MLS 419: AUBF LECTURE - These projections have small, one-way

FINALS WEEK 1 – LESSON 1 valve-like structures that allow the CSF to

Cerebrospinal Fluid enter the bloodstream of the large veins of
the head
Cerebrospinal Fluid (CSF) • Reabsorbed at the Arachnoid villae/granulations
• Discovered by Domenico Felice Cotugno • Where a volume of 90 – 150 ml is maintained
• Composition and f ormation (adults)
- CSF is the 3rd major fluid of the body • 10-60 mL in neonates
- Adult volume: 90-150 mL → CSF formation, circulation, and reabsorption into the blood
- Neonate volume: 10-60 mL make up a dynamic process that constantly turns over about
3 PROCESSES THAT GOVERN THE EXISTENCE OF CSF: 20 mL/hr if the flow path between production and
1. CSF production/f ormation reabsorption of CSF into the blood is obstructed for any
2. CSF circulation reason if there are problems in the 3 processes where CSF
3. CSF reabsorption accumulates producing hydrocephalus
→ Proper maintenance of these three processes = → Intracranial pressure – can also increase causing brain
maintains the proper volume of CSF damage, mental retardation, or death if left untreated.

1. CSF FORMATION
• CSF gates (?) the brain and spinal cord
• Produced primarily about 70% in the highly
vascularized choroid plexuses of the 4 ventricles
- The 4 ventricles: 2 lumbar ventricles, the
third ventricle, f ourth ventricle
- Choroid plexus: vascular f ringe-like f olds
present in the pia mater (directly lines the
brain and the spinal cord; the innermost
layer of the meninges)
- Ependymal cells: line the brain & spinal cord
also play a minor role in the production of CSF FUNCTIONS
CSF
• Supplies nutrients to nervous tissues
- At rate @20 ml/hr (adults)
• Removes metabolic wastes = Glutamine and
• Selective secretion/f iltration process f rom your
Ammonia
plasma under hydrostatic pressure and active
• Protects/cushions the nervous system against
transport and not as an ultraf iltration process
trauma
2. CSF CIRCULATION
BLOOD BRAIN BARRIER (Hematoencephalic barrier)
Brain and spinal cord are surrounded by 3 membranes
• Separates the brain tissues and the blood circulation
collectively termed as the meninges.
• Occurs due to tight f itting endothelial cells that
• Dura mater - the tough outermost membrane is next
prevent passage of larger molecules.
to the bone.
• Arachnoid mater - derives its name f rom its visual
resemblance to a spider web, is the middle layer.
• Pia mater - adheres to the surf ace of the neural
tissues
• CSF f lows through the subarachnoid space (space
between the arachnoid mater and pia mater), where
it gates and protects the delicate tissues of the CNS
• From its initial f ormation in the ventricles, the CSF
circulates all throughout the subarachnoid space • Parts:
towards the brain stem and spinal cord principally 1. Layer of endothelial cells – interconnected
through pressure changes caused by postural, through tight junctions and not containing
respiratory and circulatory pressures. f enestrations that is normally present in
3. CSF REABSORPTION endothelium of other tissues
• Eventually the CSF f lows f rom the subarachnoid 2. Basement membrane – consisting of the basal
space to the top outer surf ace of the brain where lamina of the astrocytes and the basal lamina of
projections of the arachnoid membrane called the endothelial cells
arachnoid granulations are present.
 3. Protrusions of astrocytes – pedicles or ➢ Routinely collected via lumbar puncture between 3 rd
vascular f eet & 4th (or lower in adults), or 4th & 5th (children) lumbar
• Functions of BBB: vertebra under sterile conditions
• Essential to protect the brain f rom pathogens ➢ Do not puncture a locally inf ected area. Avoid
• Blocks chemicals and harmf ul substances inf lamed area.
present in blood Antibodies and medications o To prevent introduction of the inf ection to the
also blocked CNS
o Larger antibodies (IgM) and medications ➢ Perf ormed aseptically af ter thorough cleansing of the
such as penicillin patient’s skin and application of a local anesthetic
o may sometimes pose a problem esp. in the ➢ Needle is advanced into the lumbar intersp ace and
selection of medication to treat brain you will hear a popping sound upon penetration of
diseases. Ensure that the drug chosen for the dura mater
medication is able to penetrate the BBB to ➢ Intracranial pressure (initial or opening pressure)
achieve therapeutic levels in the brain measurement taken bef ore and af ter the f luid is
• Restricts entry of large molecules withdrawn.
• Controls/restricts/f ilters blood components. So: - Measured by a manometer
- CSF composition is unlike blood’s - Opening pressure: 50-180 mm HG
- Theref ore, CSF is NOT an ultraf iltrate of o Slightly higher f rom individuals in sitting
plasma position
CSF dissolved substances o If pressure is in normal range – 20 mL of
Total protein 15-45 mg/dL CSF (saf ely)
Albumin 10-30 mg/dL o If less than or greater than normal – 1-2
IgG 1-4 mg/dL mL of CSF only
Glucose 50-80 mg/dL - Af ter removal of desired volume and bef ore needle
is withdrawn, physician must take the closing
Calcium 2.0-2.8 mEq/L
pressure
Chloride 115-130 mEq/L
- Closing pressure: 10 to 30 mm Hg less than the
Lactate 10-22 mg/dL
opening pressure.
Magnesium 2.4-3.0 mEq/L
Potassium 2.6-3.0 mEq/L
Sodium 135-150 mEq/L
Na, Cl, Mg higher in CSF
Potassium and Calcium Lower
Therefore, not ultrafiltrate of blood
4 Major categories of disease
1. Meningeal infections – bacterial or viral
2. Subarachnoid hemorrhage – or intracranial
CEREBROSPINAL FLUID TUBINGS
hemorrhage/stroke
• Assuming that the CSF volume collected is 20 mL or
3. CNS malignancy
enough
4. Demyelinating disease – e.g. multiple sclerosis;
Specimen collection and handling
destruction of myelin sheath that covers the axons
• Tube 1 – chemistries and serology
Indications for analysis
• To conf irm diagnose of meningitis – one of the most • Tube 2 – microbiology cultures
common • Tube 3 – hematology
• Evaluate f or intracranial hemorrhage • Tube 4 (if may excess) – f or microbiological or
serological studies
• Diagnose malignancies, leukemia
If the amount collected is only 1-2 mL
• Investigate central nervous system disorders – such
→ Prioritize the testing
as multiple sclerosis
→ Microbiology test (top priority)
CSF Fluid Collection
→ Hematology studies (priority #2)
• Lumbar puncture or spinal tap
→ Chemical and serology (last priority)
• Collected by trained physician
Precautions
Specimen collection
➢ Never use glass tubes because cells in CSF adheres to
➢ Lumbar puncture or spinal tap
the glass surf aces = f alsely decreased cell counts
Patient positioning
➢ Specimen potentially inf ectious
➢ Fetal position – to expose vertebra
➢ Testing considered STAT
 Clotted
• indicates increased f ibrinogen, usually due to
traumatic tap, but may indicate damage to blood-
brain barrier.
• Traumatic tap occurs if the needle inadvertently or
unintentionally has entered an epidural vein during
insertion. It gives f resh blood since introduction of
blood only happens during collection.

If immediate processing not possible:

• Tube 1 (chem-sero) f rozen
• Tube 2 (micro) room temp.
Pellicle formation
• Tube 3 (hema) ref rigerated
• Pellicle – thin skin or a cuticle membrane or f ilm, or
ANALYSIS OF THE CSF SAMPLE
a spiderweb f ormation
Physical examination
• in ref rigerated specimen associated with tubercular
Appearance - bedside evaluation (immediately af ter
meningitis.
collection of CSF)
o Pellicle f ormation –
➢ Normal - Crystal clear, colorless
picture at right (pellicle
➢ Descriptive Terms – hazy, cloudy, turbid, milky,
in L. tube, R is normal)
bloody, xanthochromic
o Requires overnight
- Of ten are qualif ied – slight, moderate, marked,
ref rigeration or 12
or grossly.
hours to f orm, at the
➢ Unclear specimens may contain increased lipids,
least
proteins, cells or bacteria. Use precautions.
Milky
• increased lipids
Oily
• contaminated with x-ray media

Traumatic collection vs Cerebral/Intracranial

hemorrhage
When blood is found in the CSF sample, there is need to
Xanthochromia – Yellow discoloration of supernatant (may
determine whether the blood present is due to an actual
be pinkish, or orange).
bleeding in the nervous system (cerebral hemorrhage) or due
• “xantho” = yellow; “chromia” = color to trauma during collection (traumatic tap)
• Due to unconjugated bilirubin Cerebral hemorrhage
• May also be pinkish = slight amount of → old blood in the CSF
oxyhemoglobin; or orange = heavy hemolysis 1. Even distribution of blood in the numbered tubes
• Unique to CSF samples 2. Clot f ormation improbable
• Most commonly due to presence of ‘old’ blood = long ▪ No clots in CSF
standing blood (at least 2 hrs) ▪ Fibrinogen, which is responsible for
o Lysis of blood transpires only af ter 2 hrs and clotting, is not normally f ound in
xanthochromia may only be manif ested in CSF
the CSF if hemolysis already occurred 3. Xanthochromic supernatant
• Other causes include increased serum bilirubin, - RBCs must have been in CSF @ 2+ hours
carotene, increased protein, and melanoma pigment ➢ D-dimer negative, f ibrin degradation product f rom
hemorrhage site
➢ Microscopic presence of erythrophages,
siderophages, or hemosiderin granules
o Take time to f orm
Traumatic tap
→ Fresh blood
→ During puncture
 → Puncture of adjacent blood vessels, will introduce Correction of WBC count for traumatic tap
enough f ibrinogen that will enable CSF to f orm clots contamination
→ Less than 2 hrs contact time of blood and CSF = will • Uses ratio of WBCs to RBCs in blood and compares
not allow hemolysis to happen = no hemoglobin it to same ratio (WBC/RBC) in CSF
degradation = no bilirubin = no yellow • If patient’s peripheral cell counts are normal, can
collor/xanthochromia subtract 1 WBC f or each 700 RBCs counted in CSF.
→ Positive f or D-dimer test • Great chance f or considerable error, makes this of
little value
Expected results
• Normally 0 RBCs/uL regardless of age
• WBCs – depends upon patient’s age
Adult up to 5 mononuclear WBCs/uL
Newborn up to 30 mononuclear WBCs/uL
Hemorrhage Traumatic tap Children (1-4) up to 20 mononuclear /uL
Children (5+) up to 10 mononuclear / uL
Increased numbers Pleocytosis (can be neutrophilic,
lymphocytic, or mixed cell)

NEUBAUER HEMOCYTOMETER/COUNTI NG CHAMBER

- Same as taught in Hematology; apply the same
concepts
Cerebrospinal fluid (CSF) – Procedures - General f ormula f or cell counts:
• All specimens should be examined microscopically – 𝒂𝒗𝒆 . # 𝒄𝒆𝒍𝒍𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 × 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏
hematology # 𝒔𝒒𝒖𝒂𝒓𝒆𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 × 𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒆𝒂𝒄𝒉 𝒔𝒒𝒖𝒂𝒓𝒆
• Stat priority
- RBCs and WBCs begin to lyse in 1 hour, 40%
WBC disintegrates in 2 hrs.
• Electronic counters generally unusable so…
o Manual cell count is needed because the
number of cells present in the CSF are too
low to be counted accurately using
automated cell counters
1. Total Cell Count
• Clear specimens - No dilution usually required
(use saline if needed) Quality Control for CSF processes
• TCC = WBC count + RBC count • CSF controls
2. RBC Count o Should be used regularly to verif y the
accuracy of the techniques
• not routinely perf ormed
o Biweekly – diluents by examination in a
• Perf ormed only during traumatic tap
counting chamber under 400X magnif ication
• RBC Count = TCC - WBC Count
o Monthly – speed of cytocentrif uge using
➢ Standard Neubauer hemocytometer counting
tachometer; timing should be checked with a
chamber – same lang sa Hema
stopwatch
3. WBC counts
• Check techniques
➢ 3% acetic acid can be used to lyse RBC
• Decontaminate all counting chambers in bleach
o Clear – undiluted
water f or 15 minutes.
o Slightly Hazy – 1:10
o Hazy - 1:20 • Rinse thoroughly with water
o Slightly Cloudy - 1:100 • Cleaned again with isopropyl alcohol.
▪ Example: 30 ul sample + 2,970 ul
diluent CSF Differential
o Slightly Bloody – 1:200 CSF Slide Dif f erential
o Bloody – 1:10,000 • Wrights stained smear of concentrated sediment.
➢ Methylene blue staining will improve o Because cells in the CSF are usually at low
visibility – stains the nuclear details of WBC levels
Concentration Techniques:
• Centrif ugation – 5-10 minutes
 • Sedimentation – preserves cellular morphology
• Filtration - preserves cellular morphology
• Cytocentrif uge (cytospin) – f luid is added to a conical
chamber
- Cells are f orced into a monolayer within a
6mm diameter circle on the slide
- Increases number of cells to evaluate,
however, risk of cell distortion f rom the
centrif ugation process. L – lymphocytes & macrophages
- Use of 30% albumin to: reduces cell
distortion and increase yield
mono/macro, segs and lymph

• Count and dif f erentiate 100 nucleated cells.

• Any cell f ound in peripheral blood may be seen in Eosinophils
CSF, other nucleated cells and malignant cells can • Of ten associated with parasitic / f ungal inf ections
also be f ound. • Introduction of f oreign materials into the CSF,
• Cells f rom the tissues f orming the membrane may including medications and shunts
also be seen – ex. Ependymal cells – normally f ound
in the CSF
• Entire smear should be evaluated f or:
- abnormal cells, inclusions within cells,
clusters, presence of intracellular organisms
• Normal dif ferential values
o Adults: 70% lymphocytes, 30% monocytes.
o Children/newborns: reverse is true
Choroidal cells
• Types of cells
• Epithelial lining of the choroid plexus
o Lymphocyte - Normal
• Singular or in clumps
o Neutrophils – Bacterial
o Eosinophil • Nucleoli absent
o Monocyte – Viral Tubercular Fungal • Nuclei have a unif orm appearance
o Plasma Cells - MS
o Macrophages – increase f ollowing CVA
o Ependymal cells, and normal lining cells can
also be seen.
→ Read more about these cells daw in Strasinger
ana si sir ☺
So again...
• Entire smear should be evaluated f or
o abnormal cells Ependymal cells
o inclusions within cells • Normal cells but not of ten f ound, unique to CSF
o clusters • Line the ventricles and neural canal, produce CSF
o presence of f luid
intracellular organisms • Large cell with distinct round/oval nucleus,
sometimes f ound in sheets
o Lymphocytes &
monocyte/macrophages
 • Suspicious/unclassif ied or malignant cells are • Hematoidin crystals
reported as “other” or “unclassif ied” o When the digestion process continued along
o send unstained slide to cytology/pathology f urther in the siderophage, all that will remain
or histopathology for accurate identif ication is the indigestible hematoidin crystal
• Blasts (appearance similar to peripheral blood, o Primarily composed of unconjugated
always consult with hematology bilirubin
specialist/pathologist)

1991 CAP 30 CSF

hematoidin crystal/bilirubin
crystal

CHEMISTRY
• Blood-brain barrier causes selective f iltration
• Abnormal values of CSF analytes (electrolytes,
proteins, glucose, etc.)
• Bottom are malignant cells
o f rom altered permeability
• Top are leukemic cells f ound in CSF
o Increased production
• Remember – we classif y them as ‘other’ or
o Increased metabolism
‘unclassif ied’ and take the slide to the Cerebrospinal fluid (CSF) - Protein
cytologist/pathologist
• Very low compared to serum/plasma
• Normal 15 – 45 mg/dL
o Inf ants – 150 mg/dL
o Immature – 500 mg/dL
• Decreased levels – not signif icant but may signify
CSF leakage
o Otorrhea – if CSF leaks into ear
o Rhinorrhea – if CSF leaks into nose
Cellular inclusions
• Increased levels
• Erythrophage
o Damaged BBB (as in meningitis or
o Remember that whenever there is bleeding
hemorrhage) - allow inf lux of protein towards
in the cerebral spaces, red cells will have to
CSF f rom plasma
be scavenged by macrophages
o Production of immunoglobulins within CNS
o Macrophage that has engulf ed an
(multiple sclerosis)
erythrocyte
o Decreased protein clearance
o Neural tissue degeneration
Protein Fractions
ASCP 21 CSF
• Major CSF Protein – Albumin
erythrophage, with few
iron granules forming • 2nd most prevalent – Transthyretin/Prealbumin
• Alpha globulins – Haptoglobin (f ree Hb),
Ceruloplasmin (copper)
• Beta-globulin – B2 Transf errin or TAU
• Siderophage o Carbohydrate-def icient
o An erythrophage eventually becomes a o Seen in CSF, not in serum
siderophage as the digestion of RBCs takes • Gamma – IgG and IgA
place because we will now have the • Not f ound in normal CSF – IgM (too large),
hemosiderin granules f rom the degradation Fibrinogen, beta lipoprotein
of the RBCs inside

ASCP 6 macrophage,
lymphocyte,
siderophage
CSF Protein Determination ▪ Values greater than 0.70
Total Protein indicate IgG production within
• Turbidimetric – makes use of acids to precipitate the CNS
proteins out the CSF ▪ Rules out possibility of a
o Sulf osalicylic acid (SSA) – Precipitates damaged BBB-associated
albumin more than globulin: add sodium increase of CSF IgG
sulf ate (to enhance precipitation of Electrophoresis
globulins) ➢ Another way to help establish diagnosis of MS is
o Trichloroacetic acid (TCA) – reagent of by subjecting both serum and CSF to
choice; both precipitates albumin and electrophoresis
globulin equally (no need to add extraneous ➢ CSF Electrophoresis f or MS
sodium sulf ate) • Done in conjunction with serum
• Dye-binding electrophoresis
o Used to measure CSF protein, where the • For the detection of oligoclonal bands in the
protein will couple with the dye or other gamma region
molecule that can be easily measured by • Oligoclonal bands – atleast 2 bands seen in
using spectrophotometer CSF with no corresponding bands in the
o Coomassie Brilliant Blue – CHON binds to serum
dye: Red to blue • 2 or more Oligoclonal Bands in CSF but not
▪ The blue color is proportional to the in serum may support diagnosis of MS
amount of protein
▪ f ollows Beer’s Law
o The alkaline biuret procedure has been used
but the Coomassie brilliant blue is pref erred
CSF – MS Panel
Multiple Sclerosis – increased proportion of CNS IgG
→ autoimmune disease
→ symptoms vary among patients because location
and severity can be really dif f erent
→ episodes can last f or days, weeks, or months; in
• Same pattern in: Encephalitis,
between attack, patient may have f ew or no
Neurosyphilis, Guillain-Barre Syndrome,
symptoms at all
and some other Neoplastic Disorders
→ Dif f icult diagnosis, of ten diagnosed af ter everything
• To accurately diagnose MS: correlate other
else has already been ruled out or eliminated
test results (IgG index), clinical symptoms
• In lab testing, increased CSF IgG should be determined if
and manif estations
it was a result of actual production of IgG in CNS (in MS)
or if it was just because serum blood IgG reaching the
Myelin Basic Protein
CSF which can result from a damage BBB
• Abnormal protein that indicates demyelination of
• To distinguish between these 2 probabilities, we compute neuron axons
for the CSF/serum albumin index and the IgG index
• 70% lipid, 30% protein
• CSF/Serum Albumin index - measure
• In MS, there is demyelination or destruction of the
intactness of BBB
myelin sheath releasing the MBP into the CSF
𝑪𝑺𝑭 𝒂𝒍𝒃𝒖𝒎𝒊𝒏 (𝒎𝒈⁄𝒅𝑳 )
= • Not specif ic f or MS; also produced in other
𝑺𝒆𝒓𝒖𝒎 𝒂𝒍𝒃𝒖𝒎𝒊𝒏 (𝒈⁄𝒅𝑳)
demyelinating diseases
o Index less than 9: Intact BBB
• Present only in acute exacerbation of MS
▪ Increase in CSF IgG could have
• Measurement used to monitor course of disease and
been a result of increased
ef f ectiveness of treatment against MS
production in the CNS mismo
(just as is the case of MM)
o Index of 100 indicates: total destruction
of BBB
• IgG levels (both serum and CSF)
o IgG synthesis rate
𝑪𝑺𝑭 𝑰𝒈𝑮 (𝒎𝒈⁄𝒅𝑳 )/ ⁄𝒔𝒆𝒓𝒖𝒎 𝑰𝒈𝑮 (𝒈⁄𝒅𝑳)
=
𝑪𝑺𝑭 ⁄𝑨𝒍𝒃 𝑰𝒏𝒅𝒆𝒙
Cerebrospinal Fluid (CSF) – Glucose o
Has 3 isoenzymes: CK-1.-2, -3
• Selectively transported across blood-brain barrier o
Isoenzyme CK1/ CK-BB f rom brain tissue
• Normal values: 60-70% of blood glucose Following cardiac arrest, patients’ CSF with
o
• STAT procedure, glycolysis reduces level quickly. CK-BB levels <17 mg/dL have f avorable
• Procedure perf ormed as f or and in conjunction with outcome.
blood specimen ▪ If levels are low, patient is doing well
o Measure both CSF and blood glucose DIFFERENTIAL DIAGNOSIS OF MENINGITIS
o Blood glucose must be drawn about 2 hours BY LABORATORY RESULTS
bef ore the lumbar tap to allow time for Bacterial Viral Tubercular Fungal
Increased Increased Increased Increased
equilibration between the blood and CSF
WBC count WBC count WBC count WBC count
• Decreased levels seen in dif f erent types of
Neutrophils Lymphs Lymphs & Lymphs &
meningitis (bacterial, tubercular, f ungal) Monos Monos
o Hypoglycemia Marked ↑ Mod. ↑ Mod-marked Mod-marked ↑
o Brain tumors protein protein ↑ protein protein
o Leukemias Marked ↓ Normal ↓ glucose Normal to ↓
o Damage to CNS glucose glucose glucose
• Increased levels – result of plasma elevation Lactate > 35 Lactate Lactate > 25 Lactate > 25
mg/dL normal mg/dL mg/dL
CSF Lactate + gram stains Pellicle + India
formation ink/nigrosin
inversely proportional to glucose
with
• Normal values = 11-22 mg/dL
Cryptococcus
• Increase as result: neoformans
o Bacterial Meningitis - >35 mg/dL + bacterial +
o Tubercular Meningitis - >25 mg/dL antigen tests immunological
o Fungal >25 mg/dL test for C. neo
o Normal in viral
o For monitoring of treatment f or meningitis *all have increased WBC count but WBC types differ in each
▪ Serum lactate remain elevated ✓ bacterial – neutrophils
during treatment of meningitis but it ✓ viral – lymphocytes
will f all rapidly if treatment is ✓ tubercular and fungal – combination of increased
successf ul (sensitive method to lymphocytes & monocytes
evaluate ef f ectiveness of antibiotic) *remember the inverse relationship of glucose and lactate
o Increased in Hypoxia *C. neoformans is encapsulated, use India ink/nigrosin to
o Xanthochromic and hemolyzed CSF - f alsely demonstrate capsule
elevates lactate CSF - MICROBIOLOGY
CSF Glutamine • to determine causative agent
→ product of ammonia and a-ketoglutarate by the brain cells • Gram stain - routinely perf ormed on all CSF
– a process by the body which removes the toxic metabolic suspected of meningitis
waste (ammonia) from CSF, since ammonia induces coma o Extremely important f or early diagnosis of
• Normal: 8-18 mg/dL bacterial meningitis
• Increased levels associated with liver disorders o CSF should be concentrated: 1500g f or 15
• As the concentration of ammonia in the CSF minutes
increases, the supply of a-ketoglutarate becomes ▪ To increase chance of being able to
depleted; glutamine can no longer be produced to extract the organism present in CSF
remove the toxic ammonia, and coma ensues. ▪ Sediment used f or GS and culture
• Reye’s Syndrome - swelling of the brain and • Even when using concentrated samples, 10% f alse
degeneration of the liver negatives occur thus…
CSF Enzymes • Blood culture should be taken
• Lactate dehydrogenase (LDH or LD)
o Isoenzymes 1 2 3 4 5;
▪ LD1 & LD2 are in brain tissue
▪ LD 2 and 3 = Lymphocytes
▪ LD 4 and 5 = Neutrophil
o Application: ex: in bacterial meningitis, LD4
and LD5 will be most abundant
• Creatine kinase (CK)
Organisms
Birth-1 month S. agalactiae

1 month-5 years Haemophilus influenzae

5 years-29 years
29 years
Inf ants, elderly,
immunocompromised
Immunocompromised -
Fungal
Neisseria meningitidis
S. pneumoniae
L. monocytogenes
Cryptococcus neoformans
Candida albicans,
Coccidioides, or any
opportunistic organism

Limulus Lysate Test

• Diagnosis of meningitis caused by gram-negative
bacteria
• Detection of endotoxin (remember that endotoxin is
Serology
not produced by GP bacteria)
VDRL (Venereal Disease Research Laboratory)
• Reagent: Amebocytes f rom Limulus polyphemus
• CDC recommended f or preliminary detection of
(horseshoe crab)
neurosyphilis (when T. pallidum already reached the
➢ Amebocytes release lysate in the presence of
nervous system)
GNE at 37°C 1 hour
• On CSF test it has low sensitivity, but great
➢ Result is clotting (positive result)
specif icity – it may not detect all cases of
➢ If there is clotting it means that lysate was
neurosyphilis, but a positive result is a true positive
released from the amebocytes = endotoxin is
result
present = causative agent: GN organism such as
FTA-ABS
Neisseria meningitidis
➢ What if L. monocytogenes? Answer = negative, • Fluorescent treponemal antibody absorption test
because it is a GP organism • Used to detect antibodies f ormed against T. pallidum
• also used on CSF
• more sensitive than VDRL, but its use in the
diagnosis of neurosyphilis is still controversial as it is
extremely sensitive to the ef f ects of blood
India ink contamination.
• Negative stain to view the encapsulated • Must prevent blood contamination.
Cryptococcus neoformans Rapid Plasma Reagin
- of ten AIDS/immunocompromised complication • not recommended
• remember that C. neof ormans is encapsulated, • Not that sensitive compared to VDRL
when the specimen containing the organism is mixed
w/ the india ink and when viewed under the
microscope = capsule pushes away stain
• Instead of stain, can also use dark f ield microscopy
f or same ef f ect
• Gram stain - starburst pattern
• Latex agglutination tests to detect the f ungal antigen
in serum and CSF
- More sensitive
- Should be conf irmed by culture and…India ink
- Rheumatoid f actor interf erence - False Positive
results
• Naegleria fowleri
- Observe trophozoite in CSF sample
- Motile in wet preparation
- Nonmotile in cytocentrif uged stained smears
with increased WBC
 MLS 419: AUBF LEC
FINALS WEEK 2 – LESSON 2
SYNOVIAL AND SEROUS FLUIDS
SYNOVIAL FLUID
- “Joint fluid”
PHYSIOLOGY
- Viscous fluid found in the cavities of the movable joints
or synovial joints.
- Formed as an ultrafiltrate of plasma
- Its primary functions:
o Lubricate the joints during movement
o Provides nutrients to the articular cartilage
o Provides cushion and lessens the shock of joint
compression that occurs during activities such
as walking and jogging.
- The bones are lined with smooth articular cartilage
(reduces friction) and separated by a cavity containing
the synovial fluid.
- The joint is enclosed in fibrous joint capsule lined by
the synovial membrane.
- The synovial membrane contains specialized cells –
“synoviocytes”
o The synoviocytes secrete a mucopolysaccharide
containing hyaluronic acid and a small amount
of protein → VISCOSITY
- Damage to the articular membranes (articular cartilage)
produces pain and stiffness in the joints → ARTHRITIS
o Synovial fluid can be used to determine the
pathologic cause of arthritis.
▪ This is why we examine synovial fluid as
a miscellaneous body fluid in clinical
microscopy
o Infection, inflammation, metabolic disorders,
trauma, physical stress, and advanced age are
associated with arthritis
o Types of Arthritis:
▪ Rheumatoid arthritis
▪ Osteoarthritis
- Articular cartilage – over time, as we age, articular
cartilages wear off; what remains is the end of the bones
- Once the end of the bones touches the other end, it will
result into pain and stiffness
- Synovial membrane – contains the synoviocytes; where
synovial fluid is secreted
SPECIMEN COLLECTION AND HANDLING
- In disease states, there is a tendency that the synovial
fluid will have an increased amount in joints
- Needle aspiration – “arthrocentesis”
- Normal amount is <3.5ml
- Inflammation = increased
- Amount of fluid extracted will
depend on the extent of the
condition
- Record amount collected
- Healthy joint: the articular cartilage is intact
- Arthritic joint: loss of cartilage smoothness and resiliency;
loss of synovial fluid viscosity
ARTHRITIC AREAS OF THE BODY
- Spine
- Hip
- Knee
- Foot
- Hand
- Normal synovial fluid does not clot because it is viscous
due to the presence of hyaluronic acid and a small
amount of protein. However, disease fluid may clot due
to fibrinogen.
- Needle must be moistened with heparin
- Distributed into the following sterile tubes:
o Tube 1 = heparinize for gram stain & culture
o Tube 2 = heparin/EDTA for cell count
o Tube 3 = non-anticoagulated tube for other tests
o Tube 4 = sodium fluoride for glucose test
- Powdered anticoagulant should not be used because
they produce artifacts that interfere w/ crystal analysis
- Note: It is the physician that usually collects synovial
fluid. Medical technologists assist the physicial in the
collection because we provide the tubes and perform the
diagnostic procedure
- Among the different tubes, the first that you have to
process is Tube 3 (Non-anticoagulated tube)
o Non-anticoagulated tube must be centrifuged
immediately and separated to prevent interfering
from chemical and serologic test
- Perform test ASAP to prevent cellular lysis and changes
in crystals.
- Deeper yellow = presence of inflammatory and non-
inflammatory effusions
- Greenish tinge = bacterial infection
- Turbidity = associated w/ the presence of WBC,
including cell debris and fibrin
o Healthy joint: Clear synovial fluid
- = crystals
PHYSICAL EXAMINATION / GROSS APPEARANCE - WBC count <200 cells/uL = normal, may reach
- Color and Clarity 100,000/uL in severe infection
o Colorless to pale yellow
o “Synovial” DIFFERENTIAL COUNT
▪ Latin for egg - Cytocentrifuged preparation or thinly smeared slide
▪ Resembles egg white incubated w/ hyaluronidase
- Monocytes, lymphocytes, macrophages and synovial
tissue cells are the primary cells seen.
- Normal:
o Neutrophils= <25%
o Lymphocytes = <15%
- ↑ Neutrophil = septic condition
- ↑ cell count w/ predominance of lymphocyte suggests a
non-septic condition
- Eosinophil >2% = allergic disease w/ arthritis, parasitic,
o (L→R) Normal, Class I (cloudy/slightly turbid), Class II
(very turbid), Class III (bloody w/ fibrinogen), TB, Rheumatoid arthritis, Lyme disease., Hemorrhagic
Hemorrhagic type, Gouty arthritis

- Images of Abnormal SY fluid: CRYSTALS

Inflammatory Septic - Monosodium urates (MSU) = gout (Gouty arthritis)
- Calcium pyrophosphate dehydrate (CPPD) =
pseudogout.
- Cholesterol = chronic effusion
- Corticosteroid = intra-articular injection
- Calcium oxalate = renal dialysis
- Calcium phosphate (Apatite) = osteo- and
- Bloody - Infected with inflammatory arthritis
microorganisms - Image of Crystals:
- Pus-like or milky Monosodium urates Calcium pyrophosphate

Cholesterol Calcium oxalate

CHEMICAL TEST
- Glucose = should be interpreted using serum FBS.
<10mg/dL lower than serum levels
MICROSCOPIC EXAM / CELL COUNTS - Protein = 1-3 g/dL
- Total WBC count is the most frequently performed cell - Uric acid = 6-8 mg/dL, helpful in diagnosis of gout.
count. Performed in lab that do not have polarizing microscope
- RBC count are seldom requested primarily because you
can observe the color of the synovial fluid MICROBIOLOGIC EXAMINATION
o Red = Fresh RBCs - Infectious agent can enter the synovial fluid
o Rusty brown = Disintegrated RBCs - Bacteria – Staphylococcus, Streptococcus, Neisseria,
- Counts must be performed ASAP Tuberculosis (TB)
- Very viscous should be liquefied = a pinch of - Fungi, viruses
hyaluronidase to 0.5ml of synovial fluid + 1gtt. Of - Gram stain = dilute w/ saline + centrifuge/ cytocentrifuge
0.05% hyaluronic acid in phosphate buffer/ml of fluid + smear
+ incubate at 37°C for 5 mins - Culture – to isolate the type of bacteria that might be
- Undiluted for clear samples → count all squares same w/ present
CSF
- Turbid samples → NSS is used as diluent but to lyse SEROLOGIC TEST
RBC = hypotonic NSS (3%) + saponin is an ideal DF - Determination of rheumatoid factor (RF) in serum or
synovial fluid
- Not specific because present in other diseases such as - Exudate
lupus erythematosus (LE), endocarditis, TB, syphilis, o Effusion caused by damage to mesothelial lining
viral infection, infectious mononucleosis, serum of the membranes.
sickness, etc. o Ex: infection or malignancies
- However, RF has been detected in approximately 75%
of clinically diagnosed rheumatoid arthritis cases.

ARTHRITIC EXTREMETIES

SEROUS FLUID
- Pleural, Pericardial, Peritoneal

FORM AND FUNCTION

- A fluid that is formed within the closed cavities of the body
– pleural, peritoneal, pericardial cavities.
o Pleural – circulates throughout the lung cavity
o Peritoneal – circulates in the abdominal cavity
o Pericardial – circulates in the cardiac cavity
DIFFERENTIATION OF TRANSUDATES AND EXUDATES
- Each is lined by 2 continuous membranes – parietal and
the visceral
- Mesothelial cells – secrete the serous fluid
- Serous fluid – secreted by the membrane at a constant
rate and circulates between the membranes
- VOLUME:
o Pericardial = 10-50ml
o Pleural = 1-15ml
o Peritoneal = 5-20ml, but increases during
ovulation of up to 50ml. Excess fluid is drained
by the lymphatic vessels
- Formed as ultrafiltrates of plasma. Production and
reabsorption are subject to hydrostatic and oncotic
pressure
- Its primary function is to lubricate by preventing
friction between two membranes that occurs as a
result of movement of enclosed organs.

PATHOLOGY
Criteria Transudate Exudate
- EFFUSION – is any disruption in the mechanism of
formation and reabsorption that causes an increased in Appearance Clear Cloudy
fluid production in the membranes Specific gravity <1.016 >1.016
o Brought about by several factors: Total protein <3.0 g/dL >3.0 g/dL
▪ Increased Hydrostatic pressure Cholesterol <60 mg/dL >60 mg/dL
▪ Decreased oncotic pressure Lactate
▪ Increased capillary permeability dehydrogenase <200 IU >200 IU
▪ Lymphatic obstruction (LDH)
- Two types of Effusion: Fluid : serum
<0.5 >0.5
o Transudates and Exudates protein ratio
▪ Classification is a valuable initial Fluid : serum
<0.3 >0.3
diagnostic step and aid in the course of cholesterol ratio
further laboratory testing, because it is Fluid : serum
<0.6 >0.6
usually not necessary to test transudate LDH ratio
fluids. Cell count <1000/μL >1000/μL
- Transudate Spontaneous
No Possible
o Effusion that is caused by disruption in the clotting
formation and reabsorption of fluid (systemic
disorder). SPECIMEN COLLECTION AND HANDLING
o Ex: Congestive heart failure (CHF), nephrotic - Must be collected in 3 sterile tubes
syndrome; accumulation of ascites or acetic fluid - Collected by needle aspiration from the respective
in the peritoneal cavity cavities = thoracentesis (lungs), pericardiocentesis
 (heart), paracentesis (peritoneum)
- EDTA evacuated – cell counts & differential counts;
- Sterile heparinized evacuated – microbiology &
cytology;
- Heparinized evacuated – Chemistry test
o Chemical test performed on serous fluid are
frequently compared w/ plasma concentrations.

PHYSICAL EXAM – GROSS APPEARANCE

- COLOR – clear to pale yellow
- Dark red – Haemorrhagic
- Bright red – traumatic
- – chylous or pseudochylous
- Cloudy or turbid – WBC CHEMICAL TEST
- Appearance will vary according to pathologic condition - Pleural
and type of fluid. o pH – aid in diagnosing oesophageal rupture =
6.0 or lower than 7.2 will need a intercostal tube
MICROSCOPIC EXAM drainage + antibiotic therapy
- CELL COUNT – similar to other body fluids o Glucose – decrease in rheumatoid inflammation
o Undiluted if clear – count all squares & purulent infection.
o Dilute if bloody – use DF (3% acetic acid) to o Lactate – bacterial infection
lyse RBCs and count WBC squares. o Triglycerides – chylous effusion
- Differential counts are routinely performed o Adenosine deaminase (ADA) – TB &
o Centrifuged or Cytocentrifuged malignancy
o Smear preparation o Amylase – pancreatitis, ER, malignancy
o Wright’s stain - Pericardial
o Smears examined not only for WBCs, but also o Test is directed to differentiate Transudate from
for normal or malignant tissue cells Exudate
o Any suspicious cells must be referred to the o Glucose – decrease in bacterial pericarditis &
pathologist non-septic inflammation due to rheumatoid
- Primary cells – macrophages, neutrophils, disease or malignancy.
lymphocytes, eosinophil, mesothelial cells, plasma cells, - Peritoneal
and malignant cells o Glucose – decrease in TB peritonitis,
malignancy
o Amylase – Increase in pancreatitis,
gastrointestinal perforation
o Alkaline Phosphatase – Increase in
gastrointestinal perforation
o ADA – TB peritonitis and malignancy

MICROBIOLOGIC AND SEROLOGIC EXAM

- Gram stain
- Acid-fast stain
- Culture and Sensitivity
- Pleural
o ANA& RF-
o CEA
o CA125
o CA549, CA15.3
o CYFRA 21-1
- Peritoneal
o CEA
o CA125
 MLS 419: AUBF LECTURE
FINALS WEEK 3 – LESSON 3
Amniotic Fluid Analysis
AMNIOTIC FLUID ANALYSIS
Three primary reasons:
1. To enable antenatal (before birth) diagnosis of genetic
and congenital disorders early in fetal gestation
(15-18 weeks)
a. Amniotic Fluid contains cells of the fetus ⇒
cells have DNA ⇒ use for genetic analysis
2. To assess fetal pulmonary maturity later in pregnancy
(32-42 weeks) by measuring the amount of
surfactants (lecithin, sphingomyelin, and
phosphatidylglycerol)
a. Before birth, the fetus’ lungs must be matured
already ⇒ to accommodate oxygen ⇒ to
breathe
3. To estimate and monitor the degree of fetal distress
caused by isoimmunization or infection
a. Isoimmunization: for RH compatibility
AMNIOTIC FLUID
➔ liquid medium that bathes the fetus throughout
gestation (period of time between conception and birth;
pregnancy)
➔ provides information about the metabolic process
taking place during fetal maturation
◆ Amniotic fluid: can best represent the fetus
➔ present in the amnion
◆ membrane composed of a single layer of
cuboidal cells cuboidal epithelial cells that
surrounds the fetus and is filled with amniotic
fluid
◆ metabolically active
◆ involved in the exchanges of water and
chemicals between the amniotic fluid, the fetus,
and the maternal circulation
◆ produces peptides growth factors that are
needed to support fetal growth and cytokines
such as interleukin 6 and interleukin 8
➔ fetal cells and many biochemical compounds such as
electrolytes, nitrogenous compounds, proteins,
enzymes, lipids, and hormones are present in the
amniotic fluid
AMNIOTIC FLUID FORMATION
➔ amniotic fluid formation and its composition change
throughout fetal gestations
➔ initially produced by the amnion and the placenta
➔ as gestation progresses, the fetus eventually plays
more of an active role in the composition of the fluid
➔ water and solutes exchange between the fetus and its
surrounding medium through different mechanisms:
◆ intestinal absorption following fetal swallowing of
amniotic fluid
◆ capillary exchange in the pulmonary system as
the alveoli of the fetal lungs are bathed with
amniotic fluid
◆ fetal urination
➔ Fetal CSF may reach amniotic fluid in neural tube
defects (spina bifida and anencephaly)
◆ check for 2 biochemical markers:
alpha-fetoprotein (AFP) and
acetylcholinesterase (AChE)
➔ Early gestation
◆ a transudate passes through the skin of the fetus
and makes a small contribution to the amniotic
fluid volume
➔ Later stages of pregnancy
◆ fetal swallowing and urination plays a major role
in the volume and composition of amniotic fluid
● the fetus swallows amniotic fluid removing
water and electrolytes ⇒ replace through
urination with metabolic byproducts such as
urea, creatinine, and uric acid ⇒ the fetus
swallows then the fetus urinates ⇒ the
cycle continues
◆ maternal plasma also has contributions
● maternal plasma will remove metabolic
waste products from the amniotic fluid then
the maternal circulation will replenish them
with water, nutrients, and electrolyte
(mother’s love)
● whole ongoing dynamic equilibrium results
in a complete exchange of the amniotic
fluid volume every 2-3 hours
➔ Fetal pulmonary surfactants get mixed in amniotic fluid
◆ fetal respiration
◆ fetal pulmonary surfactants produced by the
alveolar cells of the fetal lungs get mixed and can
be evaluated using amniotic fluid
❏ 1st trimester (first 3 months)
❏ 35 mL of AF is from maternal circulation
❏ Latter 3rd to half period, the fetus will secrete lung
surfactants
❏ After 1st trimester
❏ fetal urine is the major contributor to the AF
volume
❏ when fetal urine production occurs, fetal
swallowing of the amniotic fluid also begins
❏ maintains the volume
 FUNCTIONS OF AMNIOTIC FLUID ➔ Late 2nd Tri to 3rd tri
➔ protective cushion for the fetus ◆ fetal lung maturity, fetal distress, Hemolytic
➔ allows fetal movement Disease of the Newborn, and infections
➔ stabilizes temperature
➔ permits proper lung development

AMNIOTIC FLUID VOLUME

➔ balance between the production of fetal urine and lung
fluid
➔ absorption from fetal swallowing and
intramembranous flow
◆ transfer of the amniotic fluid water and solute to
the fetal vascular system
➔ volume increases steadily throughout pregnancy
◆ 25 to 50 mL at 12th week of gestation
◆ 800 to 1200 mL at 37th week
◆ gradually decrease before delivery

Polyhydramnios
➔ > 1200 mL
➔ decreased fetal swallowing, neural tube defects, and
excessive fetal urination
➔ secondarily associated with fetal structural anomalies,
cardiac arrhythmias, congenital infections, or
chromosomal abnormalities

Oligohydramnios
➔ < 800 mL
➔ membrane leakage, UT deformities, increased fetal HANDLING
swallowing, and umbilical cord compressions ➔ Test for fetal lung maturity – iced on delivery then
➔ associated with congenital malformations, premature refrigerated or frozen up to 72 hours
rupture of amniotic membranes, and umbilical cord ◆ To preserve surfactants & phospholipids
compression ⇒ results in decelerated heart rate ⇒ ➔ Cytogenetic and microbial studies
death ◆ RT or BT
➔ Test for Hemolytic Disease of the Newborn
COLLECTION (AMNIOCENTESIS) ◆ protected from light (amber bottles or cover with
➔ needle aspiration into the amniotic sac aluminum foil)
➔ methods: transabdominal & vaginal amniocentesis ➔ Fluid should be separated ASAP from cells and debris
◆ common: transabdominal amniocentesis with to prevent further metabolism
ultrasound ◆ Centrifugation
◆ risky: vaginal amniocentesis ◆ Filtration recommended for FLM methods to
➔ 10 - 20 mL (30 mL MAX) can be collected in sterile avoid loss of phospholipids
syringe
➔ discard first 2-3 mL DIFFERENTIATING MATERNAL URINE FROM AMNIOTIC
➔ transfer to plastic sterile containers after collection FLUID
◆ don’t use glass as cells in the amniotic fluid may
ANALYTE AMNIOTIC MATERNAL
adhere to the glass walls
PROTEIN + -
INDICATIONS AND HANDLING
INDICATIONS GLUCOSE + -
➔ Screening blood tests are abnormal:
◆ Maternal serum AFP UREA <30MG/DL >300 MG/DL
◆ Triple screening test
CREATININE <3.5MG/DL >10MG/DL
◆ Quadruple screening test
➔ 2nd trimester (15th-18th week) collection FERN TEST + -
◆ asses genetic defects
➔ Differentiate between amniotic fluid and maternal urine
to determine possible premature membrane rupture
which allows amniotic fluid leakage or accidental
puncture of the maternal bladder during amniocentesis
◆ physical appearance cannot differentiate the
two; use protein or glucose
◆ If the mother has diabetes mellitus and renal
disease, expected positives protein and
glucose ⇒ measure for urea and creatinine ⇒
renal function of the fetus inside is still not that
completely developed compared to an adult
➔ Fern test: evaluates premature rupture of the
membranes
◆ vaginal fluid on the glass slide
◆ air dry at RT
◆ fern-like crystals/ferning (due to the protein and
sodium chloride content) upon microscopy (AF)
◆ facilitates the production of unconjugated
bilirubin (present in amniotic fluid)
➔ uses spectrophotometer to measure absorbance or
optical density
➔ normal optical density of AF: Increased (highest) at 365
nm then decreasing linearly up to 550 nm BUT if…
◆ bilirubin present: increases at 450 nm
➔ AF bilirubin concentration = difference in the OD of
theoretic baseline and OD at 450 nm
➔ Oxyhemoglobin (410nm) and meconium (between
350nm and 400 nm) interfere with analysis

COLOR AND APPEARANCE

1. Colorless to pale yellow with slight to moderate
turbidity: normal
2. Blood-streaked: traumatic tap, intraamniotic
hemorrhage, abdominal trauma
a. Pale pink to red
b. Centrifuge immediately because hemolysis
causes the formation of oxyhemoglobin ⇒ can
interfere with several biochemical tests
3. Dark red-brown: fetal death
4. Dark green: meconium
a. newborn's first bowel movement; a gelatinous
or mucus-like material that forms in the fetal
intestine as the result of swallowed amniotic
fluid and fetal intestinal secretions
b. green: contains biliverdin
c. findings: fetal distress
5. Dark yellow/yellow: HDN (bilirubin)
➔ the absorbance or the optical density is plotted against
the wavelength
➔ OD: measured in intervals between 365 nm and 550 nm
➔ the readings are plotted on a semi-logarithmic graph
paper
➔ OD should be decreasing (diagonal line)
➔ when bilirubin is present, arise in optical density should
be seen at 450 nm
➔ the amount that the curve deviates from the straight line
at 450 nm is directly proportional to the amount of
bilirubin in the amniotic fluid
➔ the od difference will just be plotted on a lily graph

TESTS FOR FETAL DISTRESS

Refers to signs before and during childbirth indicating that
the fetus is not well

1. Amniotic Fluid Bilirubin/A450/OD450

➔ A- absorbance; 450- wavelength or change in the
absorbance at 450 nanometer
➔ test for Hemolytic Disease of the Newborn
(Erythroblastosis Fetalis) ➔ 3 zones
◆ Results when an Rh-negative mother carrying ◆ Zone I- the fetus is minimally affected
an Rh-positive child⇒ maternal antibodies will ◆ Zone II- moderate hemolysis
attack the Rh-positive RBCs of the fetus inside ◆ Zone III- severe hemolysis and may die
⇒ hemolysis ⇒ fetal anemia
2. Neural Tube Defects (NTD) 1. Lecithin/Sphingomyelin Ratio (L/S Ratio)
➔ detected by maternal serum AFP, ultrasound, ➔ Reference method
amniocentesis ➔ Lecithin:
➔ Alpha-fetoprotein (AFP) ◆ major surfactant produce
◆ major protein produced by the fetal liver during ◆ produced at a relatively low and constant rate but
early gestation production increases during 35th week
◆ found both in the maternal serum and the ➔ Sphingomyelin: constant production after 26 weeks of
amniotic fluid because of natural exchange gestation
processes ➔ Before 35th week, L/S ratio is <1.6
➔ Indications for Amniotic Fluid AFP analysis: ➔ ≥ 2.0 L/S ratio: safe pre-term delivery = Mature Fetal
◆ Maternal serum AFP is high Lung
◆ Family history of NTDs ➔ Falsely Increase: meconium or blood
➔ High AFP: anencephaly and spina bifida (most ➔ Method: Thin-Layer Chromatography (TLC)
common)
◆ No closure of the fetal skin covering the neural 2. Phosphatidylglycerol
tissue and will result in leakage of AFP into the ➔ after 35 weeks
amniotic fluid ➔ Should parallel the production of lecithin
➔ delayed in maternal DM: RDS still occurs even if L/S
ratio is 2
➔ TLC: lecithin, sphingomyelin, PG

Amniostat-FLM
➔ immunologic test for phosphatidylglycerol
➔ using polyclonal anti-PG antibodies
➔ size of agglutinates
◆ Negative - immature fetal lungs
◆ Low Positive or High Positive - mature fetal
lungs
◆ Anencephaly- a serious birth defect in which a ➔ advantage: not affected by blood or meconium
baby is born without parts of the brain and skull
◆ Spina bifida- occurs when the spine and the
spinal cord do not form properly
➔ Low AFP: Down Syndrome
➔ next step: amniotic acetylcholinesterase (more specific)
◆ NV: <2.0 MoM

TESTS FOR FETAL LUNG MATURITY (FLM)

➔ Fetal distress, whether caused by HDN or other
conditions, forces the obstetrician to consider a preterm
delivery. At this point, fetal maturity must be assessed
➔ Pulmonary system is one of the last organ systems to
mature

Respiratory Distress Syndrome (RDS)

➔ Also called hyaline membrane disease 3. Foam Shake
➔ Most common cause of death in newborn ➔ AF + 95% ethanol
➔ Results from insufficiency of lung surfactant production ➔ shake, 15 secs.
➔ Surfactants: ➔ stand, 15 mins.
◆ stored in lamellar bodies (90% phospholipid, ➔ presence of bubbles/foam around the outside edge (+)
10% protein) ◆ presence of sufficient surfactants in the
◆ produced by Type II Pneumocytes amniotic fluid will allow the amniotic fluid to form
◆ allow the alveoli of the fetus to take in and foam when shaken (foam shake)
breathe out gases without collapsing ◆ 95% ethanol- anti-foam agent
➔ Low surfactants result in collapsed alveoli ◆ Positive- still form foam despite the presence of
ethanol
➔ Modification: Foam Stability Index
 Foam Stability Index 5. Lamellar Bodies Count
➔ LB diameter is similar to platelets
◆ automated hematology analyzer
◆ impedance or optical methods
➔ > 50,000 uL: FLM, <15,000 uL: immature

ADVANTAGES OF LBC
a) Rapid turnaround time
b) Low reagent cost
c) Wide availability
d) Low degree of technical difficulty
e) Low volume of amniotic fluid required
f) Excellent clinical performance

6. Dipalmitoylphosphatidylcholine
➔ Prepare 14 test tubes
➔ DPPC test
➔ 0.42-0.55 mL (increasing ethanol)
➔ Not common
➔ Add 0.5 mL of amniotic fluid for each tube
➔ 100% sensitivity, 96% specificity
➔ Result: highest conc of ethanol with stable foam is
the FSI for that specimen
➔ Example:
◆ With stable foam: 0.42-0.43
◆ No foam: 0.44-0.55
◆ Answer - FSI: 0.43

4. Tests for Amniotic Fluid Microviscosity

➔ obsolete
➔ evaluates the amount of pulmonary surfactants relative
to albumin
➔ IDEA: the presence of phospholipids decreases
microviscosity
◆ directly related
➔ PRINCIPLE: Fluorescence Polarization assay
➔ INTERFERENCES: blood, meconium, maternal urine,
and icteric samples

Lamellar Bodies (Recall)

➔ Storage of pulmonary surfactants
➔ Secreted by Type II pneumocytes at 24th week
➔ Enter the amniotic fluid at 26th week
➔ Increases in concentration from 50,000 uL to 200,000
uL at the end of the 3rd trimester
➔ Increased lamellar bodies: increased AF phospholipids
and L/S ratio
➔ Can be measured at OD 650