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66 Cards in this Set

  • Front
  • Back

Formation and Physiology of CSF

What is CSF and where does it flow through?

Where is CSF reabsorbed?

Is CSF an ultrafiltrate?

What is the function of the blood brain barrier and what cells is it composed of?

How is CSF formed?

- CSF flows through subarachnoid space between arachnoid and pia mater

- Reabsorbed into blood in arachnoid granulations/villae (one-way valves)

- Formation by selective filtration. Hydrostatic pressure and active transport. Not an ultrafiltrate. Very tight-fitting endothelial cells, prevent filtration of large molecules-called the blood brain barrier

- CSF comes from the ventricles in the brain

Blood Brain Barrier

What fluid is this involved with?

What does this protect?

What substances cannot pass this barrier?

What are some disease that can affect this barrier and what do they do to the barrier?

There are tests for substances that pass through. What are these substances?

Blood-brain barrier

- essential to protect brain

- chemicals and harmful substances do not pass

- antibodies and medications are excluded

- CSF composition differs from plasma

- Meningitis, multiple sclerosis disrupt membrane

- Test fo substances that pass through: cells, protein, bacteria, immunoglobulins

- Meningitis occurs when meninges become inflamed

- Multiple Sclerosis breaks down the barrier

Specimen Collection and Handling of CSF

What procedure can you use to collect CSF and how do you use this procedure?

How many tubes are usually used to collect the CSF and what departments use them?

How many ml of CSF is usually extracted for each tube?

How many ml of CSF can you extract before symptoms in patient occur?

If you extract too much CSF, what could happen to the patient?

CSF collected between third to fifth lumbar vertebrae

Three sterile tubes in this order

1. Chemistry/serology

2. Microbiology (avoid skin contamination)

3. Hematology (avoid cells from tap)

- Save leftover fluid/fourth tube for additional tests

- Volume removed based on patient volume and opening pressure

Lumbar puncture collection of spinal fluid. Needle inserted into L4/L5.

Do not draw more than 10ml of CSF or else a spinal tap headache may occur in patient.

The first tube for chem and serology, spin down first tube, use supernatant for total protein and glucose, sediment from first tube is how you prepare the slides.

4th tube for extra tests or if failure of other tubes. Its a backup tube.

Apperance of CSF

What is a normal appearance for CSF?

What does a cloudy appearance indicate?

What does a milky appearance indicate?

What is considered abnormal for CSF?

What colors can a xanthrochromic CSF be?

If a patient has a xanthrochromic sample, what does this indicate in the CSF and what kind of pathologic disease can occur?

After centrifugation of bloody CSF, if the sample is yellow at the top with red sediment, what does that indicate?

After centrifugation of bloody CSF, if the tube contains red sediments on the bottom, what does this indicate?

- Crystal clear, cloudy/turbid, milky, xanthochromic, hemolyzed/bloody

- Cloudy = infection; milky = lipid or protein

- Xanthochromic

1. pink, orange, yellow

2. RBC degradation products

3. Also Jaundice, increased levels of protein, carotene

4. Pathologic = cerebral hemorrhage

- Clear and colorless is normal

- Any kind of turbidity or milky appearance is abnormal which usually indicates a bacterial infection

- Xanthochromic is old blood in spinal fluid. Yellow after centrifugation

- Fresh blood is indicated by RBC sediment after centrifugation

Traumatic Tap

What happens in a traumatic tap?

How can you differentiate this from a cerebral hemorrhage?

What does a clot usually indicate?

Why can't a hemorrhage form a clot?

What are some other causes for clot formation?

What kind of hemorrhage does xanthochromia indicate?

What test is used for Xanthochromia?

Is Xanthochromia present in a recent traumatic tap?

- Blood vessel punctured during tap

- Differentiate from cerebral hemorrhage

- Uneven blood distribution in tubes with traumatic tap

1. Erythrophagocytosis, hemosiderin granules

2. Hemorrhage = even distribution in all tubes

3. Traumatic tap = decreasing tubes 1 through 3

Clot formation

- Clots present = traumatic tap (plasma)

- Hemorrhage does not have enough fibrinogen

- Other causes of clot formation

1. Nonbloody CSF = damage to blood - brain barrier

2. TB meningitis: web-like pellicle after refrigeration


- Not present in a recent traumatic tap

- Indicates older hemorrhage

- D-dimer test for hemorrhage

Cell Count

What cells are usually counted and what cells are not really counted?

Usually, how long can you wait before doing the cell count and why do you do this cell count before the time expires?

In a normal adult, how many WBCs should you find in the count?

In neonates, what cells are found and how many indicate a normal neonate?

What automated cell counter is used to count these cells?

White Blood Cell and total cell count

Red Blood Cell count seldom done

Granulocytes lyse within 1 hour; STAT

- Normal adult 0 to 5 WBC/microliter

- Neonates up to 30 mononuclear cells/microliter

Neubauer counting chamber

- Automated cell counters can be used

1. Body fluid specific automation is available

Direct spinal fluid application to counting chamber.

- Has to be done in under an hour usually

- Doctor wants to know 10 to 20 minutes later usually

- Normal adults should find less than 5 white cells per cubic microliter

Calculating CSF Cell Count

For a total cell count, how do you load the specimen?

For a total cell count, when do you count undiluted specimens?

If you need to dilute the specimen in a total cell count, what do you use normally to dilute the specimen?

For a WBC count, what do you use to dilute the sample and why do you do this?

For a WBC count, what can you use to stain the sample to help see the cells better?

For a WBC count, what do you use to load the sample?

Usually, are white and red cells recorded together or separately?

Total cell count

- Clear specimens count undiluted unless overlapping cells are seen

- Load with transfer pipette

- Dilute with normal saline if necessary

WBC count

- Dilute with 3% acetic acid; methylene blue helps to see cells; undiluted rinse transfer pipette with acetic acid, gently rotate pipette

How many cells are in there. White and red are usually recorded separately.

Dilute acetic acid that will lyse the red cells and keep white cells intact.

Quality Control of CSF

What controls are available to use?

How often should you check diluents for contamination?

How often should you check on cytocentrifuge speed and timing?

How do you clean nondisposable chambers?

Commercial cell controls are available

Check diluents for contamination biweekly

Monthly check on cytocentrifuge speed and timing

Soak nondisposable chambers in bactericidal solution for 15 minutes; rinse; clean with isopropyl alcohol

Differential Count of CSF

When do you do a differential count?

What must you do to specimen before counting?

What number of cells should be counted, classified, and reported and how do you report these results?

Valuable diagnostic aid

Stained smear only

Must concentrate specimen

- Sedimentation, filtration, centrifugation, and cytocentrifugation

100 cells should be counted, classified, and reported in terms of percentage

Have greater than 3 or 5 cells, do differential count. Spinal fluid should not have very many cells per cubic millimeter. If more than 5, do stained differential

Cytocentrifugation of CSF

- What does a cytocentrifuge do?

- What is added to the sample to allow the cells to stick to the slide and what does this addition increase?

- What charge are these slides and what does this charge do?

- For a daily control, what is performed and why do this control?


- Forces cells onto a slide in a monolayer

- Filter paper absorbs moisture

- 0.1 ml CSF to 1 drop 30% albumin

1. albumin increases the cell yield and decreases the cellular distortion

- Positively charged slides to attract cells

- Daily control of 0.2 ml Saline and two drops of albumin stained for bacterial contamination

- Difficulty in getting the spinal fluid cells to stick to slide is a problem.

- Add 30 percent bovine albumin.

- Cheaper less approved methods is 1 drop of human serum that contains a lot of protein naturally and put in slide and it has a glue like effect.

Cellular Constituents of CSF

Overall, what are the two cellular constituents?

In adults, what is the ratio of these cellular constituents?

In children, what is the ratio of these cellular constituents?

If you find a couple neutrophils in the sample? does this indicate abnormality?

What does pleocytosis do to normal cells and what disease is indicated by pleocytosis of these normal cells?

If there is an increase of neutrophils, what does this indicate?

If there is an increase in lymphocytes, what does this indicate?

Normal lymphocytes and monocytes

Adults: normal lymphocytes:monocytes = 70:30

- children's ratio is reversed

- occasional neutrophils are normal

Pleocytosis: increased amounts of normal cells

Pleocytosis of normal cells is valuable in determining the cause of meningitis

- Neutrophils = bacterial

- Lymphocytes = viral, tubercular, fungal, parasitic

Neutrophils in CSF

What is another name for neutrophils and how can you tell if these cells are neutrophils?

What disease is this mainly a part of?

Why do some neutrophils contain vacuoles?

Primarily in bacterial meningitis

Often contain phagocytized bacteria

Increased early viral, fungal, tubercular, parasitic

Vacuoles may be present

PMNs means polymorphonuclear neutrophils which just means more than one nucleus.

Phagocytic cells, ingesting bacteria, so they have these vacuoles. Phagocytized bacteria contained in vacuoles for digestion.

Lymphocytes and Monocytes in CSF

Between the two, which is the bigger cell?

What do lymphocytes and monocytes have in common?

What two types of lymphocytes are there and when do these appear?

When do lymphocytes and monocytes appear in general?

What disease are lymphocytes involved with and how many are usually found?

Lymphs and monos in viral, fungal, tubercular

Reactive lymphocytes with viral

Multiple sclerosis has 50 or fewer lymphcytes/microliter, both normal and reactive

Seen in HIV and AIDS

Lymphocytes are smaller than monocytes but they have one thing in common, one nucleus

Eosinophils in CSF

What infections are these involved with?

To differentiate between neutrophils what stain is used and what does component of eosinophils does this stain?

Parasitic and fungal infections

- Primarily coccidioides immitis)

Medications and shunts into the central nervous system

Parasitic and fungal infections.

Can only be seen with hematological stain to stain granules red.

Type of PMN

A neutrophilic red white blood cell

Cerebrospinal Protein

What is the most common test?

What is the normal range of total protein for people and the normal range for infants and people aged 40 or greater?

What molecule is predominant, and what is second?

What is the major beta globulin?

What is used to identify CSF?

What is the major gamma globulin?

Total protein is the most common test

- Normal 15 to 45 mg/dL (mg, not grams)

1. Method dependent

2. Increased in infants and persons >40

- Albumin is predominant, prealbumin is second

- Alpha globulins-haptoglobin and ceruloplasmin

- Transferrin is major beta globulin

- TAU, carbohydrate-deficient transferrin seen in CSF, not in blood; Used to identify CSF

- IgG major gamma globulin

Present in much smaller amount than in serum. Protein.

Protein is a test you always do with spinal fluid with glucose.

Spinal fluid protein method mg per deciliter range.

Albumin is predominant.

Clinical Significance of Total Protein in CSF

What do decreased protein levels indicate?

What do elevated protein levels indicate?

What is the most common causes of increased damage to the blood-brain barrier?

Decreased protein levels = fluid leakage

Elevated levels = damage to blood-brain barrier, IG production within CNS, decreased clearance, degeneration of neural tissue

Meningitis/hemorrhage most common causes of increased damage to blood-brain barrier

Find abnormal results on clear fluid with low cell counts from neurologic disorders

elevated proteins indication of meningites

Electrophoresis and Immunophoretic Techniques of CSF

What do these techniques detect and what do these represent?

Where is this located?

What else must be simultaneousy performed?

Detection of oligoclonal bands

- Represent inflammation within the CNS

- Located in the gamma region of the protein electrophoresis

- Simultaneous serum electrophoresis must be performed

- Oligoclonal bands form a diagnosis of neurological disorders.

- Common CSF test for diagnosis

CSF Glucose

What kind of transport does this use?

What is a normal range of plasma glucose?

How much plasma glucose should there be?

How much CSF glucose should there be?

How long before a spinal tap should blood be drawn?

Elevated or decreased CSF glucose is a cause relative to what?

What are the conditions for bacterial meningitis?

What are the conditions for tubercular meningitis?

What are the conditions for Viral/fungal meningitis?

Selective transport across blood brain barrier

Approximately 60% to 70% plasma glucose

1. Plasma = 100 mg/dL; CSF = 65 mg/dL

2. Draw blood 2 hours before spinal tap


- Values that are decreased relative to plasma values

- Elevated CSF glucose values are always a result of plasma elevations

Markedly decreased CSF glucose with increased neutrophils in bacterial meningitis

Tubercular meningitis decreased CSF glucose with increased lymphocytes

Viral/fungal meningitis, normal CSF glucose and increased lymphocytes

Your glucose should be 60 to 80 percent of plasma glucose

Anything from 60 to 70 is normal in spinal fluid

Usually blood sugar is done concurrent with the spinal fluid so doctor will know plasma glucose levels close to time of tap.

CSF Lactate

How much lactate would you find in Bacterial, TB and fungal infections?

How much lactate would you find in Viral infections?

What is this more reliable than?

How long are lactate levels elevated?

What are some reasons as to why there would be a lactate buildup in the CSF?

Diagnosis and management of meningitis

- Bacterial, TB and fungal levels >25mg/dL

- Viral <25 mg/dL

More reliable than CSF glucose

Levels remain elevated until treatment becomes effective, then fall rapidly

Can result from any condition that decreases oxygen flow to the tissues

- Monitor severe head injuries

- Lactic acid metabolic byproduct of metabolism.

- Head injuries, other conditions where we might have cutoff of blood flow causing lactic acid buildup

Microbiology Tests of CSF

What is the most common microbio test for CSF and what part of the CSF is involved?

What other cultures must be drawn?

Why is it difficult to interpret gram stains and what other methods could be used?

What are the top three bacterial agents of CSF?

What are the most important bacterial agents in children in CSF?

Gram stain and cultures must be performed on sediment from centrifuged CSF; cytocentrifuge helps gram stains

Blood cultures also must be drawn

Difficult to interpret gram stains, few organisms and often debris

Organisms: Should this be strep pneumocystis or pneumoniae?

- Gram stain is the most common

- Acridine orange another one used frequently. DNA stain and fluorescence.

- number one infectious agent is n. meningitis

- number 2 is strep pneumoniae

- number 3 is staph aureus

- In children, most likely organism would be h. influenza, s pneumoniae, and others.

Parasites of CSF

What is a description of acanthamoeba and what can it cause?

What is the number one parasitic ameba found in spinal fluid and what does this cause?

Where is Naegleria fowleri found?

How does N fowleri enter the body?

What N fowleri can you find in wet preps and cytospin preps?

What is the morphological description of N. fowleri?


Naegleria fowleri

- Found in ponds, lakes, and some pools

- Enters nasal passages and migrates to the brain

- Motile amoeba seen in wet preps, nonmotile in cytospin preps

- Elongated with tapered posterior

- Acanthamoeba is something you see in the eye. Contaminated contact lense solutions. Can also get into brain and cause amebic encephalitis

- Number one parasitic ameba found in spinal fluid is naegleria fowleri, amebic encephalitis

Serologic Testing of Syphilis

What is this a primary test for?

Why is this test performed less now?

What does this test detect?

What agency produces the recommended test for specificity?

What test should this serologic testing be confirmed with?

Primary test is for neurosyphilis, third stage

Performed less now that people have been treated early with penicillin

Detect active cases within the CNS

The Venereal Disease Research Laboratories (VDRL) produces the recommended test for specificity

Should be accompanied by a positive serum Fluorescent Treponemal Antibody Absorption (FTA-ABS)

- Diagnosis of tertiary syphilis

- VDRL more effective on spinal fluid

- Screening tests must be confirmed with confirmatory test like FTA-ABS

- Spinal fluid culture for bacteria should be done in addition to spinal fluid panel

- look at total protein, glucose, lactic acid, look at if greater than 5 wbcs in spinal fluid, do differential count.

- centrifuged vs non centrifuged

Semen Physiology

What are the four components of normal Semen?

Why are tests on semen done?

Where are the paired testes located and what does the location control for sperm development?

What do the seminiferous tubules of the testes produce?

Where are Sertoli cells produced and what is the function of these cells? Where are these cells stored and what do they develop?

What component produces a majority of the seminal fluids and what percentage range are these fluids?

What is the primary sugar that is the nutrition for sperm and what component produces this?

Without this component, sperm are not motile. What is this component?

Semen consists of four components

- Contributed separately by the

1. Testes and epididymis

2. Seminal Vessels

3. Prostate

4. Bulbourethral Glands

Normal semen specimen must have all four components

Paired testes: located in scrotum

- Lower scrotum temperature optimal for sperm development

Seminiferous tubules of testes

- Produce spermatozoa

1. 5% volume

- Sertoli cells provide support and nutrients for the germ cells as they undergo spermatogenesis

- Mature and stored in the epididymis

1. Develop Flagella

Seminal Vessels

- Produce majority of fluid (60 to 70%); Transport medium

- Provide fructose and flavin for sperm metabolism

- Sperm are not motile without this fluid

- For fertility workups usually

- Fertility problems. Whats the problem with conception

- Seminal vesicles store the fluid prior to ejaculation and fertilization and thats the sample used to study this.

- Fructose is the primary sugar for seminal fluid, nutrition for sperm.

- Used to identify seminal fluid for screening test finding fructose

Semen Specimen Handling

What must be observed at all times during analysis?

How are specimens discarded?

What materials and techniques must be used?

Standard precautions must be observed at all times during analysis

Specimens are discarded as biohazardous waste

Sterile materials and techniques must be used

Fertility Evaluation of Semen

What factors should you examine to determine fertility of Semen?

Macroscopic and Microscopic





Sperm concentration and count



Liquefaction of Semen

What usually happens to normal fresh semen specimen?

What is one reason why fresh semen specimen can be abnormal?

Analysis cannot occur without this event. What is this event?

What should you do when enzymes are added to induce this event and why should you do this?

Fresh semen specimen is clotted and should liquefy within 30 to 60 minutes after collection

- Deficiency in prostatic enzymes

Analysis cannot begin until liquefaction has occurred

Enzymes added to induce liquefaction

- Document

- May affect biochemical tests, motility, and morphology

- If fresh semen does not liquefy within 30 to 60 minutes, that is an abnormal finding

Volume and Viscosity of Semen

What range is considered normal for semen?

Where do you measure volume?

Why would there be an increased volume of semen?

Why would there be a decreased volume of semen?

How do you determine viscosity and what indicates a highly viscous semen?

What are some ratings you can use for viscosity?

Normal: 2 to 5 ml

Measure in a graduated cylinder

Increased volume may be seen following periods of extended abstinence

Decreased = infertility, incomplete collection

Viscosity: droplets with thin threads from a pipette are normal

- Form threads longer than 2 cm = highly viscous

Rate 0 (watery) to 4 (gel-like) or low, normal, high

Sperm Concentration and Count

What is the normal sperm count usually and whats the lowest the sperm count can be to be considered normal?

What sperm count is borderline low?

What is the sperm concentration and count a valid measurement of?

Concentration measures what?

Count measures what?

What is the formula for total sperm count?

What is the reference value?

Valid measurement of fertility

Concentration = number sperm/mL

Count = number sperm per ejaculate

Total sperm count

- sperm concentration x specimen volume

Reference value: >20 to 250 million sperm per milliliter

- 10 to 20 million borderline

Normal count = concentration x volume

- >40 million/ejaculate

Usually 40 or 50 million is borderline low

- 250 million is usually normal

Seminal Fluid Fructose

What could be a reason for a low sperm concentration in seminal fluid?

What can a low to absent fructose level in the semen cause?

What are some reason for low abnormal levels of fructose?

To examine sperm motility, what test is usually used first?

Low sperm concentration *may be caused by lack of the support medium produced in the seminal vesicles, which can be indicated by a low to absent fructose level in the semen*

Low to absent fructose level in the semen

Lack of the support medium

- Abnormalities of the seminal vesicles

- Bilateral congenital absence of vas deferens

- Obstruction of the ejaculatory duct

- Androgen deficiency

Usually motility is assessed first off by wet mount

Antisperm Antibodies

Antisperm Antibodies are more common in males or females?

Why would antisperm antibodies appear in the body?

How are female antibodies created?

How can you differentiate between male antisperm antibodies and female antisperm antibodies?

Why is this more of a problem in women than men?

Present in both men and women

Male more common: surgery, vasectomy reversal, trauma

Sperm normally do not encounter in the immune system, so body considers them foreign

Damaged sperm create female antibodies *damaged sperm from males that enter the female may create female antibodies because the immune system looks at the damaged sperm as bad thingies*

Suspect male antibodies when clumps of sperm are seen; female no clumping

- More of problem in women than men because they are harder to detect

- Possible cause of infertility

Postvasectomy Analysis

What does the postvasectomy analysis observe?

How long does it take for sperm to be sterilized and what is this based on?

How long do you analyse the sperm?

How many sperm are required for fertilization?

If vasectomy was effective, what does this indicate?

If vasectomy was ineffective, what does this indicate?

What kind of preparation is used and what do you do when sperm is negative in this preparation?

Only concern is presence of sperm

Takes several months for all sperm to be gone, based on time and ejaculations

Begin in 2 months; continue until 2 months are negative

Wet preparation under phase; if negative, centrifuge for 10 minutes, examine again

Only one sperm is required for fertilization

If vasectomy was effective, no sperm found.

If vasectomy ineffective, sperm found.

Synovial Anatomy

What is the function and location of synovial fluid?

How do you remove synovial fluid?

What is a common joint for synovial fluid problems?

- Synovial fluid. Any joint produces fluid in joint space for lubrication

- Use arthrocentesis to get synovial fluid out.

- Knees are whats picked on most often. Excess fluid in knee causes increased inflammation in knee.

Disorders of synovial fluid

What are the four classifications of disorders with synovial fluid?

Four classifications of disorders (arthritis)

- Noninflammatory: degenerative, osteoarthritis (As body wears out, get degenerative joint disease)

- Inflammatory: immunologic, lupus erythematosus (LE)(Autoimmune disease), rheumatoid arthritis (RA), Lyme disease

1. Crystal - induced, gout and pseudogout

- Septic: microbial infection

- Hemorrhagic: trauma, tumors, coagulation deficiencies (hyper extension of knee, skiing, skateboarding, skating)

Classification and Pathologic Significance of Joint Disorders

In a normal knee joint, how much synovial fluid should there be?

What color should normal synovial fluid be?

What is the clarity of normal synovial fluid?

What is the viscosity of normal synovial fluid?

How many leukocytes and neutrophils should be in normal synovial fluid?

Are crystals present in normal synovial fluid?

How much glucose should there be in normal synovial fluid?

How much total protein should there be in normal synovial fluid?

- Normal synovial fluid should have less than 3.5 ml in a knee joint. Should be colorless to pale yellow. Very similar to urine in appearance. Should be clear and not cloudy. Should form string.

- Should not have very many leukocytes (less than 200 cells/microliter) and neutrophils should be minority (less than 25% of the differential), no crystals, glucose less than 10 mg/dl

- Total protein should be less than 3 g/dl

- Should form string 4 to 6 cm long

Specimen collection and handling of normal synovial fluid

What procedure is used to extract synovial fluid from the knee?

what is the normal knee fluid amount and the inflamed knee fluid amount?

Does normal fluid clot?

Does diseased fluid clot?

4 tubes are used to collect the samples. What are the colors of these tubes and what departments are associated with these tubes?What do these tubes contain for each department?

Why should you test synovial fluid ASAP?

Needle aspiration called arthrocentesis

Normal knee fluid amount 3.5ml

- greater than 25 ml if inflamed

Normal fluid does not clot; diseased fluid clots

Collect in

- Sterile heparinized or SPS for microbiology

- Liquid EDTA (no powdered) for hematology

- Heparinized or nonanticoagulated for other tests

- Centrifuge nonanticoagulated tube and separate

- Sodium fluoride for glucose

Test ASAP to avoid cellular lysis and changes in crystal

Synovial fluid when collected, 1 tube of purple top, 1 tube of green top, and 1 tube of red top. Sometimes 1 tube of grey top.

Viscosity of synovial fluid

What causes the viscosity of synovial fluid and what is this essential for?

How does arthritis affect viscosity?

Using the string test, how long should the string be to be a considered normal synovial fluid?

What is another name for the Ropes test and how is this test done? How do you rate the results of this test?

What do you use to identify synovial fluid?

Polymerization of hyaluronic acid

- Essential for joint movement

Arthritis decreases polymerization

4 to 6 cm string from aspirating needle = OK

Ropes (mucin clot test)

- Add fluid to 2% to 5% acetic acid to form clot

- Rate, good: solid clot; clear fluid, fair: soft formed clot; low: friable clot; poor; no clot

Use acetic acid to identify synovial fluid

Crystal Identification of synovial fluid

What cases are these involved with?

Why are some reasons as to why crystals form in synovial fluid?

Important diagnostic test; frequently performed

Acute and chronic cases

Metabolic disorders and decreased renal function

Other causes; increased blood levels, degeneration of bone and cartilage, injection of corticosteroids

Types of Crystals in synovial fluid

What are the primary types of crystals?

What are these crystals involved with?

Primary: monosodium urate (MSU) in gout; calcium pyrophosphate dihydrate (CPPD) in pseudogout

MSU: impaired purine metabolism, high purine foods, leukemia chemotherapy, decreased renal excretion of uric acid

CPPD: degenerative arthritis, disorders causing elevated calcium levels

Crystal Polarization in synovial fluid

What do MSU and CPPD do to light?

What is the difference between MSU and CPPD in polarized light?

Both MSU and CPPD crystals polarize light

MASU is highly birefringent and appears brighter than CPPD

Confirm identification using compensated polarized light

MSU Under Compensated Light

What colors do MSU and CPPD produce?

How does MSU affect the long axis and what light is impeded to produce its color?

How does CPPD affect the long axis and what light is impeded to produce its color?

MSU molecules run parallel to the long axis, aligned with slow vibration; fast light is impeded, producing a yellow color (negative birefringence)

CPPD molecules run perpendicular to long axis and impede the slow light producing a blue color (positive birefringence)

Chemistry Tests of Synovial Fluid

Are there alot of chemical tests for synovial fluid? why or why not?

How do synovial fluid values relate to plasma values?

What is the most frequent test for synovial fluid and what is the normal amount that should be analyzed in this test?

This sample should be drawn at the same time as what?

What are some causes of decreased levels of this analyte?

As an ultrafiltrate of plasma, normal values are similar to those of plasma

Few are clinically important

Most frequent is glucose

- Normal: not less than 10 mg/dL of plasma glucose

- Draw sample same time as fluid is collected

- Markedly decreased levels seen in inflammatory and septic categories

Synovial fluid is not subjected to long lists of chemistry tests.

Microbiology Tests of Synovial Fluid

What microbio tests are always done for synovial fluid?

What are the primary organisms identified in synovial fluid?

What determines fungal and TB cultures?

What is the worst organism to get in the knee? What is the second worst organism?

Infections caused by inflammation, trauma, and systemic infections

Gram stain and cultures are routinely performed

Culture must include chocolate agar

- Primary organisms are staph, strep, haemophilus influenzae, and neisseria gonorrhoeae

- Patient history determines fungal and TB cultures

- All synovial fluid always gets gram stain and culture

- Some of the worst organisms to get into synovial joint space is staph, if staph gets into knee, knee will never work the same ever again. Strep is another bad one for the knee.

Serologic Tests of Synovial Fluid

A majority of the tests are performed where?

What are the most common autoimmune causes of arthritis?

What disease is arthritis a frequent complication in and what organism is associated with this disease? Do you test the serum or the fluid for this organism?

When C-reactive protein is high, what does this indicate?

When C-reactive protein is low, what does this indicate?

Which levels of C-reactive protein are normal and abnormal?

Majority of related tests are performed on serum and fluid may only serve as confirmation

RA (rheumatoid arthritis) and LE (lupus erythematosus) are the most common autoimmune causes of arthritis

Lyme disease: arthritis is frequent complication; test serum for Borellia burgdorferi antibodies

Extent of inflammation: test for C-reactive protein and fibrinogen

- RA is more severe than non RA. Difference is absence of rheumatoid factor.

- C-reactive protein, linked to inflammatory processes. High c-reactive protein indicates inflammatory process somewhere and low c-reactive protein indicates no inflammatory process, which is a good sign.

Serous Membranes that Produce Serous Fluid

What do the serous membranes line?

The serous membranes consist of two membranes. What are these membranes and where are they located?

What is the function of serous fluid and how is it named?

Line the closed body cavities

- Pleural

- Pericardial

- Peritoneal

Two membranes

- Parietal: lines cavity wall

- Visceral: lines organs in cavity

Fluid between membranes

- Serous fluid: named for each location

- Serous fluid acts as a lubricant

Formation of Serous Fluid

What is the purpose of serous fluid?

What kind of filtrate is this compared to plasma?

How is serous fluid produced and what is involved with this method of production?

What does effusion mean?

If serous fluid is disrupted, what are possible causes of this disruption?

Purpose: provide lubrication between the two membranes

Ultrafiltrate of plasma

Produced by hydrostatic and oncotic (protein) pressure in the capillaries lining the membranes

Normally, oncotic pressure is the same on both sides of the membrane; hydrostatic pressure causes the production

Small amounts of excess fluid are absorbed by lymphatic capillaries

Disruption causes fluid buildup; termed effusion

- Causes: hydrostatic pressure increased from congestive heart failure; oncotic pressure decreased from hypoproteinemia; increased capillary permeability from inflammation, infection, malignancy; lymphatic obstruction from tumors

- Serous fluid has to be constantly formed and reabsorbed.

Specimen Collection and Handling of Serous Fluids

What method is used to collect serous fluid and what body sites are these methods commonly used in?

How much serous fluid is collected?

What kind of collection does the hematology department use?

What kind of collection does the microbio and cytology departments use?

What kind of collection does chemistry and serology use?

Collected by needle aspiration

- Thoracentesis: pleural cavity

- Pericardiocentesis: heart cavity

- Paracentesis: peritoneal cavity

Abundant fluid collected greater than 100 mL

EDTA tube: hematology

Sterile heparinized orpolyanethol sulfonae (SPS): microbiology, cytology

Clotted or heparin: chemistry, serology

Transudates and Exudates of Serous Fluid

What conditions are involved with transudates?

What conditions are involved with exudates?

How many grams of total protein are associated with transudates and exudates?

If you discover test shows transudates or exudates, which one requires little further testing or no testing at all?

Are transudates considered normal or abnormal?

Are exudates considered normal or abnormal?

Primary classification of serous fluids

Transudates: systemic disorder disrupts filtration and reabsorption, congestive heart failure, nephrotic syndrome

Exudates: conditions affecting membranes, inflammation, infection, malignancy

Differentiation important for further testing

Transudates = little further testing

Transudate protein less than 3.0gm/dL

Exudate protein greater than 3.0gm/dL

- Transudates are normal and exudates are usually abnormal

- Anything less than 3 is considered transudate and anything above 3 is considered exudate

Pleural Fluid related to Serous Fluid

What is hemothorax and hemorrhagic effusion?

How do you differentiate between the two?

How do you differentiate between chylous and pseudochylous?

Bloody: hemothorax (trauma), hemorrhagic effusion (membrane) damage

Differentiate: do a hematocrit on fluid

- Greater than 50% blood HCT is hemothorax: more blood

- Membrane damage: low blood

Differentiate chylous and pseudochylous

- Chylous is triglycerides; stain with Sudan III

- Pseudochylous is cholestrol; polarize and crystals also seen in wet bright-field view

Correlation of Pleural Fluid Appearance and Disease

What do these colors indicate?

Clear, pale yellow?

Turbid, white?






Clear, pale yellow is normal

Turbid, white is a microbial infection (tb)

Bloody is hemothorax, hemorrhagic effusion, pulmonary emboli, tb, malignancy

Milky is chylous material from thoracic duct leakage. Pseudochylous material from chronic inflammation

Brown is rupture of amoebic liver abscess

Black is Aspergillus

Viscous is Malignant mesothelioma (increased hayluronic acid)

Hematology Tests for Pleural Fluid

What are the primary cells identified and what do you find the most of in serous fluid?

What does an increased amount of neutrophils indicate?

What does an increased amount of lymphs indicate?

If eosinophils are present, what do these indicate?

Differential: primary cells are neutrophils, lymphocytes, macrophages, eosinophils, mesothelial cells, plasma cells, and malignant cells

- Macrophages (scavengers) often the highest

Increased neutrophils: bacterial infection, pancreatitis, pulmonary infarction

Increased Lymphs: TB, viral infections, autoimmune disorders, malignancy

Eosinophils: trauma introducing air and blood, allergic reactions, parasites

Chemistry Tests of Pleural Fluid

What causes glucose levels to decrease in serous fluids?

When the pH is less than 7.0, what does this indicate?

When the pH is less than 6.0, what does this indicate?

What enzyme is elevated in TB and malignancy?

What enzyme is elevated in esophageal rupture and malignancy?

Glucose: Decreased in inflammations and infections, have blood comparison

pH: <7.0 indicates need for chest tubes, <6.0 indicates esophageal rupture (gastric fluid)

Adenosine deaminase elevated in TB and malignancy

Amylase: elevated in esophageal rupture and malignancy

Significance of Chemical Testing of Pleural Fluid

What happens when glucose levels are decreased?

What happens when lactate is elevated?

What happens when triglycerides are elevated?

What happens when pH is decreased?

What happens when ADA is elevated?

What happens when Amylase is elevated?

Glucose is decreased in rheumatoid inflammation and purulent infection

Lactate is elevated in bacterial infections

Triglycerides are elevated in chylous effusions

pH is decreased in pneumonia not responding to antibiotics and esophageal rupture

ADA is elevated in TB and malignancy

Amylase is elevated in pancreatitis, esophageal rupture, and malignancy

Pericardial Fluid

What is the normal amount of Pericardial Fluid?

What are the causes of pericardial effusion?

What does trauma produce?

What are some transudates involved with pericardial fluid?

How do you detect pericardial effusion?

Normally small amount: 10 to 50mL

Permeability of membranes from infection (pericarditis, endocarditis), malignancy, trauma produces exudates

Transudates: hypothryoidism, uremia, immune disorders

Detect by cardiac tamponade (compression) heard by physician

- Most often done in trauma situation. Steering wheel. Airplane crashes. Bleeding in pericardium, physical damage to heart, need surgical intervention to repair immediately

Peritoneal Lavage

Why is this performed and what situation calls for peritoneal lavage?

How is this performed?

What indicates a blunt trauma case?

What other procedures are available?

Performed to detect early abdominal bleeding and need for surgery

Blunt trauma injuries

Normal saline injected into cavity, withdrawn, and red blood cell (RBC) count performed

RBC count > 100,000 indicates blunt trauma case

Radiographic procedures also available

Chemical Tests for Peritoneal Fluid

When glucose levels are below plasma levels, what happens?

When Amylase levels are increased, what happens?

When Alkaline phosphatase levels are increased, what happens?

When BUN, creatinine levels are increased, what happens?

Glucose: below plasma levels = peritonitis and malignancy

Increased amylase: pancreatitis, gastrointestinal perforation

Increased Alkaline phosphatase: intestinal perforation

Increased BUN, creatinine: ruptured bladder, accidental perforation

Physiology of Amniotic Fluid

What produces Amniotic Fluid?

What happens during the first trimester?

When does amniotic fluid peak?

How is increased urine regulated?

What is used as a measure of lung maturity?


- Production: Fetal urine, lung fluid, and maternal circulation

- During the first trimester, approximately 35 mL of amniotic fluid is derived primarily from the maternal circulation

- Increased amniotic fluid peak at 800 to 1200 mL in the third trimester is the result of fetal urine

- Increased urine is regulated by increased fetal swallowing

- Lung fluid adds lung surfactants to amniotic fluid; used as a measure of lung maturity

Chemical Composition of Amniotic Fluid

What is the composition similar to?

What are the cells used for?

What produces the biochemical substances in the fluid and what are some of these substances?

What does fetal urine increase?

What substance is used to determine fetal age and what are the ranges for each amount of the substance?

If a neonate is less than 36 weeks, what could this indicate about the neonate?

Composition similar to maternal plasma with sloughed fetal cells

- Cells are used for cytogenetic analysis

The fluid also contains biochemical substances that are produced by the fetus, such as bilirubin, lipids, enzymes, electrolytes, urea, creatinine, uric acid, proteins, and hormones

Fetal urine increases creatinine, urea, and uric acid

Fetal age can be estimated by creatinine

- <36 weeks = 1.5 to 2.0 mg/dL

- >36 weeks = >2.0 mg/dL

Less than 36 weeks is high risk of neonate and mortality rate goes up

Maternal Urine versus Amniotic Fluid

Why differentiate between the two?

What substances should you measure and what do each amount of substances indicate?

What is the fern test? If there is ferning, what does this indicate? If there is no ferning, what does this indicate?

Needed to determine premature membrane rupture or accidental puncture of maternal bladder from amniocentesis

Measure creatinine, glucose, protein, and urea

- Amniotic fluid has <3.5 mg/dL creatinine and <30 mg/dL urea

- Values as high as 10 mg/dL for creatinine and 300 mg/dL for urea may be found in urine

- The presence of glucose, protein, or both is associated more closely with amniotic fluid

Fern test: specimen air dries on glass slide; examine microscopically for "fern-like" amniotic fluid crystals Old Time Test

- Fern test. Ferning means amniotic fluid and if there is no ferning, it is something other than amniotic fluid

Indications for Amniocentesis

What are some indications for amniocentesis?

Abnormal screening blood tests: maternal alpha fetal protein, human chorionic gonadotropin, unconjugated estriol

Abnormal chromosome analysis and history of genetic disorders

Abnormal ultrasound for fetal body measurements

In later pregnancy for possible early delivery

- Fetal lung maturity, hemolytic disease of the newborn (HDN), infection

Collection of fetal epithelial cells in amniotic fluid indicate the genetic material of the fetus

- Examined for chromosome abnormalities by

1. karyotyping

2. fluorescence in situ hybridization

3. Fluorescent mapping spectral karyotyping

4. DNA testing

Biochemical substances that the fetus has produced

- Analyzed by thin - layer chromatography

- Assessment of fetal lung maturity. We are usually only involved with fetal lung maturity test.

Collection of Amniotic Fluid

What is amniocentesis and what part of the body is this involved with?

What is the maximum amount of amniotic fluid that is safe to extract?

How do you prevent contamination?

Why should you protect specimens from light?

What tubes should you use and how many tubes are usually used?

Amniocentesis: needle aspiration of fluid from amniotic sac

- Transabdominal amniocentesis

Maximum of 30mL collected in sterile syringes

Discard first 2 to 3 mL for contamination

Protect specimens from light for bilirubin analysis for HDN at all times: amber tubes or black plastic tube covers

Usually 2 to 3 tubes. Bilirubin degrades under light.

Specimen Handling and Processing of Amniotic Fluid

When should you perform all handling procedures and where should they be delivered?

Why should you use amber tubes to store specimen?

How should you deliver fetal lung maturity tests and how should you store them?

How are cytogenetic specimens stored?

Why should you centrifuge or filter fluid for chemical tests and what is the only test you should filter for?

Perform all handling procedures immediately, and deliver to laboratory promptly

- Amber tubes to protect bilirubin integrity

Deliver fetal lung maturity tests on ice; refrigerate or freeze up to 72 hours if needed

Cytogenetic specimens kept at room temperature or 37 degrees celsius to prolong cell life

Centrifuge or filter fluid for chemical tests to remove debris; filter only for FLM tests

Color and Appearance of Amniotic Fluid

What is the color and appearance of normal amniotic fluid?

What does a blood streaked amniotic fluid indicate?

What test should you use to differentiate fetal and maternal blood?

What color indicates bilirubin and what does this indicate about the neonate?

What color indicates meconium and what is meconium?

What color indicates fetal death?

Normal amniotic fluid is colorless, with slight to moderate turbidity from cells

Blood streaked: traumatic tap, abdominal trauma, intra-amniotic hemorrhage

- Fetal versus maternal blood: Use kleihauer-Betke

Bilirubin: bright yellow. Hemolytic disease of the newborn (HDN)

Meconium (first bowel movement): Dark green

Fetal death: dark red-brown

Tests for Fetal Distress

What is HDN and what causes this?

Is the mother Rh negative or positive?

Is the newborn Rh negative or positive?

What are some examples of neural tube defects?

What is AFP and when is it produced?

Increased levels of AFP in maternal blood or amniotic fluid indicate a possibility of what?

When are increased levels AFP found?

What should you measure first? Maternal blood or amniotic fluid?


- Most commonly Rh-negative mothers

- Other red blood cell (RBC) antigens can also produce HDN

- Fetal cells with antigens enter maternal circulation and cause production of maternal antibodies

- Maternal antibodies cross the placenta and destroy fetal cells with the corresponding antigen

Neural tube defects

- Alpha fetoprotein (AFP) produced by the fetal liver prior to 18 weeks' gestation

- Increased levels in maternal blood or amniotic fluid indicate possible anencephaly or spinal bifida

- Increased levels are found when skin fails to close over neural tissue

- Measure maternal blood first, then amniotic fluid

- Mother is Rh negative

- Newborn is Rh Positive

Fetal Lung Maturity (FLM)

What is the most common complication of early delivery?

What is the function of lung surfactant?

What happens when there is a lack of lung surfactant?

Most common complication of early deliver is respiratory distress syndrome (RDS)

Lack of lung surfactant, which keeps the alveoli open during inhaling and exhaling

Surfactant decreases the surface tension on the alveoli so they can inflate more easily

Many laboratory tests are available for FLM

Lecithin-Sphingomyelin (L/S) Ratio AKA FLM

What method is this considered?

What is lecithin and when is there an increased production of this?

What is the function of sphingomyelin and when is it produced?

What is the L/S ratio before week 35 and after week 35?

When is preterm delivery considered safe?

How is the test performed?

What are some replacements for the L/S ratio?

Considered the reference method

Lecithin is the primary component of the lung surfactants; increased production occurs after the 35th week

Sphingomyelin is produced at a constant rate after the 26th week and serves as a control for the rise in lecithin

L/S ratio is 1.6 prior to week 35 and rises to 2.0 or greater for alveolar stability after week 35

Therefore, preterm delivery is considered safe with an L/S ratio of 2.0 or higher

Test is performed using thin-layer chromatography

Many laboratories have replaced the L/S ratio with the quantitative phosphatidyl glycerol immunoassays and lamellar body density procedures

Phosphatidyl Glycerol

What is this important for?

This test is usually done with another test. What test is that?

What is amniostat-FLM involved with?

Do blood and meconium interfere with the test?

Lung surface lipid phosphatidyl glycerol is also needed for lung maturity

Normally parallels lecithin, except in diabetics, so must be included in L/S ratio

Amniostat-FLM is an immunologic agglutination test for PG using antibody specific for PG that can replace the L/S ratio (no special equipment needed)

Blood and meconium do not interfere with the test