Body Fluids Quiz Study Guide

CSF, serous, synovial fluid and BAL, Amniotic, Fecal and Seminal fluid analysis lecture slides

Hemocytometer Formula (Low and High)

LOW: (avg. # of cells counted x dilution factor)/(# of SMALL squares counted x 0.04 x 0.1) = cells/uL

HIGH: (avg. # of cells counted x dilution factor)/(# of LARGE squares counted x 0.1) = cells/uL

(Count RBCs and TNCs separately) The 2 sides must agree within 10%

A CSF sample was diluted using 1 parts CSF and 9 parts saline. 49 nucleated cells were counted in 5 small squares on side A and 51 on side B. What is the cell count per ul?

1. Avg = (49+50)/2

2. (50 x 10)/(5 x 0.04 x 0.1) = 25 x 10^3 cells/uL

When given cells in 1 large square, which ones are counted as "in"?

• 1 “small square” has 16 smaller boxes within it.

• Sometimes the “small square” is bordered by 3 lines. Use the middle line as the border

Bronchoalveolar Lavage (BAL) Fluid

• Not a naturally occurring fluid

• Not sterile site, common to see bacteria ˜

• Indications:

  • To detect microbiologic pathogens in immunocompromised patients

  • To detect malignancy

Synovial Fluid Collection (Arthrocentesis)

NEEDS hyaluronidase

• Needle inserted into joint space.

• Joint fluid dispensed into tubes based on test ordered

  1. Chemistry: no anticoagulant

  2. Hematology: Sodium Heparin/Liquid EDTA

  3. Microbiology: Heparin or SPS

  • Patient fasting: minimum 4-6 hours for accurate glucose comparisons.

  • Blood sample collected at the same time.

  • Normal fluid volume: 0.1 to 3.5 mL, typically viscous.

  • Dry tap' can occur if there's no fluid buildup.

Bronchoalveolar Lavage (BAL) Fluid Collection

Done by bronchoscopy and collection in a sterile container: Aliquots of 20-50mL of sterile saline injected, then removed (1st aliquot discarded).

• Common Cells Seen in BAL:

  • Macrophages

  • Lymphs

  • PMNs

  • Eosinophils

  • Ciliated columnar epithelial cells

• Squamous cells are contaminants

• Color and Appearance

  • Normally hazy and colorless.

  • May have mucus

  • Milky white = infection

  • Red = bloody

Serous Fluids Lining Cells are called?

Mesothelial Cells

CSF Lining Cells

• Choroid - Lining of Choroid Plexus

• Ependymal - Lining of Ventricles of neural

• Spindle - Arachnoid Membrane Lining

CSF Lining Cell

Choroid Plexus Cell

Synovial Lining Cell

Synoviocytes

• Eccentric nucleus

• “Fried Egg” appearance

• Possible debris inside cytoplasm

Serous Fluid Lining Cell

Mesothelial Cell

Bronchoalveolar Lavage Fluid (BAL) Lining Cell

Macrophage and Ciliated Cell

Malignant Cells

• Very large, basophilic cells

• Irregular shaped nuclei and nucleoli

• Uneven staining in the cytoplasm

• Various nuclear size and shape

• Vacuoles over the nucleus

Malignant Cells vs Benign Cells

What is different about each bodily fluid?

• CSF: Clear and colorless, cushions the brain and spinal cord.

• Serous Fluid: Pale yellow, lubricates surfaces between organs.

• Synovial Fluid: Viscous, provides lubrication in joints.

• BAL (Bronchoalveolar Lavage): Obtained from lungs, reflects pulmonary pathology.

• Amniotic Fluid: Surrounds fetus, cushions and facilitates movement.

• Fecal Matter: Composed of waste and undigested food.

• Seminal Fluid: Viscous secretion that contains sperm and nutrients.

Pleural, Pericardial, and Peritoneal Fluid Collection

• Sterile tubes coated w/ anticoagulant used for fluid specimens (Containing fluid or blood) - for hematology or micro

• Non-anticoagulant red-tubes (chem and immuno)

• Tubes at rm temp and sent to lab IMMEDIATELY

  • If needed for chem: centrifuge and refrigerate supernatant

• Collect a blood sample (to compare)

CSF Order of Draw

3-4 sequentially labeled tubes

#1 - Chemistry & Immunology: supernatant is refrigerated or frozen

• may contain skin cells or blood from collection

#2 - Microbiology: Room temp

• To avoid skin contamination

#3 - Hematology: Refrigerated

• Less likely to contain blood from collection, that could interfere with CSF counts

#4- Other Testing: Refrigerated

• Use Tube 4 for heme, less blood contamination

If only 1 tube for CSF, how do we proceed?

1. MICRO: minimize contamination/organisms might die 

2. HEME: count cells and make slide before cells degrade

3. CHEM & IMMUNO: chemicals and proteins well preserved

• One tube = same order as multiple tubes, but chemistry/immuno is moved back to the end of the line.

Cerebrospinal Fluid (CSF) collection

“Spinal Tap” is performed aseptically to collect CSF from the 3rd & 4th or 4th & 5th lumbar interspace, after measuring opening pressure with a manometer; normal pressure allows for 20 mL collection while abnormal pressure limits collection to 1-2 mL.

Which fluid needs hyaluronidase?

Synovial fluid →

Joint space is lined with synoviocytes:

• synthesizes hyaluronate, a mucopolysaccharide that makes synovial fluid viscous

• To aid pipetting for cell counts, synovial fluid is mixed with hyaluronidase or diluted with saline

• Hyaluronidase breaks down hyaluronate

___ crystals are found in CSF?

Hematoidin crystals (rare) and/or Hemosiderin granules during Cerebral Hemorrhage

___ crystals are found in Synovial Fluid?

Monosodium Urate Crystals (MSU)

• needle-shaped and can indicate gout when present in excess.

Monosodium Urate Crystals

Gout - buildup of MSU in joints

• Strongly negative birefringent

• Crystals are yellow (negative) when parallel to the slow axis

  of the compensator

• Crystals are blue (positive) when perpendicular to the axis of the compensator

Calcium Pyrophosphate Dihydrate (CPPD)

Causes pseudogout.

• Associated with:

  • Degenerative arthritis (e.g., osteoarthritis)

  • Hyperparathyroidism (regulates calcium levels).

• Crystals:

  • Weak positive birefringent.

  • Blue (positive) when parallel to the axis of the compensator.

  • Yellow (negative) when perpendicular to the axis of the compensator.

MSU vs CPPD

MSU crystals = gout, while CPPD = pseudogout. MSU crystals are needle-shaped and show strong negative birefringence, whereas CPPD crystals are rhomboid-shaped and show weak positive birefringence.

What crystal can resemble MSU or CPPD?

Corticosteroid Crystals

Crystal Identification

Which bodily fluid is sterile?

Cerebrospinal fluid (CSF), found in the central nervous system.

How is synovial fluid viscosity determined?

by the presence of hyaluronate, a mucopolysaccharide synthesized by synoviocytes, which increases the fluid's viscosity. → String Test

• To aid pipetting for cell counts, synovial fluid is mixed with hyaluronidase or diluted with saline, as hyaluronidase breaks down hyaluronate, reducing viscosity.

Traumatic tap vs. Hemorrhage

• Traumatic tap = blood contamination during a procedure (decreases as tubes are collected)

  • NO Xanthachromia

  • Little to no erthryophagocytosis

• Hemorrhage = actual bleeding within the fluid (level of blood stays the same)

  • Xanthachromia present

  • erthryophagocytosis along with possible hemosiderin & hematoidin crystals

Traumatic Tap

• Greatest amount of blood in tube 1, least in last tube

• After centrifugation, colorless supernatant

Hemorrhage

• Consistent amount of blood in all tubes

• After centrifugation, xanthochromic supernatant

• Macrophages with phagocytosed RBCs

• Macrophages stain positive for hemosiderin and may include hematoidin crystals

(CSF)/Serum Albumin Index Purpose

• CSF albumin comes from blood → used to assess permeability of blood-barrier

• Formula: Albumin(CSF) mg/dL / Albumin(Serum) g/dL

• <9 = normal, 9 to 14 = minimal impairment, 15 to 100 = moderate to severe; exceeding 100 complete breakdown

• Quantitation: nephelometry or reflectance spectrophotometry

Cerebrospinal Fluid (CSF) Immunoglobulin G (IgG) Index Purpose

• Reference interval for IgG index: 0.30 to 0.70

• Values > increased intrathecal production of IgG

  • Ex: multiple sclerosis or inflammatory

• Values < compromised blood-brain barrier

What differentiates Chylous vs. Pseudochylous Effusions?

• Chylous Effusion:

  • Results from lymphatic obstruction.

  • High in triglycerides, chylomicrons, and lymphocytes.

• Pseudochylous Effusion:

  • Associated with chronic inflammation.

  • Contains cholesterol and diverse cell types.

  • High cholesterol promotes inflammation

Chylous effusions

Pleural & Peritoneal effusions (b/n lungs & chest wall), High triglycerides, chylomicrons, lymphocytes

• Effusions in peritoneal cavity 

• Result from obstructions in lymphatic drainage

• Effusions = more triglycerides, chylomicrons, and lymphocytes

• Chylomicrons present 

• Triglycerides > 110 mg/dL

• Cholesterol < 200 mg/dL

• Microscopic: Lymphocytes

• Pleural effusions due to… 

  • Trauma or surgery (damage to thoracic duct)

  • Obstruction of lymphatic system: tumors (lymphomas), fibrosis

•  Peritoneal effusions due to…

  • Hepatic cirrhosis

  • Portal vein thrombosis

Pseudochylous effusions

High cholesterol (= inflammation); variety of cell types

• effusions w/ chronic inflammatory conditions

• Contains cholesterol & variety of cell types 

• High cholesterol levels promote inflammation

• Triglycerides <110 mg/dL

• Cholesterol >200 mg/dL

• Microscopic: Variety of cells, lipid-laden macrophages, cholesterol crystals 

• Seen in chronic diseases such as… 

  • Tuberculosis

  • Rheumatoid arthritis 

  • Collagen vascular disease

What differentiates Transudates vs. Exudates?

Transudates

Thin, watery fluids w/ low protein content that accumulate in body cavities due to imbalances in fluid pressure or protein levels in blood

• Increased hydrostatic pressure - accumulates in body cavity

• Clear, pale yellow, no clots

• Lower cell count 

• Mononuclear cells predominate

• Bilirubin, TP, LD, cholesterol LOWER

• Glucose equal to serum level

Exudates

Fluids, cells, or other cellular substances that are discharged from blood vessels, usually from inflamed tissues

• Increased capillary permeability

• Due to malignancy, more cells/microorganisms

• Cloudy, more color, could clot

• Early neutrophils predominate (indicates infection), mononuclear cells 

• Bilirubin, TP (total protein), LD, cholesterol ALL HIGH

• Glucose low, organisms consuming glucose

• Charcot-Leyden crystals may be seen

Which is more severe? Transudates or Exudates

Exudates

• Bilirubin, TP (total protein), LD, cholesterol ALL HIGH

• Glucose low, organisms consuming glucose

(Compared to transudates:Bilirubin, TP, LD, cholesterol LOWER, Glucose equal to serum level)

Joint disorder classification - what is different about each?

Joint Fluid Colors and Their Implications

Normal: Pale yellow or colorless, clear.

Red/Brown: Indicates trauma during collection or disorders allowing blood into the joint cavity.

Greenish/Purulent: Suggests infections.

Milky: Associated with tuberculous arthritis or systemic lupus erythematosus.

Signet ring vs. LE cell

• Signet cells = Large Vacuole, nucleus flattens to one side → many: possible malignancy

• LE cell (Lupus erythematosus) = ingested WBC within vacuole (pink blob) → autoimmune disease

Amniotic fluid Collection

• Transabdominally or vaginally with simultaneous ultrasound examination

• Using long aseptic needle into numbered tubes

• 10 to 20 mL of fluid collected by physician into numbered tubes

• Use sterile plastic containers

  • Cells adhere to glass container walls

• Protect from light

  • Bilirubin is light sensitive

• Room temp or body temp: Cell culture, genetic studies, microbiology

• Assay immediately or refrigerate - fetal lung maturity testing

Amniotic fluid appearance

• Normal amniotic fluid is colorless or pale yellow

  • Yellow or amber: bilirubin

  • Greenish: meconium

  • Pale pink to red: blood or hemoglobin

  • * Dark red/brown: maybe fetal death

• All amniotic fluid is somewhat

  turbid

  • Early pregnancy: little particulate

    matter

  • Late pregnancy: more turbid; increased fetal cells, hair, and vernix

Amniotic fluid RDS tests

• FLM results are compared to reference

  ranges based on gestational age of fetus to determine lung maturity ˜

• Fetal Lung Maturity tests:

  • L/S Ratio

  • Phosphatidylglycerol

  • Foam Stability Index

  • Lamellar Body Counts

RDS tests - L/S Ratio

• Surfactants keep alveoli open during respiration

• Lecithin: Major pulmonary surfactant.

• Sphingomyelin: Phospholipid in cell membranes; role not fully established.

• Equal production of lecithin and sphingomyelin until 33 weeks.

• From 34 to 36 weeks: Increased lecithin, constant/decreased sphingomyelin.

  • L/S ratio ≥ 2.0 indicates mature fetal lungs.

  • 30%-40% of infants with L/S ratio 1.5 to 2.0 do not develop RDS.

• Measured using thin-layer chromatography.

• Blood presence in specimen decreases mature result, increases immature result.

• Meconium-contaminated specimens are unreliable

Foam Stability Index (FSI) in RDS Testing

• Less reliable test, not frequently used.

• Stable foam produced by surfactants shaken with ethanol.

• Amniotic fluid mixed with varying ethanol concentrations.

• Highest ethanol concentration yielding stable foam indicates FSI.

• FSI ≥ 0.47 suggests fetal maturity.

• Blood and meconium contamination leads to inaccurate results.

Phosphatidylglycerol (PG) in RDS Testing

Phosphatidylglycerol is a phospholipid that enhances surfactant spread across the alveolar surface.

• Not detectable until 35 weeks' gestation.

• Techniques:

  • Thin-layer chromatography (TLC)

  • Slide agglutination using anti-PG antibodies.

• Positive tests are very specific but may have many false negatives.

• PG tests are not affected by blood or meconium contamination.

• In slide agglutination, latex beads coated with anti-PG antibodies agglutinate if PG is present.

Lamellar Body Counts for RDS Testing

• Measure pulmonary surfactants in lamellar bodies from amniotic fluid.

• Normal levels in third trimester: 50,000 to 200,000/uL.

• Level > 50,000/uL indicates fetal lung maturity.

• Use automated hematology counter's platelet channel for measurement.

• Avoid bloody specimens; they can falsely elevate lamellar counts.

• Requires small sample size

What information can you get if a bloody specimen has a low lamellar body count?

Since blood in the sample falsely increases lamellar body count due to platelet contamination, a low LB count means that the actual result is extremely low. Therefore, the fetus’ lungs are very immature.

How to differentiate amniotic fluid from urine

• Amniotic Fluid

  • Has glucose

  • Significant Protein

  • Creatinine is like plasma

  • NO UREA

• Normal Urine

  • Essentially NO glucose or protein

  • High creatinine

  • HIGH UREA

Why do we use all 4 chemicals in Urine Differentiation from Aminotic fluid?

• Late pregnancy causes an increase in amniotic fluid creatinine and urea (from fetal urine) - With renal disease, mother may have high protein and glucose in urine

• Mother might have diabetes which increases urine glucose

Neural Tube Defect Testing

• Neural tube defects: abnormalities in the brain, spine, or spinal cord (e.g., Spina bifida, Anencephaly).

• Alpha Fetoprotein (AFP): secreted in fetal serum; found in amniotic fluid; high levels indicate neural tube defects, fetal abnormalities, or distress.

• Acetylcholinesterase (AChE): enzyme in CNS, RBCs, skeletal muscle; confirms positive AFP; high levels suggest neural tube defects.

• Spina bifida: incomplete closure of spinal column; leads to nerve damage and paralysis.

• Anencephaly: underdeveloped brain and skull; results in stillbirth or death shortly after birth

Lung Maturity Testing

• Pulmonary system is one of the last organs to mature in the fetus.

• Respiratory Distress Syndrome (RDS): most common cause of death in newborns.

• Lamellar bodies release surfactants that prevent lung alveoli from collapsing.

• RDS occurs due to insufficient surfactant production in newborn's lungs.

• FLM tests are of no value before 32 weeks; indicate immaturity at that stage.

• Surfactants reduce surface tension to prevent alveolar collapse and lower pressure needed for reopening during inspiration.

Bilirubin Testing

• Blood in specimen is unacceptable (oxyhemoglobin absorbs at 410-540 nm).

• Specimens are rejected if hemolyzed, but icteric color is expected.

• Meconium contamination is not acceptable.

• Specimens must be shielded from light after collection due to bilirubin's light sensitivity.

• Icteric refers to the yellowish sample color from high bilirubin levels.

Amniotic Fluid Bilirubin: Queenan vs Liley Chart

• Queenan Chart: Preferred for assessing amniotic fluid bilirubin, especially in Rh incompatibility.

  • Specifically designed for gestational ages <27 weeks.

• Liley Chart: Less reliable <27 weeks due to insufficient data.

  • fetus more in danger due to hemolysis of RBCs as zones get higher in number

Fecal Fat Test Summary

• Two-slide qualitative test for fecal fat using Sudan III, IV, or oil red O.

• Slide 1: Detects neutral fats (triglycerides)

• Slide 2: Acidified with acetic acid and heated to estimate total fecal fat

Fecal Fat: 2 slide test interpretation

• Maldigestion: Increased neutral fats on 1st slide

  • Meat fibers present

• Malabsorption: Normal amount of fat on 1st slide with increased total fat on the 2nd slide

  • characteristic, orange red color

secretory and osmotic diarrhea definition

• Secretory

  • Enterotoxin-producing organisms

  • Damage to mucosa due to drugs or disease

• Osmotic

  • Maldigestion

  • Malabsorption

Secretory VERSUS Osmotic Diarrhea

IF DIFFERENCE IS >20 mOsm/kg = OSMOTIC, <20 mOsm/kg = SECRETORY

• Both have osmotically active solutes, but source differs

• To differentiate, measure fecal osmolality, sodium (Na) and potassium (K)

• Calculate fecal osmolality from Na and K, and compare w/ actual osmolality

Secretory Versus Osmotic Example:

• Fecal Na = 6

• Fecal K = 5

• Measured fecal osmolality = 10

Is this diarrhea secretory or osmotic?

Calculated Fecal Osmolality = 2 x [ Na(+) + K(+) ]

Osmotic: difference > 20 mOsm/kg

• 2x(6+5)=22

• 22-10=12 = Secretory (<20)

Fecal Carbohydrate Testing

• Undigested carbohydrates (e.g., lactose) lead to bloating, flatulence, and diarrhea due to fermentation.

• Stool becomes acidic: pH 5 to 6 (normal feces pH > 7).

• Clinitest tablets use copper reduction to detect reducing sugars (sucrose not detected).

Which fecal fat test are occult blood tests?

1.Guaiac-Based (gFOBT) - more common

• restrictions are required due to pseudoperoxidase or peroxidase activity of various foods and medication

• Result: Oxidized indicator (colored) + H2O

2.Immunochemical based (iFOBT)

3.Porphrin-based:

Key Points about Sperm Motility

• Immotile sperm cannot reach or fertilize an egg.

• Evaluated either subjectively or using automated methods.

• One drop of semen is analyzed under 40x with a coverslip.

  • At least 6 seen

• At least 50% of sperm should show moderate to strong forward progression.

• Temperature affects motility; testing is ideally at 37°C or room temp

Sperm Vitality

• eosin-nigrosin (Blom’s) stain, brightfield, 40x

• White sperm were alive (membrane intact)

• pink-stained sperm were dead (membrane damaged and permeable).

Sperm Count

• Semen is diluted with something that will

  immobilize the sperm

• Sperm is counted manually using a hemacytometer ˜

• Vasectomy = vas deferens is cut surgically

• Post-vasectomy sperm counts should be at least

  12 weeks after procedure; any remaining sperm

  should be nonmotile

Post Vasectomy Sperm Collection

• At least 2 samples needed for fertility assessment over 3-month period.

• Abstinence of 2 to 7 days before each sample collection.

• Entire ejaculate must be collected in a clean, sterile container.

• Lab-provided container to prevent soap interference with test results.

• Samples should be delivered to the lab within 1 hour, maintained between 20 °C and 40 °C.

• Keep collection container warm by holding it close to the body.

• Time of specimen collection is crucial for evaluating liquefaction.

Sperm Count formula

Sperm Concentration (sperm/mL) x Volume of ejaculate (mL)

Cause of Sperm Agglutination and Signficance

Causes reduced fertility & caused by IgG and IgA

• associated with the presence of sperm-agglutinating antibodies.

Vaginal fluid analysis - what are the 3 chemicals for risk of premature delivery?

1. Fetal fibronectin (fFN)

2. PAMG-1

3. IGFBP-1

Vaginal fluid analysis Summary

• Fetal fibronectin (fFN)

  • glycoprotein linking the placenta to the uterine wall, released during labor and detectable in cervicovaginal secretions

  • high levels indicate a higher risk of premature delivery.

• PAMG-1 & IGFBP-1

• Proteins in high concentrations in amniotic fluid

  • presence in cervicovaginal secretions indicates premature rupture of membranes and increased risk of premature delivery.

Sweat analysis - how Chloride sweat test works

• Pilocarpine stimulates sweat glands.

• Sweat collected on a gauze pad.

• Sweat leached into a known volume of distilled water.

• Chloridometer measures chloride concentration.

• Positive test if chloride >60 mmol/L.

• Confirmatory tests should be repeated on another date.

• Repeat sweat test + genetic test confirm diagnosis.

Cystic Fibrosis

• Autosomal recessive disease caused by mutations in the CFTR gene

• Causes viscous mucous secretions, leading to pneumonia

• Diagnosed with sweat chloride test

  • chloride stays outside = higher conc. = measured for sweat

Cerebrospinal Fluid (CSF) Reference Range + Interpretation

• Greater than normal: Indicates increased intrathecal IgG production (e.g., MS, inflammatory conditions)

• Less than normal: Suggests compromised blood-brain barrier

Synovial Fluid Reference Range + Interpretation

• Fetal maturity (Foam Stability Index):

  • FSI ≥ 0.47 indicates fetal lung maturity.

• Crystals

  • Monosodium Urate (MSU) – Gout

    • Parallel: Yellow

    • Perpendicular: Blue

    • Negative birefringence

  • Calcium Pyrophosphate Dihydrate (CPPD) – Pseudogout

    • Parallel: Blue

    • Perpendicular: Yellow

    • Positive birefringence

Amniotic Fluid Reference Range + Interpretation

• Lamellar Body Count:

  • > 50,000 /µL = fetal lung maturity

  • Normal range during 3rd trimester: 50,000 – 200,000 /µL

• L/S Ratio (Lecithin/Sphingomyelin Ratio):

  • ≥ 2.0 suggests lung maturity

  • < 1.5 suggests immaturity
    (standard reference knowledge)

• Phosphatidylglycerol (PG):

  • Presence after 35 weeks gestation = lung maturity

Chylous & Pseudochylous Effusions Reference Ranges + Interpretations

• Chylous:

  • Triglycerides > 110 mg/dL

  • Cholesterol < 200 mg/dL

  • Microscopic: Lymphocytes

• Pseudochylous:

  • Triglycerides < 110 mg/dL

  • Cholesterol > 200 mg/dL

  • Microscopic: Lipid-laden macrophages, cholesterol crystals, cell variety

Fecal Testing Reference Ranges + Interpretations

• Osmotic vs. Secretory Diarrhea:

  • Fecal osmolality difference > 20 mOsm/kg = Osmotic diarrhea

  • < 20 mOsm/kg = Secretory diarrhea

• Fecal pH:

  • Normal: >7

  • Acidic: pH 5–6 → suggests carbohydrate fermentation

• Quantitative Fecal Fat:

  • ≥ 6g fat/day = Steatorrhea