Urinalysis & Body Fluids Final Exam Review 2023

URINALYSIS & BODY FLUIDS FINAL EXAM REVIEW 2023

RENAL ANATOMY & PHYSIOLOGY

• 4 Major Processes Performed by Kidney:
  • Glomerular Filtration
  • Tubular Reabsorption
  • Tubular Secretion
  • Concentration

URINALYSIS & BODY FLUIDS

• Ultrafiltration
  • Occurs in glomerulus;
  • 100% filter, but not reabsorbed: Creatinine;
  • Not filtered: Proteins.
• Tubular Reabsorption
  • Occurs in Proximal Convoluted Tubules (PCT);
  • 80% of filtrate reabsorbed back into circulation;
  • 100% of glucose reabsorbed up to renal threshold (160-180 mg/dL).
• Tubular Secretion
  • Occurs in PCT & DCT;
  • Removal of protein-bound substances from blood to become part of urine.
• Concentration
  • In Loop of Henle, Collecting Ducts;
  • Regulated by ADH and Aldosterone.

THRESHOLD SUBSTANCES

• Some substances vary based on how much is reabsorbed back into the body:
  • Example: Water & electrolytes.
  • Low Threshold Substances: (e.g., urea & creatinine) - Waste products excreted.
  • High Threshold Substances: (e.g., glucose, amino acids) - Plasma concentration above the threshold is not reabsorbed back into bloodstream.

URINE SPECIMENS

• Collection Types:
  • Random: Collected anytime for routine UA.
  • First Voided AM: Most concentrated; used for routine UA and pregnancy testing.
  • 24 HR Specimen: Collected over 24-hour period for certain analyses.
  • Fasting: Void bladder upon waking up; collect next sample.
  • 2 hr Post Prandial: Collected 2 hours after eating; used to screen for diabetes.
  • Glucose Tolerance: Collect fasting, 0.5 hr, 1 hr, 2 hr, and 3 hr for glucose and ketones to diagnose diabetes.
  • Catheterized: Collection of urine from bladder; sterile; used for cytology, routine UA, and urine culture.
  • Mid-Stream: Collection during voiding for routine UA and urine culture.
  • Clean-catch: Collection after cleansing urethral opening; used for routine UA and urine culture.
  • Suprapubic: Collection via needle aspirate from bladder; used primarily for cytology.
  • Pediatric Samples: Special devices used for collection in young children.

SPECIMEN HANDLING PROCEDURES

• Changes that Occur in Samples Left at Room Temperature > 2 hrs:
  • Increased pH, nitrite, bacteria, turbidity.
  • Decreased glucose, ketones, bilirubin, urobilinogen.
  • Cellular destruction of RBCs and casts, especially in alkaline pH.
  • Color changes due to light, bacteria, ascorbic acid, and storage conditions on chemical tests.

URINES – CHEMICAL CHANGES

• Changes in Urine Analytes Held at Room Temperature Over 2 Hours:
  • pH: Bacteria convert urea to ammonia, and pH becomes alkaline.
  • Glucose: Bacteria use glucose, concentration decreases.
  • Ketones: Failure to cover sample leading to evaporation with loss of ketones.
  • Bilirubin: Exposure to light results in loss of bilirubin.
  • Urobilinogen: Oxidation of urobilinogen to urobilin by bacteria; loss occurs.
  • Nitrites: Bacteria reduce nitrates in sample to nitrites.
  • Bacterial Growth: Reproduction at room temperature within 2 hours.
  • Turbidity: Due to bacterial overgrowth or precipitation of crystals as sample cools.
  • Loss of RBC/Casts: RBCs and casts lost in dilute, alkaline urine.
  • Color Changes: Loss of color with loss of bilirubin; production of color (porphyrins, melanin, crystals).

URINES – SPECIMEN PRESERVATION

• Preservatives: Used to Maintain Analytes in Urine Samples:
  • Thymol: Preserves sediment, interferes with protein & glucose.
  • Formalin: Preserves sediment for cytology; not used for routine UA.
  • Toluene: Preserves biochemical analytes; used for routine UA.
  • Sodium Fluoride (NaF): Preserves glucose; used for drug screens.
  • Boric Acid: Anti-bacterial; preserves proteins, formed elements; no interference with routine UA.
  • HCl: Anti-bacterial; not acceptable for routine UA (destroys formed elements).

URINALYSIS – GROSS EXAM

• Physical or Gross Exam Includes:
  • Color
  • Character
  • Odor
  • Volume (only if not sufficient)
  • pH
  • Specific Gravity (now part of biochemical analysis).

GROSS EXAM - NORMALS

• Normal Characteristics of Urine:
  • Analyses:
  • Normal Appearance: Clear with small amount of white foam.
  • Color: Pale yellow to dark yellow due to urochrome.
  • Odor: Slightly aromatic due to urinod.
  • Volume: 24 hrs: 600-2000 ml, Average: 1200-1500 ml.
  • Taste: Slightly salty.
  • pH: 4-8.
  • Specific Gravity: 1.003–1.030 (random urine), 1.015–1.025 (24 hr urine).

URINALYSIS & BODY FLUIDS - URINE COLOR

• Normal Color (due to urochrome):
  • Straw, light to dark yellow, amber;
  • Colorless: dilute, polyuria;
  • Pink-Red: blood, aniline dyes, foods, myoglobin;
  • Dark Brown-Red: porphyrins, hemoglobin, RBCs.

URINALYSIS & BODY FLUIDS - ABNORMAL COLORS OF URINE

• Color Associations:
  • Colorless: Polyuria;
  • Amber: Increased bilirubin (jaundice);
  • Orange: Pyridium;
  • Yellow-Green: Biliverdin;
  • Blue-Green: Pseudomonas, methylene blue, dyes;
  • Pink-Red/Cloudy: Blood (RBCs);
  • Pink-Red/Clear: Hemolysis (hemoglobin or myoglobin);
  • Brown-Black on Standing: Methemoglobin, melanin, homogentisic acid;
  • Yellow-Brown: Bilirubin;
  • Yellow-Orange: Pyridium, multivitamins, riboflavin.

URINALYSIS – URINE ODOR

• Normal: Faint aromatic due to volatile acids.
• Abnormal:
  • Ammonia: Bacteria or old urine.
  • Fruity: Acetone (diabetes).
  • Pungent: Onions, garlic, asparagus.

GROSS EXAMINATION - ODOR

• Abnormal Odor Associations:
  • Ammonia: Bacterial growth; UTI.
  • Putrid: UTI.
  • Fruity: Diabetes (ketones).
  • Fecal (H2S): Feces.
  • Maple Sugar: Maple sugar urine disease (MSUD).
  • Mousy: Phenylketonuria (PKU).
  • Mercaptan: Natural gas smell associated with empty tank (genetic trait).
  • Sulfur: Cystinurias.
  • Pungent: Onions, garlic, asparagus.
  • Cabbage: Methionine;
  • Sweaty Feet: Isovalerylic and glutaric acidemias;
  • Rancid: Tyrosinemia.

URINALYSIS & BODY FLUIDS - SPECIFIC GRAVITY

• Purpose: Used to measure concentrating & diluting ability of kidney.
• Methods:
  • Refractometer: Measuring refractive index, speed of light in air vs speed of light in unknown.
  • Quality Control:
  • Distilled water (1.000);
  • 5% NaCl (1.022 $ ext{+-}$ 0.001);
  • 9% Sucrose (1.034 $ ext{+-}$ 0.001).

URINALYSIS & BODY FLUIDS – SPECIFIC GRAVITY METHODS

• Refractometer: Based on refractive index;
• Ionization of Electrolytes (reagent strips): The higher the ion concentration in urine, the more H+ are released from polyelectrolyte (test pad), resulting in change in pH leading to color change in pH indicator.
• Not affected by nonionic constituents like glucose & protein;
• Must add 0.005 to specific gravity for urine > 6.5 pH.

URINALYSIS & BODY FLUIDS – SPECIFIC GRAVITY NORMALS

• Normal Values:
  • Random: 1.003 – 1.035;
  • 24 hr urine: 1.015 – 1.025.
• Abnormal:
  • Isosthenuria (fixed sp. gr. 1.010): Suggests severe renal disease.
  • Hyposthenuria (sp. gr < 1.010 consistently): Suggests diabetes insipidus, renal disease, diuretics.
  • Hypersthenuria (sp. gr > 1.010 consistently): Due to glycosuria, heart failure, excessive water loss, X-ray media.

MACROSCOPIC EXAMINATION – SPECIFIC GRAVITY CORRECTION

• Temperature Corrections:
  • Temp > 22 C: Add 0.001 for every 3 C over.
  • Temp < 22 C: Subtract 0.001 for every 3 C under.
  • 3+ Glucose: Subtract 0.004 only if 3+ or greater.
  • 3+ Protein: Subtract 0.003 only if 3+ or greater.

URINALYSIS - BIOCHEMICAL

• Biochemical Analysis Includes:
  • pH;
  • Specific Gravity;
  • Glucose;
  • Ketone;
  • Bilirubin;
  • Urobilinogen;
  • Blood;
  • Protein;
  • WBC (leukocyte esterase);
  • Nitrite.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – PH

• Measures: H+ concentration.
• Method: Double pH indicators (Methyl red & Bromthymol blue).
• PH > 9 & Ammonia odor: Suggests old urine.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – PROTEIN

• Measures: Most sensitive to albumin.
• Method: Protein error of indicators;
• pH of 3.0 is critical to test.
• Reagent: Tetrabromophenol.
• Confirmation: 3% SSA (detects all proteins).
• Use: Indicator of renal disease; if positive, look for casts on microscopic analysis.

URINALYSIS & BODY FLUIDS – PROTEIN CONFIRMATION

• Results Interpretation:
  • NEG;
  • TRACE;
  • 1+;
  • 2+;
  • 3+;
  • 4+.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – PROTEIN FALSE RESULTS

• False Positive: Highly buffered or alkaline urine.
• False Negative: Presence of other proteins (globulins, Tamm-Horsfall), very dilute urine.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – GLUCOSE

• Measures: Sensitive for glucose.
• Method: Double sequential enzyme (Glucose oxidase/peroxidase).
• Use: Detects glucose in urine;
• Renal glucosuria: Normal blood sugar, but low reabsorption;
• Pathological glucosuria: Diabetes mellitus.
• Sensitivity: 100 mg/dl;
• False Negative: Presence of large amounts of Vit C.

BIOCHEMICAL ANALYSIS - GLUCOSE

• Chemical Principles:
  • Double sequential enzyme reaction: Glu oxidase/peroxidase;
  • Chromagen: Potassium iodide.
  • Glucose Reaction:
  • $ext{Glucose} + O<em>2 ightarrow H</em>2O_2 + ext{sugar acids}$
  • $H<em>2O</em>2 + ext{chromagen} <br /> ightarrow H_2O + ext{oxidized dye}$
  • Reactant: Glucose oxidase peroxidase.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – GLUCOSE (CLINITEST TABLET)

• Measures: Reducing substances (Cu+2 → Cu+1).
• Method: Copper reduction;
• Reaction: $CuSO<em>4 + ext{glucose (reducing substances)} ightarrow ext{sugar acids} + Cu</em>2O$
• Color Change: From blue to orange-brown.
• Use: Perform on pediatric patients for galactose & lactose;
• Sensitivity: 250 mg/dl;
• False Positive: Other reducing substances, ascorbic acid;
• Pass Through: Occurs when sugar > 4+.

GLUCOSE – CLINITEST RESULT COLOR CONCENTRATION

• Interpretation:
  • NEGATIVE: Blue (< 200 mg/dl);
  • TRACE: Blue-Green (200-250 mg/dl);
  • 1+: Pea-Green (500 mg/dl);
  • 2+: Green (750 mg/dl);
  • 3+: Yellow-Orange (1000 mg/dl);
  • 4+: Brownish-Orange (2000 mg/dl).

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – KETONES

• Measures: Ketone bodies;
  • Acetoacetic acid (20%), Acetone (2%), & B-hydroxybutyric acid (78%);
• Method: Sodium nitroprusside;
  • $ext{Na nitroprusside} + ext{diacetic acid} <br /> ightarrow ext{purple colored end product}$.
• Confirmation: Acetest tablets;
• Use: Positive seen in diabetes, malnutrition, inadequate CHO intake.

BIOCHEMICAL ANALYSIS - KETONES

• Chemical Principles:
  • Principle: Colorimetric;
  • Reagents:
  • Ames (Na nitroprusside);
  • BMC (Na nitroferrocyanide & glycine).
  • Reaction:
  • Ketone bodies + Na nitroprusside → purple colored complex;
  • $NH<em>4SO</em>4 + ext{glycine}$
• Acetone & diacetic acid reaction.

URINALYSIS & BODY FLUIDS - ACETEST

• Acetest Results:
• Color Result Concentration:
  • NEGATIVE: < 5 mg/dl;
  • TRACE: 5 mg/dl;
  • SMALL (1+): 15 mg/dl;
  • MODERATE (2+): 40 mg/dl;
  • LARGE (3+): 80 mg/dl.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – KETONES FALSE RESULTS

• False Positive: Highly pigmented urines.
• False Negative: Acetone evaporates at room temp; acetoacetic acid can be degraded by bacteria.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – BLOOD

• Hematology Definitions:
  • Hematuria: Intact RBCs in urine; urine appears pink-brown & cloudy; causes include UTI, disease outside urinary system, bleeding.
  • Hemoglobinuria: Free hemoglobin in urine; urine appears pink-brown but clear; caused by intravascular hemolysis, trauma to small blood vessels.
  • Myoglobinuria: Myoglobin in urine; urine appears pink-brown but clear; caused by muscular trauma (e.g., crush injury or heart attack).

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – BLOOD METHOD

• Method: Pseudoperoxidase activity of hemoglobin.
• Reagent: H2O2 + tetramethylbenzidine;
• Reaction: $H<em>2O</em>2 + ext{tetramethylbenzidine} <br /> ightarrow H_2O + ext{oxidized dye}.$

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – BLOOD FALSE RESULTS

• False Positive: Bleach, microbial peroxidases.
• False Negative: Large amounts of ascorbic acid (Vit C), high specific gravity.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – BILIRUBIN

• Formation: By macrophages from hemoglobin degradation:
  • Iron (returned to iron stores);
  • Globin (released into AA pool);
  • Heme $ext{(heme} <br /> ightarrow ext{biliverdin} <br /> ightarrow ext{bilirubin)}$;
• Bilirubin Transport: Transported to blood bound to albumin (unconjugated or indirect bilirubin), is not water soluble.
• In liver, albumin removed & replaced with glucuronate to become conjugated or direct bilirubin, which is water soluble.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – BILIRUBIN FORMATION

• In the RE System by Macrophages:
  • Bilirubin-Albumin: Unconjugated or indirect bilirubin.
• In Liver:
  • Bilirubin Glucuronide: Conjugated or direct bilirubin.
• In Intestines:
  • Direct bilirubin reduced to urobilinogen by intestinal bacteria.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – BILIRUBIN SIGNIFICANCE

• Significance of Bilirubin Presence:
  • Hepatocellular disease;
  • Biliary obstruction;
  • Hepatitis;
  • Pancreatic cancer.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – BILIRUBIN TEST METHODS

• Reagent: Diazonium salt;
• Reaction: Diazo reaction;
• Total Bilirubin + Diazonium Salt → Azobilirubin: Azobilirubin is red in acid pH; blue in alkaline pH.
• Confirmation: Ictotest (special test pads remove interfering substances).

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – BILIRUBIN FALSE RESULTS

• False Positive: Drug interference in highly pigmented urines.
• False Negative: Large amounts of ascorbic acid (Vit C), light exposure.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – UROBILINOGEN

• Formation: In intestines from bilirubin by microbial enzymes that reduce bilirubin to urobilinogen;
• Small Amounts: Reabsorbed into circulation & are excreted by kidneys (0.2 – 1 Ehrlich unit);
• Remainder: Reduced to urobilin and excreted in feces.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – UROBILINOGEN SIGNIFICANCE

• Increased Levels: In hemolytic anemias, intravascular hemolysis, liver disease;
• Decreased Levels: In biliary obstruction;
• False Positives: Drugs (highly pigmented urines);
• False Negatives: Exposure to light.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – UROBILINOGEN METHOD

• Method: Ehrlich’s reaction;
• Reagent: Ehrlich’s reagent (P-dimethyl-amino benzaldehyde);
• Reaction: $ext{Urobilinogen} + ext{P-DABA} <br /> ightarrow ext{red colored end product}$;
• Confirmation: None available.

CORRELATION – BILIRUBIN & UROBILINOGEN

• Health Correlations:
  • Urine Urobilinogen
  • Normal: Increased in hemolytic disease;
  • Hepatic disease: Increased;
  • Biliary obstruction: Low or absent.
  • Urine Bilirubin
  • Normal: Negative;
  • Hepatic disease: Usually positive;
  • Biliary obstruction: Positive.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – NITRITE

• Use: Determines ability of bacteria to reduce nitrates (NO₃) to nitrites (NO₂);
• Nitrite Formation: Occurs in urinary bladder and takes part in chemical reaction;
• Significance: Positive suggests urine colony count > 100,000;
  • If positive, look for bacteria in microscopic exam;
  • If sterile, midstream, clean catch, any bacteria is significant.

BIOCHEMICAL ANALYSIS - NITRITE

• Reagent: Aromatic amine + diazonium salt;
• Ames Reaction: Urinary nitrite + aromatic amine → diazo;
  • Diazo salt + dye → pink colored complex;
  • (p-Arsanilic acid or sulfanilamide).

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – NITRITE FALSE RESULTS

• False Negatives: Microbial enzyme deficiency;
  • Urine in bladder < 2-4 hrs;
  • Lack of dietary nitrates;
  • Presence of large amounts of Vit C.
• False Positive: Overgrowth in urine standing at RT > 2 hours.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – LEUKOCYTE ESTERASE

• Use: Detects neutrophils in urine, either intact or lyzed > 5 / HPF; leukocyte esterase is specific for granulocytes;
• Significance: UTI, pyelonephritis, cystitis, etc;
• False Negative: High glucose, high specific gravity, antibiotic therapy.

URINALYSIS & BODY FLUIDS - CHEMICAL TEST – LEUKOCYTE ESTERASE REAGENT

• Reagent: Amino acid esterase + diazonium salt;
• Reaction: Indoxyl carbonic acid esterase can be substituted;
• Confirmation: Urine microscopic exam;
  • $ext{Pyrrole amino (or Indoxyl carbonic) acid ester} <br /> ightarrow ext{Pyrrole}$;
  • $ext{Pyrrole} + ext{diazonium salt} <br /> ightarrow ext{purple colored complex}$ (2 mins).

BIOCHEMICAL ANALYSIS

• Analyte Reactions:
  • Protein (protein error of indicator):
  • Albumin + pH indicator → albumin-indicator complex;
  • Bilirubin (Diazo reaction):
  • Direct/conjugated bili + diazonium salt → red or blue azobilirubin (soluble in water);
  • Urobilinogen (p-dimethyl amino benzaldehyde):
  • Urobilinogen + Ehrlich’s reagent → red colored complex;
  • Nitrite (Ames reaction):
  • Nitrite + aromatic amine → diazo salt;
  • Diazo salt + dye → pink colored complex;
  • Leukocyte esterase:
  • Amino acid ester → Pyrrole (or Indoxyl);
  • Pyrrole + diazo salt → purple colored complex (2 mins);

BIOCHEMICAL ANALYSIS REACTION DETAILS

• Analyte Chemical Principles:
• pH (double indicator):
  • Indicator dye has one color in non-ionized form and changes color when ionized;
  • Methyl red changes color in acid range;
  • Bromthymol blue changes color in alkaline range.
• Specific Gravity:
  • Dissolved substances in urine combine with COOH end of polyelectrolyte releasing H+ ions that react with the pH indicator causing a color change.
• Glucose (double sequential enzyme reaction):
  • $ext{Glucose (patient urine)} + O<em>2 ightarrow H</em>2O_2 + ext{sugar acids}$;
  • $H<em>2O</em>2 + ext{chromagen} <br /> ightarrow H_2O + ext{oxidized chromagen}$;
• Ketones:
  • ext{Ketone bodies (acetone & diacetic acid)} + ext{Na nitroprusside}
    ightarrow ext{purple colored complex};
• Blood (pseudoperoxidase activity of hemoglobin):
  • $H<em>2O</em>2 + ext{chromagen} <br /> ightarrow H_2O + ext{oxidized chromagen}$;
  • $NH<em>4SO</em>4 + ext{glycine}$.

BIOCHEMICAL BACK-UP METHODS

• Backup Biochemical Reactions:
  • Protein (3% SSA): 3% sulfosalicylic acid (SSA) denatures structure of protein forming a precipitate; graded based on amount of precipitation.
  • Glucose (copper reduction): Carbonyl group (C=O) of glucose (other reducing substances) reduces Cu²⁺ in CuSO₄ to Cu¹⁺.
  • $ext{Glucose} + ext{CuSO}<em>4 ightarrow Cu</em>2O + ext{sugar acids}$;
  • Ketones: Same reaction as dipstick which is Na nitroprusside.
  • Bilirubin: Same reaction as dipstick, but reaction pad traps interfering substances allowing only bilirubin to react.
  • pH Paper: For acid (methyl red) and for alkaline (bromthymol blue);
  • Specific Gravity Refractometer: Measures refractive index (compares speed of light in air vs speed of light through urine).

MICROSCOPIC EXAMINATION – GRADING

• Grading Includes:
  • Rare: 2 per slide;
  • Occasional: 1 in every low-power field (LPF);
  • Few: 2-5 in every LPF;
  • Moderate: 5-10 in every LPF;
  • Many: >10 in every LPF;
  • TNTC: Too numerous to count.

URINALYSIS & BODY FLUIDS - CELLULAR ELEMENTS

• Normal Urine May Contain:
  • RBC (0-5/HPF);
  • WBC (0-5/HPF);
  • Squamous epithelial cells;
  • Few transitional and/or renal tubular epithelial cells (0-2/HPF);
  • Normal crystals;
  • Hyaline and granular casts (few);
  • Bacteria & yeast (skin contaminants);
  • Artifacts.

URINALYSIS & BODY FLUIDS - BODY FLUID SPECIMENS

• Specimen Collection Considerations:
  • Difficult to obtain;
  • Collected by needle aspirate from site;
  • Volume varies depending upon site; 1-3 tubes collected;
  • Store unused specimens in refrigerator;
  • Stat due to cell degradation & glycolysis;
  • Simultaneous blood samples for non-hematology testing (glucose, protein).

URINALYSIS & BODY FLUIDS - BASIC FLUID ANALYSIS – HEMATOLOGY LAB

• Physical Characteristics Include:
  • Color, clarity, volume, viscosity (synovial fluid);
  • Total cell count (hemocytometer);
  • Total RBCs and total WBCs;
  • Clear fluids counted undiluted;
  • Cloudy/bloody fluids diluted with saline and counted;
  • Correct for dilution in calculation.

CEREBROSPINAL FLUID – CSF

• Description:
  • Collected by physician;
  • 1-3 tubes with 1 ml in each;
  • Spinal aspirate (between 3rd-4th lumbar vertebrae);
  • Transported at room temp;
  • Performed stat.

CEREBROSPINAL FLUID – CSF CHARACTERISTICS

• Color: Colorless;
• Clarity: Clear;
  • Hazy, cloudy, or milky (WBCs, RBCs, bacteria, protein);
  • Bloody, fatty (fat embolism), oily (X-ray media);
  • Xanthochromic: pink, orange, yellow, brown color of supernatant after centrifugation (oxyhemoglobin is pink);
• Cause of Color Change: RBC degradation;
  • Either traumatic tap (with delay in centrifugation sample > 1 hr) or subarachnoid hemorrhage.

CEREBROSPINAL FLUID – CSF CELL COUNT

• RBCs: None; few RBCs may be due to peripheral blood contamination occurring during puncture;
  • Many RBCs → traumatic tap or true hemorrhage;
• WBCs: 0-5 per cubic microliter (lymphs & monos).

CEREBROSPINAL FLUID – CSF TRAUMATIC TAP INDICATORS

• Traumatic Tap Will:
  • Gross appearance of tube #1 to #3 decrease in red color or if see decrease in RBC count in tube #1 and #3 of > 10%;
  • Clot on standing due to fibrinogen;
  • No xanthochromia after centrifugation provided done < 1 hr after collection;
  • Not contain many macrophages or any macrophages with ingested intact RBC, hemosiderin, or hematodien crystals.

CEREBROSPINAL FLUID – CSF DISEASE STATES

• Bacterial Meningitis: Increased WBC (segs), increased protein, decreased glucose, positive gram stain.
• Viral Meningitis: Increased WBC (lymphs), increased protein, normal glucose & lactate.
• Tubercular Meningitis: Increased WBC (mixed reaction), increased protein, decreased glucose, increased lactate, may have pellicle formation.

CEREBROSPINAL FLUID – COMPARISON OF BACTERIAL VS VIRAL MENINGITIS

SYNOVIAL FLUIDS - SYNOVIAL FLUID DESCRIPTION

• Description: Visous liquid from joint cavities (due to hyaluronic acid), obtained by arthrocentesis;
• Color/Clarity: Pale yellow to colorless; clear.

SYNOVIAL FLUID – CELL COUNT METRICS

• RBCs: 0/ul;
• WBCs: < 200/ul;
• Few red cells during collection, but high numbers indicate hemorrhagic bleeding into joints;
• Cannot use acetic acid -> will clot fluid;
• Mucin serves in viscosity testing;
• Mucin CLOT: Add 2% acetic acid to test mucin.

BODY FLUIDS - SYNOVIAL FUNCTION

• Function of Synovial Fluid:
  • Reduces friction (lubrication);
  • Provides nutrients;
  • Lessens shock to joints.

BODY FLUIDS - SYNOVIAL NORMAL VALUES

• Normal Values for Synovial Fluid:
  

• Abnormal Microscopic Analysis:
  

• Monosodium Urate (MSU) – GOUT:
  • Shape: Needle-shaped crystals, extra-cellular & within neutrophils;
  • Birefringence: Yellow colored needles with compensated polarized light.

SYNOVIAL FLUID - CALCIUM PYROPHOSPHATE (CPPD) PSEUDOGOUT:

• Appearance: Rhombic-shaped, intracellular;

Cholesterol:

• Shape: Notched, rhomboid plates;
• Birefringence: Negative unstained.

BODY FLUIDS - SYNOVIAL CHEMICAL EXAMINATION OF SYNOVIAL FLUID

• Analytes and Normal / Abnormal Values:
  

• I. Non-Inflammatory: Degenerative joint disorders;
• II. Inflammatory: Immunologic—lupus, RA; Crystal-induced—gout or pseudogout;
• III. Septic: Microbial infections;
• IV. Hemorrhagic: Traumatic injury, coagulation deficiencies, malignancies.

BODY FLUID - SEROUS

• SEROUS FLUID: Also called effusions;
  • What are Effusions? Excessive fluid that forms between two layers of serous membranes due to imbalance of secretion (parietal layer) & reabsorption (visceral layer);
• Types of Effusions:
  • Transudates: Extravasated fluid collection that is basically an ultra-filter of plasma with little protein and few or no cells; is an effusion from a systemic disorder.
  • Exudates: Caused by direct membrane degeneration from infections or malignancies; extravasated fluid collection rich in protein and/or cells and usually cloudy (infection in sac or malignancy).

TRANSUDATES VS EXUDATES

PLEURAL FLUIDS - ANALYSIS

• Collection: Thoracentesis;
• Normal Characteristics:
  • Color: Pale yellow;
  • Clarity: Clear;
  • Cell Count: RBCs none; WBCs < 300/ul;
  • Differential: Lymphs, monos, mesothelial cells (lining), < 25% segs.

PLEURAL FLUID ANALYSIS – MACROSCOPIC EXAM

PLEURAL FLUID ANALYSIS – MICROSCOPIC EXAM

BODY FLUIDS - PLEURAL HEMATOLOGY |

• Mesothelial Cells: Single, small or large, round cells with abundant blue cytoplasm and round nuclei with uniform dark purple cytoplasm; benign mesothelial cells.

PLEURAL FLUID ANALYSIS – CHEMICAL ANALYSIS

PERICARDIAL/PERITONEAL FLUID - COLLECTION & CHARACTERISTICS

• Pericardial and Peritoneal Fluid:
  • Collection: Pericardiocentesis; paracentesis;
  • Normal: Same normal appearance and differential count as pleural fluid.

PERICARDIAL FLUIDS - TRANSUDATE VS EXUDATE CHARACTERISTICS

PERICARDIAL FLUID ANALYSIS - MACROSCOPIC EXAM

• Appearance and Viscosity of Pericardial Fluid Exudates:
  • Appearance Due to Cause:
  • Turbid: WBCs in infection/inflammation;
  • Bloody: RBCs from accidental cardiac puncture;
  • Milky: Chylous (fatty); triglycerides present (due to leakage);
  • Milky: Pseudo-chylous; chol & chol crystals present (due to chronic inflammation).

PERITONEAL FLUID ANALYSIS - TRANSUDATE VS EXUDATE CHARACTERISTICS

PERITONEAL FLUID ANALYSIS - ABNORMAL PERITONEAL EXUDATES

| Appearance | Cause
|
|-------------------|-------------------|
| Turbid | WBCs in infection/inflammation |
| Green | Bile from gallbladder/pancreas |
| Yellow (dark) | Bilirubin from liver |
| Bloody | Trauma, infection, malignancy |
| Milky | Chylous (fatty) from trauma or lymph blockage |

PERITONEAL FLUID – ASCITES CHEMICAL ANALYSIS

SEMINAL FLUID - GROSS EXAM

• Parameters for Examination:
  • Appearance, volume, viscosity, pH, time of liquefaction;
• Microscopic Exam:
  • Motility (% motile vs nonmotile);
  • Morphology;
  • Number of normal vs abnormal shapes / 200;
  • Viability (stain sperm with eosin-nigrosin and count # of live/dead sperm).

SEMINAL FLUID NORMAL RANGES

SPERM MORPHOLOGY EVALUATION (STRICT CRITERIA)

• Head width: 2.5-3.5 μm;
• Head length: 5.0-6.0 μm;
• Smooth oval head/shape;
• Acrosome: 40%-70% head area;
• No neck or mid-piece or tail defects;
• 'Borderline' forms = abnormal.

SPERM MORPHOLOGY - EXAMPLES

SEMINAL FLUID - SPERM COUNT CALCULATION

• Count Both Sides of Hemacytometer: Average count, report as # sperm in millions/ml;
• Chemistries:
  • Acid phosphatase;
  • Fructose concentration;
  • Serology;
  • Anti-sperm antibodies.

AMNIOTIC FLUID - DESCRIPTION AND COLLECTION

• Description: Watery fluid in membranous amniotic sac surrounding the fetus;
• Collection: By needle aspiration through abdominal wall by MD; transported stat.

AMNIOTIC FLUID TEST SIGNIFICANCE:

AMNIOTIC FLUID - FETAL DISTRESS

• Usually Due to HDN: Measure bilirubin;
• Measure Abs of Fluid: Between 325 NM – 550 NM;
• Normal Fluid Peaks: At 365 NM, then decreases;
• Abnormal Fluid Peaks: At 450 NM due to bilirubin presence;
• Height of Peak = Concentration of Bilirubin.

AMNIOTIC FLUID ANALYTE NORMAL ABNORMAL

AMNIOTIC FLUID - PHOSPHATIDYL GLYCEROL (PG)

• Production Delayed in Diabetic Mothers; Low concentration of PG indicates lack of fetal lung maturity;
  • Test Methods: Amnio-Stat FLM; Shake Test.

BODY FLUID CELL COUNTS - MANUAL CELL COUNTS

• Calculation:
  • # of Cells X VCF X Dilution Factor;
  • VCF: 1 MM³ / L x W x D x # Squares counted;
  • Dilution: Amount of solute / Total volume;
  • Total volume = Solute + Diluent;
  • Dimensions: W square = 1 MM by 1 MM; R square = 0.2 MM by 0.2 MM; Neubauer depth = 0.1 MM; Fuchs-Rosenthal depth = 0.2 MM.

SEMINAL FLUID CELL COUNTS EXAMPLE

• Calculations Example:
• A seminal fluid sample was collected on a 26-year-old male patient. Total volume of the sample was 3.5 ml. A 1:20 dilution was used, and 116 spermatozoa were counted in 2 W squares.
  • Total sperm count = # of sperm counted X VCF X Dilution Factor;
  • $116 imes 5 imes 20 = 11600 ext{sperm}/ ext{mm}^3$
  • VCF: $1 ext{mm}^3 / 1 imes 1 imes 0.1 imes 2 = 1 ext{mm}^3 / 0.2 ext{mm}^3 = 5$
  • Total count = $11600/ ext{mm}^3 imes 1000 = 11.6 ext{million/} ext{ml};$ Total count: $11.6 ext{million/ml} imes 3.5 ext{ml} = 40.6 ext{million}$.

SEMINAL FLUID CELL COUNTS - MOTILITY EXAMPLE

• If 50 spermatozoa were observed for motility, and 16 out of 50 were non-motile, what was the percent non-motile and percent motile?;
• Percent Non-Motile = rac{16}{50} imes 100 = 32 ext{% non-motile};
• Percent Motile = 100 - 32 = 68 ext{% motile}.

SEMINAL FLUID CELL COUNTS - MORPHOLOGY EXAMPLE

• If 200 spermatozoa were observed for morphology and 36 out of 200 were abnormal, what was the percent abnormal?;
• Percent Abnormal = rac{36}{200} imes 100 = 18 ext{% abnormal};
• Percent Normal = 100 - 18 = 82 ext{% normal}.

CSF CELL COUNT EXAMPLE

• A CSF that is slightly cloudy is diluted using 0.1 ml of CSF and 1.9 ml of diluent. What dilution was made and what is the dilution factor?;
• Dilution: rac{0.1 ext{ml of CSF}}{0.1 ext{ml CSF + 1.9 ext{ml diluent}}} = rac{0.1}{2.0} = 1/20 ext{ dilution}.
• Dilution Factor is: 20.

CSF CELL COUNT EXAMPLE

• A red cell count was performed on a clear CSF when red cells were noted on the undiluted specimen. An average of 72 red cells were counted on a 1:10 dilution in 4 large corner squares of a Neubauer hemacytometer.
• RBC count/cu mm = ext{# of cells counted} imes ext{VCF} imes ext{Dilution Factor} = 72 imes 2.5 imes 10 = 1800 ext{RBCs/cu mm}.
• VCF = 1 ext{ mm}^3 / ext{L} imes ext{W} imes ext{D} imes ext{# squares counted}.
• VCF: $1 ext{mm}^{3} / 1 imes 1 imes 0.1 imes 4 = 1 ext{mm}^{3} / 0.4 ext{mm}^{3} = 2.5.$

CSF CELL COUNT EXAMPLE

• A cloudy CSF is diluted 1:100, and an average of 11 white blood cells are counted in 1 W square (25 R squares) of a Fuchs-Rosenthal hemacytometer. What is the cell count?
• Cell count = ext{# of cells counted} imes ext{VCF} imes ext{Dilution Factor} = 11 imes 5 imes 100 = 5500.