AUBF LAB Module 3: Chemical Analysis of Urine Part 2

Small Result (Bilirubin)

1+

Benedict's Test.

A manual test for detection of reducing sugars in the urine. (Chemical Principle of Copper Reduction)

ROTERA'S TEST

Similar manual chemical test Also detects urine ketones This is shown through a development of a purple color.

KETONE BODIES MULTISTIX

Sodium Nitroprusside

BILIRUBIN POSITIVE RESULT

Tan, Pink, or Violet

Chromogen used for these reagents is ? (Blood)

Tetramethylbenzidine

True

The color development of Benzidine's test depends on the amount of blood present in the Urine sample. (True/False)

True

The loss of oxygen is then added to the reducing substance making it an oxidized substance. (True/False)

UROBILINOGEN CHEMSTRIP

• 4 Methoxibenzene diazoniumtetrafluoroborate

HEMATURIA seen in?

• Glomerulonephritis in Renal Calculi • Microscopic Analysis - may show intact RBCs

QUALITATIVE REPORTING KETONE BODIES

• Negative • Trace • Small (1+) • Moderate (2+) • Large (3)

SEMIQUANTITATIVE REPORTING KETONE BODIES

• Negative • Trace (5mg/dL) • Small (15mg/dL) • Moderate (40mg/dL) • Large (80-160mg/dL)

UROBILINOGEN MULTISTIX

• P-Dimethylaminobenzaldehyde

MYOGLOBINURIA seen in?

• Rhabdomyolysis Heme portion of the myoglobin proteins that is toxic to the tubules • *Pose more damage risk than hemoglobin

KETONE BODIES CHEMSTRIP

• Sodium Nitroprusside • Addition of glycine to detect acetone

Intact RBCs Results (Blood)

• Speckled / Spotted Absorbent Pad

Red/Pink Plasma

Evidence of Hemolysis Marked decreased of Haptoglobin

Plus 1 (1+)

Green solution with yellow precipitate (BENEDICT'S TEST)

Trace

Green solution without precipitate (BENEDICT'S TEST)

Negative for Blood Reagent Strip

HEMOGLOBIN

HEMOGLOBINURIA seen in?

Hemolytic Transfusion Reaction (HTR), Hemolytic Anemia, and Intravascular Hemolysis

The hydrogen peroxide will combine to the chromogen. Hydrogen peroxide will then lose its hydrogen atoms and will be incorporated to the chromogenic substance. Hemoglobin acts as the catalyst for this reaction. Peroxidase is also capable of catalyzing this reaction. Product will show the chromogen that has obtain the oxygen atoms and this is accompanied by another byproduct of water. This chemical reaction can be observed in 3 situations: Hematuria, Hemoglobinuria, and Myoglobinuria.

How does the Pseudoperoxidase of hemoglobin affects the reagents?

Ketone Bodies

are the byproduct of fatty acid metabolism due to the inability to metabolize carbohydrates Seen in Diabetes mellitus, vomiting, starvation, and malabsorption

purple or violet

color indicated the presence of acetone.

>1mg/dl Urine urobilinogen

seen in hepatic disorders and hemolytic episodes.

reduced

In the copper reduction test, cupric sulfide (oxidizing agent) is being ? by the reducing sugars that are present in the urine sample.

Positive for Blood Reagent Strip

MYOGLOBIN

BetaHydroxybutyric Acid

Major ketone body but is not detected in reagent strips

BENZIDINE TEST - FOR BLOOD, HEMOGLOBIN, AND MYOGLOBIN

Manual method for the detection of blood, hemoglobin and myoglobin

LEGAL'S TEST: MANUAL METHOD

Manual method for the detection of ketone bodies.

Negative Result (Bilirubin)

Negative

Negative (-)

No changes in color (BENEDICT'S TEST)

Bilirubin test Negative result

No emerald green ring

BLONDHEIM'S TEST (AMMONIUM SULFATE TEST) MYOGLOBIN RESULTS

No precipitation occurred; Urine is still clear red

20% TOTAL (KETONE BODIES)

Noticeable turbidity

KETONE BODIES Positive Result

Observed through the development of a Purple color

Plus 4 (4+)

Orange solution with brick red precipitate (BENEDICT'S TEST)

Blue > green > orange > red

Oxidation would result in color changes which would turn the chromogens from

PLASMA EXAMINATION MYOGLOBIN

Pale yellow

Noticeable turbidity

Parent ketone

Deep Blue RESULT (BENZIDINE TEST)

Plus four (++++)

Green RESULT (BENZIDINE TEST)

Plus one (+)

Blue RESULT (BENZIDINE TEST)

Plus three (+++)

Greenish Blue RESULT (BENZIDINE TEST)

Plus two (++)

BLONDHEIM'S TEST (AMMONIUM SULFATE TEST) HEMOGLOBIN RESULTS

Precipitated Hemoglobin at the bottom of the tube

Positive RESULT (Ketone bodies)

Presence of Purple to Violet Color

HEMOGLOBINURIA

Presence of hemolyzed RBCs

HEMATURIA

Presence of intact RBCs

MYOGLOBINURIA

Presence of myoglobin proteins from lysed or damage muscles

BLOOD Follows the Principle of?

Pseudoperoxidase Activity of Hemoglobin

UROBILINOGEN BEST ANSWER RESULTS

RED

PLASMA EXAMINATION HEMOGLOBIN

Red/Pink Plasma

Negative, Trace, Small, Moderate, Large, 1+, 2+, 3+

Reporting (Blood)

UROBILINOGEN CHEMSTRIP RESULTS

White to pink

False

With the reducing property of the sugars, oxygen atoms present in the cupric sulfate are formed, transforming cupric sulfide (oxidizing agent) into its reduced form cuprous oxide. (True/False)

Plus 2 (2+)

Yellow solution with yellow precipitate (BENEDICT'S TEST)

Plus 3 (3+)

Yellow-orange solution with orange precipitate (BENEDICT'S TEST)

BLONDHEIM'S TEST (AMMONIUM SULFATE TEST) steps

1. Urine + 2.8 grams of Ammonium Sulfate with 80% saturation 2. Filter then centrifuge. 3. Test the supernatant for blood with the reagent strip

Benedict's Test. Left tube

= control (Benedict's reagent) (blue solution).

PROCEDURE (MYOGLOBIN):

1. An ammonium sulfate (NH4)2SO4 reagent is added to the urine specimen. It will then be filtered and centrifuged. 2. The only difference with the sample is that the Protein that causes its coloration will not settle at the bottom of the tube. 3. If we expose the supernatant to the Blood Reagent Strip, there will be a development of color reaction. This indicates the presence of free myoglobin in urine also called Myoglobinuria. 4. Positive blood reagent strip : (+) Myoglobinuria

PROCEDURE (HEMOGLOBIN):

1. An ammonium sulfate (NH4)2SO4 reagent is added to the urine specimen. It will then be filtered and centrifuged. 2. The tendency for the hemoglobin to accumulate at the bottom of the tube indicate the success of ammonium sulfate reagent test. 3. We will then expose the supernatant to the blood reagent strip. 4. There is no color development on the blood reagent strip which indicates Hemoglobinuria or presence of hemolyzed red blood cells. 5. Negative for Blood Reagent Strip : (+) Hemoglobinuria.

LEGAL'S TEST: MANUAL METHOD steps

1. Mix 1 drop of phenolphthalein reagent and 5 mL well-mixed urine in an evaporating dish. 2. Add NaOH or KOH drop by drop until a permanent pale pink solution is achieved 3. Add 10 drops of Sodium Nitroprusside Reagent while swirling the evaporating dish 4. A purple or violet color indicated the presence of acetone.

BENZIDINE TEST - FOR BLOOD, HEMOGLOBIN, AND MYOGLOBIN steps

1. Mix 1 mL of Benzidine Reagent and 1 mL urine in an evaporating dish. 2. Add 0.5 mL of 3% Hydrogen Peroxide and mix by swirling. 3. Observe color development after 1 minute.

COPPER REDUCTION TEST: MANUAL Steps

1. Mix 5 mL Benedict's reagent with 1 mL aliquot of urine gently by inversion. 2. Heat preparation using a water bath for a maximum of 5 minutes. (Start the timer once the solution starts to boil) 3. Read the results immediately

BENZIDINE TEST - FOR BLOOD, HEMOGLOBIN, AND MYOGLOBIN > LABORATORY DEMONSTRATION steps

1. Obtain 1000 ul of Benzidine reagent and place in the evaporating dish. 2. Mix 1000 ul of urine to the 1000 ul Benzidine Reagent 3. Add 500 ul of 3% Hydrogen Peroxide, swirl the mixture and observe for color development after 1 minute

Bilirubin LABORATORY DEMONSTRATION:

1. Obtain 5 mL of urine from a well-mixed sample and place it in a test tube 2. Overlay the urine with a tincture of alcohol iodine 3. Observe the result and report as positive or negative

LABORATORY DEMONSTRATION LEGAL'S TEST steps

1. Place 1 drop of Phenolphthalein into the evaporating dish. ▪ Phenolphthalein is the color indicator 2. Prepare a 5 mL aliquot of urine using graduated cylinder and add it to the phenolphthalein in the evaporating dish. 3. Add 10% NaOH drop by drop to the solution, until a permanent pale pink color is achieved. Then Swirl. 4. Add 10 drops of fresh Sodium Nitroprusside to the mixture while swirling. 5. We shall look for the development of purple to violet color. 6. Purple to Violet Color indicates the presence of Acetone 7. Result: Negative

LABORATORY DEMONSTRATION of Benedicts test

1. We will use a micropipette with 1000 μL volume capacity. 2. Prepare a 5 mL Benedict's reagent and add 1 mL of urine to the test tube. 3. Cover the test tube with a paraffin film and reinvert to mix the solution. (ibabaligtad yung test tube; nasa baba yung may paraffin) 4. Using water bath, boil the test tubes; We prepared 2 test tubes. a. Left tube serves as a control b. Right tube is our patient sample 5. Incubate for a maximum of 5 minutes. Start the timer once the solution boils. 6. Notice the color changes in the right tube. 7. After the incubation, remove the test tubes and interpret the results immediately.

Moderate Result (Bilirubin)

2+

BILIRUBIN MULTISTIX

2,4 Dichloroaniline diazonium salt

BILIRUBIN CHEMSTRIP

2,6 Dichlorobenzene diazonium salt

Large Result (Bilirubin)

3+

BILIRUBIN Reading time:

30 seconds

BLOOD Reading time

60 seconds

UROBILINOGEN Reading time:

60 seconds

KETONE BODIES Reading time:

: 40 seconds

Negative RESULT (Ketone bodies)

Absence of Purple to Violet Color

COPPER REDUCTION TEST: MANUAL

Benedict's Test and Clinitest follows the priniciple of:?

Benedict's Test. Right tube

Benedict's reagent with urine sample (brick red precipitate/brick red colored solution). ▪ There is a presence of reducing sugars in the urine of the patient.

BENZIDINE TEST - FOR BLOOD, HEMOGLOBIN, AND MYOGLOBIN Reagents

Benzidine Reagent and 3% Hydrogen Peroxide

78% TOTAL (KETONE BODIES)

BetaHydroxybutyric Acid

HEMOGLOBINURIA result appearance

Clear Red Urine

MYOGLOBINURIA result appearance

Clear Red Urine

HEMATURIA result appearance

Cloudy Red Urine

Distinct turbidity with no granulation

Detected in the presence of glycine

BILIRUBIN Follows the Principle o?

Diazo Reaction

DIAZO REACTION Principle

Diazo Reaction involves the combination of Bilirubin glucuronide and diazonium salt. Under acidic conditions, it will form into azodye

BLOOD MULTISTIX

Diisopropylbenzene Dehydroperoxide

BLOOD CHEMSTRIP

Dimethyldihydroperoxyhexane

2% TOTAL (KETONE BODIES)

Distinct turbidity with no granulation

UROBILINOGEN Follows the Principle of

Ehrlich's Reaction

Bilirubin test positive result

Emerald green ring at the point of contact

Pale yellow

Increase in CK and Aldolase

ACETEST TABLET TEST

It can detect ketone bodies. It contains the following reagent a. Sodium Nitroprusside b. Disodium Phosphate c. Lactose

Phenolphthalein, NaOH, Sodium Nitroprusside

LEGAL'S TEST Reagents

KETONE BODIES Follows the Principle of?

Legal's Test or Sodium Nitroprusside Test

UROBILINOGEN MULTISTIX RESULTS

Light to dark pink

Faint green RESULT (BENZIDINE TEST)

Trace (T)

Free Hemoglobin / Myoglobin Results (Blood)

Uniform Green or Shades of Blue-Green

Blondheim's test (Ammonium Sulfate Test)

Used to differentiate Hemoglobin and Myoglobin