment
■ Increase cholesterol in your blood, there is a presence of
xanthomas or yellowish discoloration of the eyebrows so * Pettenkofer’s Test
yellow
Smith’s Test ● Urine
● Test tube
● Dropper
● Urine ● Measuring cylinder
○ Preferred: First morning urine ● Concentrated H2SO4
○ Freshly voided or random can be used ● Spatula
● Tincture of alcoholic iodine ● Sucrose
● Test tube
● *Dropper Procedure
● *Measuring cylinder 1. Take 2mL urine in a measuring cylinder from the urine sample bottle
● *Smith’s reagent 2. Pour the urine from the measuring cylinder into a test tube
3. Take some sucrose using a spatula and put the sucrose into the test
Procedure
tube containing urine
1. In a test tube, place 5 mL urine. 4. Take 2mL of H2SO4 in a measuring cylinder from teh reagent bottle
2. Overlay the urine with the tincture of alcoholic iodine. 5. Tilt the test tube and pour H2SO4 along the side of the test tube with
3. Observe the result and report as positive or negative. urine
○ Presence of bile pigments
■ EMERALD GREEN RING at point of contact
→ Granny smith apples are green so GREEN RING
*Video Demo Procedure

1. Take 1mL Smith’s reagent in a measuring cylinder from the reagent
bottle
2. Pour the Smith’s reagent from the measuring cylinder into a test =>
tube Appearance of red color indicates the presence of bile salt in urine
3. Using a dropper, take some urine from the urine sample bottle
4. Tilt the test tube and pour the urine along the side of the test tube
5. A green ring is formed at the junction of two layers indicating the UNIT 9: TEST FOR INDICAN
presence of bile
INDICAN
● Indican produces blue urine
● Urinary metabolite of tryptophan
● Physiologic – Two fates:
○ Tryptophan is reabsorbed in the intestines for protein
production
○ Tryptophan is converted to indole and excreted in the feces
■ Test for indole ring = indole is color red
● Pathologic – Intestinal disorder:
○ More tryptophan molecules are converted to indican and
indican is excreted in urine
○ Indican – colorless (initially colorless when fresh urine is
Harrison’s Spot Test passed) → (upon prolonged standing) Oxidized to dye indigo
blue by exposure by air
Reagents, Materials, and Equipment ■ Indican when oxidized is color blue
● Urine ■ Babies with this disorder - blue-stained diapers
■ more concentrated so we can detect the bile pigments Obermayer’s Test
better ● Principle: Acid hydrolysis
○ Freshly voided or random can be used ○ Indoxyl is liberated from the urine indican
● Fouchet’s reagent ● Indoxyl is then oxidized by ferric ions to become indigo
● 10% barium chloride ● Indigo dye can the be extracted with chloroform
2. Mix thoroughly and allow to stand for 2 to 3 minutes

3. Read the results. Use table below:
● Urine
Reaction Category for Sulkowitch’s Test
● Obermayer’s reagent Grade Description
● Chloroform (-) Negative No precipitate
● Test tube (+/-) Trace Barely visible precipitate
● Serological pipette (+) Positive Presence of fine white cloud
(++) Moderately heavy cloud
Procedure
(+++) Heavy cloud
1. In a test tube, place 5 mL urine and 5 mL Obermayer’s reagent
(++++) Very heavy milky precipitate
2. Mix and add chloroform and wait for it to settle.
3. Observe the result and report as positive or negative. SUPPLEMENTARY VIDEO
○ (+) Blue coloration of chloroform indicates the presence of Calcium
indican
● A soft, gray, alkaline metal with symbol Ca
Jaffe’s Test ● Found in various salts like calcium chloride, calcium nitrate, and
calcium carbonate as Ca2+ ion
Reagents, Materials, and Equipment ● In group V, calcium ions are precipitated as their carbonates by
● Urine adding ammonium carbonate to their solutions.
○ Preferred: First morning urine ● Our aim here is to test for the presence of the calcium ion in a given
○ Freshly voided or random can be used salt.
● Hydrochloric acid
● Chloroform Preliminary Tests for Calcium Ion
● Calcium hypochlorite or 0.5% KMnO4 (Potassium permanganate)
Dry Heating Test
Procedure
1. In a test tube, place a small amount of urine.
2. Add an equal volume of hydrochloric acid and chloroform in the ● Original salt in a test tube
solution. Mix. ● Test tube holder
3. Add 10 to 15 drops of chloroform. ● Bunsen burner
4. Add drop by drop a strong fresh solution of calcium hypochlorite and Procedure
shake after addition of each drop. ● Take a small quantity of salt in a test tube.
○ If 0.5% KMnO4 is used, add 5 drops with 10 mL urine. ● Heat it over the Bunsen burner.
5. Observe the result and report as positive or negative. ● A white residue glows on heating, which indicates the presence of
○ (+) Blue color indicates the presence of indican calcium ions.
UNIT 10: TEST FOR URINE CALCIUM
CALCIUM
● Cation
○ 99% in bones
■ In the form of calcium pyrophosphate
○ 1% in blood and ECF
○ Calcium is an intracellular cation Charcoal Cavity Test
● Most common specimen used: Serum Reagents, Materials, and Equipment
● Calcium in urine - seen as compounds: ● Mixture of salt and sodium carbonate in a watch glass
○ Calcium chloride ● Charcoal piece
○ Calcium sulfate ● Distilled water
○ Calcium phosphate ● Spatula
● Accurate time specimen needed for urine level measurement ● Dropper
○ Measuring urinary calcium - quantitative test ● Tongs
○ 24-hour urine specimen is needed ● Blow pipe
● Bunsen burner
Sulkowitch’s Test
Reagents, Materials, and Equipment Procedure
● Urine is mixed with buffered oxalate solution ● Take a mixture of small quantity of salt and double its quantity of
○ Producing calcium oxalate sodium carbonate in a watch glass.
● Take a charcoal piece with small cavity in it.
● Result: Turbidity
● Using a spatula, place a small quantity of the mixture in the cavity of
○ The more turbid the solution, the more calcium concentration in
the charcoal piece.
urine
● 12-hr or 24-hr/timed urine
● Test tube
● Sulkowitch reagent
Procedure
1. In a test tube, place equal volumes of urine and Sulkowitch reagent
Ammonium Oxalate Test

● Precipitate obtained in the group analysis
● Hot dilute acetic acid
● Ammonium hydroxide
● Ammonium oxalate solution
● Glass rod
● Using a dropper, add a few drops of distilled water to the mixture to ● Test tube
moisten it. ● Droppers
● Using the tongs, hold the charcoal piece in front of the reducing
Procedure
flame of the Bunsen burner.
● Take the precipitate obtained in the group analysis. To this, add a
● Now direct the reducing flame on the cavity by means of the blow
small quantity of hot dilute acetic acid using a dropper.
pipe and heat strongly for some time.
● White residue glows on heating, which indicates the presence of

calcium ions
● Calcium carbonate reacts with acetic acid to form soluble calcium
acetate.
● Now transfer this solution into a test tube.
● Using another dropper, add a small quantity of ammonium oxalate
solution to the test tube. To this, add a small quantity of ammonium
hydroxide solution using a dropper.
. ● Now scratch the inner sides of the test tube using the glass rod.
Analysis of Calcium Ion
● Salt solution in a test tube
● Ammonium chloride
● Ammonium hydroxide
● Ammonium carbonate solution
● Droppers
● Spatula
● Test tube holder
● Bunsen burner
Procedure
● Take a small quantity of salt solution in a test tube. To this, add a
small amount of ammonium chloride using a spatula.
● Boil the contents of the test tube over the Bunsen burner and cool.
● Using a dropper, add ammonium hydroxide to the test tube till the
solution smells of ammonia. To this, add ammonium carbonate
solution using another dropper.
● A white precipitate of calcium oxalate is formed due to the reaction

of soluble calcium acetate with ammonium oxalate.
● Calcium ions in the solution react with ammonium carbonate to form
a white precipitate of calcium carbonate. Flame Test
● Original salt in a watch glass
● concentrated hydrochloric acid
● Dropper
● glass rod
● platinum wire loop
● Bunsen burner
Procedure
● Take a small quantity of the salt in a watch glass. To this, add a few
drops of concentrated hydrochloric acid using a dropper.
Confirmatory Tests for Calcium Ion
● Mix the contents of the watch glass using a glass rod to make a ○ (+): White clouded precipitate indicates presence of chlorides
paste. (qualitative only)
● Take a small amount of the paste on the platinum wire loop and 4. If positive, proceed to the quantitative estimation
introduce it into the oxidizing flame of the Bunsen burner. ○ Spectrophotometric analysis
● Brick red flame indicates the presence of calcium ions. ■ Higher amount of precipitate present in the solution =
higher absorption
Precautions
● Handle hydrochloric with proper care because it is highly corrosive.
● Heating should be done very carefully.
Video:
https://drive.google.com/drive/u/1/folders/1_U8m6FLSHW4z01rR3LJmLG
dYbELsMGad or https://www.youtube.com/watch?v=uKy424Vf_44
UNIT 11: TEST FOR URINE CHLORIDE
CHLORIDE
● In the blood, it is one of the major extracellular cations
● Major inorganic chemical found in urine (predominant / most
abundant)
○ Cl → Na → K
● Chloride ingested in food follows this tract:
○ Absorption in the intestinal tract
○ Filtration in the glomerulus
○ Passive reabsorption by the proximal convoluted tubules, with
sodium
● In some conditions such as cystic fibrosis: Excess chloride is
excreted in sweat and urine
○ That is why one way to detect if the patient has cystic fibrosis is
the test (screening test) for sweat chloride
● In some disorders of the kidney tubules, we test for chloride to
differentiate the different types of disorders
● Preferred specimen: 24-hour/timed urine
Precipitation
● Most common principle
● Principle: precipitate chloride
○ More precipitate = more chloride concentration there is
● Precipitated as silver chloride upon addition of silver nitrate and
nitric acid in urine

● Urine
○ Preferred: first morning urine
○ Freshly voided or random urine can be used
● 6 N nitric acid
● Silver nitrate solution
● Test tube
● Pasteur pipette
Procedure
1. In a test tube, place 5 mL urine and add 3 to 4 drops of 6 N nitric
acid. Centrifuge
2. To the filtrate, add 2 to 3 drops of silver nitrate solution
3. Record results as positive or negative
Mr. Joemarie T. Malana
MT UNIT 15: Microscopic Examination of Urine Dr. John Henrick Uy LAB
2022 MT6328: Analysis of Urine and Body Fluids 2nd Shifting | Medical Technology | University of Santo Tomas
OUTLINE ○ Maliit ang RBC, mahirap bilangin if you use lpf so hpf dapat
ROUTINE URINALYSIS 1 ● Normal value
- Three step analysis ○ 0-2 / hpf
SIGNIFICANT FINDINGS IN THE URINARY SEDIMENT 2
○ 0-3 / hpf
- Hematuria
- Pyuria Clinical Significance
- Epithelial Cells
- Cylindruria ● Damage to the glomerular membrane
- Crystalluria ○ Glomerulonephritis
LEGEND ○ Post-streptococcal glomerulonephritis
BLACK TEXT
Based from ppt
PURPLE COLORED TEXT
Based from lecture proper
BLUE COLORED TEXT
Based from Strasinger 6E
MAROON COLORED TEXT
Based from reinforcement
● Vascular injury within the genitourinary tract
○ Vehicular trauma patients
ROUTINE URINALYSIS ○ Stab wound
● Three step analysis ● Macroscopic hematuria
○ First: Physical characteristics of urine are noted and recorded ○ Grossly bloody urine
■ Perform this using a well mixed sample ○ Advanced glomerular damage
○ Second: Series of chemical test is run ○ May also indicate menstrual contamination
■ Performed using the reagent strips ● Microscopic hematuria
○ Third: Urine sediment is examined under a microscope to ○ Minute but significant amount of RBC
identify the components of sediments. ○ Early diagnosis of glomerular disorders
■ We should know how to report the sediments ○ May also be seen in patients with renal calculi
■ Does it use high power field (hpf) or low power field (lpf)
■ Should it be quantitative or semiquantitative
■ Centrifuge tube should be closed and the centrifuge
should be set at the right speed
Pointed structure is the RBC. We can see that the cell is still intact
which is why it is hematuria. As opposed to hemoglobinuria wherein
the RBC has already lysed.
● Hemoglobinuria: Red and Clear Urine
● Hematuria: Red and Turbid Urine
Purple Structure: Squamous epithelial cell; Red blood cell is almost

SIGNIFICANT FINDINGS IN THE URINARY SEDIMENT the same size of the nucleus of the epithelial cell. Other structures which
may appear as RBCS:
● Significance depends on the amount of the urinary sediment
● Yeast cells
● Oil droplets
HEMATURIA
● Air bubbles
● Hematuria - presence of intact red blood cells in the urine
Appearance PYURIA
● The appearance of RBCs are dependent on the environment they ● Pyuria - increase in urinary WBC
are subjected to ● Also known as leukocyturia
○ In isosthenuric urine: RBC is biconcave or normal
Appearance
● Crenated or irregularly shaped (hypersthenuric)
● Contain granules and multilobed nuclei
○ Hypersthenuric urine: concentrated
● Neutrophil - predominant WBC found in the urine sediment
● “Ghost cells” (hyposthenuric)
○ Signifies an infection in the patient
○ Examined under reduced light
○ Commonly: UTI
○ Hyposthenuric urine: diluted
● Neutrophils lyse rapidly in dilute alkaline urine and begin to lose
○ In hyposthenuric urine, water will go into the cell which causes
nuclear detail.
the RBC to swell and eventually burst.
○ Similar to exposure of WBCs or RBCs to hyposthenuric urine
■ Only the outline of the RBC will be seen since the cell is
● “Glitter Cells”
already lysed.
○ Neutrophils exposed to hypotonic urine
● Dysmorphic RBC
○ Light blue in Sternheimer-Malbin stain
○ Associated to Glomerular bleeding
○ No pathologic significance
■ Dysmorphic RBCs should be quantitated to determine if it
■ Granules appear to be undergoing Brownian Movement
is significant in number
■ Dysmorphic cells may also be seen in hyposthenuric urine Manner of Reporting
■ Ice tea colored or cola colored urine ● average number seen in 10 hpfs
○ Resembles: Acanthocyte with multiple protrusion ● Normal value:
○ 0-5 / hpf
Manner of Reporting ○ 0-8 / hpf
● Average number seen in 10 hpfs
○ RBCs are 6-8 microns
Clinical Significance ■ Malignancy (Transitional cell carcinoma) or viral

● Presence of an infection or inflammation in the genitourinary system infection
● BACTERIAL:
○ Pyelonephritis (infection of the upper genitourinary tract) ● Renal Tubular Epithelial Cells (RTE)
cystitis, prostatitis, and urethritis ○ Most clinically significant epithelial cell
● NON-BACTERIAL: ○ Origin:
○ glomerulonephritis, lupus erythematosus, interstitial nephritis, ■ nephron
and tumors. ■ Especially in the tubules
● EOSINOPHILURIA: ○ Shape:
○ Drug-induced interstitial nephritis ■ rectangular, polyhedral, cuboidal or columnar with
■ Common example is methicillin (no longer commonly eccentric nucleus
used in practice) due to adverse effect of causing ○ Interpretation:
interstitial nephritis ■ Average number / 10 hpf
■ Hansel stain ■ > 2 RTE /hpf: TUBULAR INJURY
→ If more than 1% seen = significant → Renal tubular acidosis
● Lymphocytes: → Renal tubular necrosis
○ smallest WBC; RBC-look alike; early stages of renal transplant ■ RTE + Oval fat body: Nephrotic Syndrome
rejection → Nephrotic syndrome: voluminous amount of
proteinuria
→ Anasarcic yung patient or very edematous all over
the body
■ Bubble Cell: Acute Tubular Necrosis
→ The tubules do not work; the urine becomes
isosthenuric
→ Specific gravity is the same as the glomerular fluid
Manner of Reporting
RARE FEW MODERATE MANY

0-5 5 - 20 20 - 100 > 100
EPITHELIAL CELLS
Appearance
● Squamous Epithelial Cell
○ abundant, irregular cytoplasm and prominent nucleus
○ It indicates how clean your “catch” is
■ Rare or Few: Clean catch
■ Moderate or Many: Poor collection
○ “Clue cells”
■ pathognomonic for Gardnerella vaginalis (bacterial
vaginosis)
○ Rare-Few-Moderate-Many/ lpf
Can you highlight the difference?
Identify the pointed structure

Case: Patient with nephrotic syndrome
Clue cells - epithelial cells which are studded (may dot dot). These are
your bacteria or Gardnerella vaginalis.
● Transitional Epithelial Cell (Urothelial cell)

○ Can be seen in the bladder - expands and contracts
○ Spherical, polyhedral or caudate with centrally located
nucleus
○ Singly, in pairs, in clumps (syncytia)
○ Catheterization This is your oval fat body. Similar to RTE cell but has absorbed fat
○ Interpretation: loules.Lipids are highly refractile
○ Rare-Few-Moderate-Many/ lpf (semiquantitative)
○ ↑ TEC with abnormal morphology:
CYLINDRURIA 2. Cellular Cast

● Cylindruria - presence of urinary casts ● RBC Cast
● All casts are made out of proteins called ○ Bleeding within nephron
○ Tamm-Horsfall proteins / Uromodulin ○ Glomerulonephritis
● Usually significant but it depends on the number of casts found ○ dysmorphic RBCs can also be observed
● Casts are reported : per LPF ○ casts studded with RBCs
● Parang tinapay if palaman mo is peanut butter, you call it a peanut ● WBC Cast
butter sandwich ○ Inflammation within nephron
○ So pag may WBC sa cast, it is called WBC cast ○ Pyelonephritis
○ If your cast contains oval fat bodies, it is called fatty casts ■ Primary marker for distinguishing pyelonephritis (Upper
● Casts evolve from coarsely granular casts to a finely granular and UTI) from cystitis (lower UTI)
eventually to a broad cast which indicates an end stage renal ■ WBC casts with symptoms of UTI such as difficulty in
failure urinating and fever are usually associated with this
1. Hyaline Cast disease
● Prototype cast → If it is cystitis you don’t get high grade fever
● Normal Value: ■ It is important to diagnose this because we are aggressive
○ 0-2 / lpf with the treatment for pyelonephritis to prevent renal
● Light Pink - Sternheimer-Malbin scarring
● Casts have a high refractive index so it is hard to visualize ● Epithelial (RTE) Cell Cast
them especially these hyaline casts. It still needs to be stained. ○ Advanced tubular destruction
● Casts are very large and usually cluster at the edge of the ○ Renal tubular damage
coverslip and so it is important to check the edge of the cover ○ Cylindrical, spherical, eccentrically located nucleus
slip during the preparation of slides in normal conventional
methods.
● Clinical Significance (SHED)
○ Strenuous exercise
○ Heat exposure
○ Emotional Stress
○ Dehydration
○ Glomerulonephritis, pyelonephritis, congestive heart failure,
chronic renal disease
■ Glomerulonephritis - usually associated with RBC casts
■ Pyelonephritis - common with WBC casts; infection of
the upper urinary tract
■ Chronic renal disease - broad casts and different types of
casts
3. Granular Cast
● Derived from lysosomes of RTE cells
○ cells have been disintegrated and therefore, lysosomes are
released and become granular casts
○ there are no cells that can be seen
● Glomerulonephritis
● Pyelonephritis
● Stress
● Strenuous exercise
● Types:
○ Coarse Granular Cast
○ Fine Granular Cast
● These cellular casts will eventually evolve to granular casts and
progress into coarse and fine granular casts then onto broad casts
which are end stage casts.
● Strasinger: it is not important to differentiate the two types of
granular casts because the treatment is the same
4. Fatty Cast
● Not stained with Sternheimer-Malbin stain
● Lipid globules on surface
● RBC in background
● Polarized Maltese Cross
○ not exclusive to oval fat bodies but also starch from powdered
gloves (artifact); malarial parasite (Babesia)
Starts with hyaline, then cells attach to the hyaline cast, then granular ● Composition:
casts. Broad casts pertain to severe tubular damage because casts take ○ TG (neutral fat): Lipid stain
up the shape of the kidney tubule. ■ Need to use Sudan 3 or Oil Red O stain to stain fats
(triglycerides)
○ Cholesterol: Polarizing Microscope
■ Cholesterol crystals can polarize light and show a blue AMORPHOUS URATES or AMORPHOUS PHOSPHATES
color and has a positive birefringence
○ Nephrotic syndrome: lipiduria
■ Excretion of fat in urine
○ Toxic tubular necrosis, diabetes mellitus, and crush
injuries.
Solubility:
● Amorphous urates - dissolved upon heating
○ When you refrigerate urine and you see a tinge of red, this is
uroerythrin. This is caused by the precipitation of amorphous
urates and so if you heat this, it will dissolve
● Amorphous phosphates - dissolve in dilute acetic acid
5. Broad Cast ○ acid siya so sino madidisolve? opposite which is Amorphous
● aka Renal Failure Cast phosphates
● Indicates destruction or widening of the tubular walls
○ The shape of the tubular walls are not intact and the holes are CALCIUM OXALATE
enlarged which indicates that there is a destruction or widening
of the tubular walls
● Most common type: Granular and Waxy
● Waxy Cast
○ stain a homogenous, dark pink in supravital stain
○ Final degenerative form of all types of cast
■ it is the end and become clear because they are
disintegrated
■ from the RBC or WBC casts → coarse and fine granular
CALCIUM OXALATE TWO FORMS:
casts → waxy casts ● Dihydrate (Wheddelite)
○ Brittle, highly refractile ○ octahedral envelope: two pyramids joined at their bases
○ Fragmented with jagged ends
○ Extreme urine stasis ● Monohydrate (Whewellite)
■ Prognosis is not favorable ○ dumbbell shaped or oval
○ Chronic Renal Failure ○ Ethylene glycol poisoning → antifreeze that is colorless,
sweet-smelling
Note:
1. This is the ONLY crystal seen in acidic - neutral and alkaline
urine
● Visible in a wide range of pH
2. Birefringent under polarized light
3. Majority of renal calculi are composed of calcium oxalate (CaOx)
4. Foods high in oxalic acid: tomatoes and asparagus / ascorbic acid -
oxalic acid is an end product of ascorbic acid metabolism
● Ascorbic acid (Vit C) is metabolized to oxalic acid and it
would combine with calcium to form calcium oxalate
Are all these the same?
CRYSTALLURIA
Factors that Contribute to Crystal Formation
● It is important to know these because we would know how to
prevent the precipitation of crystals which presents symptoms of
these pathologies.
1. pH
● Uric acid crystals in patients with gout
○ A basic pH should be maintained to prevent the
precipitation of uric acid in the kidneys of the patient
● One should know when the crystals are soluble or
insoluble at which pH
2. Temperature
● Does the crystal dissolve in heat or not? Figure. Uric Acid crystals
3. Solute Concentration ● Barrel shaped
● In what environment does the crystal dissolve ● Rosette shaped
● Six-sided
○ Kamukha ng cystine crystals but this is your uric acid crystal
Manner of Reporting
● Diamond shaped
1. Normal Crystals: rare, few, moderate, or many /hpf ● Rhombic
2. Abnormal Crystals: average / lpf ● Hexagonal
● Whetstone (4-sided flat plate) Calcium sulfate crystals in

● Wedge urine - Cigarette-butt
● Board exam question:
○ Sino sa crystals and nag eexist na may pinakamaraming
morphology?
■ Answer: Uric acid crystals
● Significance:
○ Seen in patients with hyperuricemia, gout
○ A condition or defect wherein there is a lack of enzyme in the
salvage pathway in the purine metabolism
Calcium phosphate - Rosette
Lesch-Nyhan Syndrome Apatite
● Also known as Nyhan’s syndrome,
Kelley-Seegmiler syndrome and
Juvenile gout
● It is a hereditary disorder of purine
metabolism, characterized by
mental retardation, self-mutilation of
the fingers and lips by biting,
impaired renal function, and
abnormal physical development
● It is a recessive disease that is Calcium carbonate -
linked to the X chromosome Dumbbell (effervescence after
HAc) - presence of
effervescence after adding
acetic acid could indicate that
● It is caused by a deficiency of the enzyme hypoxanthine-guanine the crystal is of calcium
phosphoribosyltransferase (HGPRT) carbonate
○ Function of this enzyme: this enzyme is responsible for
salvaging the products of your purine metabolism in order for
you to create your IMP and GMP which is used to synthesize
your nucleic acid Note: Calcium phosphate is mistaken as sulfonamide.
○ However, if you lack the HGPRT, the product of purine ● Sulfonamide appears as rosette too
metabolism will not be salvaged, and uric acid accumulates ● To differentiate: (Aside from asking the patient if he/she takes
● Can be seen early on in infants sulfonamide)
○ Diaper: may orange sand (sandy/gritty texture) 1. Calcium phosphate is soluble in acetic acid
● People affected with this syndrome can manifest with self 2. Lignin test (25% HCl): [+] yellow for sulfonamide (if the crystal
mutilation of the fingers, lips is from sulfonamide)
○ Biting
○ Other signs/symptoms: Uric Acid (normal) or Cystine (abnormal)
■ Mental retardation
■ Abnormal physical development
TIP
● Usually ang mga mode of inheritance ng mga enzyme deficiency
disorders ay ang X-linked recessive or autosomal recessive
● There are a few exceptions
Crystalluria: Normal Crystals Point of Differentiation Uric Acid Cystine

● Uric acid Color Yellow-brown Colorless
● Amorphous urates Ammonia Soluble Soluble
● Calcium oxalate Dilute HCl Insoluble Soluble
● Amorphous phosphates Like-like does not dissolve
● Calcium phosphate Birefringence Birefringent Non birefringent
● Triple phosphate Cyanide-nitroprusside Negative Positive
● Ammonium biurate reaction
● Calcium carbonate
Ammonium biurate or Triple phosphate
Calcium Crystals ● Ammonium biurate - Thorny
Apple Crystal
Calcium Crystals
○ Clinical significance:
Calcium oxalate crystals in ■ Usual pH: alkaline
urine - Enveloped
■ Usually seen in old
Dihydrate calcium oxalate
Monohydrate and dihydrate specimen
have their own clinical ■ Clinical significance:
significance (please see ask patient for a
book) new specimen
■ When urine stands, it becomes more alkaline due to the
breakdown of ammonium to ammonia
● Triple phosphate (struvite crystals) - Coffin-Lid Crystal (prism
shape with feathery appearance upon disintegration)
○ Associated with Proteus spp. bc it alkalizes the urine using Leucine Crystals
urease allowing for the precipitation of phosphate → crystals ● Acidic urine
○ Why is it called triple phosphate? ● Spheroids with concentric striations
■ The term “triple phosphate” stems from early chemical ● Dense
analyses of the stones which demonstrated the presence ● Highly refractile
of calcium, magnesium, ammonium and phosphate ● Yellowish brown
(i.e., three cations and one anion) ● “Pseudo” maltese cross
○ Clinical significance: ● Seen in severe liver disease
■ Common in alkaline urine, especially in cases of UTI ● Hereditary amino acid metabolic disorders
(caused by urease producing organism such as Proteus) ● Can be seen with tyrosine crystals
● Resembles parasite egg
● Clue: kapag crystals na amino acids, usually hepatic diseases
talaga
Tyrosine Crystals
● Colorless or yellow, fine silky needles in
sheaves or clumps
● Seen in liver disease and tyrosinemia
(an inborn error of metabolism)
● Dissolve in alkali
Sulfonamide Crystals
● Yellow-brown sheaves of wheat with
central bindings, round forms with radial
striations etc.
● Occurs following sulfonamide therapy
● Soluble in acetone (unique characteristic)
● NOTE: request form - you should know the
medications being taken by the patient in
order to correlate it with the microscopic
findings
Cholesterol
● Staircase crystal: Notched plate
Crystalluria: Abnormal Crystals ● Nephrotic syndrome
● Cystine ● Soluble in chloroform
● Cholesterol ● Commonly confused with
● Leucine radiographic dye
● Tyrosine ○ Differentiate using polarizing
● Bilirubin microscope
● Sulfonamides ■ Cholesterol crystal is positively birefringent
● Radiographic dye
● Ampicillin Radiographic dye
● Cause of HIGH specific gravity in the
Bilirubin Crystals urine
● Acidic urine ● Refractometer: >1.040
● Pigmented yellowish ● Chemical strip not affected by
brown granules radiographic dye
● Pathologic appear in a ● Soluble in 10% NaOH
wide variety of
hepatobiliary (problem sa
pagprocess ng bilirubin)
and hematopoietic
diseases
Fatty Cast
● Yellowish tinge
● Contains large spherical, highly refractile fat droplets
● Polarized light show “Maltese-cross” pattern
● Oval fat body is associated with marked proteinuria and nephrotic
syndrome
Figure. Fatty casts
Parasites in Urinary Sediment
Structures exhibiting Maltese Cross

● Not exclusive for oval fat body
● Starch Granule
● Fatty Cast ● First picture: Trichomonas vaginalis
● Babesia ○ Jerking; tumbling motion
● Leucine crystals - pseudo maltese cross formation ○ Causative agent of ping pong disease / strawberry cervix
■ Ping pong disease because you need to treat both of the
partners (because if you only treat one of the partners,
papasahan ulit siya, parang ping pong lang)
■ Does this have a cyst stage? No cyst stage.
● Second picture: Schistosoma haematobium
○ With ova na may terminal spine
○ Not common in the Philippines
○ Causing egyptian hematuria
Figure. Maltese cross under polarized light ● Third picture: Enterobius vermicularis
○ D shaped ova
Starch Granule ○ Pinworm
● Colorless ○ Pruritus ani
● Irregularly round with dark striation to the center ○ Indication of fecal contamination
● Asymmetric “Maltese cross” in polarized light ○ Recollection
○ Can be confused with fat bodies
● Frequent contaminant
Figure. Starch polarized Figure. Fat droplet polarized

Urine Artifacts Glass Fragments

● Glass fragments
● Oil droplet
● Air bubbles - look like RBC
○ How to differentiate?
■ Manipulate fine adjustment knob
■ Usually air bubbles and oil droplets exist in the same
plane even though you have adjusted the fine
adjustment knob, compared to the RBC na nawawala
kapag inadjust mo yung fine adjustment knob
● Fibers
○ Can look like casts
○ Possible contamination from underwear of patient
○ May be mistaken as worm
● Plant material
○ That’s why specimen container should be clean
● Pollen grain
● Spores
● Starch granules
○ From powdered gloves
○ Maltese cross formation
Talcum Powder Particles

● Talcum powder particles
● Talcum powder particles

○ If patient uses powder in genital area
MT UNIT 16: Methods and Procedures of Diagnosing Metabolic Disorders Dr. John Henrick Uy | Mr. Joemarie Malana LAB
OUTLINE ○ To compensate, ketoacids will be released in the urine causing

Abnormal Metabolic Conditions Detected 1 mousy odor and reaction with ferric chloride.
Overflow Versus Renal Disorders ● For hartnup disease and cystinuria: you have defects in the
Newborn Screening (RA9288) 2 reabsorption of specific substances.
Aminoacidopathies
Porphyrias ○ Hartnup: problem with tryptophan metabolism.
Mucopolysaccharides ○ Cystinuria: problem with reabsorption of not just cystine, but a
Inborn Purine Metabolism Defects total of four amino acids: C,O,L,A
Inborn Defects in Metabolism of Other Reducing Sugar
■ Cystine, ornithine, lysine, and arginine
LEGEND
BLACK TEXT PURPLE COLORED TEXT BLUE COLORED TEXT
Based from ppt Based from lecture proper Based from Strasinger 6E NEWBORN SCREENING ACT OF 2004 (RA9288)
● Vision
ABNORMAL METABOLIC CONDITIONS DETECTED ○ The National Comprehensive Newborn Screening System
envisions all Filipino children will be born healthy and well, with
COLOR ODOR CRYSTALS
an inherent right to life, endowed with human dignity; and
Homogentisic acid Phenylketonuria Cystine Reaching their full potential with the right opportunities and
Melanin Maple syrup urine Leucine
Indican disease Tyrosine accessible resources.
Porphyrins Isovaleric acidemia Lesch-Nyhan disease ● Mission
Cystinuria ○ To ensure that all Filipino children will have access to and avail
Cystinosis of total quality care for the optimal growth and development of
Homocystinura their full potential.
● Color ● Goal
○ Black: Melanuria, Homogentisic acid, Alkaptonuria ○ To reduce preventable deaths of all Filipino newborns due to
○ Blue: Indicanuria (Blue Diaper) more common and rare congenital disorders through timely
○ Port wine: Porphyrin (Hereditary porphyrias) screening and proper management
● Odor ● Republic Act No. 9288 or the Newborn Screening Act of 2004
○ Mousy: Phenylketonuria ○ Administrative Order No. 2018-0025: National Policy and
○ Burnt sugar: Maple Syrup Urine Disease Strategic Framework on Expanded Newborn Screening for
■ Defect in the metabolism of your branched chain amino ● 2017-2030
acids ○ Administrative Order No. 2014-0045 or the Guidelines on the
● Crystals Implementation of the Expanded Newborn Screening Program
○ Cystine, Leucine, Tyrosine: Problem in the metabolism of ● Current state-mandated screening for as many as 29 inborn errors
these amino acids of metabolism
○ Lesch-Nyhan Disease: Orange-sand diaper due to the ● Urine tests are primarily for follow-up
presence of uric acid crystals ● Heel stick blood tests are used for testing
○ Performed before infant leaves the hospital
OVERFLOW vs RENAL DISORDERS ○ Metabolites appear first in the blood
Overflow ● Confirmed by tandem mass spectrophotometry, MS/MS
● Breakdown in a normal metabolic pathway causing accumulation ● Importance: Early detection of inborn errors of metabolism and
of previous metabolite to initiate the early treatment
○ Due to the accumulation of the metabolites, it would eventually ○ Inborn Errors of Metabolism are generally treatable
be secreted out of the urine ■ Restrict certain amino acids in the diet so that there will
○ So it causes an overflow in the urine not be an accumulation
● Overrides Transport maximum/Impaired normal reabsorption ○ Early Detection: prevent mental retardation which is irreversible
● Inherited lack of specific enzyme for protein, fat, or carbohydrate Expanded Newborn Screening Panel
● metabolism — Inborn Error of Metabolism ● Notes:
1. Number of disorders in the panel may increase as per approval
Renal by the Advisory Council for Newborn Screening
● Functional defect
2. The expanded newborn screening fee does NOT cover
○ Congenital or acquired problem in reabsorption of certain
Confirmatory for Endocrine Disorders and G6PD
amino acids which is why it appears in the urine
Deficiency.
OVERFLOW METABOLIC RENAL 3. The expanded newborn screening fee does NOT cover
INHERITED
treatment and management for Endocrine Disorders, G6PD
Phenylketonuria Infantile tyrosinemia Hartnup disease Deficiency, and Hemoglobinopathies.
Tyrosinemia Melanuria Cystinuria
4. The treatment mentioned herein only covers medications,
Alkaptonuria Indicanuria
Maple syrup urine 5-Hydroxyindoleacetic medical foods, catheter for peritoneal, a nd hemodialysis
disease acid needed for the treatment of acute crisis management i.e.
Organic acidemias Porphyria dialysis for MSUD, Propionic Acidemia, Urea Cycle Defects.
Cystinosis 5. Argininosuccinic Aciduria (ASA) was added into the
Porphyria expanded newborn screening panel in November 2018
Mucopolysaccharides
● Phenylketonuria: you lack an enzyme to metabolize phenylalanine

in milk, so what happens is that the metabolite proximal to the
enzyme would accumulate resulting in ketones.
○ Increase in ketones may cause acidosis, ketosis, seizures,
and mental retardation.
Disorder Disorder Metabolite Tested ○ Nagsasama yung parents that have the same defective gene,
Group so the child has a higher chance of inheriting the gene from
Congenital Hyperthyroidism CH Thyroid Stimulating both parents, and present with the disorder.
Endocrine Hormone (TSH) ● Described by Ivan Folling
Disorder Congenital Adrenal CAH 17-hydroxy-progeste ● Failure to inherit the gene to Phenylalanine hydroxylase
Hyperplasia rone (17 a-ohp) ● Tyrosine becomes essential in these individuals due to the absence
Homocystinuria HCY Methionine of phenylalanine.
Hypermethioninemia/Methioni MAT Methionine ○ The milk formula of patients with PKU should not have
ne Adenosine Transferase phenylalanine, but should be supplemented with tyrosine.
Amino Deficiency ● Patients with PKU will have white skin, because melanin will not be
Acid
Maple Syrup Urine Disease MSUD Leucine formed (see Fig 1 - right blue box connected to tyrosine)
Disorder
Phenylketonuria PKU Phenylalanine
Tyrosinemia Type I TYR Succinylacetone
Tyrosinemia Type II, III (SA) Tyrosine
Carnitine Palmioyltransferase CPT1 Hexadecanoyl
I Deficiency carnitine+ CPT ratio
Carnitine Palmioyltransferase CPT2 Hexadecanoyl
II Deficiency carnitine+ CPT ratio
Carnitine Uptake Deficiency CUD Free carnitine
Fatty Acid Glutaric Acidemia Type II GA II

Butyrylcarnitine+Isov
Disorder alerylcarnitine
Long Chain Hydroxyacyl-CoA LCHAD 3-Hydroxyhexadecan
Dehydrogenase Deficiency oylcarnitine
Medium Chain-Acyl-CoA MCAD Octanoylcarnitine
Dehydrogenase Deficiency
Very Long Chain-Acyl-CoA VLCAD Tetradecanoylcarniti
Dehydrogenase Deficiency ne
Tri-functional Protein TFP Hydroxyhexadecano
Deficiency ylcarnitine
3-Methylcrotnyl CoA 3MCC 3-Hydroxyisovalerylc
Carboxylase Deficiency arnitine
Beta Ketothiolase Deficiency BKT Hydroxyisovalerylcar
Phenylalanine hydroxylase converts phenylalanine to tyrosine.
nitine
Organic Since there is an absence of phenylalanine hydroxylase, there is an
Glutaric Acidemia Type I GA I Glutarylcarnitine
Acid accumulation of phenylalanine which will be converted to phenylketones
Isovaleric Acidemia IVA Isovalerylcarnitine (phenylketonuria) and eventually to phenylpyruvic acid.
Methylmalonic Acidemia MMA Propionylcarnitine Phenylalanine → Phenylketones → Phenylpyruvic acid
Multiple Carboxylase MCD 3-
Deficiency Hydroxyisovalerylcar
Phenylpyruvic acid will accumulate in the blood
nitine+Pronionylcarni
tine ● symptoms: seizure, mental retardation, weakness, lethargy,
failure to thrive.
Propionic Acidemia PA Propionylcarnitine
● Since there is an excess, it will be excreted in the urine with
Cycle Citrullinemia CIT Citrulline
mousy odor.
Defect Argininosuccinic Aciduria ASA Citrulline
Alpha Thalassemia HgB Hemoglobin Phenylpyruvic acid will form instead of tyrosine because
Hemoglobi Beta Thalassemia
phenylalanine hydroxylase is missing.
nopathies Hemoglobin C
Hemoglobin D ● There will be a decrease in Tyrosine.
Hemoglobin E ● Phenylalanine is an essential amino acid, while Tyrosine is
Sickle Cell Disease not essential in normal patients.
Galactosemia GAL Total Galactose ● But since patients with phenylketonuria cannot process and
Glucose-6-Phosphate G6PD G6PD enzyme should not be given phenylalanine, tyrosine becomes
Dehydrogenase Deficiency Def activity essential.
Other
Cystic Fibrosis CF Immunoreactive
Trypsine (IRT) Rule of thumb for all metabolic disorders:
Biotinidase Deficiency BTND Biotinidase enzyme ● Anything BEFORE t he enzyme will increase
activity ● Anything AFTER the enzyme will decrease
Aminoacidopathies Phenylalanine-Tyrosine Pathway PKU Screening

● Phenylketonuria (PKU) Ferric Chloride Test - Nonspecific Test
● Tyrosinuria
1. Place 1 mL of urine in a tube
● Alkaptonuria
2. Slowly add 5 drops of 10% ferric chloride
3. Observe for a permanent blue green color
PHENYLKETONURIA (PKU)
● Non-specific because it is also used in other disorders; iba iba lang
● Autosomal recessive
yung color results sa ibang test
○ Normally seen in marriages in the same consanguinity or same
family (Magka-mag anak sila)
Figure 2. Blue green color signifies a positive Figure. Guthrie blood test
reaction Guthrie blood test used to
be the confirmatory
test, but now we use
the tandem mass
spectrometry to
confirm PKU; tandem
mass spectrometry is
also used in other
metabolic disorders
Guthrie blood test

Diphenylhydrazine Test (DNPH Test)
1. Media containing beta-2-thienylalanine, an inhibitor of Bacillus
1. Place 2 mL of urine in a test tube
subtilis, is streaked with the bacteria
2. Add equal volume of DNPH reagent
2. Blood-impregnated discs placed on the agar
3. Mix the solution
3. Phenylalanine counteracts the inhibitor, and bacteria grow around
4. Wait for 10 Minutes
the disc.
5. Observe for a yellow precipitate: hydrazone
● Suspected PKU patients have their blood placed on the disk which
is placed on the agar with the bacteria
○ Beta-2-thienylalanine is an inhibitor of Bacillus subtilis
○ But in the presence of phenylalanine, beta-2-thienylalanine is
neutralized
○ Which in turn allows the growth of the bacteria that is on the
agar
● In a PKU patient mataas ang phenylalanine so, maneutralize yung
inhibitor ng bacteria (beta-2-thienylalanine) which results to bacterial
growth
● phenylalanine high = neutralizes bacterial inhibitor =
● (+) bacterial growth = (+) guthrie blood test TYROSINEMIA / TYROSYLURIA
● Confirmatory test: Tandem Mass Spectrometry; commonly used ● Accumulation of excess tyrosine in the urine
for all metabolic disorders ● Tyrosinemia - accumulation of excess tyrosine in the plasma
● Three Types
Guthrie Procedure ○ Type I
1. Take a little blood of the newborn ■ Deficiency of the enzyme fumarylacetoacetate
2. Put it on a blotting paper hydrolase (FAH)
3. Dry and send out to the post ○ Type II
● this test measures the amino acid that can’t be metabolized ■ Deficiency of the enzyme tyrosine aminotransferase
4. Once received by the laboratory, punch the paper with the blood (TAT)
sample on it ○ Type III
5. Put the bit of paper on a bacterial plate ■ Deficiency of the enzyme 4-hydroxyphenyl pyruvate
6. Bacteria grows when treated with phenylalanine dioxygenase (HPPD)
● Halo of growth in the plate represents bacterial growth
From: https://www.youtube.com/watch?v=i5FgGrMQGkI
Figure. Blood Blotting
Paper for Guthrie Test
Halo of growth in the plate

represents bacterial
growth
Amino acid present has
spread around and
allowed the bacteria Phenylalanine production is not restricted because there is enough
Phenylalanine Hydroxylase. There will be a source of Tyrosine.
to grow
In cases of Tyrosinemia, you have to restrict not only Tyrosine but also
Phenylalanine because it is also a source of Tyrosine.
Causes of Tyrosinemia
Metabolic Defects
● Premature transient tyrosinemia
● Underdevelopment of liver function
● Acquired severe liver disease
○ liver impairment that halts the metabolism of the amino acid
Hereditary Defects
● Type I: FAH deficiency
○ Produces a generalized renal tubular disorder and
progressive liver failure in infants soon after birth
● Type II: TAT deficiency

○ Corneal erosion and lesions on the palms, fingers, and soles
of the feet believed to be caused by crystallization of tyrosine in
the cells
● Type III: HPPD deficiency

○ Results in mental retardation if dietary restrictions of
If the homogentisic acid oxidase is not present, homogentisic acid will
phenylalanine and tyrosine are not implemented
accumulate and maleyl acetoacetic acid will decrease.
Tyrosinemia / Tyrosyluria Screening Alkaptonuria Screening

Nitroso-napthol test Ferric Chloride Test - Nonspecific Test
1. Place 5 drops of urine in a test tube 1. Place 1 mL of urine in a tube
2. Add 1 mL of 2.63N nitric acid 2. Slowly add 5 drops of 10% ferric chloride
3. Add 1 drop of 21.5% sodium nitrite 3. Observe for a blue color
4. Add 0.1 mL 1-nitroso-2-napthol
5. Mix the solution Homogentisic Acid Test
6. Wait 5 minutes 1. Place 4 mL of 3% silver nitrate in a tube
7. Observe for an orange – red color 2. Add 0.5 mL of urine
3. Mix the solution
ALKAPTONURIA 4. Add 10% NH4O H by drops 5.
● Enzyme deficiency is homogentisic acid oxidase ● Ammonium hydroxide causes the solution to become alkaline
● Black alkaline urine, possible black-stained diapers ● Freshly voided urine is acidic so initially, you will think that the
○ Urine will initially appear normally colored when released, but urine of the infant is normal but upon alkalinization of urine,
when urine is left to stand, the urine becomes more alkaline there is a presence of a black color.
leading to the formation of black-stained diapers. 5. Observe for black color
● Manifests later in life with brown pigment deposits in tissues
Clintests Tablet
● (+) yellow precipitate
● Quantitative tests available
● aside from detecting sugars it can also be used to screen
alkaptonuria
● yellow precipitate is formed as the homogentisic acid is able to
reduce the clinitest.
Brown pigment deposits in tissues
MELANURIA
● Second pathway for tyrosine
○ Melanin, thyroxine, epinephrine
Melanin
● Pigment for dark hair and skin
● Absence results in Albinism
● Increased in conditions like malignant melanoma

○ Presence of malignant melanin cells that continuously produce
melanin which is why melanin is increased
● Dark urine from oxidation of melanogen
○ Similar to alkaptonuria, but for alkaptonuria, dark colored urine
is attributed to alkaline condition of the urine
○ For melanuria, it is due to exposure to the air or oxidation of
the colorless melanogen to brown pigment - melanin
Melanuria Screening
Thormahlen Test (Sodium nitroprusside test)
1. Add 0.2 ml aqueous sodium nitroprusside to 5ml urine
2. Add 0.5 ml of 40% sodium hydroxide.
3. Observe for a Red Color
4. Add 3.5ml of 30% glacial acetic acid.
5. Observe for a blue or dark color
Ferric Chloride Test - Nonspecific Test
2. Slowly add 5 drops of 10% ferric chloride
3. Observe for a gray or black precipitate
AMINOACIDOPATHIES BRANCHED CHAIN AMINO ACID

DISORDERS Accumulation of ketone bodies will ensue, because these three
● Maple Syrup Urine Disease (MSUD) amino acids (leucine, isoleucine, and valine) are capable of
● Organic acidemias ketogenesis.
● Purely ketogenic amino acid: leucine and lysine
MAPLE SYRUP URINE DISEASE (MSUD) ● Ketogenic and Glucogenic: isoleucine a nd valine
● Autosomal-recessive; Inborn error of metabolism
● First week: failure to thrive Isovaleric Acidemia Screening
● Urine: strong odor of maple syrup, and thick, dark appearance p-Nitroaniline Test
(Burnt Sugar Odor) 1. Place 1 drop of urine in a tube
○ Due to the defect in the metabolism of branched chain amino 2. Add 15 drops of 0.1% p-nitroaniline in 0.16 M HCl
acids: Leucine, Isoleucine, Valine 3. Add 5 drops of 0.5% sodium nitrite
● Common manifestations: Failure to thrive, and Burnt sugar odor 4. Mix 5. Add 1 mL of 1 M sodium acetate buffer at pH 4.3
● Dietary regulation - important to avoid mental retardation due to the 5. Boil for 1 min
accumulation of ketone bodies 6. Add 5 drops of 8 N NaOH
● Positive urine ketones 7. Observe for emerald green color
● Confirmatory test: Tandem Mass Spectrometry
AMINOACIDOPATHIES TRYPTOPHAN DISORDERS

● Indicanuria
● 5-Hydroxindole Acetic Acid (5-HIAA)
MSUD Screening
2,4-Dinitrophenylhydrazine (DNPH) Test
2. Add 10 drops of 0.2% 2,4-DNPH in 2N HCl
3. Wait 10 minutes
4. Observe for yellow or white precipitate Green box: Normally, tryptophan is metabolized into indole and
● Also has DNPH test, similar with PKU secreted in the feces
Blue box: In abnormal conditions such as an intestinal disorder
ORGANIC ACIDEMIAS: (Hartnup’s disease):
Isovaleric, Propionic, Methylmalonic acidemias ● There would be an accumulation of excess indole and is
● Organic Acidemia metabolized by the liver to produce indican
● Indican is readily excreted in the urine and exposure to the
● Early: severe vomiting, acidosis, hypoglycemia, ketonuria
air would change indican to indigo blue.
● Isovaleric: “sweaty feet odor” ● Diaper is blue stained; blue stained diaper syndrome
● Deficiency of isovaleryl coenzyme A
Orange box: On the other side, in patients with malignant tumors Cystinuria Screening
● These tumors continuously produce 5-hydroxytryptophan ● Cyanide-Nitroprusside Test
● It will then be converted to serotonin (derived from the amino 1. Place 3 mL or urine in a tube
acid - tryptophan). 2. Add 2 mL sodium cyanide
● Excess serotonin would then be converted to 5-HIAA which is 3. Wait 10 minutes
then excreted in the urine.
4. Add 5 drops 5% sodium nitroprusside
● 5-HIAA levels can be tested in tumor patients, and if it is
elevated in either serum or urine, it is possible that they have a 5. Observe for red-purple color (+)
malignant tumor secreting 5-hydroxytryptophan
CYSTINOSIS
INDICANURIA ● Inborn error of metabolism
● Normal metabolism of indican: ● Three variations: nephropathic (infantile, later life), and
○ Tryptophan enters intestine non-nephropathic
○ Reabsorbed or is converted to indole by bacteria ○ Infantile: rapid progression to renal failure
○ Exits in the feces ○ Late onset: gradual progression to renal failure
● Intestinal disorders and Hartnup disease cause increased ○ Non-nephropathic: benign, some ocular problems
tryptophan conversion to indole ● Defect in lysosomal membranes prevents release of cystine into
● Increased indole reabsorbed, excreted by kidney (through urine) cytoplasm for metabolism → crystalline cystine deposits in body
on its way to the liver ○ If not released from the lysosomes, cystine would accumulate
● Exposure of urine to air (through standing) yields an indigo blue in your cells → crystallize in your body (not in the urine) →
color clinical findings: deposits in cornea, bone marrow, lymph
● Hartnup disease “blue diaper syndrome” nodes, renal tubules
○ Inherited disorder affects intestinal reabsorption of indole ● Deposits found in corneas, bone marrow, lymph nodes, organs
○ Fanconi syndrome - renal tubular reabsorption ○ Cornea - causing blurring of vision
○ Requires dietary supplements ○ Bone marrow - can cause cytopenia as it interferes with blood
● Screening Test: Ferric Chloride Test production
○ Urine: blue or violet color with ferric chloride that can be ○ Lymph nodes - can make you susceptible to infections
extracted into chloroform ○ Renal tubules - causing Fanconi syndrome
● Renal tubules are affected resulting to Fanconi syndrome
5-HYDROXYINDOLE ACETIC ACID (5-HIAA) ● Laboratory - aminoaciduria, reducing substances, cystine crystals
● Tryptophan produces serotonin ○ In the laboratory you could appreciate aminoaciduria. In the
● Tryptophan is produced by the intestinal argentaffin cells and is microscopic examination of urine, you could see cystine
carried in the body to the muscles by platelets crystals. You could also test for reducing substance, because it
● Excess excreted in the urine as 5-HIAA will yield a positive result in your reducing substances.
● Argentaffin cell tumors = ↑↑ 5-HIAA in urine form excess serotonin ● Treatment: Renal transplants and cystine-depleting medications
produced ○ Cystine-depleting medications to prevent crystallizing of the
○ If there is a tumor in the intestinal argentaffin cells = high amino acids
serotonin = excess excreted in urine as 5-HIAA ● Positive clinitest for reducing substances
● Screening test:
○ Nitrous acid and 1-nitroso-2-naphthol produce p urple to
black color (+)
● 5-HIAA can be quantitated in the blood
● Normal values: 2-8 mg/day
● Disease states: >25 mg/day
● Random specimens are acceptable
● Patient instructions:
○ No bananas, pineapples, tomatoes, phenothiazines, and
acetanilids for 72 hours
○ also asparagus since they are a good source for serotonin -
the happy hormone Figure. Clinical features of cystinosis: corneal cystine crystals, excessive
○ 24-hour urines must be preserved with HCl or boric acid thirst/dehydration, excessive urination, rickets, failure to thrive, Fanconi
syndrome, kidney failure, elevated cystine in white blood cells,
AMINOACIDOPATHIES CYSTINE DISORDERS hypothyroidism
● Cystinuria
● Cystinosis
HOMOCYSTINURIA
● Homocystinuria ● Defect in metabolism of methionine
● Increased methionine levels in the blood
CYSTINURIA ● Failure to thrive, cataracts, mental retardation, thromboemboli
● Elevated cystine in the urine because there is a defect in resulting to premature death
reabsorption ○ When homocysteine accumulates, it causes failure to thrive,
● It is not just the cystine that is affected in this condition, it also
cataracts, mental retardation, and predisposition to
affects your lysine, arginine and ornithine
thromboembolism resulting to premature death
● Two modes of inheritance:
○ In some studies, elevated levels of homocysteine among
○ Cystine, ornithine, lysine, and arginine (COLA) or
individuals predisposes one to cardiac pathologies such as
○ Cystine and lysine are NOT reabsorbed myocardial infarction or heart attack
● If you are unable to reabsorb the cystine, it will accumulate in the ■ Homocystinuria has been an interest of some
renal system → it will precipitate out / crystallize → appreciating / researches because can be correlated to risk for
seeing hexagonal plates in the urine (urine sediment)
cardiovascular diseases such as myocardial ● Per enzyme deficiency in each set, there is a corresponding
infarction porphyria
● Treatment: Requires diet modification ● Clinical signs and symptoms can be grouped
○ Restrict patient’s diet of methionine to lessen accumulation of ○ First 5 products/metabolites of heme synthesis: any
homocysteine, causing less symptoms enzyme defect before the formation of first ring (ringed
metabolite), the common clinical manifestations are seizures,
behavioral disorders, mental retardation
○ Disorders coming after the first ring form (first heme ring):
common manifestation are photosensitivity, skin disorders (skin
lesions, hypertrichosis)
● All of them produce the classic port wine urine
PORPHYRIA
● Porphyrins are the intermediate compounds in the production of
heme
● Three primary porphyrins: uroporphyrin, coproporphyrin,
protoporphyrin
● Precursors: a-aminolevulinic acid (ALA) and porphobilinogen
● Urine: ALA, porphobilinogen, urobilinogen
Figure. Methionine metabolism ● Feces/bile: coproporphyrin, protoporphyrin
Thick line - problem: Homocysteine accumulates ● Blood: free erythrocyte protoporphyrin for lead poisoning
● In the more advanced testing for this product, you could already
Homocystinuria Screening determine what specific type of porphyria the patient is suffering
● Silver Nitroprusside Test from by testing the specific enzymes
1. Place 1 mL of urine in a test tube ○ Colorimetric method is not needed as it is not specific
2. Add 2 drops concentrated NH4OH ● Porphyrias can be inherited or acquired from erythrocytic and
3. Add 0.5 mL 5% silver nitrate hepatic malfunctions or exposure to toxic agents
4. Wait 10 minutes ○ Inherited Porphyrias: classified by clinical symptoms as
5. Add 5 drops sodium nitroprusside neurologic/psychiatric, cutaneous/photosensitivity, or both
6. Observe for a red-purple color (+) ■ Neurologic/psychiatric - before the ring form of heme
synthesis
PORPHYRIN DISORDERS (book) - DEFECTS IN HEME SYNTHESIS ■ Cutaneous/photosensitivity - more terminal phases of
● Hereditary Porphyrias heme synthesis
○ Acquired (more common): lead poisoning, alcoholism, iron
deficiency, chronic liver and renal disease
■ Lead exposure affects aminolevulinic acid (ALA)
synthetase and ferrochelatase
→ Ferrochelatase incorporates iron in ferrous form into
the protoporphyrin ring - nagkakaroon na ng heme
● Urine: port wine color after air exposure
○ Referred to as porphyrinuria
● Ehrlich reaction: positive only for ALA and porphobilinogen (in
the urine)
○ Convert ALA to porphobilinogen by adding acetyl acetone
● Fluorescence under ultraviolet light used for other porphyrins
○ Extract into glacial acetic acid and ethyl acetate
○ Violet, pink, red, based on concentration
○ Color of the fluorescence would be based on the amount of
porphyrins contained in the specimen
Figure. Symptoms of Porphyria: seizures, high blood pressure, heart

palpitations, red or brown urine, severe abdominal pain, vomiting,
insomnia, anxiety
Historical Note: Vampires in Old Europe

Did you ever wonder how the legend of vampires got started? Think
about the previous discussion on the symptoms and inheritance of
Figure. Pathway of Heme Synthesis porphyrias.
● Photosensitivity → avoidance of sunlight 3. Vigorously shake both tubes

● Pale coloring → anemia caused by heme disorder 4. Place in a rack for layers to settle
● Port wine-colored urine, red-stained teeth (gums and oral 5. Observe both tubes for red color in the layers
area) → drinking blood
● Psychiatric symptoms → abnormal behavior Interpretation:
● Inherited disorder → familial association, small gene pool ● Tube 1
○ Upper layer = urine
Dracula (Vlad the impaler) is associated with Transylvania, now ■ if colorless = urobilinogen
Romania. Porphyria was a common disease of early royalty in Europe ■ if red = porphobilinogen or Ehrlich-reactive compounds
as a result of intermarriage among the royals of different countries. King ○ Bottom layer = chloroform
George III reportedly died blind, deaf, and mad from porphyria. ■ if colorless = porphobilinogen or Ehrlich-reactive
compounds
Werewolves ■ If red = urobilinogen
● Hypertrichosis - mabuhok ○ If both layers are red, re-extract the urine layer from tube 1
■ Place 2 mL of urine layer from Tube 1 and 2 mL
Watson-Schwartz Differentiation Test chloroform and 4 mL sodium acetate into a new tube.
Repeat procedure.
The classic test for differentiating between urobilinogen, ■ Interpretation:
porphobilinogen, and Ehrlich-reactive compounds is the → Upper layer - urine colorless
Watson-Schwartz test. The test is performed as follows: → Bottom layer - chloroform - red = excess
1. To each tube add: urobilinogen
Tube 1 Tube 2 → Both layers red = porphobilinogen and urobilinogen
2 mL urine 2 mL urine ● Tube 2
2 mL chloroform 2 mL butanol ○ Upper layer = butanol
4 mL sodium acetate 4 mL sodium acetate ■ if red = urobilinogen or Ehrlich-reactive compounds
■ if colorless = porphobilinogen
2. Observe the color of the layers ○ Bottom layer = urine
3. Interpretation:
● The addition of chloroform to Tube 1 results in the extraction
of urobilinogen into the chloroform (bottom) layer, producing
colorless urine (top) layer, and a red chloroform layer on the
bottom. Neither porphobilinogen nor other Ehrlich-reactive
compounds are soluble in chloroform. Porphobilinogen is also
not soluble in butanol; however, urobilinogen and other
Ehrlich-reactive compounds are extracted into butanol.
Therefore, the addition of butanol to Tube 2 produces a red
(upper) butanol layer if urobilinogen or Ehrlich-reactive
compounds are present and a colorless butanol layer if
porphobilinogen is present. As shown in the figure,
urobilinogen is soluble in both chloroform and butanol, and
porphobilinogen is soluble in neither. If both urobilinogen and
porphobilinogen are present, both layers appear red. Before
reporting the test as positive for both substances, an
additional chloroform extraction should be performed on the
red urine (upper) layer in Tube 1 to ensure that the red color is
not due to excess urobilinogen.
Figure. Watson-Schwartz reactions
Hoesch Screening Test for Porphobilinogen

The Hoesch test is used for rapid screening or monitoring of urinary
porphobilinogen.
1. Two drops of urine are added to approximately 2 mL of Hoesch
reagent (Ehrlich reagent dissolved in 6 M HCl)
2. Immediately observed the top of the solution for the appearance of
a red color that indicates the presence of porphobilinogen
3. Shake the tube
Interpretation:
When the tube is shaken, the red color is seen throughout the solution.
The test detects approximately 2 mg/dL of porphobilinogen, and
urobilinogen is inhibited by the highly acidic pH. High concentrations of
methyldopa and indican, and highly pigmented urines, may produce
false-positive results.
Figure. Watson-Schwartz reactions MUCOPOLYSACCHARIDOSES (MPS)

● Hurler Syndrome
Watson-Schwartz Test ● Hunter Syndrome
1. Label two tubes #1 and #2 ● Sanfilippo Syndrome
2. To each tube add:
Tube 1 Tube 2 MUCOPOLYSACCHARIDOSES
● Inherited disorders preventing the metabolism of
2 mL urine 2 mL urine
glycosaminoglycans (gags) in the connective tissue
2 mL chloroform 2 mL butanol
4 mL sodium acetate 4 mL sodium acetate ● Incompletely metabolized polysaccharides accumulate in connective
tissue → producing signs and symptoms
● Substances found in urine are dermatan sulfate, keratan sulfate, and INBORN METABOLIC DEFECTS IN PURINE METABOLISM
heparin sulfate ● Lesch-Nyhan Disease
Types of Mucopolysaccharidosis LESCH-NYHAN DISEASE
Type Name of the syndrome Gag accumulated ● Inherited sex-linked recessive
MPS Type I Hurler’s syndrome/scheie Dermatan, heparan sulfate ● Defect in the enzyme hypoxanthine guanine
syndrome phosphoribosyltransferase (HGPRT)
MPS Type II Hunter’s syndrome Dermatan, heparan sulfate ○ HGPRT enzyme is absent
MPS Type III Sanfilippo syndrome Heparan sulfate ● Massive excretion of uric acid
MPS Type IV Morquio syndrome Keratan sulfate, chondroitin ● Normal development 6-8 months
sulfate ● Orange sand in diaper
MPS Type VI Maroteaux lamy syndrome Dermatan, chondroitin sulfate
○ Grainy
MPS Type VII Sly syndrome Dermatan/heparan/chondroiti
● Clinical manifestations:
n sulfate
MPS Type IX Natowicz syndrome Hyaluronan ○ Self-mutilative disorder
● Each type affects a different enzyme ○ Aggression
● Hurler’s and Hunter’s syndrome have same manifestations ○ Mental retardation
○ Very hard to distinguish ○ Kidney stones
○ One distinguishing feature between the two: ■ Due to uric acid accumulation
■ Hurler’s syndrome - corneal clouding, blurring of vision ○ Arthritis
■ Hunter’s syndrome - no corneal involvement ■ Due to gout
→ TIP: Pag hunter, dapat malinaw ang mata; So sa ○ Spasticity
hunter’s syndrome walang blurring of vision ○ Choreoathetosis
● One common feature of these MPS: all have mental retardation ○ Hyperreflexia
except MPS Type IV or Morquio syndrome ○ Orange or red urine
○ TIP: Morquio has more IQ so wala siyang mental retardation ○ Gout
● Confirmatory test:
○ Test for the activity of the enzyme (?)
○ Screening: Test levels of uric acid in the patient - elevated
● Laboratories should be alert for the presence of increased uric acid
crystals in pediatric urine specimens
CARBOHYDRATE DISORDERS (book) - [INBORN] DEFECTS IN

METABOLISM OF OTHER REDUCING SUGARS
● Galactosuria
● Fructosuria
● Lactosuria
Figure. Mucopolysaccharidosis ● Pentosuria
● General clinical presentations of patients with Inborn Defects in Metabolism of Other Reducing Sugars
mucopolysaccharidosis: ● Melituria; frequently inherited
○ Facial features like flat nasal bridge and thick lips ○ Melituria - presence of increased urinary sugar, most frequently
○ Short torso / hearing loss due to an inherited disorder
○ Dysplasia or abnormal bone size
○ Developmental delays and mental retardation
○ Enlarged organs like heart, liver, and spleen
■ Internal organ involvement because Internal organs
contain connective tissue, and if these substances
accumulate in the tissues, they could produce
organopathies
○ Heart diseases / respiratory issues
MPS Screening
Figure. Galactose metabolism
Cetyltrimethylammonium Bromide (CTAB) Test ● Two types:
1. Place 5 mL of urine in a tube ○ Defect in first enzyme - GALK enzyme
2. Add 1 mL 5% CTAB in citrate buffer ■ Usually no manifestation
3. Read turbidity (+) in 5 minutes
■ Asymptomatic
○ Defect in second enzyme - GALT enzyme
Mucopolysaccharide Paper Test
■ Mas nagiging toxic yung patient
1. Dip filter paper into 0.59% azure A dye in 2% acetic acid
■ May manifest with brain damage, cataract, jaundice if not
2. Dry the filter paper
avoided early
3. Add 1 drop of urine to the filter paper
● Treatment: Removal of lactose from diet
4. Wash with 1 mL acetic acid + 200 mL methanol diluted to a liter
○ Galactosuria, indicating the inability to properly metabolize
5. Observe for a blue color (+)
galactose to glucose. The resulting galactosemia with toxic
intermediate metabolic products results in infant failure to
thrive, combined with liver disorders, cataracts, and severe
mental retardation. Early detection of galactosuria followed
by removal of lactose (a disaccharide containing galactose
and glucose) from the diet can prevent these symptoms
PENTOSURIA
● Ingestion of large amounts of fruit
● Monosaccharides are Clinitest positive

○ They are reducing and is free to react with copper
● Positive copper reduction test result with a negative reagent
strip for glucose is strongly suggestive of a disorder of
carbohydrate metabolism
○ May not be glucose
○ May be other sugars such as galactose, fructose, or lactose
LACTOSURIA
● Seen in pregnancy and lactation
FRUCTOSURIA
● Parenteral feeding
Figure. Fructose metabolism

● Fructokinase and Aldolase B
○ Fructokinase deficiency - benign manifestation
■ If your fructokinase is absent, you still have your
hexokinase enzyme, which will take the role of the
fructokinase - hence, benign manifestation only
○ Aldolase B deficiency - more toxic presentation
■ Mental retardation, cataract, seizure
Fructose Screening (Seliwanoff Test)

2. Add 5 mL of 25% HCl
3. Boil 5 min
4. Add 5 mg resorcinol
5. Boil 10 seconds
6. Observe for a red precipitate (+)
Figure. Seliwanoff’s Test
MT UNIT 17: CSF Analysis Mr. Joemarie Malana LAB
OUTLINE ■ Known to have contamination from cellular components

INTRODUCTION TO CSF ANALYSIS 1 which is why it cannot be used for hematology (false
CHEMICAL EXAMINATION OF CSF increase in number of cell)
- CSF Total Protein Determination
■ First flow of CSF may have bacterial contaminants from
- CSF Glucose Determination 2
MICROBIOLOGICAL SCREENING TESTS the skin which is why it is not used for microbiology
- Sample Preparation for Stained Smear 3 ○ Tube #2: Microbiology (Room Temperature)
- Gram Staining ○ Tube #3: Hematology (Cell Count)
- Acid-Fast Staining (Ziehl-Neelsen Method)
- India Ink Staining ■ Last to be aspirated so it is expected or known to not
SEROLOGIC TESTING contain any cells that may interfere with the results
CELL COUNTING IN THE FOLLOWING SAMPLES ● Storage: 2 weeks, except for requests, specimens, and results in
- Clear 4
- Hazy question requiring not discarding the specimen until such issues or
- Turbid concerns are settled.
CSF DIFFERENTIAL COUNT ○ If there is excess CSF, it should be stored (frozen) for further
- Cytocentrifuge
- CSF Cellular Constituents use or additional testing such as for molecular testing
- CSF Differential Counts in Meningitis ○ Practical setting: can be stored longer than 2 weeks
- Differential Diagnosis of Meningitis 5
QUICK GUIDE FOR CSF ANALYSIS (NORMAL VALUES)
CHEMICAL EXAMINATION OF CSF
LEGEND ● Measurement of CSF constituents such as CSF protein and
Based from ppt Based from lecture proper glucose by the use of spectrophotometer
● Protein quantification in CSF
INTRODUCTION TO CSF ANALYSIS ○ Identifies increased permeability of BBB (Blood Brain Barrier)
Materials, Reagent, and Equipment to plasma proteins
○ Detects increased intrathecal secretions of Immunoglobulins
● Compound Microscope ● Glucose Liquicolor Kit ● CSF Glucose:
● Watch Glass ● Total Protein Reagent Kit ○ Used to rule out bacterial meningitis
● Graduated Cylinder ● WBC diluting fluid ○ In cases of bacterial, tubercular, and fungal meningitis, the CSF
● Pasteur pipette ● RBC diluting fluid of glucose may significantly be reduced
● Cuvette ● Centrifuge
○ For viral meningitis, CSF is normal
● Glass slide and coverslip ● Neubauer Counting Chamber
● WBC Thoma pipette ● Centrifuge Tube ● Aside from the IgG index, the cesar serum albumin index, glucose
biospectrophotometric and total protein biospectrophotometric
● Compound microscope: for microscopic examination analysis, is done in our teaching lab
● WBC Thoma pipette: for cell counting ● Electrophoresis and the determination of indices are performed in a
● Glucose Liquicolor Kit: for glucose determination more complexed laboratory like the actual clinical lab
● Total Protein Reagent Kit: for total protein determination ○ Used when patients are suspected of multiple sclerosis
Cerebrospinal Fluid Analysis CSF TOTAL PROTEIN DETERMINATION

● Sterile fluid that is clear and colorless, free from any coagulum
Procedure - Spectrophotometer
and pellicle
1. Label cuvettes “BLANK”, “STANDARD”, “SAMPLE”.
○ Sterile: there should be no contaminants that pass through the
● Blank = Reagent Blank
blood brain barrier that can contaminate the CSF
● Standard = from which we will be able to compute the
■ No microorganisms present
concentration of the sample
■ Chemical analytes should be within the normal range
● Sample = CSF sample or Unknown
○ Pellicle: web-like clots which are particularly present when
2. The reagents must be dispensed to the following cuvettes shown
there is tubercular meningitis
below:
● Contains a carbohydrate-deficient transferrin fraction referred to
● BLANK = 1.0 mL of reagent
as “tau”
● STANDARD = 0.02 mL of Total Protein standard
○ Tau – index to differentiate it from other body fluids
● SAMPLE = 0.02 mL of CSF sample
○ Unique in to the CSF body fluid
3. Pipette 1.0 mL of Total Protein reagent into all vials
● Formed by the brain’s third choroid plexus
4. Mix all the vials by inversion
● Ultrafiltrate of plasma
5. Stand for 10 minutes
○ Similar to the fluid that leaves the glomerulus (ultrafiltrate)
6. Measure the absorbance of the sample and the standard against
○ Not all components of the blood or plasma are seen in the CSF
reagent blank at 520-580 nm
● Functions
7. Compute for the total protein concentration of the sample
○ Supplies nutrients to the nervous tissue
○ Protects the brain and spinal cord against trauma and
pressure change
○ Channel for the removal of waste in and around the CNS
● Specimen collection of CSF is done by performing lumbar
● Au = absorbance of unknown
puncture.
● As = absorbance of the standard
○ There are other ways to collect CSF, but lumbar tap or spinal
● 8,000 mg/dL = concentration of the standard; taken from the
tap is the most common method
reagent insert
○ Ex. Cisternal Tap
● Samples must be placed into a sterile, numbered, screw-capped
Other Known Methods for CSF Protein Determination
tubes.
Total Protein Methods
● Order of draw:
1. Turbidimetric Method
○ Tube #1: Chemistry and Serology (Frozen)
A. Trichloroacetic Acid (TCA)
● precipitates both albumin and globulin
B. Sulfosalicylic Acid (SSA) Myelin Basic Protein

● precipitates albumin only ● Protein component of the lipid-protein complex that insulate the
● to precipitate globulins, add sodium sulfate (Na2SO4) nerve fibers
2. Dye-Binding Method ● Presence of MBP in CSF indicates destruction of myelin sheath
A. Coomassie Brilliant Blue ● Used to monitor the course of MULTIPLE SCLEROSIS
● Protein binds to dye - dye turns from red to blue ● MULTIPLE SCLEROSIS – demyelinating disorder
● ↑ Protein = ↑ Blue color ○ Findings in multiple sclerosis:
Note: ALBUMIN is also the main protein found in the CSF ■ (+) Anti-myelin sheath autoantibody
→ performed using immunofluorescence - similar to
Protein Fractions
fluorescent antinuclear antibody test
1. CSF/Serum Albumin Index
→ use of fluorescent labelled antibodies to detect
● Assess the integrity of BBB
autoantibodies
● CSF albumin (mg/dL); Serum albumin (g/dL)
→ we view the results using the fluorescence
● Normal value = < 9
microscope
● Abnormal = > 9
→ (+) fluorescence = (+) autoantibody
○ Correlates the degree of damage
■ (+) Oligoclonal bands in CSF but not in serum
○ An index of 100 would indicate complete damage of the
■ (+) MBP
BBB
■ ↑ IgG Index
CSF GLUCOSE
2. IgG Index ● Done in conjunction with blood glucose
● Assess conditions with IgG production within the CNS (Ex. ● Specimen for blood glucose should be drawn 2 hours prior to
Multiple sclerosis) spinal tap
● CSF IgG (mg/dL); Serum IgG (g/dL) ● Normal Values = 60-70% of blood glucose (50-80 mg/dL)
● CSF albumin (mg/dL); Serum albumin (g/dL) ○ Ex: if blood glucose is 100mg/dL you would expect the CSF
● Normal value = < 0.70 glucose to be around 65 mg/dL
● Abnormal = > 0.70 ● ↑ CSF Glucose = due to increased plasma glucose
○ Indicative of IgG production within the CNS ● ↓ CSF Glucose = Meningitis (bacterial, tubercular, fungal)
● Normal in Viral meningitis
Procedure
1. Label cuvettes “BLANK”, “STANDARD”, “SAMPLE”.
CSF Electrophoresis 2. The reagents must be dispensed to the following cuvettes shown
● Done in conjunction with serum electrophoresis below:
● The detection of oligoclonal bands in the Gamma region indicates ○ BLANK = 1.0 mL of reagent
immunoglobulin production ○ STANDARD = 0.01 mL of glucose standard
○ Oligoclonal bands represent inflammation within the CNS ○ SAMPLE = 0.01 mL of CSF sample
○ The presence of 2 or more oligoclonal bands in CSF but not 3. Pipette 1.0 mL of Glucose Liquicolor reagent into all vials
in serum is valuable for the diagnosis of MULTIPLE 4. Mix all the vials by inversion
SCLEROSIS 5. Stand for 10 minutes
● Other conditions with oligoclonal banding in CSF but not in serum: 6. Measure the absorbance of the sample and the standard against
1. Encephalitis reagent blank at 500 nm
2. Neurosyphilis 7. Compute for the glucose concentration of the sample
3. Guillain-barre syndrome
4. Neoplastic disorders
● 100 mg/dL is lifted from the package insert of the kit used in this
determination
MICROBIOLOGICAL SCREENING TESTS

● Rapid detection for the presence of microorganisms in the CSF is
very important.
○ CSF should be a sterile fluid and since it cushions the central
nervous system the presence of microorganisms would be
detrimental to it.
● The following tests are useful:
○ Gram staining
■ detects the presence of bacteria
■ can observe the morphology of the organism whether it
is rod shaped or a cocci
■ gram negative diplococci - Neisseria
○ Acid-fast staining
■ rule out tubercular meningitis (caused by mycobacteria
especially Mycobacterium tuberculosis)
■ acid fast bacilli appear reddish rods in the smear
○ India ink staining
■ presumptive test for C. neoformans ● Acid-fast staining (especially in sputum) is no longer recommended
■ especially useful if the patient is immunocompromised by the CDC as a screening test for mycobacterial infection → Gene
(HIV infection) X-pert na especially for CSF infections by mycobacterium
○ Chemical analysis
■ Glucose - very useful in tandem with microbiological Procedure
screening test so that even without the identification of the 1. Stain the smear using Acid-fast staining (Ziehl-Neelsen method)
microorganism, the doctor can already provide treatment ● Stain: Carbolfuchsin
for the patient ● Decolorizer: Acid Alcohol
■ eg. detect bacteria in gram stain with low glucose = ● Counterstain: Methyl blue / Malachite green
bacterial HIV 2. Blot dry and examine under oil immersion objective.
○ Culture of CSF 3. Draw and record the presence of acid-fast organism
■ after it is gram positive, we inject the CSF in the blood
culture bottle directly INDIA INK STAINING
■ Blood culture bottles are not limited for blood samples but 1. Using a sterile loop get some sediment and place it on a glass
also for CSF and other serous fluids slide.
2. Add a drop of India ink, mix completely.
SAMPLE PREPARATION FOR STAINED SMEAR 3. Air dry.
1. Pour 1-2 mL of fresh undiluted spinal fluid coming from Vial #2 into a 4. Examine under the microscope.
conical centrifuge tube. Spin the sample for 10 minutes at 2,500 5. Report for the presence of capsulated organism
RPM. ● (+) C. neoformans = unstained capsule over a black
● Vial #2 - microbiological studies background
● purpose is to sediment or pellet or concentrate microorganisms
that can be detected in the CSF Cryptococcus neoformans
2. Following centrifugation, remove the supernatant fluid with ● Gram stain = “Starburst pattern”
Pasteur pipette and either save at 4⁰C to 6⁰C or freeze just in case ● Lateral Flow Assay (LAF)
additional tests is needed. ○ Rapid method, utilizes a reagent strip coated with monoclonal
● supernatant can be used in serological studies such as the Ab that react with the cryptococcal polysaccharide capsule
detection of antigens and antibodies ○ uses immunochromatographic strips
3. Resuspend the sediment by gently tapping the tip of the centrifuge ■ detection of cryptococcal polysaccharide capsule
tube. ■ solid phase embedded on the strip - monoclonal Ab
4. Prepare two smears:
a. Gram’s staining
b. Acid-fast method
5. Transfer a small amount of the resuspended sediment onto a glass
slide and smear out as if preparing a blood smear. Air-dry and heat
fix.
6. Proceed to Gram staining and Acid-fast staining
GRAM STAINING
1. Stain the smear using Gram’s staining method.
● Stain: Crystal Violet SEROLOGIC TESTING
● Mordant: Gram’s Iodine ● used specifically when the patient is expected to have
● Decolorizer: Acid Alcohol neurosyphilis
● Counterstain: Safranin 1. Venereal Disease Research Laboratories (VDRL)
2. Blot dry and examine under oil immersion objective. ○ Recommended by CDC for the detection of neurosyphilis
3. Draw and record all significant structures observed. 2. Fluorescent treponemal antibody-absorption (FTA-ABS)
● eg. gram positive cocci; gram positive rods ○ Sensitive than VDRL
● agents of bacterial meningitis is associated with certain age group ○ Prone to contamination with blood
○ 1 month old - Streptococcus agalactiae 3. Rapid Plasma Reagin (RPR)
○ 1 mths - 5 yrs old - Haemophilus influenzae ○ Not recommended; less sensitive than VDRL
○ 5 yrs - 29 yrs old - Neisseria meningitidis 4. LAT and ELISA
■ Gram negative diplococci ○ Serologic methods for the detection of bacterial antigens in
○ Infants, elderly, and immunocompromised may be found with CSF
Listeria monocytogenes ○ LAT - Latex Agglutination Tests
○ ELISA - Enzyme-linked Immunosorbent Assay
ACID-FAST STAINING : Ziehl-Neelsen Method
● used to detect Mycobacterium tuberculosis or other mycobacteria CSF CELL COUNTING
that can be present in the CSF ● CSF Vial #3 - used for cell counting
○ CSF will have a pellicle or web-like clot that will form within ○ expected to have the least or no contamination by the blood
12 to 24 hrs after incubation cells introduced during the spinal tap or lumbar tap
● Acid fast bacilli appears thin and red bacilli ● Take note of the following:
● If the CSF bacterial load is low, there is a high chance that it will not ○ Volume
be observed ○ Color
● The basic practice now is to order tests for molecular studies ○ Transparency
(Gene X-pert) in addition to routine microbiology ○ Presence of coagulum or pellicle formation
○ There can be a + Gene X-pert with a -- AFB ● Leukocyte Count (WBC)
○ routinely performed on CSF specimen
○ After this, depending on the number of cells counted, there can Turbid CSF Samples
be a necessity for differential counting because the 1. Draw CSF first up to the 0.5 mark of WBC pipette then diluting fluid
percentages of the cells in CSF may contribute to the doctor’s up to mark 11
diagnosis in correlating the results of the microbiologic and 2. Close the ends of the pipette and shake vigorously, then stand for
chemical analysis of CSF 15-20 minutes
● RBC counts 3. Shake pipette before discarding 2-3 drops and then fill the counting
○ performed ONLY in traumatic tap CSFs or when correction chamber
is needed for leukocyte or protein ○ Dilution: 1:20
○ calculated by a total cell count -- WBC count ■ 1 part CSF : 19 parts WBC diluting fluid
● Clarity of the specimen determines the counting technique that 4. Count the total number of WBC in the 4 large squares. The total
will be utilized number of WBC is then expressed as WBC count per cubic
○ Clear, hazy, and turbid have different counting techniques millimeter
○ A sample that is clear is easier to read than a turbid sample
○ There are additional measures applied for turbid samples
● Any cell count should be performed IMMEDIATELY
○ WBCs and RBCs begin to lyse within 1 hour
○ 40% WBCs disintegrate within 2 hours
○ General rule for CSF samples: It should be STAT especially ● NOTE: If there are a total WBC count of 35/cumm and above,
microbiologic and CSF cell counts make a differential count
■ Release the result of Gram stain immediately to the doctor ○ Using the sediment used in Vial #1 or #2, prepare a smear, air
to determine presence of bacteria dry. The dry smear is then stained with Giemsa without any
○ Results from microbiological screening and molecular chemical fixative. Classify 100 WBC and report in percent.
screenings should be released immediately and given to the ■ Vial #1 is not optimal for use if it is contaminated with cells
physician from the spinal tap
○ Gene-Xpert has a turnaround time of 2 hours and 30 minutes ■ Vial #3 is best to use if it is still available
● Dilutions require at least a calibrated automatic pipette or the use
of Traditional Thoma WBC pipette may be used to employ the said CSF DIFFERENTIAL COUNT
procedure ● Performed on stained smear
Clear CSF Samples ● Specimen should be concentrated (sediment) before smearing by
1. With the use of a non-heparinized capillary tube draw a well mixed using the following methods:
sample and charge it to the Neubauer counting chamber 1. Cytocentrifugation (cytospin)
2. Let it stand for 2-3 minutes. This will allow the cells to settle 2. Centrifugation
3. Count the WBC in the entire ruled area (9 large squares) 3. Sedimentation
4. Report directly the number of cells counted. It is usually expressed 4. Filtration
as WBC per cubic millimeter
Cytocentrifuge
Hazy CSF Samples ● Most commonly used in histopathology
1. Draw WBC diluting fluid up to 1 mark of Thoma WBC pipette and ● Fluid is added to conical chamber
draw CSF sample up to 11 mark ● Cells are forced into a monolayer with a 6mm diameter circle on the
○ Presumably: withdraw CSF sample up to the 1 mark, and slide
withdraw WBC diluting fluid up to the 11 mark (Sir Malana will ○ Good results due to presence of monolayer
still confirm) ● Addition of albumin:
2. Close the ends of the pipette and shake vigorously, then stand for ○ Increases cell yield / recovery
15-20 minutes ○ Decreases cellular distortion
3. Shake pipette before discarding 2-3 drops and then fill the counting ● If there is no cytocentrifuge, since it is not common in the lab,
chamber common centrifugation is employed, or sedimentation or filtration.
4. Count the total number of WBC in the entire areas. The total ○ Most commonly used in routine laboratory: common
number of WBC is then expressed as WBC count per cubic centrifugation
millimeter ○ Cytocentrifuge is performed only when it is available - as it is
better than the ordinary centrifugation
CSF Cellular Constituents: Predominant Cells in CSF
● Predominant: Lymphocytes and Monocytes
● Occasional: Neutrophils
● Correction: (WBC counted/9) x 10 x (10/1) = WBC/cumm ● Adult (70:30 ratio)
○ Will still be clarified - but this formula will be used for the ○ 70% Lymphocytes
meantime ○ 30% Monocytes
○ Dilution factor = 10 ● Neonates (inversed ratio) - 30% lymphocytes / 70% monocytes
■ EXPLANATION: You fill the pipette with CSF up to the 1 ○ Up to 80% monocytes is considered normal
mark, and fill with diluting fluid up to the 11 mark, then ● Pleocytosis
discard 2-3 drops → laman ng bulb is only 10. Hence, the ○ Denotes leukocytosis in CSF
dilution is 1:10 (1 part CSF, 9 parts diluting fluid) ○ Increase in cells in the CSF
● Nice to know: ○ Abnormal condition
○ In Hematology: (WBC count x VCF) = WBC/cumm
■ VCF = 1/(volume of square x number of squares used)
→ Volume = area x depth = 1 x 0.1 = 0.1
CSF Differential Counts in Meningitis

Type of Glucose Protein WBC count WBC Comment
infection differential
Bacterial <40 >100 1,000-10,000 PMN Partially
mg/dL predominate treated
infections
may be
lymphocyte
predominant
Viral Normal 20-100 50-500 PMN on early Decreased
mg/dL onset, glucose is
Lymphocyte characteristic
on late of HSV
infection
Fungal and <50 20-100 50-500 Lymphocyte
mycobacterial mg/dL predominate
● Bacterial meningitis
○ Glucose: Decreased
○ Proteins: >100 mg/dL
■ Huge impact
○ WBC count: significantly increased compared to viral and
fungal
○ Polymorphonuclear cells such as neutrophils are predominant
● Viral meningitis
○ Glucose: Normal
○ Proteins: 20-100 mg/dL
● Fungal and mycobacterial meningitis
○ Glucose: Decreased
○ Proteins: 20-100 mg/dL
Differential Diagnosis of Meningitis
Bacterial Viral Tubercular Fungal
Predominant Neutrophil Lymphocyte Lymphocyte Lymphocyte
Monocyte Monocyte
WBC
Protein Increased Increased Increased Increased
(Damage of BBB)
Glucose Decreased Normal Decreased Decreased
Lactate Increased Normal Increased Increased
Other (+) Gram stain Common agents: Mycobacterium Cryptococcus
(+) Culture Enteroviruses tuberculosis neoformans
Information (+) Limulus lysate Polioviruses (+) AFB stain (+) GS = “starburst
test Echovirus (+) Pellicle/weblike pattern”
Coxsackievirus clot formation after (+) India ink =
12-24 hr capsule
refrigeration (unstained);
background
(Black)
(+) Immunologic
test for C.
neoformans
● Nowadays, molecular studies are very important when it comes to
determining the cause of meningitis, especially if viral
○ Enteroviruses, Polioviruses, Echovirus, and
Coxsackievirus cannot be determined using the routine
methods in detecting bacterial, mycobacteria, and fungal
elements
■ Most useful method: RT-PCR (rapid method)
QUICK GUIDE FOR CSF ANALYSIS NORMAL VALUES

NORMAL VALUES
Volume Adult: 140-170 mL Crystals None present
Neonates: 10-60 mL
Color Colorless Bacteria None present
Clarity Crystal clear Glucose 60-70% that of
plasma glucose
Viscosity None viscous Lactate <25 mg/dL
Also no clots
WBC count Adult: 0-5/cumm Total Protein <15-45 mg/dL
Newborn: 0-30/cumm
Neutrophils 2% of the differential Glutamine 8-18 mg/dL
Lymphocytes 62% of the differential LDH <40 IU/L
Monocytes 36% of the differential Creatine Kinase <5 IU/L
● No crystals and bacteria because it is sterile fluid
MT UNIT 18: Seminal Fluid Analysis Mr. JM M. Luna LAB
2022 MT6328: Analysis of Urine and Body Fluids 3rd Shifting | Medical Technology | University of Santo Tomas
OUTLINE ○ Typical to ovum penetration is the enzyme contained in the

SEMINAL FLUID ANALYSIS acrosomal cap located at the peak of the head
Introduction 1 ■ Encompases approximately half of the head
- Normal Morphology of a Sperm Cell
■ Covers approximately ⅔ of the sperm nucleus (yung red
Seminal Fluid
- Composition na dot sa picture)
- Clinical Significance ○ Anomalies in the head are associated with poor ovum
- Specimen Handling and Collection 2 penetration because the acrosome is essential to ovum
Seminal Fluid Analysis
- Materials, Equipment, and Reagent penetration
Physical Examination of Seminal Fluid 3 ● Long flagellar tail
- Appearance ○ Approx 45um long
- Liquefaction
- Volume and Viscosity ● Midpiece
- pH ○ Approx 7um long
Examination of Motility ○ Sometimes considered as a part of the tail
- Motility Grading 4
Examination of Morphology ○ Attaches the head of the sperm to the tail
- Normal appearance ○ Thickest part of the tail because it is surrounded by
- Abnormal Sperm Morphology mitochondrial sheath that produces the energy required for
Determination of Sperm Cell Concentration 5
- Methods of Counting
tail movement
Test for Sperm Cell Viability 6 ○ Anomalies in the midpiece will affect motility of the sperm
- Supravital Staining
- Other Tests SEMINAL FLUID
Quick Guide 7 ● Fluid where the sperm cells are contained
LEGEND
Composition
Based from ppt Based from lecture proper ● secretions from the following: testes, epididymis, seminal vesicle,
prostate gland, and bulbo-urethral glands
INTRODUCTION
Part Process Involved
● Seminal Fluid or Semen (Left)
○ Fluid in which you can find the cells together with the other Testes Sperm cell (spermatogenesis)
compounds that make up the entire seminal fluid (Left) Epididymis Flagella (maturation)
○ Specimen that is submitted and analysed in the laboratory Ductus deferens Propels sperm
● Sperm Cell or Sperm (Right) (Vas deferens)
○ Sperm is the cell Seminal vesicles Fructose, flavin, and other proteins
○ Individual cells that we look at under the microscope Prostate gland ACP, citric acid, zinc, proteolytic enzymes
● Do not use the terms interchangeably Bulbo-urethral glands Alkaline mucus
Review: Normal Morphology of a Sperm Cell
● Testes
○ Contains the seminiferous tubules for the secretion of sperm
○ There are germ cells for the production of spermatozoa, they
are located in the epithelial cells of the seminiferous tubule
○ The testes hangs externally or outside the body in a sac
called the scrotum
■ This is essential because a temperature lower than the
body is required for optimum sperm development
● Epididymis
○ After spermatogenesis which occurs in the testes, the
immature or the non-motile sperm enters the epididymis
○ The epididymis is where the sperm develops flagella;
becomes flagellated
○ And where they are stored until ejaculation
○ The entire process of spermatogenesis takes about 90 days
● Vas Deferens
○ During ejaculation the sperm cells travel through the vas
deferens, also known as ductus deferens
● Oval shaped head ○ Through which they are propelled to the ejaculatory duct
○ Approx 5um long, 3 um wide where they receive the contribution of the seminal vesicle
IRINEO, Ritzl Mei | TAN, Kashlee Gwyn | TENG, Pamela Joyce | 3I-MT Hustle kahit Hassle 💯 1
● Seminal Vesicle ● Kept at room temp, tested within 1 hour

○ The seminal vesicle produces most of the fluid in the semen or ● Collected by masturbation
the seminal fluid; 60-70% ○ but an alternative is prostatic massage but this will likely
○ The fluid is high in fructose and flavin result to EPS or expressed prostatic secretions
■ Fructose: needed for energy in propelling the sperm ○ Prostatic massage will trigger the entire ejaculation cascade
■ Flavin: responsible for the gray color of the semen therefore releasing semen or sperm in the process
■ Other proteins: responsible for the coagulation of the ○ Normal values will be different, as most values are
ejaculate standardized by self collection
● Prostate Gland ○ Follow masturbation as the method of collection
○ Contributes 20-30% of the seminal fluid ● Sterile container
○ Milky acidic fluid which contains: ● Properly labeled
■ Acid phosphatase ○ Time and date of collection
■ Citric acid ○ Including period of abstinence, time of collection and receipt
■ Zinc ● Should be examined within 1 hour after collection
■ Proteolytic enzymes involved in coagulation and ● May be collected directly or using non lubricant-containing
liquefaction rubber or polyurethane condoms
● Bulbo-urethral glands ○ Ordinary condoms are not acceptable (spermicides)
○ Contributes 5% of the seminal fluid ■ Anti-contraceptive and contains spermicides
○ Alkaline mucus ● Handle the specimen with caution
■ Alkaline because the vaginal vault is acidic, so the ○ Semen is a body fluid with living cells therefore it is a reservoir
alkalinity will help neutralize the acidity of the vagina of infectious diseases. It is considered as a biological hazard.
■ No neutralization: motility will be impaired ○ Always use gloves and other protective equipments in cases of
HIV and Hepatitis
Clinical Significance ● Potential reservoir for HIV and hepatitis virus
● Infertility
○ Most common Cause Result
○ In every couple that is perceived to be infertile or unable to Prolonged abstinence Higher volume
conceive, it it always the male factor that’s first worked up Decreased motility
○ Because it is easier to perform and less costly
First part missing Decreased count
● Post vasectomy Increased pH
○ To determine if the vasectomy is successful Will not liquefy
○ Vasectomy is the process or procedure wherein the vas Last part missing Decreased volume
deferens is ligated or cut Will not clot
○ So the sperm will not be able to travel through the entire male
reproductive system
● Resolving forensic or medico-legal cases
○ Usually the conventional rape
○ But right now, rape is a term that encompasses activities not
involving ejaculation
○ If there is a sperm cell in a urine or a genetically 46XX female
or genetically 46XX child or prepubertal child (male or female)
finding a sperm cell becomes questionable
Specimen Handling and Collection
● Abstinence Invalid specimen as it does not have time and date of collection, period
○ Infertility evaluation - 3 days of abstinence, and time of collection on the label
○ Post vasectomy - none
○ Abstinence prior to analysis is recommended SEMINAL FLUID ANALYSIS
■ This is done to standardize the results
○ On the average it is recommended to abstain for 2-7 days Materials, Equipment, and Reagent
● Compound microscope
■ Prolonging the abstinence will result to higher volume
● Watch glass
and decreased motility
● Graduated cylinder
○ WHO recommends 2-3 samples, 7 days to 3 weeks apart
● Pasteur pipette
with 2 abnormal samples considered significant for infertility
● Timer
testing
● Glass slide and coverslip
○ Complete collection is essential
● Thoma WBC pipette
■ When the first part is missing: testes and other parts of
● Counting chamber
the reproductive tract that is on the initial portion of
● Reagents
spermatogenesis
○ Diluting Fluids
→ Sperm count will decrease
■ 0.5% chlorazene or
→ pH will be increased; since there is no acid
■ 1% formalin in 3% trisodium citrate or 5.0% sodium
secretions from the prostate
bicarbonate or
→ It will not liquify
■ Saline and Chilled water
■ When the last part is missing
■ Eosin stain
→ The volume will be decreased; the volume is
→ So that it is easier to visualize the cells under the
contributed by the seminal vesicle
microscope on the counting chamber
→ The specimen will not clot
○ Gram’s staining
● Collected in a room provided by the laboratory or
■ Crystal violet
■ Gram's Iodine
■ 95% ethyl alcohol or acetone-alcohol
■ Safranin
PHYSICAL EXAMINATION OF SEMINAL FLUID

● Before you do any examination, you have to check the specimen for
culture
● Perform any culture prior to other tests in seminal fluid analysis
● Just like any other body fluids such as urine, CSF, etc you check the
physical appearance of the fluid.
1. Take note of the appearance/ color, volume, odor, viscosity, pH clotted semen that has failed to liquefy
of the specimen and take note liquefaction time.
2. Viscosity can be estimated by observing the formation of droplets Volume and Viscosity
that form when the fluid is expelled with a Pasteur pipette
Volume
● Normal volume – 2 to 5 mL
A normal semen specimen should liquify approximately 30 minutes ○ use a graduated cylinder to measure the volume
after ejaculation ○ Increased volume
■ may be due to prolonged abstinence
Appearance ■ abstinence should only be 2-7 days or approximately in
● Normal color of the semen has a gray-white color, appears average 3 days
translucent, and has a characteristic musty odor → an abstinence of 10 days may cause the rejection of
● Clear - if the sperm concentration is very low the specimen
● Turbid indicates the presence of WBC ○ Decreased volume
● Red - RBC ■ usually associated with infertility, or defect in the
● Yellow function of the seminal vesicles.
○ Urine contamination from urinary pigments, prolonged → Seminal vesicles are the part of the reproductive tract
abstinence, medication that contributes the 60-70% of the seminal fluid
→ Urine is toxic to sperm hence motility will be affected if the ■ May also be due to incomplete collection
specimen is contaminated with urine
Viscosity
● Refers to the consistency of the fluid
● Normal semen should be easily drawn into a pipette
● Rating can be 0 (watery) to 4 (gel-like)
pH
● Should be measured within 1 hour of ejaculation
○ due to the loss of carbon dioxide that occurs
● Normal pH is alkaline (7.2 to 8.0)
○ to neutralize the acidity of the vagina
● Increased pH in infections
● Decreased pH in
○ increased prostatic fluid
Liquefaction
○ ejaculatory duct obstruction
● Fresh semen is clotted
○ poorly developed seminal vesicles
● Liquefies within 30 to 60 minutes after collection
● Analysis cannot begin until liquefaction has occurred
● Failure to liquefy EXAMINATION OF MOTILITY
● Important because a viable sperm but not able to move forward or
○ Deficiency in prostatic enzymes
propel itself, will more likely to not succeed in reaching the ovum or
○ Improper collection
fertilization
● If after two hours and the specimen has not liquefied,
● Not only the sperm appearance, other fluids, and compartments
○ an equal volume of physiologic Dulbecco’s
should be normal because if the sperm does not move, it cannot
phosphate-buffered saline or proteolytic enzymes such as
reach the ovum which is not in the entrance of the vagina
alphachymotrypsin or bromelain may be added to induce
● If you place a moving sperm in the vagina, it will be able to travel
liquefaction and allow the rest of the analysis to be performed.
into the female tract and into the ovum
■ These treatments may affect biochemical tests especially
● Within one hour of collection, check the percentage of motility and
sperm motility and morphology so their use must be
repeat after four hours.
documented
1. The slide is prewarmed to 37ºC.
■ The dilution of semen with bromelain must be accounted
2. Place a drop of seminal fluid on a slide and cover with a cover slip.
for when calculating sperm concentration.
● Wait for the specimen to settle of approximately 1 minute
● Jelly-like granules (gelatinous bodies) may be present in liquefied
● It is important to use a well-mixed liquefied specimen
semen specimens and have no clinical significance.
3. Examine under HPO and report the percentage (%) of motile and
● Mucus strands, if present, may interfere with semen analysis.
non-motile sperm cells.
○ At least 20 fields are evaluated.
○ Describe the motility on a 0-4+ scale
● Alternative method: Examine 200 cells per slide and then count the
percentage of the different motile categories using a manual cell
counter
Motility Grading
GRADE WHO SPERM MOTILITY ACTION
4+ a Rapid, straight line motility
3+ b Slower speed, some lateral movement
2+ b Slow forward progression, noticeable lateral
movement
1+ c Mobile, no forward progression
0 d No movement
● A minimum motility of 50% with a rating of 2 after an hour is

considered normal.
● Normally used in the laboratory
Alternative Sperm Motility Grading Criteria Figure. Most Common Abnormal Variants
● The WHO Laboratory Manual for the Examination and Processing of ● Head, acrosomal cap, neckpiece, and tail
Human Semen (2010) currently recommends a simpler system for ● Amorphous head - no form
grading motility that does not include speed because of the difficulty ● Pinhead - small head
in standardized reporting ● Spermatid - immature sperm, has not acquired a flagella tail;
cannot travel the entire female reproductive tract
DESCRIPTION SPERM MOTILITY ACTION
Progressive Motility (PM) Sperm moving linearly or in a large circle ● Abnormality in the head = ovum penetration impairment; not able
Nonprogressive Motility Sperm moving with an absence of to fertilize properly
(NP) progression ● Abnormality in the midpiece = movement is impaired
Motility (IM) No movement ● Abnormality in the tail = cannot move forward
Computer-Assisted Semen Analysis (CASA) Normal Appearance

● Instrumentation/ automation ● Oval-shaped head
○ May reduce the subjectivity of the analysis ○ Approx 5um long, 3 um wide
○ Can still be operator dependent just like other automation ● Long, flagellar tail
system ● Acrosome cap
● Objective determination of both velocity and trajectory ○ For ovum penetration
● Can also include sperm concentration ○ Encompass half of the head
○ Covers approx. 2/3 of the nucleus
Abnormal Sperm Morphology
EXAMINATION OF MORPHOLOGY
1. Prepare thin smear of seminal fluid.
○ Dry and fix by heat.
○ Stain the smears by Gram’s method.
2. Examine under OIO and count 200 sperm cells, counting both the
normal and abnormal forms. Spermatozoan with double head, hematoxylin-eosin (x1000)
○ Also report any epithelial cells, testicular cells, red blood cells, Example of an abnormal spermatozoa.There is a tail and a neck but
white blood cells, and crystals. there are two heads.
3. Report normal and abnormal forms in percentage
4. Use the illustration provided to describe the morphology of the
sperm cells in a given sample
*Normal values for sperm morphology depend on the method of

examinations used:
Routine Criteria: greater than 50% of normal forms
Kruger’s Strict Criteria: greater than 30% of normal forms
● Kruger’s Strict Criteria requires the use of a stage micrometer or

morphometry Figure. Spermatozoan with amorphous head, hematoxylin-eosin (x1000)
○ At present, evaluation of sperm morphology using this
criteria is not routinely performed in the clinical laboratory
○ However, it is recommended by WHO
○ Is an integral part of the assisted (?) reproductive
technology
Figure. Spermatozoan with amorphous head, hematoxylin-eosin (x1000)
● Mature sperms are counted in the 5 central squares and use the 4
corners and the center square of the RBC squares. Charge and
count on both sides and then settle for 3-5 minutes before
counting. An average of two counts are used. If the counts do not
agree, you should repeat the dilution.
● Take note: Only fully developed cells are counted. You will not
count immature cells.
○ If you observe that there are multiple immature cells that
Figure. Immature spermatozoa interfere with the counting, you count it separately:
■ > 1,000,000 spermatids = disrupted
spermatogenesis
DETERMINATION OF SPERM CELL CONCENTRATION
1. Using a Thoma WBC pipette, draw semen to the 0.5 mark and ● While counting, if there is WBC and the count exceeds
diluting fluid to the 11 mark (Dilution 1:20) 1,000,000/mL, that means that there might be a presence of
● This dilution will immobilize the cells before counting infection.
● Diluting fluid - 0.5% chlorazene or 1% formalin in 3%
Two WBC Counting Squares
trisodium citrate or 5.0% sodium bicarbonate or chilled water
and eosin stain ● Count the cells in the two WBC counting squares (WBC’s) using
2. Shake for 2 minutes. LPO.
3. Discard the first few drops and charge the Neubauer Counting ● The total sperm count per ejaculate is calculated by multiplying the
Chamber. number of sperm cells per mL of the specimen volume.
4. Count the sperm cells
Methods of Counting 𝑆𝑝𝑒𝑟𝑚 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
● Using five central RBC squares
2
● Using two WBC counting squares 2𝑚𝑚 (𝑎𝑟𝑒𝑎) 𝑥 0.1𝑚𝑚 (𝑑𝑒𝑝𝑡ℎ) 𝑥 1/20 (𝑑𝑖𝑙𝑢𝑡𝑖
Five Central RBC Squares
● Count the cells in the five squares (RBC’s) in the large center using Multiply the answer by 1000
HPO.
● The total sperm count per ejaculate is calculated by multiplying the ● 1/20 = 0.05
number of sperm cells per mL of the specimen volume. ● 0.1 mm depth is constant
● 2 mm2 - 2 WBC counting chamber
○ 1mm2 / area
Sperm cells/mL = cells counted x 1,000,000
● 2 x 0.1 x 0.05 = 0.01
● Example: ● Example:
○ Counted 75 cells in total: ○ If you have counted 750 cells
■ Sperm cells/mL = 75,000,000 ■ 750 / 0.01 = 75,000
○ Total number of cells in an ejaculate (submitted to the lab):
■ Multiply the answer by 1000: 75,000 x 1000 = 75,000,000
■ (Number of cells x volume of sample submitted)
■ 75,000,000 x 2 mL = 150,000,000 per 2 mL (total) cells/mL
■ To get the total number of cells submitted to the
laboratory (similar to computation in RBC squares)
= (number of cells / mL)(total volume submitted)
● NOT a routine test

Additional Ex.: Calculating Sperm Concentration & Sperm Count ● Qualitative method
○ Resorcinol test (+) orange
○ Resorcinol will produce an orange color in the sample if there is
presence of fructose
○ Confirms presence of fructose
● Quantitative method
○ Spectrophotometry: Normal is ≥ 13 umol/ejaculate
● Tested within 2 hours to prevent fructolysis
○ Example: Baka naman nag-test ka after 5 hours na, wala na
talagang fructose. Nag-lyse na siya.
Antisperm Antibodies
● Usually performed for post-vasectomy cases
○ Vasectomy - patient undergoes surgery, damaging the vas
deferens and blood-testis barrier
○ Body eventually develops antibodies against sperm
■ May also be due to infection or trauma
■ Sometimes, if there are sperm produced (if any), they will
already be damaged upon production
■ There are times that this may cause antibody production
● NOTE: Area of 1 WBC square = 1mm2 even in the female partner - but it is usually seen in
males
TEST FOR SPERM CELL VIABILITY ● Physical indication: semen or sperm cell samples that are clumped
● Test for living versus dead cells ● Useful in fertility work-up
● Vitality should be assessed within 1 hour of collection ● Agglutination and immunobead test
Supravital Staining ○ Serologic tests used to determine presence of ASA
1. Mix an equal amount of eosin stain and seminal fluid. Microbial Testing
2. Prepare a thin smear from the mixture of eosin and seminal fluid ● 1 M/mL WBC
○ Sometimes, eosin-nigrosin stain is used ○ Possibility of infection
3. Air dry. No washing required. ● Useful in diagnosing infections
4. Examine 100 sperm cells. ● Usual organisms detected in microbial testing for seminal fluid:
5. Get the percentage of sperm cells that are stained and unstained. ○ Chlamydia trachomatis
● PRINCIPLE: Living cells - NOT infiltrated by dye ○ Ureaplasma urealyticum
○ Non viable or non vital cells will be stained ○ Mycoplasma hominis
Chemical Testing
● Detect anomalies in the tract
● Helpful in determining whether a specimen is semen / if semen is
present in the specimen.
○ Also used in medico-legal
■ Disputed rape case
→ May prove that there is a contamination of
ejaculation or sperm in the vaginal vault if a
specimen obtained is presumptive to have sperm or
semen through the use of chemical testing
→ Chemical components of the sperm can prove the
presence of semen in the specimen
Figure. Nonviable spermatozoa demonstrated by the eosin-nigrosin stain ■ Microscopic testing may also be performed
(x1000) → For alleged rape, you will be able to recover motile
● They are already heat fixed, which means all cells are probably or moving sperm cells in the vaginal vault up to 24
dead hours after the ejaculation process
● However the principle is, if they are alive before the staining, they → Up to 3 days after it happened, you will still be able
will not take up the dye to pick up non motile cells
→ Up to 7 days, you will still be able to retrieve the
heads of the cells
Other Tests ⇒ May last up to 10 days, but usually only up to 7
● Seminal Fluid Fructose
days
● Antisperm Antibodies
● How can it detect anomalies in the tract?
● Microbial Testing
○ Chemicals produced by the different parts of the male
● Chemical Testing
reproductive tract
Seminal Fluid Fructose
● Fructose is added to the fluid by the seminal vesicles Defects in prostate gland ↓ zinc
○ So any anomaly prior to the sperm switching to the area Lack of prostatic fluid ↓ citric acid
after the seminal vesicle, for example ejaculatory duct ↓ acid phosphatase
obstruction or the vas deferens, will result in a decreased Disorder in the epididymis ↓ α-glucosidase
seminal fluid fructose ↓ l-carnitine (sometimes)
QUICK GUIDE
PARAMETER NORMAL VALUES

Volume 2-5 mL
Viscosity Pours in droplets
pH 7.2-8.0
Sperm count/mL 20-160 million/mL
Motility >50% within 1 hour
Grade of Motility 2+
Morphology >30% normal forms (Kruger’s Strict Criteria)
>50% normal forms (Routine Criteria)
Sperm Cell Viability >75% unstained
● Volume: >2-5 mL may be due to prolonged abstinence
● Viscosity: pours in droplets when using Pasteur pipette
● pH
○ <7.2-8.0 (acidic): there is an increase in prostatic secretions
○ >7.2-8.0 (alkaline): indicate infection
● Sperm count/mL (not the total volume submitted): 20-160
million/mL
● Sperm cell viability: >75% unstained
○ REMEMBER: unstained = nonviable / dead sperm cells
○ Living cells DO NOT take up the eosin-nigrosin stain
MT UNIT 19: Fecal Analysis with Fecal Occult Blood Test Ms. Ruth Bangaoil LAB
OUTLINE
FECAL ANALYSIS WITH FECAL OCCULT BLOOD TEST
Introduction 1
Fecal Analysis
- Feces
- Specimen Collection
- Types of Fecal Samples
Macroscopic Examination
- Macroscopic Stool Characteristics
Microscopic Examination
- Procedure 2
- Fecal Fat Determination (Quantitative)
- Muscle Fibers
- Fecal Leukocytes
Chemical Testing of Feces 3 Figure. Stool sample container
- Fecal Fat Determination (Quantitative)
- Fecal Occult Blood Test Types of Fecal Samples
- APT Test 4
● Random specimen
- X-ray Film Test
- Fecal Carbohydrates ● Timed specimen (3-day collection)
Diarrhea
- Classifications
Random Specimen
- Secretory Diarrhea
- Osmotic Diarrhea ● Can be collected any time of the day
- Altered Motility Diarrhea 5 ● For qualitative testing
- Common Fecal Tests for Diarrhea
● Leukocytes, muscle fibers, and fecal fats
- Summary of Fecal Screening Tests
LEGEND
Times Specimen (3-day collection)
Based from ppt Based from lecture proper Based from Strasinger 6E ● 3 day collection specimen; 72 hour collection specimen
● For quantitative testing for fecal fats
INTRODUCTION
● Fecal analysis is a routine test done in the laboratory MACROSCOPIC EXAMINATION
● Fecal occult blood is a rapid diagnostic test for the detection of ● Color
colorectal cancer ○ Normal color is brown due to presence of urobilin
○ Not routinely done in the laboratory, performed only when it is ● Consistency
specifically requested by the physician ● Color and consistency is reported in the final result
● The first indication of GI disturbances can often be indicated by
FECAL ANALYSIS the changes in the brown color and formed consistency of the
● Also known as stool analysis or stool exam normal stool.
● An integral part of medical examination on the account that feces is ● The appearance of abnormal fecal color may also be caused by
the end-product of body metabolism ingestion of highly pigmented foods and medications, so a
● Includes macroscopic, microscopic, and chemical analyses for differentiation must be made between this and a possible pathologic
early detection of gastrointestinal (GI) bleeding, liver and biliary duct cause
disorders, malabsorption syndromes, and inflammation
● Should be examined immediately, or within 1 hour from the time Macroscopic Stool Characteristics
Color/Appearance Possible cause
of collection
Upper gastrointestinal bleeding
Feces Iron therapy (supplements)
Black
● It is the solid organic refuse of the body composed of: Charcoal
○ 75-80% Water Bismuth (antacids)
○ 20-25% (Remaining Fraction): Organic Solids Lower gastrointestinal bleeding
● Composition: Red Beets and food coloring
Rifampin
○ Bacteria, cellulose, and other undigested foodstuffs,
Bile-duct obstruction
gastrointestinal secretions, bile pigments, cells from intestinal Pale yellow, white, gray
Barium sulfate
walls, electrolytes, and water Biliverdin/oral antibiotics
● Around 100-200g of stool is passed per day Green
Green vegetables
Specimen Collection Bile-duct obstruction
Bulky / frothy
● Should be collected in a clean bedpan, then transfer an ample Pancreatic disorders
Ribbon-like Intestinal constriction
amount in a screw-capped disposable container
Colitis
○ Similar to specimen collection container for urine, but a spatula
Dysentery
is included Mucus / blood-streaked mucus
Malignancy
● Should not be contaminated with urine or any other body Constipation
secretions
○ Because this may alter the results of the stool exam MICROSCOPIC EXAMINATION
● Properly labelled ● Done to detect presence of leukocyte, ova, cyst, and trophozoite
● NOTE: Containers that contain preservative for ova and of parasite which may be associated with diarrhea
parasites must NOT be used to collect specimens for other tests ● Microscopic screening of fecal smears is performed to detect the
presence of leukocytes associated with microbial diarrhea and
undigested muscle fibers and fats associated with steatorrhea
● Qualitative Fecal Fats
● Muscle Fibers
● Fecal Leukocytes
Procedure Split Fat Stain (Fatty acids)

1. Mark the glass slide on the middle, place drop of NSS on one slide, ● Emulsified stool + 36% acetic acid + Sudan III
1 drop of Lugol’s iodine on the other side ○ 36% acetic acid → orange droplets (fatty acids)
2. Using an applicator stick, place a guava seed size of stool sample
in each solution GRADE DESCRIPTION
3. Make a fecal emulsion; put a cover slip Normal 100 droplets (<4µm)
○ Two coverslips are used; one for the NSS side and another for Slightly Increased 100 droplets (1-8µm)
the Lugol’s iodine side Increased 100 droplets (6-75µm)
4. Examine under the LPO, then shift to HPO
5. Report the structures seen as follows: ● Procedure:
○ Epithelial cells, macrophages, vegetable fibers, muscle 1. Mix emulsified stool with one drop of 36% acetic acid
fibers, vegetable spirals, fungi and yeast cells, and 2. Add two drops saturated Sudan III
bacteria are reported as: 3. Mix and coverslip
GRADE DESCRIPTION 4. Heat gently almost to boiling
(+/-) Occasional 5. Examine under high power
(+-) Few 6. Count and measure the orange droplets per high-power field
(++) Moderate
(+++) Many or abundant II. Muscle Fibers
● Creatorrhea - abnormal excretion of muscle fibers in feces
○ RBCs and WBCs are reported as: ranges / hpf
■ Ex: 0-1/hpf; 1-3/hpf Muscle Fiber Determination
○ Ova, cyst, and trophozoites should be reported with ● Patient should include meat in his diet
complete scientific name and the stage ● Emulsified stool + 10% eosin → put coverslip (stand for 3 min) →
count the number of undigested fibers/hpf
I. Fecal Fat Determination (Qualitative)
● Fats GRADE DESCRIPTION
○ Steatorrhea - increased fat excretion in the stool (>6 g/day) Completely digested No striations
○ Exocrine pancreatic insufficiency (EPI), celiac disease, and Partially digested Striation in 1 direction
tropical sprue
● Tests:
1. Screening test: microscopic exam of feces for fat globules
2. Definitive test: fecal fat determination Undigested Striation in BOTH direction
Abnormal 10 undigested muscle fibers

(Presumptive diagnosis for:
Biliary obstruction; Cystic fibrosis)
III. Fecal Leukocytes

● Primarily neutrophils
● >3 neutrophils/hpf - invasive condition
● Seen in condition that affect the intestinal mucosa such as
ulcerative colitis and bacterial dysentery
● Microscopic screening is performed as a preliminary test to
determine whether diarrhea is being caused by invasive bacterial
pathogens
This figure is graded as +3 for fats ● When the patient has diarrhea but without leukocytes, the physician
requests for stool culture
○ Microbiology specimens should be the first to be analyzed and
Neutral Fat Stain (Triglycerides)
so follow-up tests are not accepted
● Used for Triglyceride
○ Stool culture should be done within 1 hour of collection and
● Main difference
should not be contaminated
○ Split fat stain uses 36% acetic acid
○ Neutral Fat Stain uses 95% ethanol,
Diarrhea WITH leukocytes Diarrhea WITHOUT leukocytes
○ but we use Sudan III for both
Salmonella Toxin-production:
● Stool suspension + 95% ethanol + Sudan III Shigella S. aureus
○ 95% ethanol → orange droplets (neutral fats.triglycerides) Yersinia Vibrio spp
Enterovasive E. coli Viruses (HIV)
≥ 60 droplets / hpf = Steatorrhea Campylobacter Parasites
Fecal Leukocyte Determination
● Procedure: ● Wet preparation:
1. Homogenize one part stool with two parts water ○ stool + Loeffler’s methylene blue
2. Mix emulsified stool with one drop 95% ethyl alcohol on slide ○ Faster, but more difficult to interpret
3. Add two drops saturated Sudan III in 95% ethanol ○ Procedure:
4. Mix and coverslip 1. Place mucus or a drop of liquid stool on a slide
5. Examine under high power ● Disposable plastic pipette is used
6. Count orange droplets per high-power field 2. Add two drops Loffler methylene blue
3. Mix with a wooden applicator stick
4. Allow to stand for 2-3 minutes

𝐹𝑎𝑡𝑡𝑦 𝑙𝑎𝑦𝑒𝑟 𝑙𝑒𝑛𝑔𝑡ℎ 𝑖𝑛 𝑐𝑚
5. Examine for neutrophils under high power
𝐹𝑎𝑡𝑡𝑦 𝑙𝑎𝑦𝑒𝑟 𝑙𝑒𝑛𝑔𝑡ℎ 𝑖𝑛 𝑐𝑖𝑚 + 𝑠𝑜𝑙𝑖𝑑 𝑙𝑎𝑦𝑒𝑟 𝑙𝑒𝑛𝑔𝑡ℎ
x 100
● Dried preparation:
○ stool + Wright’s / Gram stain
○ Provide permanent slides for evaluation Acid steatocrit is reported in percent
■ can be read and checked again
■ we do not report significant findings immediately but refer ● The fecal fat in grams per 24 hours for adults is quantified as
to the pathologist first before reporting follows:
○ Observation of gram (+) or gram (-) bacteria
● Lactoferrin latex agglutination test [0.45 x (acid steatocrit in percent as a whole
○ Lactoferrin = 2º granules of neutrophils number)] - 0.43
○ (+) invasive bacterial pathogen
○ >3 neutrophils/hpf - invasive condition ○ An acid steatocrit value <31% was considered normal
○ A value >31% indicated steatorrhea in adults
CHEMICAL TESTING OF FECES
● Quantitative Fecal Fat Testing
● Occult Blood ● The fecal fat for children up to the age of 15 years is as follows:
○ Guaiac-Based Fecal Occult Blood Test
○ Immunochemical Fecal Occult Blood Test [0.1939 x (acid steatocrit in percent as a whole
○ Porphyrin-Based Fecal Occult Blood Test number)] - 0.2175
● APT Test (Fetal Hemoglobin)
● Fecal Enzymes
○ Acid steatocrit is higher in infants and dropped with age
○ Analysis of feces focuses primarily on the proteolytic enzymes
○ An acid steatocrit of <10% is indicative of steatorrhea in
trypsin, chymotrypsin, and elastase I
children
● Carbohydrates
Fecal Fat Determination (Quantitative) Near-infrared reflectance spectroscopy (NIRA)

● A rapid procedure for fecal fat
Van de Kramer titration ● 48- to 72-hour stool collection to exclude day-to-day variability
● Gold standard for fecal fat determination ● Quantitates water, fat, and nitrogen in grams per 24 hours
● For definitive diagnosis of steatorrhea
● Sample: 3-day stool specimen (72 hrs, store in ref temp) Summary - (slight)
○ Refrigerating the specimen prevents any bacterial degradation
● Titration with NaOH (sodium hydroxide) Tests, Materials, and Instrumentation for Fecal Fat Analysis
○ In the titration method, fecal lipids are converted to fatty acids Procedure Materials, Instrumentation
and titrated to a neutral endpoint with sodium hydroxide.
GRADE DESCRIPTION Sudan III Sudan stain, microscopy
Normal 1g - 6g fats/day Steatocrit and acid steatocrit Hematocrit centrifuge, gravimetric
Steatorrhea > 6g fats/day assay
Fecal elastase-I Immunoassay ELISA technique
Acid steatocrit
● Gravimetric assay Near-infrared reflectance NIRA spectrophotometer
spectroscopy (NIRA) Wavelengths Range 1400-2600 nM
● Rapid test to estimate the amount of fat excretion
Computer software for processing
● Similar to microhematocrit test spectra
● More convenient than 72-hour stool collection
● Reliable to monitor patient’s response to therapy Van de Kamer Fecal fat extraction and titration of long
chain fatty acid by sodium hydroxide
● Screen steatorrhea in pediatrics
● Procedure:
1. 0.5 g of feces from a spot collection is diluted 1 to 4 with Chemical Testing: Fecal Occult Blood Test
● Rapid screening test for the early detection of colorectal cancer
deionized water
● Also known as guaiac test
2. Vortex for 2 minutes to homogenize the specimen
● Normally, a person loses about 0.5-1.5 mL of blood into the
3. A volume of 5 N perchloric acid equal to 20% of the
gastrointestinal tract daily
homogenate volume is added and the mixture is then vortexed
○ In association with shedding of epithelial cells
for 30 seconds. Confirm the pH to be <1
● Significant: > 2.5 mL blood/150g stool
4. Place the acid-homogenate mixture in a 75 microliter plain
○ 150 g of stool - normal amount of excreted stool daily
hematocrit capillary tube. Seal the end with wax.
○ If there is about 2.5 mL of blood in the stool, this is already
5. The capillary tube is centrifuged horizontally at 13,000 rpm for
significant
15 minutes in a microhematocrit centrifuge.
● Sample: central portion of the stool
This separates fat as an upper layer overlying a solid fecal
● Principle: pseudoperoxidase activity of hemoglobin
layer.
6. The length of the fat and solid layers are measured using a
magnifying lens.
7. Calculate the acid steatocrit in percent
8. Calculate the fecal fat in grams per 24 hours ● Chromogens
1. Benzidine - most sensitive
■ Usually not permitted by the FDA because it is
● The acid steatocrit in percent carcinogenic
2. Guaiac - preferred
3. Ortho-toluidine DIARRHEA
● Any alteration in the metabolism or physiologic function of the body
may result in diarrhea
● A change in the usual bowel habit
○ More frequent, looser stools
● An increase in daily stool weight above 200 g with increased
liquidity and frequency of more than three times per day
● Diagnosis of diarrhea starts with a thorough history to characterize
the constitution
○ Is the diarrhea bland (?) or bloody?
○ Other constitutional symptoms
Figure. Positive fecal occult blood test ○ What is the duration of the illness?
● Stool is applied on one side and hydrogen peroxide is dropped on
the other Classifications
● Positive (+): Blue ● Duration
● Negative (-): Colorless ● Mechanism
● Severity
Summary of Occult Blood Testing Interference ● Stool characteristics
False-Positive False-Negative
● Aspirin and anti-inflammatory ● Vitamin C >250 mg/d Duration
medications ● Iron supplements containing ● Acute diarrhea: < 4 weeks
● Red meat vitamin C ● Chronic diarrhea: > 4 weeks
● Horseradish
● Raw broccoli, cauliflower, Mechanism
radishes, turnips ● Secretory
● Melons ● Osmotic
● Menstrual and hemorrhoid ● Altered Motility
contamination ● Tests to differentiate the mechanisms:
● Patient is instructed NOT to eat red meat for 3 days and to ○ Fecal electrolytes (sodium, potassium)
DISCONTINUE aspirin and anti-inflammatory medications ○ Fecal osmolality
○ Instructed by physician ○ Stool pH
Chemical Testing: APT Test (Fetal Hemoglobin)
● Differentiates fetal blood from maternal blood Osmotic Gap
● Specimen: infant stool / vomitus Stool/fecal osmotic gap - measurement of differences in solute types
● NOTE: between serum and feces; used to distinguish among different causes of
○ Hemoglobin F is alkali resistant diarrhea
○ Hemoglobin A is denatured by NaOH Normally, feces is in osmotic equilibrium with the blood serum: between
● Procedure: 290-300 mOsm/kg
○ Emulsified stool → centrifuge → add 1% NaOH to supernatant
● Interpretation of results Osmotic Gap = 290-[2(fecal sodium + fecal potassium)]
○ Pink solution: (+) fetal blood
Osmotic diarrhea: > 50 mOsm/kg
○ Yellow-brown supernatant: (+) maternal blood Secretory diarrhea: < 50 mOsm/kg
Chemical Testing: X-ray film test
● Detects trypsin enzyme Secretory Diarrhea
● (-) trypsin - seen in clinical conditions associated with cystic ● ↑ secretion of water and electrolytes
fibrosis ● ↓ absorption of electrolytes
● Procedure: ● Causes: bacterial, viral, and protozoan infections
○ Emulsify stool + xray film ○ E. coli, Clostridium, Vibrio cholerae, Salmonella, Shigella,
● Interpretation of results Staphylococcus
○ Clearing of film: (+) trypsin ● Other causes:
○ No clearing of film: (-) trypsin ○ Drugs, stimulant laxatives, hormones
○ Inflammatory bowel disease (Crohn disease, ulcerative colitis,
Historical Note: Screening Test for Fecal Trypsin
lymphocytic colitis, diverticulitis)
Historically, absence of trypsin has been screened for by exposing x-ray paper to
stool emulsified in water. When trypsin is present in the stool, it digests the gelatin ○ Endocrine disorders (hyperthyroidism, Zollinger-Ellison
on the paper, leaving a clear area. Inability to digest the gelatin indicates a syndrome, vipoma)
deficiency in trypsin production. The gelatin test is an insensitive procedure that ○ Neoplasms, and collagen vascular disease
detects only severe cases of pancreatic insufficiency. In addition, false-negative
results may occur as the result of intestinal degradation of trypsin and the possible Osmotic Diarrhea
presence of trypsin inhibitors in the feces. The proteolytic activity of bacteria
● Retention of water and electrolytes in the large intestine due to
enzymes may produce false-positive results in old specimens.
incomplete breakdown or reabsorption of food
Chemical Testing: Fecal Carbohydrates ● Causes:
● Most valuable in assessing cases of infant diarrhea (i.e. lactose ○ Maldigestion (impaired food digestion)
intolerance) ○ Malabsorption (impaired nutrient absorption)
1. Clinitest ○ Disaccharidase deficiency (lactose intolerance), poorly
○ Test for reducing sugars absorbed sugars, laxatives, antacids, amebiasis, antibiotics
○ >0.5 g/dL = carbohydrate intolerance
2. pH
○ Normal stool pH = 7.0-8.0
○ CHO disorders = pH < 5.5
Altered Motility Diarrhea

● Enhanced (hypermotility) or slow motility (constipation)
● Causes:
○ Irritable bowel syndrome (IBS) - functional disorder in which
the nerves and muscles of the bowel are extra sensitive,
causing cramping, bloating, flatus, diarrhea, and constipation
○ Rapid (accelerated) gastric emptying (RGE) dumping
syndrome
Common Fecal Tests for Diarrhea
Secretory Osmotic
● Stool cultures ● Microscopic fecal fats
● Ova and parasite examinations ● Muscle fiber detection
● Rotavirus immunoassay ● Qualitative fecal fats
● Fecal leukocytes ● Trypsin screening
● Microscopic fecal fats
● Muscle fiber detection
● Quantitative fecal dats
● Clinitest
● D-xylose tolerance test
● Lactose tolerance test
● Fecal electrolytes
● Stool pH
● Fecal osmolality
Summary of Fecal Screening Tests

Test Methodology/Principle Interpretation
Examination for Microscopic count of Three per high-power field
neutrophils neutrophils in smear indicates condition
stained with methylene affecting intestinal wall
blue, Gram stain, or
Wright’s stain
Qualitative fecal fats Microscopic examination of 60 large orange-red
direct smear stained with droplets indicates
Sudan III malabsorption
Microscopic examination of 100 orange-red droplets

smear heated with acetic measuring 6-75 µm
acid and Sudan III indicates
malabsorption
Occult blood Pseudoperoxidase activity of Blue color indicates
hemoglobin liberates gastrointestinal
oxygen from hydrogen bleeding
peroxide to oxidize guaiac
reagent
APT test Addition of sodium hydroxide Pink color indicates
to hemoglobin-containing presence of fetal blood
emulsion determines
presence of maternal or
fetal blood
Trypsin Emulsified specimen placed Inability to digest gelatin
on x-ray paper determines indicates lack of trypsin
ability to digest gelatin
Clinitest Addition of Clinitest tablet to Reaction of 0.5 g/dL
emulsified stool detects reducing substances
presence of reducing suggests carbohydrate
substances intolerance
MT UNIT 20: Vaginal Secretions Mr. Joemarie Malana LAB
OUTLINE ● Specimens for T. vaginalis should be processed

VAGINAL SECRETIONS 1 within 2 hours
- Vaginal Secretions Analysis ○ Refrigerate
- Specimen Collection and Transport
■ Chlamydia trachomatis
- Color and Appearance
DIAGNOSTIC TESTS ■ Herpes simplex virus
- pH Test
- Microscopic Procedures 3 Color and Appearance
OTHER DIAGNOSTIC TESTS
- Culture
- POCT Normal findings in vaginal secretions
- Fetal Fibronectin Test PARAMETER DESCRIPTION
- Amnisure Test
VAGINAL DISORDERS 3 Appearance White, flocculent discharge
- Bacterial Vaginosis pH 3.8 to 4.2
- Trichomoniasis
- Desquamative Inflammatory Vaginitis Amine (Whiff) test Negative
- Atrophic Vaginitis WBCs 2+
LEGEND Lactobacilli Predominant
BLACK TEXT COLORED TEXT Clue Cells Absent
Other cells Absent (except RBCs during menses)
Other Organisms Other lactobacilli subgroups, occasional yeast
AUBF LAB Vaginal Discharge Lecture Video
Abnormal findings in vaginal secretions
COLOR of DISCHARGE SIGNIFICANCE
Thin, homogenous white-to-gray discharge Bacterial vaginosis
VAGINAL SECRETIONS
White “cottage-cheese” like discharge Candida infections
Vaginal Secretions Analysis
Increased yellow-green frothy, adherent
● Indications Trichomonas vaginalis
discharge
○ Diagnosis of infection
■ Vaginitis or inflammation of vagina is one of the most Yellow, opaque cervical discharge Chlamydia trachomatis
common conditions in females of childbearing age.
■ The causes would include bacterial vaginosis (BV), DIAGNOSTIC TESTS
vulvovaginal candidiasis, or trichomoniasis A. pH Test
○ Determination of pregnancy ● normal pH is around 3.8 to 4.5 due to glycogen metabolism of
■ in determining ruptured fetal membranes or the Lactobacilli forming lactic acid
assessment of risks of preterm delivery ○ Acidic pH suppresses overgrowth of infectious organism
○ Forensic testing ○ Some Lactobacilli also produce hydrogen peroxide which is
■ done in cases of sexual assault toxic to pathogens hence serves as protection from urogenital
infections
Specimen Collection and Transport ● done during pelvic examination and before swab is placed in saline
1. A speculum moistened with warm water is used to visualize the or KOH solution
vaginal fornices ● uses pH test paper that can evaluate pH values in 4.5 range
● Lubricants should NOT be used as it may contain ● used to differentiate causes of vaginitis
antibacterial agents especially if you are to perform
microbiological examinations for the specimen. B. Microscopic Procedures
2. Swab the vaginal walls and pool to collect epithelial cells along
with the secretions using one or more sterile, polyester-tipped 1. Wet Mount
swabs on plastic shaft or manufacturer-designed swabs ● use of saline on a slide and cover slip
● Cotton swabs should NOT be used because cotton is toxic to ● LPO (10x)
Neisseria gonorrhoeae ○ For scanning of even distribution of cellular components, types
● Wood in wooden shaft may be toxic to Chlamydia and number of epithelial cells, clumping of epithelial cells, and
trachomatis the presence of budding yeast and hyphae
● Calcium alginate inactivates Herpes simplex virus (HSV) for ● HPO (40x)
viral culture ○ Counting of organisms and cells
3. Gross examination (including pH) is performed immediately Quantification Scheme for Microscopic Examination
4. Put a swab sample in slide with a drop of 10% KOH Rare Less than 10 organisms or cells/ slide
5. Place the swab (different from 4) in a tube containing 0.5 to 1 mL of 1+ Less than 1 organism or cell/ HPF
NSS 2+ 1-5 organisms or cells/ HPF
6. Transport properly labeled specimens in a biohazard bag to the 3+ 6-30 organisms or cells/ HPF
laboratory 4+ >30 organisms or cells/ HPF
● Based on the laboratory setting, vaginal swab specimens are COMMON CELLS AND ORGANISMS
collected by the obstetrician-gynecologists
○ Even female medical technologists are not allowed to collect ● Squamous ● RBC ● Bacteria
the swab because the procedure is a delicate one Epithelial Cells ● Parabasal Cells ● Trichomonas
● Analysis should be done immediately, however in case of delay, ● Clue cells ● Basal Cells vaginalis
storage depends on the pathogen of interest ● WBC ● Yeast Cells
○ Room temperature
■ recovery of Neisseria gonorrhoeae
💯
■ preserve the motility of Trichomonas vaginalis
IRINEO, Ritzl Mei | TAN, Kashlee Gwyn | TENG, Pamela Joyce | 3I-MT Hustle kahit Hassle 1
Squamous Epithelial Cell Bacteria

● Exhibit polygonal a. Lactobacillus species
“flagstone” appearance ○ Absent or decreased
with prominent centrally numbers of lactobacilli
located nucleus relative to the number of
● Originates from linings of epithelial cells may
vagina and urethra indicate alteration of
normal flora
b. Anaerobic Streptococci
c. Diphtheroids
d. Coagulase-negative
Clue Cells Staphylococci
● squamous epithelial cells e. Alpha-hemolytic Streptococci
bombarded with
Gardnerella vaginalis Trichomonas vaginalis
● appears granular, irregular ● Oval shaped protozoan with 4 anterior flagella and undulating
described as “shaggy” membrane that covers half of body
appearance ● Exhibits “jerky” motility on wet mount
● Causes strawberry cervix
● Quickly loses viability after collection, hence needs to be examined
within 2 hours
White Blood Cells Yeast Cells

● Normally present in rare to ● Candida albicans and non-Candida species:
scanty numbers ○ most common fungal infections but may be part of normal flora
● Presence in more than 3+ ● May appear as budding cells (see figure) or as hyphae
may indicate vaginitis
2. Gram Stain
● Considered as gold standard
in determining cause of
vaginitis
● We use the Nugent’s gram
stain criteria to diagnose
Red Blood Cells bacterial vaginosis
● Can appear distorted in ○ The types of bacterial
secretions morphophytes are
● Not usually present but evaluated and scored, for
may appear in cases of example:
menstruation or ■ Lactobacillus morphophytes are the predominant bacteria
desquamative in normal vaginal flora. Therefore, if 4+ Lactobacillus
inflammation processes morphophytes are present in the gram stain
● May be confused with ■ And Garderella and Bacteroides spp. Morphophytes (0)
yeast cells ■ And curved gram-variable rods are absent
■ The score is 0
Parabasal Cells ○ Nuget’s Scoring interpretation:
● round to oval cells with ■ 0-3: Normal vaginal flora
marked basophilic ■ 4-6: Intermediate
granulation or amorphic ■ 7 or more: Diagnostic of bacterial vaginosis
basophilic structures (blue
Nugent’s Gram Stain Criteria to Diagnose Bacterial Vaginosis
blobs)
● has nucleus to cytoplasmic Gardnerella and Curved
Lactobacillus Bacterois spp. Gram-Variable rods Points
ratio of 1:1 to 1:2
Morphophytes (Mobiluncus spp.)
● located in luminal
4+ 0 0 0
squamous epithelium of
3+ 1+ 1+ or 2+ 1
vaginal mucosa
● rare to find but increased presence indicates desquamative 2+ 2+ 3+ or 4+ 2
inflammatory vaginitis 1+ 3+ 3
0 4+ 4
Basal Cells 3. Amine (Whiff) Test
● Located deep in the basal layer of vaginal stratified epithelium ● Principle: increased numbers of anaerobic bacteria produce volatile
● Rounded cells with nuclear to cytoplasmic ratio of 1:2 polyamines that are released in vaginal secretions and produce
● Not normally found but if present and accompanied by WBCs may “fishy” odor when mixed with KOH
indicate desquamative inflammatory vaginitis ● Positive result indicates bacterial vaginosis caused by G.
vaginalis in conjunction with Mobiluncus spp. and T. vaginalis
4. KOH Preparation ○ Indicates fetal membrane rupture when seen at high levels
● Done by adding 10% KOH ● Used to determine rupture of fetal membranes
● Used to detect presence of fungal infection
● 10% glycerin may be added to preserve slide VAGINAL DISORDERS
● KOH preparation is usually performed after the Amine (Whiff) Test 1. Bacterial Vaginosis (BV)
○ So after the amine test, just place a cover unto the preparation 2. Trichomoniasis
and that will already be the KOH slide 3. Desquamative Inflammatory vaginitis (DIV)
○ This is because the Amine test is performed by mixing the 4. Atrophic Vaginitis
secretion with KOH. So whiff, then put the cover slip.
○ To preserve the slide, simply put 10% glycerin Bacterial Vaginosis (BV)
● Associated with new or multiple sex partners, frequent douching,
OTHER DIAGNOSTIC TESTS use of intrauterine devices, pregnancy and lack of protective
lactobacilli
A. Culture
● Treatment: metronidazole or clindamycin cream
● gold standard test in detecting yeast and Trichomonas
● time consuming and tedious Amsel’s Diagnostic Criteria
● Diamond’s medium is a special culture medium used for ● Thin, white, homogeneous discharge
Trichomonas vaginalis ● Vaginal fluid pH greater than 4.5
● We also have a culture pouch system for the detection of ● Positive amine test
Trichomonas ● Presence of clue cells
○ Using this culture pouch system, specimen is inoculated into ● 3 out of 4 criteria present indicates positivity to bacterial vaginosis
the pouch within 30 minutes of collection
○ And then incubated for 5 days at 37C in CO2 atmosphere Trichomoniasis
○ Then the pouch is examined microscopically for motile ● Caused by parasitic protozoan Trichomonas vaginalis
Trichomonas. ● Transmission: sexual intercourse
● Causes vaginitis in women and sometimes, urethritis in men
B. POCT ● Most men are asymptomatic carriers
1. OSOM Trichomonas Rapid Test (Genzyme Diagnostics, Cambridge, MA)
● immunochromatographic test for detection of T. vaginalis antigen in Desquamative Inflammatory Vaginitis (DIV)
● Syndrome characterized by profuse purulent vaginal discharge,
vaginal secretions in 10 mins
vaginal erythema, and dyspareunia
2. OSOM BVBLUE (Genzyme Diagnostics, Cambridge, MA)
Atrophic Vaginitis
● Detects vaginal fluid sialidase
● Syndrome found in menopausal women
○ Sialidase is an enzyme associated with bacterial vaginosis
● Caused by thinning of the vaginal mucosa due to reduced
cause by Giardirella, Bacteroides, and Mobiluncus
estrogen production and decreased glycogen production
● Positive: blue or green color change
● Vaginal environment changes and the balance of normal flora is
● Negative: Yellow color
altered
3. VS-Sense Pro Swab and FEMEXAM pH

● Measures elevated vaginal pH to determine bacterial vaginosis
and Trichomoniasis
4. Amines TestCard
● Amine test system for determination of bacterial vaginosis
C. Fetal Fibronectin Test

● Fetal Fibronectin: adhesive glycoprotein in the extracellular matrix
at the maternal and fetal interface within the uterus
● Elevates during the first 24 weeks of gestation but then
diminishes
● persistence after 24 weeks up to 34 weeks of gestation is
associated with pre-term delivery
● Methods:
a. Enzymatic (Solid-Phase ELISA)
■ The vaginal sample is incubated with FDC-6, a
monoclonal antibody specific for fFN, and the presence or
absence of the fFN is determined spectrophotometrically
at a wavelength of 550 nm
b. Rapid fFN assay (Lateral flow, solid-phase ICT or
immunochromatographic test)
D. AmniSure Test
● Qualitative rapid test that uses immunochromatographic device (ICT
kit)
● Detects presence of high levels of PAMG-1 (Placental alpha-1
microglobulin)
Clinical Features and Laboratory FIndings in Vaginitis

Desquamative
Findings Bacterial Vaginosis Candidiasis Trichomoniasis Atrophic Vaginitis
Inflammatory Vaginitis
Appearance Thin, homogenous, White, curd-like vaginal Yellow-green frothy Excessive purulent Excessive purulent
white-to-gray vaginal discharge adherent vaginal vaginal discharge, vaginal vaginal discharge,
discharge discharge increased in erythema vaginal erythema
volume
pH >4.5 3.8 to 4.5 >4.5 >4.5 >4.5
WBCs Rare or absent 3+ or 4+ 2+ to 4+ 3+ to 4+ 3+ to 4+
Lactobacilli Rare or absent Present Absent or present Absent or reduced Decreased
Clue Cells >20% Absent Absent or present
Other Cells Large clumps of Occasional parabasal or Occasional parabasal or
epithelial cells basal cells basal cells
>1+ RBCs >1+ RBCs
Amine Positive Negative Positive Negative Negative
(Whiff) test
Other tests Confirmatory tests: Confirmatory tests: Confirmatory tests:
DNA probe, proline DNA probe, OSOM DNA probe or culture,
aminopeptidase, OSOM BVBLUE Rapid Test OSOM Trichomonas
BVBLUE Rapid Test Rapid Test
MT UNIT 21: Bronchoalveolar Lavage Mr. Joemarie Malana LAB
OUTLINE ● Transport and Specimen Handling

BRONCHOALVEOLAR LAVAGE 1 ○ Keep specimens at room temperature during transport to the
- Fluid Color laboratory and process them immediately.
- White and Red Blood Cell Counts
○ When delivery to the laboratory is delayed for longer than 30
- Leukocytes
- Erythrocytes minutes (eg. specimen is coming from other institutions or
- Epithelial Cells labs/ sent out to bigger laboratories)
- Fungi, Viruses, Bacteria ■ transport the specimens on ice (4°C)
- Cytology
○ Specimens that will not be analyzed immediately should be
LEGEND
■ centrifuged
Based from ppt Based from lecture proper Based from Strasinger 6E ■ the cells resuspended in a nutrient-supplemented
medium, and refrigerated at 4°C for up to 24 hours.
○ Specimens are UNACCEPTABLE for testing after 24 hours.
AUBF LAB Bronchoalveolar Lavage Lecture Video ○ Cell counts
■ should be performed within 1 hour
■ are stable for up to 3 hours if the fluid is in a
nutrient-supplemented medium.
BRONCHOALVEOLAR LAVAGE
○ Samples can be filtered through loose gauze (50 to 70 µm
● Method for obtaining cellular, immunologic, and microbiologic
nylon filter) to remove mucus, phlegm, and dust.
information from the lower respiratory tract
● Diagnostic tests on BAL fluid
● particularly useful in evaluating:
○ include a cell count with differential count
○ Immunocompromised patients
○ microbiologic studies (culture and gram stain)
○ Interstitial lung disease
○ cytopathology especially if malignancy is expected.
■ infectious, non-infectious, Immunologic, or malignant
● Macroscopic observation
○ Airway diseases
○ is recorded describing the color and clarity of the specimen.
○ Suspected alveolar hemorrhage
● The appearance of the BAL fluid can provide valuable diagnostic
○ Pulmonary alveolar proteinosis
information.
○ Langerhans cell histiocytosis
○ BAL fluid color can be clear (colorless), milky white, light
○ Dust exposure
brown-beige, and red.
● Often used in conjunction with high-resolution computerized
○ BAL fluid clarity may be described as clear, hazy, cloudy, or
tomography (HRCT), medical history, and physical examination to
turbid.
determine the need for a surgical biopsy
● Fiber-optic bronchoscope is guided into a selected
Fluid Color
bronchopulmonary segment (right middle or lingular lobe)
● Bloody
○ Done during bronchoscopy
○ with increasing intensities during the sequential aliquots
○ Target areas are better defined using HRCT before the
○ indicates acute diffuse alveolar hemorrhage
procedure.
● Orange-red
Bronchoalveolar Lavage ○ result of an older hemorrhagic syndrome
Optimal targets Areas of alveolar ground glass opacity, ○ evaluated for intracellular iron content by cytochemistry
more prominent nodular profusion, ● Milky or light brown-beige
fine reticulation ○ accumulation of phospholipid-protein complexes derived
Sterile NSS Instilled into the alveolar spaces through from pulmonary surfactant in the lung alveoli
the bronchoscope to mix with the ○ strongly suggests pulmonary alveolar proteinosis
bronchial contents and are aspirated ○ The BAL fluid should be centrifuged if it looks milky. The
for cellular examination and culture presence of clots should be noted.
Instillation volume between 100 and 300 mL of sterile ○ The fluid volume of the BAL should also be measured.
saline in 20- to 50-mL aliquots
White and Red Blood Cell Counts
Transport temperature RT or 4ºC
● May be performed with dilution to facilitate counting using a
Diagnostic tests Cell count with differential, microbiologic hemocytometer
studies, and cytopathology ○ If cell concentration is less than the automated instrument’s
Macroscopic observation Color and clarity of the specimen linearity specifications, hemocytometer should be used
● Cell viability - determined by adding Trypan blue
Color Clear (colorless), milky white, light
● Counts may be automated, as long as the analyzer to be used is
brown-beige, red
validated for the cell counting employing the BAL specimen
Clarity Clear, hazy, cloudy, or turbid ○ There is no specific cell counter or analyzer that is solely used
for cell counting in the BAL
● Instillation volume ○ In actual practice, we don’t automate cell counting of serous
○ The segment lavaged should be recorded on the requisition fluids, and the hemocytometer is still used
form ■ This is because in validation studies there is a
○ The first aliquot is discarded then, the remaining aliquots are discrepancy when you use the automated cell counter
either sent individually for analysis or pooled for further analysis
○ The desired fluid volume for analysis is 10 to 20mL (minimal
volume is 5mL)
○ Optimal sampling retrieves greater than 30% with a typical
recovery range of 50% to 70%.
○ Low volume recovery (less than 25%) caused by fluid
retention in the lung may appear in chronic obstructive lung
diseases (COPD) and should be noted on the requisition form.
Leukocytes Fungal, Viruses, and Bacteria

● Evaluating the predominant inflammatory cellular pattern, provides ● We can also do microbiologic studies using the bronchoalveolar
valuable information to the clinician in determining a differential lavage specimen, implying that we can test for determination of
diagnosis fungi, viruses, and bacteria
○ The morphologic appearance of cells and particles such as the ● Organisms identified include:
morphology of macrophages in extrinsic allergic alveolitis ○ Pneumocystis carinii
and sarcoidosis ■ No longer called as such in the hospital
○ Or the detection of dust particles in occupational exposure ■ Now called as Pneumocystis jirovecii
conditions can provide diagnostic information ■ Due to lack of virology lab or highly specialized lab to
● Differential slides: prepared by cytocentrifugation using routine culture the virus (in our routine lab): Normally processed
procedures using molecular methodologies such as PCR
● Stain: Wright-Giemsa or May Grunwald-Giemsa ○ Toxoplasma gondii
● At least 300 cells but often 500 to 1000 cells are counted and ○ Strongyloides stercoralis
classified ■ Parasite; especially on its erratic migration
● Cells seen in BAL fluid: ○ Legionella pneumophila
○ Macrophages ○ Cryptococcus neoformans
○ Lymphocytes ■ Specifically attributed or normally associated with
○ Ratio of CD4+ and CD8+ lymphocytes (CD4/CD8 ratio) immunocompromised patients such as HIV patients
■ For the CD4+ and CD8+ lymphocytes, a special procedure ○ Histoplasma capsulatum
must be performed which is immunophenotyping using ○ Mycobacterium tuberculosis
flow cytometry ○ Mycoplasma pneumoniae
■ So we stain the cells with antibodies against the CD4 and ■ Causes atypical pneumonia
CD8 markers of the lymphocytes ○ Influenza A and B viruses
○ Neutrophils ■ Due to lack of virology lab or highly specialized lab to
○ Eosinophils culture the virus (in our routine lab): Normally processed
○ Ciliated columnar bronchial epithelial cells using molecular methodologies such as PCR
○ Squamous epithelial cells ○ Respiratory syncytial virus
● Quantitative or semiquantitative cultures: useful for
ventilator-associated pneumonia (VAP) and can diagnose the
infection if the organism is identified
○ Ventilator-associated pneumonia (VAP) - group of hospital /
healthcare associated infection or nosocomial infection
○ With the increasing concern about nosocomial infections and
antibiotic-resistant microorganisms, BAL is more frequently
performed on ventilator-assisted patients to detect infection and
monitor antibiotic therapy
Figure. Bronchoalveolar lavage: Normal macrophages and lymphocytes (x1000)
Erythrocytes
● Erythrocytes: indicates an acute alveolar hemorrhage
● Phagocytosed erythrocytes:
○ an alveolar hemorrhage has occurred within the past 48
hours
○ Phagocytosed by macrophages
● Hemosiderin-laden macrophages:
○ indicate an alveolar hemorrhage older than 48 hours Figure. Bronchoalveolar lavage: Figure. Bronchoalveolar lavage:
Amorphous material associated with P. Characteristic cup-shaped organisms
Epithelial Cells carinii when examined under low power indicating P. carinii (x1000)
● Ciliated columnar bronchial epithelial cells (x100)
● For bronchoalveolar lavage specimens, it is expected that these
ciliated columnar bronchial epithelial cells are lesser as compared to Cytology
the bronchial wash specimens because of the more vigorous ● Observing sulfur granules, hemosiderin-laden macrophages,
washing technique Langerhans cells, cytomegalic cells
○ Bronchoalveolar lavage specimens < Bronchial wash ● Oil Red O: fat droplets seen in fat embolism
specimens ● Sudan III: lipid-laden alveolar macrophages
● Lavage specimen: 4% to 17% ● Periodic acid Schiff staining or Oil Red O: pulmonary alveolar
proteinosis or aspiration
● Dust particle inclusions: pneumoconioses or asbestos exposure
● Malignancy: evaluation by a pathologist
○ If malignancy in the pulmonary system is suspected, there
should be a careful evaluation done by a pathologist on the
slides prepared by the medical technologist
Figure. Bronchoalveolar lavage: Ciliated bronchial epithelial cells; notice the

eosinophilic bar (x1000)
MT UNIT 22: Urinalysis Automation Mr. Joemarie Malana LAB
OUTLINE surface of the pad or by using a light emitting diode (LED) to

URINALYSIS AUTOMATION 1 provide a specific wavelength needed for each test pad color
- Goal of Urinalysis Automation reaction
- Advantages of Urinalysis Automation ○ The light is reflected to a photodetector and then analogue or
- Measurement Technology Methods in Automated Urinalysis
digital converter
- Types of Urinalysis Automation
○ The instrument compare the amount of light reflection with that
LEGEND of known concentrations then display or print concentration
Based from ppt Based from lecture proper Based from Strasinger 6E units or transmit data to the laboratory information system or
LIS
● We also have other principles employed such as the Light
transmission or light scatter refractive index, turbidimetry, and later
AUBF LAB Urinalysis Automation Lecture Video on flow cytometry specially for sediment analysis
Types of Urinalysis Automation

URINALYSIS AUTOMATION ● Semiautomated Chemistry Analyzers:
○ depend on an operator for specimen mixing, test strip dipping,
Goal of Urinalysis Automation
and microscopic results input
● Improve reproducibility and color discrimination
● Fully Automated Chemistry Analyzers:
○ manual urinalysis is prone to operator or technologist
○ tubes of urine are placed on a rack or carousel and moved
subjectivity
automatically through the instrument
● Increasing productivity and standardization for reporting
○ there is no need to manually dip the strips in the urine tubes
urinalysis results (since subjectivity is eliminated)
● Automated Urine Cell Analyzers:
○ do more in a shorter period of time
○ mix, aspirate, dilute, and stain urine to classify urine
sediment particles
Advantages of Urinalysis Automation
● Standardize sample processing (analysis of test strips or rating of ○ analyzers used for microscopic analysis or sediment analysis
test strips, urine sediment analysis including the identification of ● Automated Urine Systems:
cells) ○ perform a complete urinalysis that includes the physical,
● Report results with consistent quality and reduced hands-on time chemical, and microscopic parts of a routine urinalysis
● Instruments are user-friendly and include visual and audio prompts ○ most complete
for operation
○ user-friendly - can be plug and play with minimal operator or Semi-Automated Urine Chemistry Analyzers
technologist intervention needed ● Test for chemical components of urine
● Online computer capability with LIS (laboratory information system) ● The instruments read and interpret the reagent strip results
interface consistently
● Bar coding ○ thereby standardizing the interpretation of reagent strip results
● Manual entry of color, clarity, and microscopic results to be included ○ and eliminating personal or technologist color bias and
on the printed report timing discrepancies
● Flagging abnormal results ● Depending on the instrument and reagent strip used, the following
○ thereby triggering manual verification by the medical Tests can be performed:
technologist ○ Leukocyte ○ Urobilinogen
● Storing patient and control results ○ Nitrite ○ pH
○ we can review archives or backtrack patient results and control ○ Protein ○ Specific gravity
data when there is a need ○ Blood ○ Color
● Minimal calibration, cleaning, and maintenance ○ Glucose ○ Creatinine
○ Ketone ○ Protein-to-creatinine ratio
Measurement Technology Methods in Automated Urinalysis ○ Bilirubin
● Well-suited for small- and medium-volume laboratories and
Urine Measurement Technology
physician’s offices
Manufacturer Color Clarity Specific Gravity
○ But, In the Philippines, since we are bound to comply with the
ARKRAY, Inc Photometry Light scatter Refractive index provisions of the clinical laboratory law and its implementing
Iris Diagnostics Light Light Refractive index rules and regulations, diagnostic testing like urinalysis
transmission/light transmission/light
should only be done in DOH licensed clinical laboratories.
scatter scatter
Thus, it should not be done in physician’s offices
Roche Reflectance Turbidity Refractometry
Diagnostics photometry ● Self-calibrating
● Perform automatic checks (Auto-Checks)
Siemens Reflectance Light Refractive index
Healthcare photometry transmission/light ○ to identify strip type and humidity exposure
Diagnostics, Inc. scatter ● For semi-automated instruments:
● The principles of testing depends on the manufacturer offering the ○ The reagent strip is manually dipped and placed on the strip
automated platform reader
● eg. Roche Diagnostics color identification is done through the use of ○ The reaction pads are read at the correct time
the principle of reflectance photometry ○ and the strip is moved to the waste container
● Reflectance photometry ○ The results are displayed, printed, or transmitted to an LIS
○ principle: light reflection from the test pads decreases in and patient identification and specimen color and clarity may be
proportion to the intensity of the color produced by the entered manually or a barcode reader can be used to identify
concentration of the test substance samples
○ monochromatic light source is directed toward the reagent pads ○ Positive results are flagged to indicate a patient sample that
by placing a filter between the light source and the reflective requires additional confirmation testing
■ Example: microscopic evaluation in high result for blood Fully Automated Chemistry Instruments
Figure. Clinitek Advantus Machine Manufacturer
semi-automated urine chemistry Clinitek Atlas Siemens Healthcare Diagnostic, Inc.
analyzer Urisys 2400 system Roche Diagnostics
Aution Max AX-4030 U.S. ARKRAY
IChem Velocity Iris Diagnostics
Figure. Clinitek Atlas automated

urine chemistry analyzer by
Siemens
- Has a carousel that contains
● The semi-automated instrument requires the operator to: more than one urine tubes,
1. Dip the reagent strip into a well-mixed urine sample so that you can perform
2. Blot the strip to remove excess urine chemistry testing with a
3. Place the strip onto the reagent strip platform simultaneous loading of
4. Press the analyze/enter button several urine samples
Automated Urine Microscopy Analyzers / Cell Analyzers

Semiautomated Chemistry Instruments
● Provide efficient standardized results in less than 1 minute as
Machine Manufacturer compared with approximately 6 minutes using the manual method,
Clinitek Advantus Siemens Healthcare Diagnostic, Inc. markedly improving turnaround times
Clinitek Status Siemens Healthcare Diagnostic, Inc. ○ Only samples that are flagged by the machine with
Urisys 1800 system Roche Diagnostics abnormalities will undergo microscopic analysis
COBAS u411 Roche Diagnostics Automated Microscopy

DiaScreen 50 U.S. ARKRAY Machine Manufacturer
iChem 100 Iris Diagnostics UF-1000i Urine Cell Analyzer Sysmex Corporation
iQ 200 Automated Urine Microscopy Iris Diagnostics
Fully Automated Urine Chemistry Analyzers
Urine Analyzer (iQ 200 Sprint) Iris Diagnostics
● Designed for a high-volume urinalysis laboratory with user
walk-away capability ● Sysmex UF-1000i
○ Walk-away capability means is a system that decreases the ○ Uses laser-based flow cytometry that measures forward light
need for manual intervention scatter, side scatter, fluorescence staining characteristics,
○ After loading the sample, you can already do other jobs and adaptive cluster analysis to identify stained urine
● Can load many samples on a carousel or a rack at one time with sediment particles
the capability to insert a STAT specimen during the run
● Color change for the pads of chemical parameters: reflectance
photometry
○ Tests are measured by the dry pad test using reflectance
photometry to detect color change and taking readings at the
appropriate time and wavelength
○ For each specific test, analytes measured vary by the
Figure. Sysmex UF1000i urine chemistry analyzer
instrument and may include:
■ Leukocytes ■ pH
■ Ketones ■ Bilirubin
■ Protein ■ Color
■ Glucose ■ Clarity
■ Nitrite ■ Creatinine
■ Blood ■ Protein low
■ Urobilinogen
● Urine color: reflectance photometry or spectrophotometry at
multiple wavelengths
● Specific gravity: refractive index methodology
● Clarity: measurement of transmitted or scattered light
● The instruments use integrated barcode sample identification
and allow abnormal ranges to be selected
○ so that samples that require microscopic examination or
confirmatory testing can be identified and flagged
■ Flagged - the screen would give visual signals (big
exclamation mark or color coding: red)
● Patient results, quality control results, and calibrators are stored for
Figure. Diagram of urine particle analysis in the Sysmex UF1000i
visual display, print out, or transmission to a laboratory computer
● Standardized controls are run as set by laboratory protocol ● iQ 200
○ Uses digital imaging and auto-particle recognition (APR)
software to automatically analyze and pre-classify urine
particles in uncentrifuged urine based on size and shape
○ The digital video camera takes 500 pictures as the specimen UC-3500: Fully automated urine chemistry analyzer
passes through the flow cell. The digital images are sent to the
computer, where the actual analysis will take place.
○ Employs a camera that would specifically capture the image of
the particle or the sediment and compare it to the library of
sediments encoded using the APR software
Figure. iQ 200 microscopy analyzer
UF-5000: Fully automated urine flow cytometer

Figure. Diagram of the iQ 200 digital flow capture process
Automated Urinalysis Systems

● Combined automated urine chemistry analyzers and automated
urine cell analyzers to create completely automated urinalysis
systems
● Combining the chemistry and the microscopic automation, this would
create a significantly improved turnaround times for routine
urinalysis
● Technologists’ hands-on time has been significantly reduced
● Samples are easily transferred from one instrument to the next,
providing a complete walkaway capability with minimal specimen
handling from sampling through results
○ Brought about by the sample racks and moving the sample
racks on a conveyor system
○ For example, in a sample rack you have 10 urine tubes from
distinct patients, you don't need to manually transfer the rack
from the chemistry analyzer to the automated microscopy
analyzer - the conveyor belt will do it for you
● Interfacing with the LIS, bar-coded samples are automatically
identified and processed according to the requested tests
● Systems can independently perform both physical and chemical
testing, microscopy analysis, and a combination of both
● Complete urinalysis report can be sent directly to the LIS or printed
out, thereby reducing clerical error
○ Machine can readily transmit the results to the laboratory
information system once the analysis is finished
Automated Urinalysis Systems

iRICELL Urinalysis Systems Iris Diagnostics UD-10: Fully automated urine particle digital imaging device
(iRICELL 3000plus, iRICELL
2000plus, iRICELL 3000pro,
iRICELL 2000pro, iRICELL 1500)
CLINITEK AUWi System Siemens Healthcare Diagnostics, Inc
Other urinalysis systems:

● Siemens Atellica Systems: used as a urinalysis system in our
diagnostic setting in the Philippines
VIDEO: UN-Series - Intelligent urinalysis workflow

[https://www.youtube.com/watch?v=gPUosFt2o0E]

MT6328 - AUBF LAB (Comprehensive)

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OUTLINE ● Information on label:

■ Color Random Specimen

IRINEO | TAN, K. | TENG | 3I-MT Hustle kahit Hassle 💯 2

Timing Schedule Example (Timed Specimens)

IRINEO | TAN, K. | TENG | 3I-MT Hustle kahit Hassle 💯 4

Figure 5. ​Digital refractometer

Historical Significance: Urinometer

Figure 6. ​Urinometer set-up; graduated cylinder (left) weighted​ ​float

Figure 7. ​Parts of a Urinometer

Figure 8. ​Harmonic Oscillation Densitometry

OUTLINE UNIT 3: QUALITATIVE TESTS FOR ALBUMIN

UNIT 6: TESTS FOR OTHER SUGARS Procedure

UNIT 8: TESTS FOR BILE PIGMENTS Video

Reporting SSA Turbidity

Video 1 (kasama nito yung Heller’s Test)

Sulfosalicylic Acid (SSA) Test

Reagents, Materials, and Equipment

Samples Only black lines are visible

Equipment (3+) = Both newsprint and

Reagents, Materials, and Equipments

Reagents, Materials, and Equipments

UNIT 4: QUANTITATIVE TEST FOR ALBUMIN

KWILECKI’S MODIFICATION OF ESBACH’S METHOD

Reagents, Materials, and Equipment

Robert’s Test KINGSBURY AND CLARK METHOD

Procedure ● Fehling’s Solution B

Reagents, Materials, and Equipments

Reagents, Materials, and Equipment

Interpretation For Nylander’s Test

Benedict’s Quantitative Method

Reagents, Materials, and Equipment

Reagents, Materials, and Equipment

Reagents, Materials, and Equipment

Reagents, Materials, and Equipment

3. Boil for less than 30 seconds. PENTOSES

8. Yellow ethereal layer ​will confirm the presence of fructose.

Reagents, Materials, and Equipment

3. Using a dropper, add a small amount of dilute sodium

5. Now, add a small amount of dilute sodium hydroxide solution

4. Shake the test tube well

* Sodium Nitroprusside Test

→ B1 - indirect bilirubin​; bound to albumin so it cannot ● Test tube

*Video Demo Procedure

2. Mix thoroughly and allow to stand for 2 to 3 minutes

UNIT 10: TEST FOR URINE CALCIUM

Ammonium Oxalate Test

● White residue glows on heating, which indicates the presence of

● A white precipitate of calcium oxalate is formed due to the reaction

UNIT 11: TEST FOR URINE CHLORIDE

Reagents, Materials, and Equipment

Purple Structure: Squamous epithelial cell; Red blood cell is almost

Clinical Significance ■ Malignancy (Transitional cell carcinoma) or viral

RARE FEW MODERATE MANY

Can you highlight the difference?

Identify the pointed structure

● Transitional Epithelial Cell (Urothelial cell)

CYLINDRURIA 2. Cellular Cast

Are all these the same?

● Whetstone (4-sided flat plate) Calcium sulfate crystals in

Crystalluria: Normal Crystals Point of Differentiation Uric Acid Cystine

Figure. Fatty casts

Figure 5. Digital refractometer

Figure 6. Urinometer set-up; graduated cylinder (left) weighted float

Figure 7. Parts of a Urinometer

Figure 8. Harmonic Oscillation Densitometry

8. Yellow ethereal layer will confirm the presence of fructose.

→ B1 - indirect bilirubin; bound to albumin so it cannot ● Test tube

● Phenylketonuria: you lack an enzyme to metabolize phenylalanine

● Type II: TAT deficiency

● Type III: HPPD deficiency

● Increased in conditions like malignant melanoma

Figure. Symptoms of Porphyria: seizures, high blood pressure, heart

● Photosensitivity → avoidance of sunlight 3. Vigorously shake both tubes

Figure. Watson-Schwartz reactions

Figure. Watson-Schwartz reactions MUCOPOLYSACCHARIDOSES (MPS)

● Monosaccharides are Clinitest positive

Figure. Fructose metabolism

Figure. Seliwanoff’s Test