Clinical PathologyPractical 2

Review

Special thanks to Pat Siao

Detection of ACETONE in urine using Acetone Powder

Acetone Powder:•Sodium nitroprusside•Ammonium sulfate•Sodium carbonate, anhydrous

Detection of Acetone in urine using Acetone Powder

VIOLET color =Presence of ACETONE in urine(+) Ketosis or Diabetes Mellitus

No violet color =(-) ACETONE in urine

Analysis #21: Quantitative Determination of Total PROTEIN

Tests for: Incipient Renal Disease•Pyrogallol red-molybdate complex (absorbance 467nm)•PRM + protein purple color inc. absorbance (598nm)

Quantitative Determination of Protein

Example Practical Question:• Au = .20• As = .10• Cs = 50 mg/dL• Urine Volume = 10 dL• = 1000 mg/24 hrs or 1g/24 hrs• Normal = 0.28 – 1.41 g/24 hrs

)(dLvolCsAs

AuTp

Analysis #22: Micral Test

• Immunological detection of human albumin (IgG)

• Absorbed urine on strip binds antibody-gold-conjugate to urine albumin

• Color produced is directly related to albumin content

• Ideal specimen: First morning urine

(+) Positive when 2/3 morning urine specimens > 20 mg/L = persistent microalbuminuria(-) negative < 20 mg/L = physiologic urine albumin concentration

Analysis #23: Blood Urea Nitrogen (Modified Berthelot)

(630nm) echromophor indophenoloramineQuinonechl

oramineQuinonechlChloramine

ChloramineAmmonium

AmmoniumCOUrea

Salicylate

Salicylate

cyanidenitroferri & tehypochlori

2Urease

Analysis #23: Blood Urea Nitrogen (Modified Berthelot)

stdCAs

AuBUN

Example Practical Question:• Au = .20• As = .10• Conc. Of Standard = 25 mg/dL• = 50 mg/dL (high BUN)• Normal = 8-18 mg/dL

High BUN Low BUN

Impaired kidney function

Liver disease

Kidney disease Malnutrition

dehydration overhydration

Analysis #25: Creatinine Clearance

Unknown:•1 mL urine•1.5 mL 10% NaOH•20 mL picric acid•77.5 mL distilled water

Standard:•1 mL Creatinine Std•1.5 mL 10% NaOH•20 mL picric acid•77.5 mL distilled water

Tests for: kidney functionSpecimen: 24 hour urine specimenCreatinine + Picric acid forms red color proportional to amount of creatinine

Analysis #25: Creatinine Clearance

stdCRS

RU Creatinine Urine

Example Practical Question:• RU = .15• RS = .10• Conc. Of Standard = 100 mg/dL• = 150 mg/dL

Analysis #25: Creatinine Clearance

(mg/dL) Creatinine Serum

(cc/min) urine of x vol.(mg/dL) Creatinine Urine Clearance Creatinine

Example Practical Question:• Urine Creatinine = 200 mg/dL• Serum Creatinine = 2 mg/dL• Total Volume = 2 cc/min• = 200 cc/min (high CC)• Normal = 55 – 118 cc/min

Microscope Findings in Urine

Uric Acid Crystals

Renal stones, gout, high purine metabolism, acute febrile conditions, chronic nephritis

Calcium Oxalate Crystals

Renal stones, DM, liver disease, chronic renal disease, can be found in normal individuals

Triple Phosphate

Renal Canaliculi, Chronic Pyelitis, Enlarged prostate, UTI

Hyaline Cast

Severe renal disease with increased numbers, healthy individuals after heavy exercise

Granular Cast

Nonpathologic – strenuous exercise/stress; Pathologic – Glomerulonephritis, Pyelonephritis

Bacteria

With increased WBC UTI

Squamous Epithelial Cells

Occur in urethra and vagina; Vaginal contamination

Yeast Cells (Budding Yeast)

UTI especially from diabetic parents, immunosuppressed patients, skin or vaginal inefections

Amorphous Urates

Pus Cells

RBC

Can originate from any part of the urinary tract, Glomerulonephritis, Trauma, Renal diseases

Analysis #27Hema-screen Slide Test

• Tests for: fecal occult blood• Hemoglobin comes in contact with guaiac.• Peroxide developing solution guaiac-peroxidase reaction

occurs making blue-green color

Positive (+) Negative (-)

Any trace of blue color, within or on the outer rim of the specimen, is positive for occult blood.

(+)Normal in Infants

Abnormal in Adults

(-)Abnormal in Infants

Normal in Adults

Infants:•GI is sterile•↑ transit time due to shorter bowel•(-) = Abnormal

•Obstruction•Cystic Fibrosis

Analysis #28: Neutral Fat Estimation (Sudan III Stain)

Neutral Fat DropletIndicated by yellow-orange red globules

NORMAL< 50 fat globules/hpf

Positive (+)Normal

Negative (-)Abnormal

Analysis #29: Fecal Trypsin Determination

•Screens for: Cystic Fibrosis and Pancreatic Insufficiency•X-ray filmstrip immersed in diluted fecal emulsion•Trypsin & Chymotrypsin digest gelatin on x-ray strip•(+) trypsin complete digestion of gelatin (blue tint)•(-) trypsin gelatin remains intact

Pea-sized fecal material2% Sodium Carbonate

Cystic Fibrosis• Trypsin and chymotrypsin are proteolytic

enzymes. Their job is to digest protein in the small intestine. Normally, their precursors (their inactive forms: trypsinogen and chymotrypsinogen) are produced in the pancreas and transported to the small intestine.

• In Cystic Fibrosis, mucous plugs can block the pancreatic ducts that lead into the small intestine, preventing trypsinogen and chymotrypsinogen from reaching the intestine.

• (-) trypsin in stool suggests Cystic Fibrosis or pancreatic insufficiency

(+) for BILE!

Analysis #30: Harrison’s Spot TestGreen = biliverdinBlue = bilicyaninYellow = choletelin

Tests for: liver function or blockage of bile duct•Filter: Barium Chloride + Urine --> add 2 drops of Fouchet’s reagent to filter paperGreen = bilirubin is presentBarium Chloride precipitates bilirubin oxidized to biliverdin by ferric chloride

It’s NORMAL to see It’s NORMAL to see a cherry red ringa cherry red ring

< 1:20< 1:20Cherry red ring in tubes > 1:20 Cherry red ring in tubes > 1:20

means means hemolytic jaundicehemolytic jaundice

Analysis #31: Ehrlich’s test(Quantitative Test for Urobilinogen)

•Ehrlich’s reagent reacts with urobilinogen to form colored urobilinogen-aldehyde•10% calcium chloride – removes bilirubin

Check the tubes at an angle…

Cherry Red = POSITIVE!

No Cherry Red = NEGATIVE!

Analysis #31: Ehrlich’s test(Quantitative Test for Urobilinogen)

Slight Positive in tube 1:20

Negative in the greatest dilution (1:320)

Analysis #32: Van Den Bergh Reaction(Qualitative Test for Bilirubin)

Violet Ring @ point of contact =(+) for Direct Bilirubin

If you see…If you see…

(+)Urobilinogen

(+)Harrison’s

Partial Obstructive JaundicePartial Obstructive Jaundice

If you see…If you see…

(-)Urobilinogen

(+)Harrison’s

Complete Obstructive JaundiceComplete Obstructive Jaundice

Example Practical Set Up

Jaundice Qualitative Harrison’s Spot Test

Normal(no jaundice)

(+)(<1:20)Quantitative

(-)

Partial (+) (+)

Complete (-) (+)

Hemolytic (+)(>1:20)Quantitative

(-)

Microscope Findings in Feces

Vegetable Cells

Vegetable Cells

Vegetable Cells

Vegetable Fibers

Muscle Fibers

Muscle Fibers

Muscle Threads

Yeast Cells

RBC

Pus Cells

Fat Globules

Analysis #33: Blood Grouping

• Forward typing determines antigens on patient's or donor's cells– Cells are tested with the

antisera reagents anti-A, anti-B

• Reverse typing determines antibodies in patient's or donor's serum or plasma

Anti-A SeraAnti-A Sera

A w/ AgglutinationA w/ Agglutination

A w/ no agglutinationA w/ no agglutination

Anti-B SeraAnti-B Sera

B w/ AgglutinationB w/ Agglutination

B w/ no agglutinationB w/ no agglutination

Forward Typing Table

Anti-A Anti-B

Blood Type

(-)(-) (-)(-) OO(+)(+) (+)(+) ABAB(+)(+) (-)(-) AA(-)(-) (+)(+) BB

Reverse TypingKnown A Reagent CellsKnown A Reagent Cells

KA w/ AgglutinationKA w/ Agglutination

KA w/ no agglutinationKA w/ no agglutination

Known B Reagent CellsKnown B Reagent Cells

KB w/ AgglutinationKB w/ Agglutination

KB w/ no agglutinationKB w/ no agglutination

Reverse Typing Table

Known A Known B Blood Type

(-)(-) (-)(-) ABAB(+)(+) (+)(+) OO(+)(+) (-)(-) BB(-)(-) (+)(+) AA

Rh Typing

• Agglutination = Rh(+)