Analysis of Urine and Body Fluids

LABORATORY | Midterms | Week 3-5

CEREBROSPINAL FLUID
▪ If received another tube, Tube 4, it is the
INTRODUCTION additional test for Microbiology/IS. Most
common additional test for CSF is Micro.
Because CSF is formed by filtration of the plasma one
o Save leftover fluid for additional tests
would expect to find the same low-molecular weight ▪ One attempt only on getting CSF. Once na
chemicals in the CSF that are found in the plasma. natapon, you will be the one responsible as
Abnormal values result from alterations in the medtech. It must be handled with care
o Volume removed based on patient volume and
permeability of the blood-brain barrier or increased opening pressure
production or metabolism by the neural cells in ▪ Usually 20 mL of CSF collected from
response to a pathologic condition. normally individuals who have enough
amount of CSF. Already enough for 3 tubes.
CEREBROSPINAL FLUID ▪ Normal pressure for CSF collection: 50 –
• This is from spinal tap wherein we collect sample on 200 mm of H2O
the lumbar puncture. o Often small volume is collected
• As medtechs, we do not collect sample only practice
or skilled physicians, we only handle and process
the sample
• CSF was first recognized by Cotugno in 1764
• It is the 3rd major body fluid
o 1st is blood
o Major body fluid is found and secreted in body

SPECIMEN COLLECTION AND HANDLING

• Adults: between 3rd and 4th ventricle
o The space between 3rd and 4th ventricle
where the needle is inserted to be
punctured and aspirate CSF.
• Children: between 4th and 5th ventricle
• What if we only receive only 1 tube of CSF in the
• POSITION: Fetal position/ lateral decubital position
lab? This case happens in patients who are hard to
• CSF collected between 3rd, 4th, 5th ventricles
get CSF. As medtechs, first test, we deliver the tube
• Referred to as a spinal fluid tap- CSF from spinal
in micro. Second test, we deliver in Hema. After the
cord or spinal column
second test, we can deliver now the tube in CC/IS.
• In normal individual who can produce enough
(MICRO, HEMA, CC/IS)
amount of CSF, 3 tubes are required
o Rationale behind this is that Micro and Hema is
• Three sterile tubes in this order:
done first because it requires enough volume of
o 1. Chemistry/serology
the sample. What we do on micro is that CSF is
▪ Tube 1 is usually having blood presence to
place and streak on agar plate which requires
be expected. Aspiration is done 90 degrees
enough volume of the sample. CC/IS is done
angle (venipuncture is 15-30 degrees).
last in only 1 tube because it only requires low
Addition of blood can alter result in
amount of CSF.
hematology that’s why it is placed to tube
• Usually STAT requests
3.
o Results must be released within 30 minutes
▪ This is the least interference of false
• Handle carefully/avoid repeat taps
positives and negatives
o Only physician is responsible to this
o 2. Microbiology (avoid skin contamination)
• Preservation:
▪ Possible of getting natural flora or normal
o Chemistry/serology- (Frozen); -20 degree
flora like Staphylococcus epidermidis and
Celsius
can be mistaken as cause of disease in CSF
o Microbiology- (Room temperature)
▪ Why do we collect CSF? We are trying to
▪ Room temp to culture or isolate bacteria
identify the cause of meningitis which is the
specifically for Haemophilus influenzae and
inflammation of meninges.
Neisseria meningitidis. These 2 bacteria that
▪ Meningitis can be bacterial, viral, or fungal
causes meningitis cannot live in frozen or
meningitis.
ref temp.
o 3. Hematology (avoid cells from tap)
o Hematology- (Refrigerate); 2-6 degree Celsius
▪ Can contaminate with blood
▪ Cells can be distorted and cannot identify if
placed in frozen or room temp.

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 CSF

• Extra fluid is frozen • Clot formation

• Always wear gloves; meningitis can be very o Clots present = traumatic tap (plasma)
contagious; centrifuge capped tubes o Hemorrhage does not have enough fibrinogen
o Tubes must be capped because of possible (no clot formation)
aerosol transmission o Other causes of clot formation
▪ Nonbloody CSF = damage to blood-brain
APPEARANCE barrier
o Normal color: Colorless ▪ TB meningitis – formation of web-like
o Clarity: Crystal clear pellicle after refrigeration caused is
o Viscosity: Same as water Tubercular meningitis.
• Terminology: • How can you say that your CSF is Bloody or rich in
o Crystal clear, cloudy/turbid, milky, blood CSF? > 6,000 per/uL
xanthochromic, hemolyzed/bloody
o Cloudy = infection; milky = lipid or protein CELL COUNT
• Xanthochromic/ xanthochromia
o Pink= very slight amount of oxyhemoglobin • Count presents of RBCs, WBCs and Predominant
o Orange= heavy hemolysis Blood cells. Help in identifying the cause of
o Yellow= there is a conversion of meningitis, Bacterial, viral or fungal.
oxyhemoglobin to unconjugated bilirubin o White blood cell (WBC) and total cell count; red
o Due to RBC degradation products blood cell (RBC) count seldom done
o Also jaundice (yellow CSF), ↑ ↑ protein, carotene o Granulocytes lyse within 1 hour; STAT
(Orange CSF) o Normal adult 0-5 WBCs (Predominant CSF cells:
o Pathologic = cerebral hemorrhage Lymphocyte, Ratio is 70 lymphocytes :30
▪ Presence of blood is due to: monocyte)
▪ Traumatic tap or, o Neonates up to 30 mononuclear cells
▪ Skin part is puncture in collection (Predominant CSF cells: Monocyte, Ratio is 70
causing to bleed and put in a sample or monocytes :30 lymphocyte)
▪ There is bleeding in cerebral o If increased of monocytes in adult, it is
pathologic caused.
o Standard Neubauer formula used for counts

CALCULATING CSF CELL COUNTS FOR

HEMATOLOGY

X- xanthochromic
RBC- volume 0.2 mm
TRAUMATIC TAP
• Blood vessel punctured during tap WBC- volume 1 mm
• Differentiate from cerebral hemorrhage
• Uneven blood distribution in tubes with traumatic Modification:
tap
o D-dimer test for hemorrhage
Count 5 large squares on both sides of the chamber =
o Erythrophagocytosis, hemosiderin granules
o Hemorrhage = even distribution in all tubes 10 squares
o Traumatic tap = decreasing tubes 1 through 3
• Xanthochromia 10 squares x 0.1 = 1 μL (volume counted)
o Not present in a recent traumatic tap
o Indicates older hemorrhage Number of cells counted x dilution x 1 μL = cells/μL in
CEREBRAL HEMORRHAGE VS. TRAUMATIC TAP CSF
CEREBRAL TRAUMATIC TAP
HEMORRHAGE
Distribution Even distribution Uneven distribution
of blood
Clot Negative or Absent Positive or Present
formation
Supernatant Xanthochromic or Clear or Not
Common common
Erythrophage positive negative
D-dimer positive negative

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 CSF

Clear No dilution Increased Neutrophils- indicates bacterial

Slightly Hazy 1:10 meningitis and possible cerebral hemorrhage
Hazy 1:20
Slightly cloudy 1:100 Increased Macrophage- due to RBCs in spinal
Cloudy or slightly bloody 1:200 fluid or due to contrast media
Bloody or Turbidity 1:10,000
Increased Blast cells- indicates acute leukemia
• Diluting fluids:
o Total cell count- normal saline or NSS 0.85% Increased Plasma cell- indicates multiple
NaCl sclerosis and lymphocyte reaction
o WBC (routine cell count)- 3 % acetic acid
because it lyses the RBCs Increased Ependymal, choroidal and spindle
o RBC- total cell count subtract to WBC count, shape cells- due to the presents of diagnostic
then that is your RBC count. procedures
• Total cell count
o Clear specimens count undiluted unless
Malignant Cells- due to metastatic carcinoma or
overlapping cells are seen
o Load with transfer pipette primary CNS.
o Dilute with normal saline if necessary
• WBC count
o Dilute with 3% acetic acid; methylene blue (to
check WBC cells) helps to see cells; undiluted
rinse transfer pipette with acetic acid, gently
rotate pipette (To not lyse WBC)

QUALITY CONTROL
• Commercial cell controls are available
• Check diluents for contamination biweekly (twice a
week).
• Soak no disposable chambers in bactericidal
solution for 15 minutes; rinse; clean with isopropyl
alcohol
o Why chamber need to rinse? Need to sterile the
glass because of meningitis
• Monthly check on cytocentrifuge speed and timing

DIFFERENTIAL COUNT
• Very important
• Do not use counting chamber (Used automated or
cytocentrifuge)
• Must centrifuge specimen
• Cytocentrifuge
o Forces cells onto a slide in a monolayer
o Filter paper absorbs moisture
o Positively charged slides to attract cells
o 0.1 mL CSF and 1 drop 30% albumin CHEMISTRY TESTS
(Purpose: in order to have cell yield to have a
greater chance of reading enough cells) CEREBROSPINAL PROTEIN
• Albumin increases cell yield • Normal values differ from blood because of blood-
• Daily control of 0.2 mL saline and 2 drops of albumin brain barrier (very specific; selective; protect the
stained for bacterial contamination brain from any substances that enter the brain;
• Cytocentrifuge produces some cellular distortion small amount of substances enter from blood;
• Count and classify 100 cells if possible circulation is the flow into our veins, vessels located
in the body but pagdating sa brain (blood brain
If increased lymphocyte in CSF- viral, tubercular, barrier) selective lang ang pumapasok sa utak.
fungal meningitis, normal in adult. Hindi pwedeng sobrang daming blood na
nagcicirculate sa brain dahil magkakaroon ng brain
bleeding/damage. Ample/enough amount of
Monocyte- indicates viral, tubercular, fungal
volume/substances is only needed to flow to your
meningitis, normal in children brain)

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CSF
• Total protein is common test
o Normal 15-45 mg/dL (mg, not grams) through
to all proteins in CSF
o Fractions differ, prealbumin (transthyretin) –
transporter of thyroid hormone is second major
protein
o The major protein located in CSF is the
ALBUMIN
o Alpha globulin
▪ Protein present in CSF:(Haptoglobin and
Ceruloplasmin)
o Beta globulin
▪ Protein present in CSF: [Transferrin (the
most predominant in CSF or major
globulin)]
o Transferrin is major beta globulin
o TAU, carbohydrate-deficient transferrin seen in
CSF, not in blood; used to identify CSF
▪ distinctive protein only located in CSF
▪ If we compare CSF in blood sample, the CSF
has TAU protein. This TAU protein is a
carbohydrate deficient transferrin
o IgG major gamma globulin in CSF
▪ Immunoglobulin present in CSF (IgA)
▪ There are protein substances that are not
present in CSF. So, if they are found in CSF
there is an indication that possibly may
nakapasok sa blood brain barrier (there is a
leakage of blood components in the brain).
▪ Proteins that are not located normally in
CSF are:
• IgM
• Fibrinogen
• Beta- lipoprotein
CLINICAL SIGNIFICANCE
• Decreased protein levels = fluid leakage
o (leakage of plasma or blood components in our
blood brain barrier)
• Elevated levels = damage to blood-brain barrier, Ig
production within CNS (this happens in cases of
inflammation, infection possibly the meningitis),
decreased clearance (limited substances present in
CSF and they are cleared inside the CSF because
they are too dangerous in the brain), degeneration
of neural tissue
• Meningitis/hemorrhage most common causes of
increased damage to blood-brain barrier
(nagpapakuha tayo ng CSF in order to determine
what type of meningitis has cause the patient)
• Find abnormal results on clear fluid with low cell
counts from neurologic disorders
METHODOLOGY
• Turbidity/ Turbidimetric measurement
o In a sample magkakaroon ng turbidity because
of the presence of substances or analytes. If the
sample is turbid there is a presence of proteins
o Protein is not specific
o USED 2 SOLUTIONS:
1. SSA (sulfosalicylic acid)
▪ Combined with sodium sulfate and albumin
will contribute more to the turbidity than
globulin
▪ Precipitate more albumin
2. TCE (trichloroacetic acid)
▪ Reagent of choice because it will precipitate
both albumin and globulin
o Trichloroacetic acid precipitates both albumin
and globulin
• Dye – binding
o Coomassie blue dye uses protein error of
indicators principle
o Red to blue: measure intensity of blue color. If
the color changes from red to blue, it indicates
that it has protein in the CSF Protein is not
specific
• Automated instrumentation available
o Pyrogallol red-molybdate
o Specific
*We can also use protein fractions to detect what
fraction of protein is present in CSF which is more
specific. If we want to specify what protein fraction
are present in CSF, we may perform other test such as
Electrophoresis, Radio immunodiffusion, and
Nephelometry
PROTEIN FRACTIONS
• CSF/serum albumin index
o Signifies blood-brain barrier integrity
FORMULA FOR CSF TO ALBUMIN INDEX
CSF albumin (mg/dL) = Index
Serum albumin (g/dL)
Normal value is less than 9 = no damage
If greater than 9 = the blood brain barrier is not
functioning well
• CSF IgG index
o Measure IgG synthesis of immunoglobulin G
(major gamma globulin) in CNS. If sobra sobra
yung production ng IgG it indicates that there is
already an infection happening in CNS.
o Multiple sclerosis (if sobrang daming IgG in
CNS)
o Stimulates immunocompetent cells in CNS
• Values for CSF albumin and globulin available on
automated instruments
FORMULA FOR IGG INDEX
CSF IgG (mg/dL) / serum IgG (g/dL)
CSF alb (mg/dL) / serum albumin (g/dL)
=Index
Normal value = >0.77/ 0.70(latest) indicates normal IgG
production in CNS
If less than 70 = indicates the production of IgG is not
enough in CNS
Variables: IgG, Serum and albumin
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CSF
PROTEIN ELECTROPHORESIS
• Primary purpose or detection for multiple
electrophoresis, comparing the serum and CSF.
• Detection of oligoclonal bands (protein fractions
that migrates in gamma portion and has a gamma
band in CSF electrophoresis) gamma globulins are
the immunoglobulins.)
o If maraming nagmamigrate sa gamma portion
o Many migrations in gamma part it indicates
inflammation within the CNS
o Must run serum electrophoresis
o Multiple sclerosis (MS) = no bands in serum
while have bands in CSF
o Leukemia, lymphoma, viral infection, HIV:
bands in both serum and CSF
o Primary purpose for MS, compare to IgG index
also
o Encephalitis, neurosyphilis, Guillain-Barre, and
neoplasms may give same pattern
o Other diseases associated with oligoclonal
bands MS NENG (Multiple Sclerosis,
Neurosyphilis, Encephalitis, Neoplasm, Guillain-
Baree)
• CONSIDER SYMPTOMS
a. MYELIN BASIC PROTEIN
i. Presence in CSF indicates demyelination of
myelin sheathe (serve as a conductor other
that protection – the nerve impulses from
one neuron to another this myelin sheath
helps the transmission of nerve impulse
becomes faster) around axons of neurons.
If myelin sheath is absent your
reaction/what you feel is delayed. Nauubos
din in some diseases
ii. Monitors the course of multiple sclerosis
iii. Effectiveness of treatment
iv. Immunoassay procedures available (Radio
immunodiffusion/ Radio immuno assay)
CSF GLUCOSE
• Glucose is one of the basic substances needed for
metabolic activity or energy of the cells. The glucose
only enters CSF by selective transport. Mas madami
yung glucose sa blood compared sa CSF.
• Selective transport across blood-brain barrier
Kokonti lang yung glucose na pumapasok sa CSF
fluid
• Approximately 60%-70% plasma glucose
120 mg/dL blood/plasma glucose (compute it: 60-
70 mg/dL present glucose in CSF). Kukunin yung 60-
70% nung glucose to compute it
o Plasma = 100 mg/dL; CSF = 65 mg/dL
o Draw blood 2 hours before tap (draw blood 2
hours before lumbar puncture/ 2 hours before
the doctor collect ng CSF
SIGNIFICANCE
• Very low levels in bacterial meningitis, altered
transport and use by neural cells, not bacteria
• TB meningitis also lowers glucose values and
lymphocytes
• Viral/fungal meningitis, normal glucose and
lymphocytes
CSF LACTATE
• Valuable or useful for antibiotic therapy
• More reliable because it directly measures in the CSF
sample
• Diagnosis and management of meningitis
• TB and fungal levels >25 mg/dL
• Bacterial >35 mg/dL
• Viral <25 mg/dL
• More reliable than glucose
• Levels remain elevated until treatment becomes
effective, then fall rapidly
• No need to compare with plasma
CSF GLUTAMINE
• Produced by brain cells from ammonia and α-
ketoglutarate to remove toxic ammonia. Excess
amount of ammonia to the brain could lead to brain
damage.
• Normal value: 8-18 mg/dL
• Elevated in liver disease
• Determines ↑ ammonia in CSF; after α-
ketoglutarate is depleted = coma
• >35 mg/dL = disturbance of consciousness possible
namacomatose yung patient
• More reliable than blood ammonia
• Increase CSF glutamine is seen in children with
Reye syndrome
CSF ENZYME
1. Lactate dehydrogenase (LDH)
o It depends saang tissue siya located
o LD1-LD6
o Brain tissue = LD1 & LD2
o Lymphocytes = LD2 & LD3
o Neutrophils = LD4 & LD5
2. Creatinine kinase
o CK-BB (enzyme of creatinine in brain)
MICROBIOLOGY TESTS
• Use to know if the cause of meningitis is viral,
bacterial, or fungal
• Gram stain and cultures must be performed on
sediment from centrifuged CSF; cytocentrifuge
helps Gram stains (bacterial meningitis)
• Blood cultures also must be drawn
• Difficult to interpret Gram stains, few organisms and
often debris (mahihirapang magdetect if it is gram
positive or negative dapat enough yung sample)
kaya inuuna yung micro kapag iisang tube lang for
CSF para madetect ng mabuti)
• Organisms: S. pneumocystis, H. influenza, E.
coli, N meningitidis, Listeria monocytogenes, S.
agalactiae
• Cultures also plated on chocolate agar
• TB (very hard to culture) smears very important
because of growth time delay
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CSF

• Latex agglutination tests are available for

o Group B streptococcus, H. influenza, S. pneumocystis, N. meningitidis, and E. coli
• Gram stain is the best for detection
• Compare with hematology and chemistry results to be confident in releasing the results. That’s why we
need to check carefully the correlations between other sections of the laboratory
• Cryptococcus neoformans
o Major causation of fungal meningitis
o Frequent complication of AIDS
o Gram stain produces a starburst pattern
o India ink stain (called Negative stain) on CSF to detect the capsule and budding yeast; beware of air bubbles

o Latex agglutination tests and immunoassay procedures are the most sensitive detection method
o Latex agglutination primary interference is from rheumatoid factor (the rheumatoid factor is positive in latex
agglutination test)
o Always confirm immunologic results with culture in Hematology, IS and CC in order to be confident enough to
release the results

SEROLOGIC TESTING
• Primary test is for neurosyphilis, third stage
• Less performed 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 and
VDRL is recommended for neurosyphilis
• Should be accompanied by a positive serum FTA-antibodies (neurosyphilis)
• Additionally, we can used latex agglutination & ELISA for bacterial antigen

TYPES OF GLUCOSE OTHERS CSF Lactate

MENINGITIS CONCENTRATION

VIRAL Normal Increased WBC <25 mg/dl

(lymphocytes)

TUBERCULAR Decreased Increased WBC >25 mg/dl

(lymphocytes)

BACTERIAL Decreased Increased WBC >35 mg/dl

(neutrophils)
FUNGAL Normal to (+) India ink for >25 mg/dl
decreased C. neoformans

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