AUBF - MICROSCOPIC EXAMINATION OF URINE SEDIMENT PART 2

diba?!?!?!?!?!?!?! nakakaputangina!!!!

False - all 3 are performed

In routine urinalysis, only physical and chemical test are done, microscopic test not needed

Clinical and Laboratory Standards Institute

Meaning of CLSI?

1. Requested by a physician

2. specified patient population is being tested (e.g. pregnant women, pediatrics, geriatrics, diabetics, immunocompromised, etc.)

3. Abnormal physical and chemical result

CLSI recommended to perform microscopic examination during?

Blood (intact RBCs)

Correlations (Macroscopic screening, chemical and microscopic examinations)

Color: Red, Cloud or turbid?

• Squamous EC (non-pathologic)

• WBCs (pathologic)

• Confirm pathologic or non-pathologic cause of turbidity

Correlations (Macroscopic screening, chemical and microscopic examinations)

Clarity: cloudy or turbid?

RBCs / RBC casts

Correlations (Macroscopic screening, chemical and microscopic examinations)

(chemical examination) significance of blood?

Yeast

Correlations (Macroscopic screening, chemical and microscopic examinations)

(chemical examination) significance of glucose?

• WBCs intact or lysed

• WBC cast (bactreria in UTI)

Correlations (Macroscopic screening, chemical and microscopic examinations)

(chemical examination) significance of Leukocyte esterase?

Bacteria (WBCs in UTI)

Correlations (Macroscopic screening, chemical and microscopic examinations)

(chemical examination) significance of Nitrite?

Increased casts and cells

Correlations (Macroscopic screening, chemical and microscopic examinations)

(chemical examination) significance of protein?

• Myoglobinuria

• Hemoglobinuria

Correlations (Macroscopic screening, chemical and microscopic examinations)

What is the clinical significance of clear color urine

Low pH; Increased concentration of glucose

Yeast grows best at ? pH with an increased concentration of ?

Bacteria and WBCs

Correlations (Macroscopic screening, chemical and microscopic examinations)
True UTI = ?

Bacteria

Correlations (Macroscopic screening, chemical and microscopic examinations)

Contamination =?

WBCs only

Correlations (Macroscopic screening, chemical and microscopic examinations)

Inflammation = ?

Tamm-Horsfall protein

Casts are made up of what?

Physical: turbidity, red color

Chemical: (+) Blood, (+) protein

Exceptions: Number hemolysis

Microscopic elements: RBCs

Physical: ?

Chemical:?

Exceptions:?

Physical: Turbidity

Chemical: (+) Protein, (+) Nitrite (+) LE

Exceptions: Number lysis

Microscopic elements: WBCs

Physical: ?

Chemical:?

Exceptions:?

Physical: turbidity

Chemical: -

Exceptions: number

Microscopic elements: Epithelial cells

Physical: ?

Chemical:?

Exceptions:?

Physical: -

Chemical: (+) protein

Exceptions: Number

Microscopic elements: casts

Physical: ?

Chemical:?

Exceptions:?

Physical: turbidity

Chemical: (↑) pH (+) nitrite (+) leukocytes

Exceptions: Number and type

Microscopic elements: Bacteria

Physical: ?

Chemical:?

Exceptions:?

Physical: Turbidity, color

Chemical: pH (+) bilirubin

Exceptions: number and type

Microscopic elements: Crystals

Physical: ?

Chemical:?

Exceptions:?

Easy identification of WBC and RBC

Fresh and/or properly preserved specimen = ?

True

True or False: Bring specimen to room temperature before centrifugation

• amorphous materials

• Crystals (precipitate

What forms when urine centrifuged in refrigerator temp?

I

RBCs and WBCs will settle at the bottom of the tube

What will happen if specimen is not mixed properly before being transferred?

10-15 mL (ave: 12 mL)

Specimen volume??

pediatric patient

Less volume is usually found on what patient?

True

True or false: Note the volume on the report form and how much of the specimen was used

perform corretion

What is an alternative instead of reporting how much specimen was obtained and used?

5 mins

Centrifugation time?

400 RCF

Relative centrifugal force

1500-2500 RPM

Revolutions per minute???

RCF is better

What is better, RCF or RPM?

diameter

RCF is better because RPM depends on the ? of the centrifuge.

How is RPM converted to RCF

False - False (-)

True or False: Braking or abruptly stopping the centrifuged will lead to false (+) result

will disturb the sediment - when you decant, pwedeng siyang mawala, thus producing a false negative result

Why does the sudden stop of centrifuge lead to false (-) result?

Paraffin film

What is used as an alternative if cap is not available for centrifugation?

0.5 - 1 mL

? should remain in the tube after decantation

• Bottom’s up and don’t hesitate, upright, then flick the tube (from Lab demo ni Ma’am Gia)

• It is better to pipette off, however, it is not performed in the laboratory because it tends to disturb the sediment.

How to decant?

mix the sediment

Why do you tap the lower part of the tube after decanting?

vortex mixer

What is used to have an equal distribution of microscopic elements?

Concentration factor (formula)

0.02 mL (20uL) / 1 drop; then place coverslip

How much of the sediment (urine) should be examined?

Cen slide

• commercial system/ closed system

• Fast, clean, accurate, microscopic urine analysis

• Specially designed tube

• Direct reading of the urine sediment

Plastic

What is the Cen slide made of ?

Provides a more clear image; made of plastic and medjo malabo

Why to some MTs still prefer using glass slides?

True

true or false about cen slide: Excess sediments tend to form clumps and are unevenly distributed

No

Does Cen slide requires decanting, re-suspending, pipetting, sediment transfer, microscope slides, and coverslip

weeks and months

Cen slide can preserve the microscopic elements up to ?and ?

10 - 20 fields low (10x) and high (40x) power

how many fields is needed in examining sediments under the microscope?

detecting

LPO - use for detecting or identifying cast?

near the edges of the coverslip

Cast are usually found where under the microscope?

identifying the type of cast

HPO use for detecting or identifying cast?

Bright-field microscope

type of microscope used for examining sediment?

Subdued light

how much light is used in examining sediments?

Epithelial cells

What is used as a point of reference in examining sediment (in the correct plane)?

females

Squamous EC are usually seen in who?

LPF

Squamous epithelial cells - LPF or HPF??

semi-quatitatively

Squamous epithelial cells - qualitatively, quantitatively, or semi-quantitatively?

• none

• rare

• few

• moderate

• many

Squamous epithelial cells manner of reporting?

• Rare: 0 - 5

• Few: 5 - 20

• Moderate: 20 - 100

• Many: >100

• none - 0

• rare - ?

• few - ?

• moderate -?

• many - ?

LPF

Cast - LPF or HPF??

quantitatively

Cast - qualitatively, quantitatively, or semi-quatitatively?

• None

• 0 - 2

• 2 - 5

• 5 - 10

• >10

Cast - manner of reporting

HPF

RBCs or WBCs - LPF or HPF??

Quantitatively

RBCs or WBCs - qualitatively, quantitatively, or semi-quantitatively?

Average number per 10 HPFs

RBCs or WBCs manner of reporting?

• None:

• 0 - 2

• 2 - 5

• 5 - 10

• 10 - 25

• 25 - 50

• 50 - 100

• >100 (TNTC)

RBCs or WBCs manner of reporting (numerical value)?

HPF

Crystals - LPF or HPF?

semi-quantitatively

Crystals - qualitatively, quantitatively, or semi-quantitatively?

LPF

Abnormal crystals - LPF or HPF?

quantitatively

Abnormal crystals - qualitatively, quantitatively, or semi-quantitatively?

• none

• rare

• few

• moderate

• many

Crystals - manner of reporting?

Abnormal Crystals ave / LPF

Abnormal crystals manner of reporting?

• rare - 0-2

• few -2-5

• moderate - 5-20

• many - >20

Crystals manner of reporting (numerical values)?

• none - 0

• rare - ?

• few -?

• moderate - ?

• many - ?

True

True or False: Most crystals in urine are found in a normal urine specimen

quantitatively

If reported as crystals only (without specific type), report as ?

HPF

Bacteria - LPF or HPF

quantitatively; estimated

Bacteria - qualitatively, quantitatively, or semi-quantitatively?

• none

• rare

• few

• moderate

• many

Bacteria manner of reporting?

• Rare: 0 - 10

• Few: 10 - 50

• Moderate: 50 - 200

• Many: >200

Bacteria manner of reporting (numerical value)

• none - 0

• rare - ?

• few - ?

• moderate - ?

• many - ?

Noting the presence

Manner of reporting of the following?

• Budding yeast

• Mycelial elements

• Trichomonas (vaginalis)

• Sperm*

• RBCs

• WBCs

• RTE

• Casts

sediments that are reported quantitatively?

• squamous EC

• crystals

• yeast

• bacteria

Sediments that are reported semi-quantitatively?

• RBCs

• WBCs

• Crystals

• RTEs

• Yeast

• Bacteria

• Transitional EC

sediments that uses HPF?

• squamous EC

• casts

Sediments that uses LPF?

False - mL

True or false: some laboratories report in L rather than LPF or HPF

0-2

Reference intervals

RBC?

0-5

Reference intervals

WBC?

0-2

Reference intervals

Cast (hyaline)

Few

Reference intervals

Squamous EC

few

Reference intervals

Transitional

0-2

Reference intervals

Rnal EC

Negative

Bacteria and yeast

1. calculation of the area of an LPF or HPF

   • 𝐴𝑟𝑒𝑎 = 𝝿 𝑟 ²

   • Diameter of HPF = 0.35 mm

   • Radius = 0.175 (kalahati ng diameter)

   • 3.14 x 0.175 ² = 0.096 mm2 π = 3.14

2. Calculation of the maximum number of LPFs pr HPFs in the viewing area

   • Area under a 22 mm x 22 mm coverslip = 484 mm²

   • 484/0.096 = 5040 HPFs

3. calculation of the number of HPFs per mL of urine tested using the concentration factor and the volume of sediment examined

   • 540/0.02mL x 12 = 5040/0/24 = 21,000 HPF/mL of urine

   • 5040 = maximum number of LPFs or HPFs in the viewing area

   • 0.02 mL = amount of sediment placed on the slide

   • 12 = concentration factor

4. calculation of the number of formed elements per mL of urine by multiply the number of HPFs per mL by the average number formed elements per field

   • 4 𝑊𝐵𝐶/𝐻𝑃𝐹 𝑥 21, 000 = 84, 000 𝑊𝐵𝐶/𝑚L

steps in converting ave number/LPF or HPF to Number / mL

• Sternheimer-Malbin stain

• 0.5% Toluidine Blue

• Oil Red O and Sudan III

• Hansel

• Prussian blue

• Gram stain

Urine sediment stains

Sternheimer-Malbin stain

• Provides a more detailed visualization of the internal structure of WBCs, epithelial cells and casts

• Delineates structure and contrasting colors of the nucleus and cytoplasm