CHAPTER

CEREBROSPINAL, SYNOVIAL,
SEROUS BODY FLUIDS, AND 29 
ALTERNATIVE SPECIMENS
Donald S. Karcher, Richard A. McPherson

CEREBROSPINAL FLUID, 481 Transudates and Exudates, 498 Specimen Collection, 504
Specimen Collection and Opening Recommended Tests, 498 Recommended Tests, 504
Pressure, 481 Gross Examination, 499 Gross Examination, 504
Indications and Recommended Microscopic Examination, 499 Microscopic Examination, 505
Tests, 482 Chemical Analysis, 500 Chemical Analysis, 505
Gross Examination, 482 Immunologic Studies, 501 Microbiological
Microscopic Examination, 483 Microbiological Examination, 506
Chemical Analysis, 486 Examination, 502 ALTERNATIVE SPECIMENS, 507
Microbiological Examination, 491
PERICARDIAL FLUID, 502 Saliva, 507
SYNOVIAL FLUID, 493 Specimen Collection, 502 Meconium, 507
Specimen Collection, 494 Gross Examination, 502 Hair and Nails, 507
Recommended Tests, 494 Exudates and Transudates, 502 Breath Testing, 507
Gross Examination, 494 Microscopic Examination, 502 Tissue Aspirates, 507
Microscopic Examination, 494 Chemical Analysis, 503 Billing for Tests in Nonstandard
Chemical Analysis, 497 Immunologic Studies, 503 Specimens, 508
Immunologic Studies, 497 Microbiological CHEMICAL MEASUREMENTS IN
Microbiological Examination, 497 Examination, 503 BODY FLUIDS, 508
PLEURAL FLUID, 498 PERITONEAL FLUID, 503 SELECTED REFERENCES, 508
Specimen Collection, 498 Transudates and Exudates, 503

transported to the cells of the median eminence; and (5) it maintains

KEY POINTS
• Determining the etiologic cause of fluid accumulation in various body
cavities (i.e., joints, chest, abdomen) is critical for proper treatment
of these disorders.
• Appropriate laboratory examination of these fluids is therefore critical
for the diagnosis of numerous diseases (i.e., bacterial, viral, and
fungal infections; distinction between various arthritides; primary
[i.e., mesothelioma] and metastatic malignancies; among others).
• Accurate test interpretation depends on appropriate specimen
collection, physician/laboratory communication, analytically sound
laboratory methods, and reliable reference values.
CEREBROSPINAL FLUID
In adults, approximately 500 mL of cerebrospinal fluid (CSF) is produced
each day (0.3 to 0.4 mL/min). The total adult volume varies from 90 to
150 mL, about 25 mL of which is in the ventricles and the remainder in
the subarachnoid space. In neonates, the volume varies from 10 to 60 mL.
Thus, the total CSF volume is replaced every 5 to 7 hours (Wood, 1980).
An estimated 70% of CSF is derived by ultrafiltration and secretion
through the choroid plexuses. The ventricular ependymal lining and the
cerebral subarachnoid space account for the remainder. CSF leaves the
ventricular system through the medial and lateral foramina, flowing over
the brain and spinal cord surfaces within the subarachnoid space. CSF
resorption occurs at the arachnoid villi, predominantly along the superior
sagittal sinus.
The CSF has several major functions: (1) It provides physical support
because a 1500-g brain weighs about 50 g when suspended in CSF; (2) it
confers a protective effect against sudden changes in acute venous (respira-
tory and postural) and arterial blood pressure or impact pressure; (3) it
provides an excretory waste function because the brain has no lymphatic
system; (4) it is the pathway whereby hypothalamus releasing factors are
central nervous system ionic homeostasis.
The concept of the blood-brain barrier (BBB) is derived from dye-
exclusion (tryphan blue) studies. It consists of two morphologically distinct
components: A unique capillary endothelium held together by intercellular
tight junctions; and the choroid plexus, where a single layer of specialized
choroidal ependymal cells connected by tight junctions overlies fenestrated
capillaries. The CSF ionic components (e.g., H+, K+, Ca++, Mg++, bicarbon-
ate) are tightly regulated by specific transport systems, whereas glucose,
urea, and creatinine diffuse freely but require 2 hours or longer to equili-
brate. Proteins cross by passive diffusion at a rate dependent on the
plasma-to-CSF concentration gradient and inversely proportional to their
molecular weight and hydrodynamic volume (Fishman, 1992). Thus, the
BBB maintains the relative homeostasis of the central nervous system
environment during acute perturbations of plasma components.
SPECIMEN COLLECTION AND  
OPENING PRESSURE
Cerebrospinal fluid may be obtained by lumbar, cisternal, or lateral cervical
puncture or through ventricular cannulas or shunts. Details of the perfor-
mance of lumbar puncture are described elsewhere (Herndon & Brum-
back, 1989; Ward & Gushurst, 1992). Respiratory compromise may occur
in infants if the head is flexed (Ward & Gushurst, 1992).
A manometer should be attached before fluid removal to record the
opening pressure. CSF pressure varies with postural changes, blood pres-
sure, venous return, Valsalva maneuvers, and factors that alter cerebral
blood flow. The normal opening adult pressure is 90 to 180 mm of water
in the lateral decubitus position with the legs and neck in a neutral posi-
tion. It may be slightly higher if the patient is sitting up and varies up to
10 mm with respiration. However, the pressure may be as high as 250 mm
of water in obese patients. In infants and young children, the normal range
is 10 to 100 mm of water, with the adult range attained by 6 to 8 years of
age (Fishman, 1992). Opening pressures above 250 mm H2O are diagnos-
tic of intracranial hypertension, which may be due to meningitis,

481
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 intracranial hemorrhage, and tumors (Seehusen et al, 2003). If the opening BOX 29-1 
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
pressure is greater than 200 mm H2O in a relaxed patient, no more than Diseases Detected by Laboratory Examination of CSF
2.0 mL should be withdrawn.
Idiopathic intracranial hypertension is most commonly seen in obese High Sensitivity, High Specificity*
women during their childbearing years. When an elevated opening pres- Bacterial, tuberculous, and fungal meningitis
sure is noted, CSF must be removed slowly and the pressure carefully High Sensitivity, Moderate Specificity
monitored. Additional CSF should not be removed if the pressure reaches
Viral meningitis
50% of the opening pressure (Conly & Ronald, 1983).
Subarachnoid hemorrhage
Elevated pressures may be present in patients who are tense or straining
Multiple sclerosis
and in those with congestive heart failure, meningitis, superior vena cava Central nervous system syphilis
syndrome, thrombosis of the venous sinuses, cerebral edema, mass lesions, Infectious polyneuritis
hypoosmolality, or conditions inhibiting CSF absorption. Opening pres- Paraspinal abscess
sure elevation may be the only abnormality in cryptococcal meningitis and
Moderate Sensitivity, High Specificity
pseudotumor cerebri (Hayward et al, 1987). Decreased CSF pressure may
be present in spinal-subarachnoid block, dehydration, circulatory collapse, Meningeal malignancy
and CSF leakage. A significant pressure drop after removal of 1 to 2 mL Moderate Sensitivity, Moderate Specificity
suggests herniation or spinal block above the puncture site, and no further Intracranial hemorrhage
fluid should be withdrawn. Viral encephalitis
Up to 20 mL of CSF may normally be removed. Prior to collecting Subdural hematoma
the sample, the clinician should be aware of the quantity of CSF required
for the requested tests to ensure that a sufficient sample is submitted. In From American College of Physicians, Health and Public Policy Committee: The
diagnostic spinal tap, Ann Intern Med 104:880, 1986, with permission.
addition, the clinician should always provide an appropriate clinical history CSF, Cerebrospinal fluid.
to the laboratory. The sample site (e.g., lumbar, cisternal) should be noted *Sensitivity is the ability of a test to detect disease when it is present; specificity is
because cytologic and chemical parameters vary at different sites. The the ability of a test to exclude disease when it is not present.
necessity for a simultaneous serum glucose should also be considered. This
is best obtained 2 to 4 hours before lumbar puncture because of the delay BOX 29-2 
in serum-CSF equilibrium.
Recommended CSF Laboratory Tests
The CSF specimen is usually divided into three serially collected sterile
tubes: Tube 1 for chemistry and immunology studies; tube 2 for microbio- Routine
logical examination; and tube 3 for cell count and differential. An addi- Opening CSF pressure
tional tube may be inserted in the No. 3 position for cytology if a Total cell count (WBC and RBC)
malignancy is suspected. However, under certain conditions, some varia- Differential cell count (stained smear)
tions are critical. For example, if tube 1 is hemorrhagic because of a Glucose (CSF/plasma ratio)
traumatic puncture, it should not be used when protein studies are the Total protein
most important aspect of the analysis (i.e., suspected multiple sclerosis). Useful Under Certain Conditions
Indeed, tube 3 should be examined for the major purpose of CSF collec-
Cultures (bacteria, fungi, viruses, Mycobacterium tuberculosis)
tion. Perhaps the only definite statement one can make is that tube 1
Gram stain, acid-fast stain
should never be used for microbiology because it may be contaminated
Fungal and bacterial antigens
with skin bacteria. If questions arise, communication between the labora- Enzymes (LD, ADA, CK-BB)
tory and the clinician before CSF analysis is critical. Lactate
Glass tubes should be avoided because cell adhesion to glass affects the Polymerase chain reaction (TB, viruses)
cell count and differential. Specimens should be delivered to the laboratory Cytology
and processed quickly to minimize cellular degradation, which begins Electrophoresis (protein, immunofixation)
within 1 hour of collection. Refrigeration is contraindicated for culture Proteins (C-reactive, 14-3-3, τ, β-amyloid, transferrin)
specimens because fastidious organisms (e.g., Haemophilus influenza, Neis- VDRL test for syphilis
seria meningitidis) will not survive, and for samples in which flow cytometry Fibrin-derivative d-dimer
is likely to be needed for detection of leukemia or lymphoma cells, as Tuberculostearic acid
refrigeration may affect expression and/or detection of certain surface
antigens on these cells. Modified from Kjeldsberg CR, Knight JA: Body fluids: laboratory examination of amni-
otic, cerebrospinal, seminal, serous, and synovial fluids, ed 3, Chicago, 1993, ©
American Society for Clinical Pathology, with permission.
INDICATIONS AND RECOMMENDED TESTS ADA, Adenosine deaminase; CK-BB, creatine kinase-BB; CSF, cerebrospinal fluid;
LD, lactate dehydrogenase; RBC, red blood cell; TB, tuberculosis; VDRL, Venereal
Indications for lumbar puncture can be divided into four major disease Disease Research Laboratories; WBC, white blood cell.
categories: meningeal infection, subarachnoid hemorrhage, primary or
metastatic malignancy, and demyelinating diseases (American College of the unaided eye by observing for Tyndall’s effect (Simon & Abele, 1978).
Physicians, 1986). Identification of infectious meningitis, particularly bac- Here, direct sunlight directed on the tube at a 90-degree angle from the
terial, is the most important indication for CSF examination (Box 29-1). observer will impart a “sparkling” or “snowy” appearance as suspended
Recommended laboratory tests are directed toward identification of these particles scatter the light.
disorders (Box 29-2). CSF examination for other diseases is generally less Clot formation may be present in patients with traumatic taps, com-
helpful but often provides supportive evidence of a clinical diagnosis or plete spinal block (Froin’s syndrome), or suppurative or tuberculous men-
helps to rule out other diseases (Irani, 2009). Limited routine studies fol- ingitis. It is not usually seen in patients with subarachnoid hemorrhage.
lowed by reflexive ordering of more focused tests (as needed) on the stored Fine surface pellicles may be observed after refrigeration for 12 to 24
specimen have been advocated as a way of improving test efficiency hours. Clots may interfere with cell count accuracy by entrapping inflam-
(Albright et al, 1988). matory cells and/or by interfering with automated instrument counting.
Viscous CSF may be encountered in patients with metastatic mucin-
producing adenocarcinomas, cryptococcal meningitis due to capsular poly-
GROSS EXAMINATION saccharide, or liquid nucleus pulposus resulting from needle injury to the
Normal CSF is crystal clear and colorless and has a viscosity similar to that annulus fibrosus.
of water. Abnormal CSF may appear cloudy, frankly purulent, or pigment Pink-red CSF usually indicates the presence of blood and is grossly
tinged. Turbidity or cloudiness begins to appear with leukocyte (white bloody when the RBC count exceeds 6000/µL. It may originate from a
blood cell [WBC]) counts over 200 cells/µL or red blood cell (RBC) counts subarachnoid hemorrhage, intracerebral hemorrhage, or cerebral infarct,
of 400/µL. However, grossly bloody fluids have RBC counts greater than or from a traumatic spinal tap.
6000/µL. Microorganisms (bacteria, fungi, amebas), radiographic contrast
material, aspirated epidural fat, and a protein level greater than 150 mg/ Xanthochromia
dL (1.5 g/L) may also produce varying degrees of cloudiness. Experienced Xanthochromia commonly refers to a pale pink to yellow color in the
observers may be able to detect cell counts of less than 50 cells/µL with supernatant of centrifuged CSF, although other colors may be present

482
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TABLE 29-1  CV is about 48%. A Neubauer hemocytometer with nine 1-mm2 squares
Xanthochromia and Associated Diseases/Disorders
CSF Supernatant Color Associated Diseases/Disorders
Pink RBC lysis/hemoglobin breakdown products
Yellow RBC lysis/hemoglobin breakdown products
Hyperbilirubinemia
CSF protein >150 mg/dL (1.5 g/L)
Orange RBC lysis/hemoglobin breakdown products
Hypervitaminosis A (carotenoids)
Yellow-green Hyperbilirubinemia (biliverdin)
Brown Meningeal metastatic melanoma
CSF, Cerebrospinal fluid; RBC, red blood cell.
(Table 29-1). To detect xanthochromia, the CSF should be centrifuged and
the supernatant fluid compared with a tube of distilled water. Xanthochro-
mic CSF is pink, orange, or yellow owing to RBC lysis and hemoglobin
breakdown. Pale pink to orange xanthochromia from released oxyhemo-
globin is usually detected by lumbar puncture performed 2 to 4 hours after
the onset of subarachnoid hemorrhage, although it may take as long as 12
hours. Peak intensity occurs in about 24 to 36 hours and then gradually
disappears over the next 4 to 8 days. Yellow xanthochromia is derived from
bilirubin. It develops about 12 hours after a subarachnoid bleed and peaks
at 2 to 4 days, but may persist for 2 to 4 weeks.
Visible CSF xanthochromia may also be due to the following: (1) oxy-
hemoglobin resulting from artifactual red blood cell lysis caused by deter-
gent contamination of the needle or collecting tube, or a delay of longer
than 1 hour without refrigeration before examination; (2) bilirubin (bil-
irhachia) in jaundiced patients; (3) CSF protein levels over 150 mg/dL,
which are also present in bloody traumatic taps (>100,000 RBCs/µL) or
in pathologic states such as complete spinal block, polyneuritis, and men-
ingitis; (4) disinfectant contamination; (5) carotenoids (orange) in people
with dietary hypercarotenemia (i.e., hypervitaminosis A); (6) melanin
(brownish) from meningeal metastatic melanoma; and (7) rifampin therapy
(red-orange).
Although spectral absorbance scans provide an objective record of
xanthochromia, careful gross CSF inspection has comparable sensitivity
(Britton et al, 1983). Spectrophotometry can also help to differentiate
hemoglobin-derived substances from other xanthochromic pigments with
different maximal absorption peaks.
Differential Diagnosis of Bloody CSF
A traumatic tap occurs in about 20% of lumbar punctures. Distinction of
a traumatic puncture from a pathologic hemorrhage is, therefore, of vital
importance. Although the presence of crenated RBCs is not useful, the
following observations may be helpful in distinguishing the two forms of
bleeding.
1. In a traumatic tap, the hemorrhagic fluid usually clears between the
first and third collected tubes but remains relatively uniform in sub-
arachnoid hemorrhage.
2. Xanthochromia, microscopic evidence of erythrophagocytosis, or
hemosiderin-laden macrophages indicate a subarachnoid bleed in the
absence of a prior traumatic tap. RBC lysis begins as early as 1 to 2
hours after a traumatic tap. Thus, rapid evaluation is necessary to avoid
false-positive results.
3. A commercially available latex agglutination immunoassay test for
cross-linked fibrin derivative D-dimer is specific for fibrin degradation
and is negative in traumatic taps (Lang et al, 1990). However, false-
positive results might be expected in disseminated intravascular coagu-
lation, fibrinolysis, or trauma from repeated lumbar punctures.
MICROSCOPIC EXAMINATION
Total Cell Count
Although the traditional manual method for cell counting in CSF samples,
using undiluted CSF in a manual counting chamber, continues to be a
useful approach, because of the low cell counts frequently encountered in
CSF, the precision of manual counting in these samples is inherently
limited (Barnes et al, 2004). For example, using 18 large squares (1 mm2
each) in a Fuchs-Rosenthal–type chamber with a depth of 0.2 mm, a total
volume of 3.6 µL (18 × 0.2 µL/square) is examined. With 5 cells/µL, a
total of 18 cells is counted. The coefficient of variation (CV), is 24%; ±2
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with a depth of 0.1 mm has a CV of 45% (+90% for 2 CV) with the same
cell concentration. Improvements in the hardware and software in flow
cytometers now allow reliable use of these instruments in performing
automated total WBC counts and WBC differential counts (Hoffman &
Janssen, 2002; Aune et al, 2004), and even in detecting bacteria (Nanos &
Delanghe, 2008) in CSF samples. Although these instruments are increas-
ingly used clinically to perform these counts on CSF samples, the low
clinical decision levels for total WBC count in CSF and persistent techni-
cal limitations of flow cytometers at low WBC levels and with certain
WBC types continue to be cause for concern (Hoffman & Janssen, 2002;
PART 3
Andrews et al, 2005; Glasser et al, 2009; Kleine et al, 2009). When issues
of turnaround time and cost are considered, however, automated total
WBC and WBC differential counts in CSF samples may be reasonable
alternatives to manual counting (Zimmerman et al, 2011; Li et al, 2014),
with added manual examination of the sample to avoid missing pathologic
cell types as a useful cross-check (Strik et al, 2005). When considering use
of automated cell counters with CSF samples, laboratories should carefully
follow manufacturer and Clinical and Laboratory Standards Institute
guidelines (CLSI Approved Guideline H56-A, 2006) when implementing
these automated methods.
The normal leukocyte cell count in adults is 0 to 5 cells/µL. It is higher
in neonates, ranging from 0 to 30 cells/µL, with the upper limit of normal
decreasing to adult values by adolescence. No RBCs should be present in
normal CSF. If numerous (except with a traumatic tap), a pathologic
process is probable (e.g., trauma, malignancy, infarct, hemorrhage).
Although RBC counts have limited diagnostic value, they may give a useful
approximation of the true CSF WBC or total protein in the presence of a
traumatic puncture by correcting for leukocytes or protein introduced by
the traumatic puncture. To be valid, all measurements (WBC, RBC,
protein) must be performed on the same tube. This procedure also assumes
that the blood is derived exclusively from the traumatic tap. The corrected
WBC count is as follows:
WBCcorr = WBCobs − WBCadded
where
WBCadded = WBCBLD × RBCCSF RBCBLD
and
WBCobs = CSF leukocyte count
WBCadded = leukocytes added to CSF by traumatic tap
WBCBLD = peripheral blood leukocyte count
RBCCSF = CSF erythrocyte count
RBCBLD = peripheral blood erythrocyte count
An analogous formula may be used to correct for added total protein
(TP):
TPadded = [ TPserum × (1 − HCT )] × RBCCSF RBCBLD
In the presence of a normal peripheral blood RBC count and serum
protein, these corrections amount to about 1 WBC for every 700 RBCs
and 8 mg/dL protein for every 10,000 RBCs/µL. This latter RBC correc-
tion factor is reasonably accurate as long as the peripheral WBC count is
not extremely high or low.
An observed/expected (added) WBC count ratio greater than 10 has a
sensitivity of 88% and a specificity of 90% for bacterial meningitis. When
the predicted WBC is below the observed count, the probability of bacte-
rial meningitis appears to be low (Mayefsky & Roughmann, 1987; Bonadio
et al, 1990).
Differential Cell Count
Suggested differential count reference ranges are presented in Table 29-2.
A differential performed in a counting chamber is unsatisfactory because
the low cell numbers give rise to poor precision, and identifying the cell
type beyond granulocytes and “mononuclears” is difficult in a wet prepara-
tion. Direct smears of the centrifuged CSF sediment are also subject to
significant error from cellular distortion and fragmentation.
483
 TABLE 29-2 
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
CSF Reference Values for Differential Cytocentrifuge Counts
Cell type Adults, % Neonates, %
Lymphocytes 62 ± 34 20 ±
Monocytes 36 ± 20 72 ±
Neutrophils 2±5 3±
Histiocytes Rare 5±
Ependymal cells Rare Rare
Eosinophils Rare Rare
CSF, Cerebrospinal fluid.
Figure 29-1  Cerebrospinal fluid cytology (lymphocyte to monocyte distribu-
tion ratio 70 : 30).
The cytocentrifuge method is rapid, requires minimal training, and
allows Wright’s staining of air-dried cytospins. It is the recommended
method for differential cell counts in all body fluids (Rabinovitch & Corn-
bleet, 1994). Cell yield and preservation are better than with simple cen-
trifugation. From 30 to 50 cells can be concentrated from 0.5 mL of
“normal” CSF. Variable artifactual distortions may be seen, but they are
minimized when the specimen is fresh, albumin is added to the specimen
(2 drops of 22% bovine serum albumin), and the cell concentration is
adjusted to about 300 WBCs/L prior to centrifugation (Kjeldsberg &
Knight, 1993). Manual differential cell counting on a cytocentrifuge prepa-
ration of CSF continues to be the most reliable method, even with low
cell numbers. Although automated differential counts may be safely per-
formed on CSF samples using flow cytometers (Hoffman & Janssen, 2002;
Aulesa et al, 2003; Li et al, 2014), confirmation of the automated differ-
ential count by manual examination of a cytocentrifuged smear is recom-
mended with some instruments (Aune et al, 2004; Strik et al, 2005) and is
a requirement with specimens at risk for containing neoplastic cells. Effec-
tive detection of neoplastic cells, such as leukemic cells, using the cytocen-
trifuge method on CSF appears to be highly instrument-dependent
(Huppmann et al, 2012).
Filtration and sedimentation methods are too cumbersome for routine
use. Filtration does, however, allow concentration of large volumes of CSF
for cytologic examination or culture, while retaining the fluid filtrate for
additional studies.
In adults, normal CSF contains small numbers of lymphocytes and
monocytes in an approximate 70 : 30 ratio (Fig. 29-1). A higher proportion
of monocytes is present in young children, in whom up to 80% may be
normal (Pappu et al, 1982). Erythrocytes due to minor traumatic bleeding
are commonly seen, especially in infants. Small numbers of neutrophils
(PMNs) may also be seen in “normal” CSF specimens, most likely as a
result of minor hemorrhage (Hayward & Oye, 1988) and improved
cell concentration methods. No general consensus regarding an upper
limit of normal for PMNs has been established. Many laboratories accept
up to 7% neutrophils with a normal WBC count. Over 60% neutrophils
has been reported in high-risk neonates without meningitis (Rodriguez
et al, 1990). The number of PMNs may be decreased by as much as 68%
within the first 2 hours after lumbar puncture owing to cell lysis (Steele
et al, 1986).
484
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18
22
5
4
Figure 29-2  Choroid plexus cells in cerebrospinal fluid.
Figure 29-3  Cluster of blastlike cells in cerebrospinal fluid from a premature
newborn. (From Kjeldsberg CR, Knight JA: Body fluids: laboratory examination of
amniotic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993, ©
American Society for Clinical Pathology, with permission.)
Traumatic puncture may result in the presence of bone marrow cells,
cartilage cells, squamous cells, ganglion cells, and soft tissue elements. In
addition, ependymal and choroid plexus cells may rarely be seen (Fig.
29-2). Moreover, blastlike primitive cell clusters, most likely of germinal
matrix origin, are sometimes found in premature infants with intraven-
tricular hemorrhage (Fig. 29-3).
Increased CSF neutrophils occur in numerous conditions (Box 29-3).
In early bacterial meningitis, the proportion of PMNs usually exceeds
60%. However, in about one quarter of cases of early viral meningitis, the
proportion of PMNs also exceeds 60%. Viral-induced neutrophilia usually
changes to a lymphocytic pleocytosis within 2 to 3 days. A total PMN
count of over 1180 cells/µL (or more than 2000 WBCs/µL) has a 99%
predictive value for bacterial meningitis (Spanos et al, 1989). Persistent
neutrophilic meningitis (over 1 week) may be noninfectious or due to less
common pathogens such as Nocardia, Actinomyces, Aspergillus, and the zygo-
mycetes (Peacock et al, 1984).
Increased CSF lymphocytes have been reported in various diseases/
disorders (Box 29-4). Lymphocytosis (>50%) may occur in early acute
bacterial meningitis when the CSF leukocyte count is under 1000/µL
(Powers, 1985). Reactive lymphoplasmacytoid and immunoblastic variants
may be present, particularly with viral meningoencephalitis. Blastlike lym-
phocytes may be seen admixed with small and large lymphocytes in the
CSF of neonates.
Plasma cells, not normally present in CSF, may appear in a variety of
inflammatory and infectious conditions (Box 29-5), along with large and
small lymphocytes, and in association with malignant brain tumors
(Fishman, 1992). Multiple myeloma may also rarely involve the meninges
(Oda et al, 1991).
Although eosinophils are rarely present in normal CSF, they may
be increased in a variety of central nervous system (CNS) conditions
BOX 29-3  BOX 29-6 
Causes of Increased CSF Neutrophils Causes of CSF Eosinophilic Pleocytosis
Meningitis Commonly associated with:
Bacterial meningitis Acute polyneuritis
Early viral meningoencephalitis CNS reaction to foreign material (drugs, shunts)
Early tuberculous meningitis Fungal infections
Early mycotic meningitis Idiopathic eosinophilic meningitis
Amebic encephalomyelitis Idiopathic hypereosinophilic syndrome
Other infections Parasitic infections
Cerebral abscess
Infrequently associated with:
Subdural empyema

PART 3
AIDS-related CMV radiculopathy Bacterial meningitis
Following seizures Leukemia/lymphoma
Following CNS hemorrhage Myeloproliferative disorders
Subarachnoid Neurosarcoidosis
Intracerebral Primary brain tumors
Following CNS infarct Tuberculous meningoencephalitis
Reaction to repeated lumbar punctures Viral meningitis
Injection of foreign material in subarachnoid space (e.g., methotrexate,
Modified from Kjeldsberg CR, Knight JA: Body fluids: laboratory examination of amni-
contrast media)
otic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993, ©
Metastatic tumor in contact with CSF American Society for Clinical Pathology, with permission.
CNS, Central nervous system; CSF, cerebrospinal fluid.
AIDS, Acquired immunodeficiency syndrome; CMV, cytomegalovirus; CNS, central
nervous system; CSF, cerebrospinal fluid.

BOX 29-4 
Causes of CSF Lymphocytosis
Meningitis
Viral meningitis
Tuberculous meningitis
Fungal meningitis
Syphilitic meningoencephalitis
Leptospiral meningitis
Bacterial due to uncommon organisms
Early bacterial meningitis where leukocyte counts are relatively low
Parasitic infestations (e.g., cysticercosis, trichinosis, toxoplasmosis)
Aseptic meningitis due to septic focus adjacent to meninges
Degenerative Disorders
Subacute sclerosing panencephalitis
Multiple sclerosis
Drug abuse encephalopathy
Guillain-Barré syndrome
Acute disseminated encephalomyelitis
Figure 29-4  Eosinophils in cerebrospinal fluid from a child with malfunction-
Other Inflammatory Disorders ing ventricular shunt.
Handl syndrome (headache with neurologic deficits and CSF
lymphocytosis)
Sarcoidosis plasma cells. This pattern is seen in tuberculous and fungal meningitis,
Polyneuritis chronic bacterial meningitis (i.e., Listeria monocytogenes and others), lepto-
CNS periarteritis spiral meningitis, ruptured brain abscess, Toxoplasma meningitis, and
amebic encephalomeningitis. A mixed cell pattern without neutrophils is
CNS, Central nervous system; CSF, cerebrospinal fluid.
characteristic of viral and syphilitic meningoencephalitis. Macrophages
with phagocytosed erythrocytes (erythrophages) appear from 12 to 48
BOX 29-5  hours following a subarachnoid hemorrhage or traumatic tap. Hemosiderin-
laden macrophages (siderophages) appear after about 48 hours and may
Inflammatory and Infectious Causes of CSF Plasmacytosis persist for weeks (Fig. 29-5). Brownish yellow or red hematoidin crystals
Acute viral infections may form after a few days.
Guillain-Barré syndrome Morphologic cerebrospinal fluid examination for tumor cells has mod-
Multiple sclerosis erate sensitivity and high specificity (97% to 98%) (Marton & Gean, 1986).
Parasitic CNS infestations Sensitivity depends on the type of neoplasm. CSF examination of leukemic
Sarcoidosis patients has the highest sensitivity (about 70%), followed by metastatic
Subacute sclerosing panencephalitis carcinoma (20% to 60%) and primary CNS malignancies (30%). Sensitiv-
Syphilitic meningoencephalitis ity may be optimized by using filtration methods with larger fluid volumes
Tuberculous meningitis or by performing serial punctures in patients in whom a neoplasm is
strongly suspected. Processing of CSF samples using liquid-based thin-
CNS, Central nervous system; CSF, cerebrospinal fluid.
layer methods also increases sensitivity in the detection of neoplastic cells
and enhances preservation of these cells for potential immunocytochemical
(Box 29-6). For example, eosinophilia is frequently mild (1% to 4%) in a analysis (Sioutopoulou et al, 2008). These liquid-based methods are now
general inflammatory response, but in children with malfunctioning ven- commonly used for cytopathologic examination of CSF and other body
tricular shunts, it may be marked (Fig. 29-4). A suggested criterion for cavity fluid specimens.
eosinophilic meningitis is 10% eosinophils (Kuberski, 1981); parasitic Leukemic involvement of the meninges is more frequent in patients
invasion of the CNS is the most common cause worldwide. Coccidioides with acute lymphoblastic leukemia (Fig. 29-6) than in those with acute
immitis is a significant cause of CSF eosinophilia in endemic regions of the myeloid leukemia (Fig. 29-7); both are significantly more common than
United States (Ragland et al, 1993). CNS involvement in the chronic leukemias. A leukocyte count over 5
Increased CSF monocytes lack diagnostic specificity and are usually cells/µL with unequivocal lymphoblasts in cytocentrifuged preparations is
part of a “mixed cell reaction” that includes neutrophils, lymphocytes, and commonly accepted as evidence of CSF involvement. The incidence of

485
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29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
Figure 29-5  Hemosiderin-laden macrophages (siderophages) from the cere-
brospinal fluid of a patient with subarachnoid hemorrhage. Hemosiderin crystals
(golden-yellow) are also present. (From Kjeldsberg CR, Knight JA: Body fluids: labora-
tory examination of amniotic, cerebrospinal, seminal, serous and synovial fluids, ed 3,
Chicago, 1993, © American Society for Clinical Pathology, with permission.)
Figure 29-6  Acute lymphoblastic leukemia in cerebrospinal fluid. Note uni-
formity of the blast cells.
Figure 29-7  Acute myeloid leukemia in cerebrospinal fluid.
CNS relapse in children with lymphoblasts but cell counts lower than 6
cells/µL appears to be low and is not significantly different from cases in
which no blasts are identified (Odom et al, 1990; Gilchrist et al, 1994;
Tubergen et al, 1994). As noted previously, the ability to detect leukemic
cells in CSF using the cytocentrifuge method is highly instrument-
dependent, and low levels of these cells may be missed with certain instru-
ments (Huppmann et al, 2012).
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Figure 29-8  Burkitt’s lymphoma in cerebrospinal fluid. The cells are character-
ized by blue cytoplasm with vacuoles and a slightly clumped chromatin pattern.
(From Kjeldsberg CR, Knight JA: Body fluids: laboratory examination of amniotic,
cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993, © American
Society for Clinical Pathology, with permission.)
Non-Hodgkin’s lymphomas involving the leptomeninges are usually
high-grade tumors (lymphoblastic, large cell immunoblastic, and Burkitt’s
lymphomas) (Fig. 29-8); low-grade lymphomas and Hodgkin’s lymphoma
are significantly less common (Bigner, 1992; Walts, 1992). T cells pre-
dominate in normal and inflammatory conditions, whereas most lympho-
mas, especially those occurring in immunocompromised hosts, are of B
cell lineage. Lymphoblastic lymphoma, the most common T-cell lym-
phoma to involve the CSF, can be detected by terminal deoxynucleotidyl
transferase stain.
Multiparameter flow cytometric immunophenotypic studies, DNA
analysis by polymerase chain reaction (PCR), and more recently DNA
sequence analysis have been shown to significantly improve diagnostic
sensitivity and specificity in CSF samples involved by leukemic or lym-
phoma cells (Rhodes et al, 1996; Finn et al, 1998; Scrideli & Queiroz,
2004; Bromberg et al, 2007; Quijano et al, 2009; Martinez-Laperche et al,
2013; Lacayo et al, 2013). Considering their greater sensitivity in detecting
leukemia or lymphoma cells in CSF samples, whenever available these
advanced technologies should be part of the complete evaluation in
patients being worked up for these conditions.
Amebas, fungi (especially Cryptococcus neoformans), and Toxoplasma gondii
organisms may be present on cytocentrifuge specimens but may be difficult
to recognize without confirmatory stains.
CHEMICAL ANALYSIS
Reference values for lumbar cerebrospinal fluid in adults are listed in
Table 29-3.
Proteins
Total Protein
More than 80% of the CSF protein content is derived from blood plasma,
in concentrations of less than 1% of the plasma level (Table 29-4).
Prealbumin (transthyretin), transferrin, and small quantities of nerve
tissue–specific proteins are the major qualitative differences that normally
exist between CSF and plasma proteins. Although some authors have
argued against routine measurement of total protein (American College of
Physicians, 1986), it is the most common abnormality found in CSF. Thus,
an increased CSF protein serves as a useful, albeit nonspecific, indicator
of meningeal or CNS disease.
Reference Values.  CSF total protein reference values vary consider-
ably among laboratories owing to differences in methods, instrumentation,
and type of reference standard used. CSF protein levels of 15 to 45 mg/
dL have long been accepted as the “normal” reference range (Silverman
& Christenson, 1994). Other studies using different methods have shown
generally higher reference ranges, approximately 15 to 60 mg/dL (Lott &
Warren, 1989).
Although discrepancies in gender and in those older than 60 years of
age have been reported, the differences are probably not significant.
However, infants have significantly higher CSF protein levels than older
children and adults. Thus, mean levels of 90 mg/dL for term infants and
TABLE 29-3  BOX 29-7 
Adult Lumbar CSF Reference Values Conditions Associated with Increased CSF Total Protein
Analyte Conventional Units SI Units Traumatic Spinal Puncture
Increased Blood-CSF Permeability
Protein 15-60 mg/dL 0.15-0.60 g/L
Arachnoiditis (e.g., following methotrexate therapy)
Prealbumin 2%-7%
Meningitis (bacterial, viral, fungal, tuberculous)
Albumin 56%-76% Hemorrhage (subarachnoid, intracerebral)
α1-Globulin 2%-7% Endocrine/metabolic disorders
α2-Globulin 4%-12% Milk-alkali syndrome with hypercalcemia
Diabetic neuropathy
β-Globulin 8%-18%

PART 3
Hereditary neuropathies and myelopathies
γ-Globulin 3%-12% Decreased endocrine function (thyroid, parathyroid)
Electrolytes Other disorders (uremia, dehydration)
Osmolality 280-300 mOsm/L 280-300 mmol/L Drug Toxicity
Sodium 135-150 mEq/L 135-150 mmol/L Ethanol, phenothiazines, phenytoin
Potassium 2.6-3.0 mEq/L 2.6-3.0 mmol/L CSF Circulation Defects
Chloride 115-130 mEq/L 115-130 mmol/L Mechanical obstruction (tumor, abscess, herniated disk)
Carbon dioxide 20-25 mEq/L 20-25 mmol/L Loculated CSF effusion
Calcium 2.0-2.8 mEq/L 1.0-1.4 mmol/L Increased Immunoglobulin (Ig)G Synthesis
Magnesium 2.4-3.0 mEq/L 1.2-1.5 mmol/L Multiple sclerosis
Lactate 10-22 mg/dL 1.1-2.4 mmol/L Neurosyphilis
Subacute sclerosing panencephalitis
pH
Lumbar fluid 7.28-7.32 Increased IgG Synthesis and Blood-CSF Permeability
Cisternal fluid 7.32-7.34 Guillain-Barré syndrome
Collagen vascular diseases (e.g., lupus, periarteritis)
pCO2 Chronic inflammatory demyelinating polyradiculopathy
Lumbar fluid 44-50 mm Hg
CSF, Cerebrospinal fluid.
Cisternal fluid 40-46 mm Hg
pO2 40-44 mm Hg
Other Constituents Elevated CSF protein levels may be caused by increased permeability
Ammonia 10-35 µg/dL 6-20 µmol/L of the blood-brain barrier, decreased resorption at the arachnoid villi,
Glutamine 5-20 mg/dL 0.3-1.4 mmol/L mechanical obstruction of CSF flow due to spinal block above the puncture
Creatinine 0.6-1.2 mg/dL 45-92 µmol/L site, or an increase in intrathecal immunoglobulin (Ig) synthesis. Common
Glucose 50-80 mg/dL 2.8-4.4 mmol/L conditions associated with elevated lumbar CSF protein values (>65 mg/
dL) are summarized in Box 29-7.
Iron 1-2 µg/dL 0.2-0.4 µmol/L
Low lumbar CSF total protein levels (<20 mg/dL) normally occur in
Phosphorus 1.2-2.0 mg/dL 0.4-0.7 mmol/L some young children between 6 months and 2 years of age and in patients
Total lipid 1-2 mg/dL 0.01-0.02 g/L with conditions associated with increased CSF turnover. These include the
Urea 6-16 mg/dL 2.0-5.7 mmol/L following: (1) Removal of large CSF volumes; (2) CSF leaks induced by
Urate 0.5-3.0 mg/dL 30-180 µmol/L trauma or lumbar puncture; (3) increased intracranial pressure, probably
Zinc 2-6 µg/dL 0.3-0.9 µmol/L
due to an increased rate of protein resorption by the arachnoid villi; and
(4) hyperthyroidism (Fishman, 1992).
CSF, Cerebrospinal fluid; pCO2, partial pressure of carbon dioxide; pO2, partial pres- Protein electrophoresis of concentrated normal CSF reveals two dis-
sure of oxygen. tinct differences from serum: a prominent transthyretin (prealbumin) band
and two transferrin bands. Transthyretin is relatively high because of its
TABLE 29-4  dual synthesis by the liver and the choroid plexus. The second transferrin
band, referred to as β2-transferrin, migrates more slowly than its serum
Mean Concentrations of Plasma and CSF Proteins
equivalent owing to cerebral neuraminidase digestion of sialic acid
Protein CSF, mg/L Plasma/CSF Ratio residues.
Methods.  Turbidimetric methods, commonly based on trichloroace-
Prealbumin 17.3 14 tic acid (TCA) or sulfosalicylic acid and sodium sulfate for protein
Albumin 155.0 236 precipitation, are popular because they are simple and rapid, and require
Transferrin 14.4 142 no special instrumentation. However, they are temperature sensitive
Ceruloplasmin 1.0 366 and require much larger specimen volumes (about 0.5 mL). A false
protein elevation may be observed using TCA methods in the presence
Immunoglobulin (Ig)G 12.3 802
of methotrexate (Kasper et al, 1988). Benzethonium chloride and
IgA 1.3 1346 benzalkonium chloride have been used as precipitating agents in auto-
α 2-Microglobulin 2.0 1111 mated methods and micromethods (Luxton et al, 1989; Shephard &
Fibrinogen 0.6 4940 Whiting, 1992).
IgM 0.6 1167 Colorimetric methods include the Lowry method, dye-binding,
β-Lipoprotein 0.6 6213 methods using Coomassie brilliant blue (CBB) or Ponceau S, and the
modified Biuret method. The CBB method is rapid and highly sensitive,
Adapted from Felgenhauer K: Protein size and cerebrospinal fluid composition, Klin and can be used with small sample sizes. Immunochemical methods
Wochenschr 52:1158, 1974, with permission. measure specific proteins, require only 25 to 50 µL of CSF, and are
CSF, Cerebrospinal fluid. relatively simple to perform once conditions and reagents have been
standardized. Automated methods are now commonly used and
115 mg/dL for preterm infants were reported; the upper levels were usually show good correlation with traditional standard methods (Lott &
150 mg/dL and 170 mg/dL, respectively (Sarff et al, 1976). Others have Warren, 1989).
noted that the CSF protein concentration falls rapidly from birth to 6
months of age (mean levels, 108 mg/dL to 40 mg/dL), plateaus between Albumin and IgG Measurements
3 and 10 years of age (mean, 32 mg/dL), and then rises slightly from 10 The permeability of the blood-brain barrier may be assessed by immuno-
to 16 years of age (mean, 41 mg/dL) (Biou et al, 2000). chemical quantification of the CSF albumin/serum albumin ratio in grams

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 per deciliter (g/dL). The normal ratio of 1 : 230 (Tourtellotte et al, 1985)
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
yields an unwieldy decimal of 0.004, which prompted the use of the CSF/
serum albumin index, which is arbitrarily calculated as follows:
CSF albumin ( mg dL )
CSF Serum albumin index = (Eq. 29-1)
Serum albumin ( g dL )
An index value less than 9 is consistent with an intact barrier. Slight
impairment is considered with index values of 9 to 14, moderate impair-
ment with values of 14 to 30, and severe impairment with values greater
than 30 (Silverman & Christenson, 1994). The index is slightly elevated
in infants up to 6 months of age, reflecting the immaturity of the blood-
brain barrier, and increases gradually after 40 years of age. A traumatic tap
invalidates the index calculation.
Increased intrathecal IgG synthesis is reflected by an increase in the
CSF/serum IgG ratio:
CSF IgG ( mg dL )
CSF Serum IgG ratio = (Eq. 29-2)
Serum IgG ( g dL )
The normal ratio is 1/390 or 0.003 (Tourtellotte et al, 1985). Similar
to the albumin index, the CSF/serum IgG index may be obtained by using
milligrams per deciliter for the CSF IgG value. The CSF/serum IgG index
normal range is 3.0 to 8.7.
The CSF/serum IgG index can be elevated by intrathecal IgG syn-
thesis or increased plasma IgG crossover from breakdown of the blood-
brain barrier. Ig derived from plasma crossover may be corrected by
dividing the CSF/serum IgG index by the CSF/albumin index to yield the
CSF IgG index.
CSF IgG ( mg dL ) Serum IgG ( g dL )
CSF IgG index =
CSF albumin ( mg dL ) Serum albumin ( g dL )
(Eq. 29-3)
or
CSF IgG ( mg dL ) × Serum albumin ( g dL )
CSF IgG index =
Serum IgG ( g dL ) × CSF albumin ( mg dL )
(Eq. 29-4)
The reference range for the IgG index varies, reflecting variations in
determination of the four index components. A reasonable normal upper
limit is 0.8 (Souverijn et al, 1989). However, each laboratory should deter-
mine its own critical ratio.
The IgG synthesis rate is calculated by an empirical formula (Tourtel-
lotte et al, 1985):
IgG synthesis rate ( mg day ) = [(CSF IgG − Serum IgG 369)
− (CSF albumin − Serum albumin 230)
× (Serum IgG Serum albumin )
× 0.43 × 5 dL day]
(Eq. 29-5)
All protein concentrations are expressed in milligrams per deciliter.
The first bracketed term represents the difference between measured CSF
IgG and the IgG expected from diffusion across a normal blood-brain
barrier; 369 is the normal serum/CSF ratio. The second bracketed term
represents the difference between measured CSF albumin and expected
albumin if the blood-brain barrier is intact; 230 is the normal serum/CSF
albumin ratio. The CSF albumin excess is multiplied by the IgG/albumin
ratio and the molecular weight ratio of IgG to albumin (0.43) to correct
for changes in CSF IgG due to increased barrier permeability. The number
5 converts the result from a concentration to a daily amount, assuming an
average daily CSF production of 500 mL (i.e., 5 dL). The formula does
not consider variations in CSF production or Ig consumption. It assumes
that the IgG/albumin ratio remains constant over various degrees of blood-
brain barrier impairment—a concept that may lead to variable error
(Lefvert & Link, 1985). The reference interval for the synthesis rate is –9.9
to +3.3 mg/day. Values greater than 8.0 mg/day indicate an increased rate
(Silverman & Christenson, 1994).
CSF IgG is normally 3% to 5% of total CSF protein, but in multiple
sclerosis (MS), the concentration approaches that of plasma (15% to 18%)
(Hersey & Trotter, 1980). The CSF IgG index and the IgG synthesis rate
have a sensitivity of 90% in patients with definite MS, but the sensitivity
is lower in patients with possible MS, in whom accuracy is most needed
(Marton & Gean, 1986). In addition, the specificity for MS is only
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moderate because increased intrathecal IgG synthesis occurs in many other
inflammatory neurologic diseases.
The Ig index and synthesis rate calculations may also be applied to IgM,
IgA, Ig light chains, and specific antibodies to infectious microorganisms.
For example, increased synthesis of IgM and free κ light chains have been
suggested as markers for MS (Rudick et al, 1989; Lolli et al, 1991).
Electrophoretic Techniques.  Although the diagnosis of MS is ulti-
mately a clinical one, significant advances have been made in laboratory
testing for this disorder. CSF total protein is increased in less than 50%
of patients with MS. Indeed, if the CSF protein exceeds 100 mg/dL, the
patient probably does not have MS. However, the γ-globulin fraction, as
determined by CSF electrophoresis, is often increased in MS. Thus, the
CSF total protein/γ-globulin ratio exceeds 0.12 in about 65% of cases
(Johnson & Nelson, 1977). Using electroimmunodiffusion, a CSF IgG/
albumin ratio greater than 0.25 is present in about 75% of cases (Tourtel-
lotte et al, 1971). Levels greater than the mean CSF IgG index + 3SD are
present in 80% to 85% of MS cases, although the upper reference level
varies significantly among laboratories.
High-resolution agarose gel electrophoresis of concentrated CSF
from patients with MS often shows discrete populations of IgG, the oli-
goclonal bands. Although these discrete IgG populations are normally
absent, two or more bands are necessary to support the diagnosis of MS;
a single band is not considered a positive result. Using this technique,
oligoclonal bands have been reported in 83% to 94% of patients with
definite MS, 40% to 60% of those with probable MS, and 20% to 30%
of possible MS cases. However, they are also frequently present in patients
with subacute sclerosing panencephalitis, various viral CNS infections,
neurosyphilis, neuroborreliosis, cryptococcal meningitis, Guillain-Barré
syndrome, transverse myelitis, meningeal carcinomatosis, glioblastoma
multiforme, Burkitt’s lymphoma, chronic relapsing polyneuropathy,
Behçet’s disease, cysticercosis, and trypanosomiasis, among others (Trotter
& Rust, 1989; Chalmers et al, 1990; Fishman 1992; Hall & Snyder, 1992).
Subsequent studies have shown that agarose gel electrophoresis sensitivity
for MS is less than previously reported (see later).
Oligoclonal light chains (both κ and λ) are present in about 90% of
MS patients (Gallo et al, 1989; Sindie & Laterte, 1991). They have also
occasionally been identified in the CSF of those who are negative for IgG
oligoclonal bands. Detection of free light chains in CSF and comparison
to serum has been shown to be a sensitive marker for intrathecal immu-
noglobulin synthesis (Fischer et al, 2004).
Coomassie brilliant blue or paragon violet stains can resolve oligo-
clonal bands in only 5 µg of IgG (Silverman & Christenson, 1994).
However, silver staining is 20 to 50 times more sensitive than CBB and
can be used on unconcentrated CSF. It is important to note that these
electrophoretic techniques must be simultaneously carried out on the
patient’s serum to be certain that a polyclonal gammopathy is not present
(e.g., liver disease, systemic lupus, rheumatoid arthritis, chronic granulo-
matous disease) because these disorders may be accompanied by Ig diffu-
sion into the CSF, yielding false-positive results.
Immunofixation electrophoresis (IFE) is more sensitive than agarose
gel electrophoresis and does not require CSF concentration (Cawley et al,
1976). Another study reported a sensitivity of 74% using this technique
compared with 57% for agarose gel electrophoresis (Cavuoti et al, 1998).
More recently, using a semiautomated immunofixation-peroxidase tech-
nique, the sensitivity was 83% and the specificity was 79% in patients with
clinically definite MS (Richard et al, 2002).
Isoelectric focusing and IgG immunoblotting (IgG-IEF) per-
formed on paired CSF and serum samples is the most sensitive and now
the recommended method for the detection of oligoclonal bands
(Andersson et al, 1994; Fortini et al, 2003; Keren, 2003). One study
showed that IgG-IEF detected 100% of definite MS, but only about
50% were positive by agarose electrophoresis (Lunding et al, 2000).
Others detected 91% of MS cases but only 68% with agarose (Seres
et al, 1998). Similarly, a semiautomated IgG-IEF technique identified
90% of MS cases compared with 60% for agarose electrophoresis
(Fortini et al, 2003). In 2005, an international consensus standard for the
diagnosis of MS established IgG-IEF as the method of choice for quali-
tative detection of oligoclonal IgG bands as evidence of intrathecal syn-
thesis of IgG (Freedman et al, 2005). This method is more sensitive and
specific than quantitative methods.
In summary, the diagnosis of MS, as with many other neurologic dis-
orders, is ultimately a clinical one based on neurologic history and physical
examination. Nevertheless, advanced laboratory results in CSF, such as
elevated IgG indices and particularly the detection of oligoclonal IgG
bands, as well as neuroimaging techniques, have proved invaluable in the
diagnosis of MS.
TABLE 29-5  bacterial meningitis (Corral et al, 1981; Abramson et al, 1985; Stearman
CSF Proteins and Central Nervous System Diseases
Protein Major Diseases/Disorders
α 2-Macroglobulin Subdural hemorrhage, bacterial meningitis
β-Amyloid and τ proteins Alzheimer’s disease
β2-Microglobulin Leukemia/lymphoma, Behçet’s syndrome
C-reactive protein Bacterial and viral meningitis
Fibronectin Lymphoblastic leukemia, AIDS, meningitis
Methemoglobin Mild subarachnoid/subdural hemorrhage
Myelin basic protein Multiple sclerosis, tumors, others
Protein 14-3-3 Creutzfeldt-Jakob disease
Transferrin CSF leakage (otorrhea, rhinorrhea)
AIDS, Acquired immunodeficiency syndrome; CSF, cerebrospinal fluid.
Other CSF Proteins
Approximately 300 different proteins have been identified in CSF using
two-dimensional electrophoresis, the first dimension being isoelectric
focusing and the second polyacrylamide gel in the presence of sodium
dodecyl sulfate (Harrington et al, 1986). Using this technique, four abnor-
mal proteins were identified in patients with Creutzfeldt-Jakob disease
(CJD). Two of these proteins (molecular mass about 40 kilodaltons [kDa]
each) were present in some, but not all, patients with herpes simplex
encephalitis, Parkinson’s disease, Guillain-Barré syndrome, and schizo-
phrenia. They were not present in various other neurologic disorders, nor
in 100 normal CSF control specimens. However, these and two other
proteins (molecular masses about 26 and 29 kDa) were present in all cases
of CJD and in 5 of 10 cases of herpes simplex encephalitis. Neither of these
latter proteins was present in any other neurologic disease or in controls.
Increased concentrations of various specific CSF proteins have been
associated with several CNS diseases (Table 29-5).
Myelin Basic Protein.  Myelin basic protein (MBP), a component of
the myelin nerve sheath, is released during demyelination as a result of
various neurologic disorders, especially MS. Thus, MBP has been shown
to positively correlate with CSF leukocyte count, intrathecal IgG synthesis,
and the CSF/serum albumin concentration quotient (Sellebjerg et al,
1998). These results supported the use of MBP in CSF as a surrogate
disease marker during acute MS exacerbations. Others have found that
analysis of antibody against MBP in patients with a clinically isolated
syndrome is a rapid and precise method for predicting early conversion to
clinically definite MS (Berger et al, 2003). However, increased CSF levels
have also been reported in Guillain-Barré syndrome, lupus erythematosus,
subacute sclerosing panencephalitis, and various brain tumors, and follow-
ing CNS irradiation and chemotherapy (Mahoney et al, 1984; Brooks,
1989). Measurement of CSF MBP levels has also been proposed as a
prognostic marker in patients with serious head injury (Noseworthy et al,
1985). Recently, the clinical utility of MBP measurement in patients with
MS has been questioned, based on similarity of abnormal results to other
established laboratory methods, such as presence of oligoclonal bands on
protein electrophoresis, for diagnosing MS (Greene et al, 2012).
α2-Macroglobulin.  Except for a small amount transported across the
BBB in pinocytic vesicles, α2-macroglobulin (A2M) is normally excluded
from the CSF because of its large size. The number of these vesicles is
increased in certain polyneuropathies, resulting in an increased CSF A2M
level. Significant elevation reflects subdural hemorrhage or breakdown of the
BBB, as occurs in bacterial meningitis. A2M measurement alone, or in rela-
tionship to albumin and IgG, may assist in the evaluation of neurologic dis-
orders and increased CSF protein, and in the rapid differentiation between
bacterial and aseptic meningitis (Meucci et al, 1993; Kanoh & Ohtani, 1997).
β2-Microglobulin.  This protein is part of the human leukocyte
antigen class I molecule on the surfaces of all nucleated cells. CSF levels
above 1.8 mg/L are associated with leptomeningeal leukemia and lymphoma
but are not highly specific (Weller et al, 1992), in that they have a maximal
positive predictive value of 78% in cases with a positive cytology (Jeffrey
et al, 1990). β2-microglobulin (B2M) has also been shown to be a marker
for neuro-Behçet’s syndrome (Kawai & Hirohata, 2000). Viral infections,
including human immunodeficiency virus (HIV)-1, other inflammatory con-
ditions, and various malignancies have also been associated with elevated
levels. The measurement of B2M remains primarily investigational.
C-Reactive Protein.  Early studies indicated that CSF C-reactive
protein (CRP) is useful in differentiating viral (aseptic) meningitis from
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& Southgate, 1994). Others reported that CSF CRP is a more useful
screening test for viral versus bacterial meningitis, especially in children
(Sormunen et al, 1999). A meta-analysis of CRP studies since 1980 sug-
gested that a normal CSF or serum CRP has a high probability of ruling
out bacterial meningitis (i.e., negative predictive value about 97%) (Gerdes
et al, 1998). Another study found not only that CSF CRP levels were
increased in bacterial meningitis, but that these levels were significantly
higher in patients with gram-negative bacterial meningitis than in those
with gram-positive bacterial meningitis (Rajs et al, 2002). This assay has
been found to be more specific than blood CRP levels in the laboratory
PART 3
diagnosis of meningitis in children (Malla et al, 2013).
Fibronectin.  This large glycoprotein (molecular mass about 420 kDa)
is normally present in essentially all tissues and body fluids. Its primary
function is its role in cell adhesion and phagocytosis (Ruoslahti et al, 1981).
Thus, cell adhesion allows leukocytes to adhere to and pass through the
vascular endothelia and migrate to the inflammatory site.
In children with acute lymphoblastic leukemia, elevated CSF fibronec-
tin levels are associated with a poor prognosis, presumably due to leukemic
involvement of the CNS (Rautonen et al, 1989). Significant CSF eleva-
tions have also been reported in Burkitt’s lymphoma (Rajantie et al, 1989),
some metastatic solid tumors, astrocytomas, and bacterial meningitis
(Weller et al, 1990; Torre et al, 1991). Decreased levels have been reported
in viral meningitis and in the acquired immunodeficiency syndrome
(AIDS)–dementia complex (Torre et al, 1991; 1993). Detection of fibro-
nectin with alternatively spliced extra domain A is found in lower amounts
in CSF from children with bacterial meningitis as opposed to viral men-
ingitis or without meningitis (Pupek et al, 2013).
β-Amyloid Protein 42 and τ Protein.  The diagnosis of Alzheim-
er’s disease (AD) is based on the presence of dementia and a specific clinical
profile (i.e., from medical history, clinical examination) suggestive of AD,
together with the exclusion of other causes of dementia. Pathologically,
the disease is characterized by the presence of neurofibrillary tangles and
amyloid plaques.
Studies indicate that measurement of biochemical markers increases
diagnostic accuracy, especially early in the course of the disease, when
clinical symptoms are mild and vague and overlap with cognitive changes
that accompany aging and ischemic dementia. Thus, increased CSF levels
of microtubule-associated τ protein and decreased levels of β-amyloid
protein ending at amino acid 42 have been shown to significantly increase
the accuracy of AD diagnosis (Andreasen et al, 2001; Riemenschneider
et al, 2002; Sunderland et al, 2003). The positive predictive value for early
AD is greater than 90% (Andreasen et al, 2001). Others have found that
the calculated ratio of phosphorylated τ protein to β-amyloid peptide is
superior to either measure alone (Maddalena et al, 2003). The results were
as follows: Distinguishing patients with AD from healthy controls—
sensitivity 96%, specificity 97%; patients with AD from those with non-AD
dementia—sensitivity 80%, specificity 73%; and patients with AD from
those with other neurologic disorders—sensitivity 80%, specificity 89%.
These assays all appear to have a place in the biochemical diagnosis of early
AD (Blennow & Zetterrberg, 2009).
Protein 14-3-3.  The transmissible spongiform encephalopathies
constitute a group of uniformly fatal neurodegenerative diseases. Of these,
CJD is the major spongiform disease in humans. Two proteins, designated
130 and 131, have been detected in low concentrations in CSF from CJD
patients. These proteins have the same amino acid sequence as protein
14-3-3 (Hsich et al, 1996). In patients with dementia, a positive immunoas-
say for the 14-3-3 protein in CSF strongly supports a diagnosis of CJD.
In a subsequent study of patients with suspected CJD, the sensitivity of
the 14-3-3 protein determined by immunoassay was 97%, and the specific-
ity was 87% (Lemstra et al, 2000). False-positive results were seen primar-
ily in patients with stroke and meningoencephalitis.
Others, using a modified Western blot technique, reported a 94.7%
positive predictive value and a 92.4% negative predictive value for CJD
(Zerr et al, 1998). False-positive results were seen in patients with herpes
simplex encephalitis, atypical encephalitis, metastatic lung cancer, and
hypoxic brain damage.
Detecting elevation of protein 14-3-3, in combination with elevation
of other brain-derived proteins, such as τ protein, in CSF appears to be
useful in the diagnosis of CJD (Sanchez-Juan et al, 2006).
Transferrin and CSF Leakage.  Cerebrospinal fluid leakage usually
presents as otorrhea or rhinorrhea following head trauma, in some cases
beginning months to years after the injury. Recurrent meningitis is a
serious complication, making accurate identification of the leaking fluid
very important. In this regard, protein and glucose measurements are too
nonspecific to be of value. Transferrin, an iron-binding glycoprotein with
489
 a molecular mass of about 77 kDa, is synthesized primarily in the
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
liver. However, two transferrin isoforms are present in the CSF; the
major isoform (β1-transferrin) is present in all body fluids. The second
isoform (β2-transferrin), present only in the central nervous system, is
produced in the central nervous system by the catalytic conversion of
β-1-transferrin by neuraminidase. Immunofixation electrophoresis readily
identifies both isoforms.
Detection of β2-transferrin in CSF by protein electrophoresis with
transferrin immunofixation is a noninvasive, rapid, and inexpensive test of
high sensitivity and specificity that requires as little as 0.1 mL of fluid
(Ryall et al, 1992; Normansell et al, 1994). Several reports have demon-
strated the value of this technique in the diagnosis of CSF otorrhea and
rhinorrhea—conditions in which both isoforms are readily identified
(Irjala et al, 1979; Rouah et al, 1987; Zaret et al, 1992). Others have
stressed the importance of β2-transferrin identification in both CSF and
inner ear perilymphatic leakage, as well as possible sources of error due to
the presence of a transferrin allelic variant (Skedros et al, 1993a, 1993b;
Sloman & Kelly, 1993). β2-transferrin is sufficiently stable in CSF stored
at room temperature and/or when mixed with nasal mucus that a negative
result in a patient-collected nasal fluid specimen up to 1 week old is a
reliable negative finding for CSF leakage (Bleier et al, 2011).
Methemoglobin and Bilirubin.  Although most cases of subarach-
noid and intracerebral hemorrhage are readily identified by computed
tomography (CT), patients with mild subarachnoid hemorrhage, small
subdural or cerebral hematomas, and blood seepage from an aneurysm or
neoplasm and from small cerebral infarcts often are not identified by this
technique. In these cases, CSF spectrophotometric analysis has been
shown to detect methemoglobin in colorless CSF (<0.3 µmol/L)
(Trbojevic-Cepe et al, 1992). An increase in CSF bilirubin is also recog-
nized as a key finding supporting the diagnosis of subarachnoid hemor-
rhage (UK National External Quality Assessment Scheme for
Immunochemistry Working Group, 2003). A single net bilirubin absor-
bance cutoff point of >0.007 absorbance units is recommended in the
decision tree for interpretation and reporting of results.
Glucose
Derived from blood glucose, fasting CSF glucose levels are normally 50
to 80 mg/dL (2.8 to 4.4 mmol/L)—about 60% of plasma values. Rigorous
evaluation of CSF and serum glucose levels in a large number of children
confirmed that the CSF glucose level is normally approximately 60% of
the serum glucose level during childhood (Nigrovic et al, 2012). Results
should be compared with plasma levels, ideally following a 4-hour fast, for
adequate clinical interpretation. The normal CSF/plasma glucose ratio
varies from 0.3 to 0.9, with fluctuations in blood levels caused by the lag
in CSF glucose equilibration time.
CSF values below 40 mg/dL (2.2 mmol/L) or ratios below 0.3 are
considered to be abnormal. Hypoglycorrhachia is a characteristic finding
of bacterial, tuberculous, and fungal meningitis. However, sensitivity can
be as low as 55% for bacterial meningitis (Hayward et al, 1987), so a
normal level does not exclude these conditions. Some cases of viral menin-
goencephalitis also have low glucose levels, but generally not to the degree
seen in bacterial meningitis. Meningeal involvement by a malignant tumor,
sarcoidosis, cysticercosis, trichinosis, ameba (Naegleria), acute syphilitic
meningitis, intrathecal administration of radioiodinated serum albumin,
subarachnoid hemorrhage, symptomatic hypoglycemia, and rheumatoid
meningitis may also produce low CSF glucose levels (Fishman, 1992).
Decreased CSF glucose results from increased anaerobic glycolysis in
brain tissue and leukocytes and from impaired transport into the CSF.
Bacteria are usually present in insufficient quantities to be a major con-
tributor to the use of glucose in CSF. CSF glucose levels normalize before
protein levels and cell counts during recovery from meningitis, making it
a useful parameter in assessing response to treatment.
Increased CSF glucose is of no clinical significance, reflecting
increased blood glucose levels within 2 hours of lumbar puncture. A trau-
matic tap may also cause a spurious increase in CSF glucose.
Lactate
CSF and blood lactate levels are largely independent of each other. The
reference interval for older children and adults is 9.0 to 26 mg/dL (1.0 to
2.9 mmol/L) (Knight et al, 1981). Newborns have higher levels, ranging
from about 10 to 60 mg/dL (1.1 to 6.7 mmol/L) for the first 2 days, and
from 10 to 40 mg/dL (1.1 to 4.4 mmol/L) for days 3 to 10 (McGuinness
et al, 1983). Elevated CSF lactate levels reflect CNS anaerobic metabolism
due to tissue hypoxia.
Lactate measurement has been used as an adjunctive test in differentiat-
ing viral meningitis from bacterial, partially-treated bacterial, mycoplasma,
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fungal, and tuberculous meningitis, in which routine parameters yield
equivocal results (Cunha, 2012). In patients with viral meningitis, lactate
levels are usually below 25 mg/dL (2.8 mmol/L) and are almost always less
than 35 mg/dL (3.9 mmol/L), whereas bacterial meningitis typically has
levels above 35 mg/dL (Bailey et al, 1990; Cameron et al, 1993). Using 30
to 36 mg/dL as the cutoff value for bacterial meningitis, the sensitivity and
specificity are about 80% and 90%, respectively. Viral meningitis, partially
treated bacterial meningitis, and tuberculous meningitis often have inter-
mediate lactate levels that overlap each other, limiting the use of lactate
measurements in this differential diagnosis.
Persistently elevated ventricular CSF lactate levels are associated with
a poor prognosis in patients with severe head injury (DeSalles et al, 1986).
F2-Isoprostanes
F2-isoprostanes are increased in diseased regions of the brain in patients
with Alzheimer’s disease (AD) (Pratico et al, 1998) and are markers of free
radical brain injury associated with advanced age and latent AD
(Montine et al, 2011). Compared with age-matched controls, CSF
F2-isoprostanes are elevated in patients with probable AD (Montine et al,
1999). Therefore, in conjunction with CSF τ and β-amyloid protein, the
measurement of CSF F2-isoprostanes appears to enhance the accuracy of
the laboratory diagnosis of AD (Montine et al, 2001) and by itself appears
to correlate with the degree of cognitive decline in some AD patients
(Duits et al, 2013).
Enzymes
A wide variety of enzymes derived from brain tissue, blood, or cellular
elements have been described in the CSF. Although CSF enzyme assays
are not commonly used in the diagnosis of CNS diseases, there are
diseases/disorders in which they may prove useful.
Adenosine Deaminase
Adenosine deaminase (ADA) catalyzes the irreversible hydrolytic deamina-
tion of adenosine to produce inosine. Because ADA is particularly abun-
dant in T lymphocytes, which are increased in tuberculosis, its measurement
has been recommended in the diagnosis of pleural, peritoneal, and men-
ingeal tuberculosis. Higher ADA levels are present in tuberculous infec-
tions than in viral, bacterial, and malignant diseases (Blake & Berman,
1982; Mann et al, 1982, Choi et al, 2002), and measuring ADA in CSF
appears to be a sensitive and specific assay in diagnosing tuberculous
meningitis (Xu et al, 2010). ADA appears to have limited utility in HIV-
associated neurologic disorders (Corral et al, 2004).
Creatine Kinase
Brain tissue is rich in creatine kinase (CK) because it participates in main-
taining an adequate supply of adenosine triphosphate. Increased CSF CK
activity has been reported in numerous CNS disorders, including hydro-
cephalus, cerebral infarction, various primary brain tumors, and subarach-
noid hemorrhage, among others (Savory & Brody, 1979). In patients with
head trauma, CSF CK levels correlate directly with the severity of the
concussion (Florez et al, 1976).
CSF CK-MM and CK-MB are not normally present; when identified,
they are due to blood contamination (CK-MM) and an equilibrium
between CK-BB and CK-MM to produce CK-MB. Because the CK-BB
isoenzyme accounts for about 90% of brain CK activity and mitochondrial
CK (CKmt) the other 10%, CK isoenzyme measurements are more spe-
cific than total CK for CNS disorders (Chandler et al, 1984).
CSF CK-BB is increased about 6 hours following an ischemic or anoxic
insult. Global brain ischemia following respiratory or cardiac arrest results in
diffuse cerebral injury with peak CK-BB levels in about 48 hours (Chandler
et al, 1986). Here, CSF CK-BB activity less than 5 U/L (upper normal level)
indicates minimal neurologic damage; 5 to 20 U/L indicates mild to moderate
CNS injury; and levels between 21 and 50 U/L are commonly correlated with
death. Death occurs in essentially all patients with levels above 50 U/L.
Increased CSF CK-BB levels also correlate with the outcome following a
subarachnoid hemorrhage (Coplin et al, 1999). A CK-BB level greater than
40 U/L increases the chance of an unfavorable early or late outcome to 100%.
Lactate Dehydrogenase
Lactate dehydrogenase (LD) activity is high in brain tissue, with a pre-
dominance of the electrophoretically fast-moving isoenzyme fractions LD1
and LD2. Total LD activity of 40 U/L is a reasonable upper limit of normal
for adults and 70 U/L for neonates (Donald & Malan, 1986; Engelke et al,
1986). LD is useful in differentiating a traumatic tap from intracranial
hemorrhage because a current traumatic tap with intact RBCs does not
significantly elevate the LD level (Engelke et al, 1986). Sensitivity and
specificity are about 70% to 85% depending on the cutoff value. As with
lactate, LD activity is significantly higher in bacterial meningitis than in
aseptic meningitis (Donald & Malan, 1986; Engelke et al, 1986). Using a
cutoff of 40 U/L, the sensitivity is about 86% and the specificity about
93%.
Total CSF LD levels are also increased in patients with CNS leukemia,
lymphoma, metastatic carcinoma, bacterial meningitis, and subarachnoid
hemorrhage (Kjeldsberg, 1993). CSF LD isoenzymes have been shown to
add considerable specificity in the evaluation of various metastatic brain
tumors (Fleisher et al, 1981). The LD5/total LD ratio is increased (i.e.,
above 10% to 15%) in patients with leptomeningeal metastases from
carcinoma of the breast and lung and malignant melanoma. LD isoenzyme
analysis is also useful in detecting CNS involvement by leukemias and
lymphomas (Lossos et al, 2000). Isoenzyme analysis shows a distinct
pattern in young children with infantile spasms (Nussinovitch et al, 2003a)
and febrile convulsions (Nussinovitch et al, 2003b). Compared with con-
trols, both disorders are characterized by decreased LD1, increased LD2
and LD3, and no changes in LD4 and LD5.
CT is of limited value in estimating recovery potential and neurologic
outcome during the early stages of ischemic brain injury. However, com-
pared with controls (mean LD, 11.2 U/L), patients with an early stroke
had a mean level of 40.9 U/L, and those with a transient ischemic attack
(TIA) had a mean value of 11.8 U/L (Lampl et al, 1990). In patients with
hypoxic brain injury, an increased LD level 72 hours following resuscita-
tion indicates a poor prognosis (Karkela et al, 1992).
Lysozyme
Lysozyme (muramidase) catalyzes the depolymerization of mucopolysac-
charides. Because the enzyme is particularly rich in neutrophil and mac-
rophage lysosomes, its activity is very low in normal CSF. However, CSF
lysozyme activity is significantly increased in patients with both bacterial
and tuberculous meningitis. Thus, discriminant analysis demonstrated that
97% of patients with bacterial meningitis had increased lysozyme levels
(Ribeiro et al, 1992). Others found that patients with tuberculous menin-
gitis had significantly higher CSF lysozyme levels than those with bacterial
meningitis, partially treated bacterial meningitis, and controls (Mishra
et al, 2003). The diagnostic sensitivity and specificity for tuberculous men-
ingitis were 93.7% and 84.1%, respectively. Increased levels are also
present in cerebral atrophy, various CNS tumors, multiple sclerosis, intra-
cranial hemorrhage, and epilepsy (Kjeldsberg, 1993).
Ammonia, Amines, and Amino Acids
CSF ammonia levels vary from 30% to 50% of blood values. Elevated
levels are generally proportional to the degree of existing hepatic encepha-
lopathy but are difficult to quantify. Moreover, because hepatic encepha-
lopathy generally correlates with blood ammonia levels, the measurement
of CSF ammonia has little, if any, clinical value. However, cerebral gluta-
mine, synthesized from ammonia and glutamic acid, serves as the means
for CNS ammonia removal. Thus, CSF glutamine levels reflect the con-
centration of brain ammonia. Glutamine reference intervals are method
dependent; the upper reference level is about 20 mg/dL. Values over
35 mg/dL are usually associated with hepatic encephalopathy (Fishman,
1992). Elevated CSF glutamine levels have also been reported in patients
with depression (Levine et al, 2000) and encephalopathy secondary to
hypercapnia and sepsis (Mizock et al, 1989).
A major etiologic theory of schizophrenia involves dopamine. The
cornerstone for this theory is the fact that neuroleptic drugs that block
dopamine receptors are effective in the treatment of this disorder. Thus, it
has been reported that CSF levels of homovanillic acid (HVA), a metabolite
of the biogenic amines, are related to the severity of schizophrenic psychosis
(Maas et al, 1997). However, HVA concentration varied as a function of
psychosis rather than being related to the diagnosis of schizophrenia per se.
Others have reported decreased CSF levels of 5-hydroxyindoleacetic acid, a
metabolite of serotonin, in schizophrenic patients with suicidal behavior
(Cooper et al, 1992). This report adds support for a possible relationship
between suicide and CNS serotonin metabolism.
Although free CSF amino acids are relatively high in infants younger
than 30 days of age, their concentration is further increased in those with
febrile convulsions and bacterial meningitis. γ-Aminobutyric acid (GABA),
a major inhibitory brain transmitter, is significantly decreased in basal
ganglia neurons, and is very low or undetectable in the CSF of patients
with Alzheimer’s disease and Huntington’s disease (Achar et al, 1976;
Dubowitz et al, 1992). CSF GABA was detected in all patients with
migraine attacks, but not in those with tension headaches or in the control
group without headaches (Welch et al, 1975). Infants with startle disease,
a rare inherited autosomal dominant disorder characterized by seizures or
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the so-called stiff baby syndrome, have significantly decreased CSF
GABA levels (Dubowitz et al, 1992; Berthier et al, 1994).
Electrolytes and Acid-Base Balance
There are no clinically useful indications for the measurement of CSF
sodium, potassium, chloride, calcium, or magnesium. Measurements of
CSF pH, partial pressure of carbon dioxide (pCO2), and bicarbonate are
also not practical for patient care (Fishman, 1992).
Tumor Markers
Numerous studies have shown that various tumor markers are increased
PART 3
in the CSF of patients with both primary and metastatic tumors.
Carcinoembryonic Antigen
Carcinoembryonic antigen (CEA) is an oncofetal protein produced by a
variety of carcinomas. An early study found increased CEA levels in 44%
of patients with metastatic brain tumors (Suzuki & Tanaka, 1980). Others
reported that CSF levels of CEA have a sensitivity of only about 31%,
although the specificity is about 90% for detecting metastatic carcinoma
of the leptomeninges (Klee et al, 1986; Twijnstra et al, 1986). CSF levels
of CEA in patients with benign, primary malignant, and metastatic brain
tumors are approximately 0.31 ng/mL, 0.92 ng/mL, and 6.3 ng/mL,
respectively (Batabyal et al, 2003). CSF CEA levels are particularly sensi-
tive in detecting leptomeningeal carcinoma metastasis (Kang et al, 2010).
Other oncofetal proteins include human chorionic gonadotropin
(hCG), produced by choriocarcinoma and malignant germ cell tumors
with a trophoblastic component, and α-fetoprotein, a glycoprotein pro-
duced by yolk sac elements of germ cell tumors. Both β-hCG and
α-fetoprotein may be useful in the diagnosis and monitoring of the
response to therapy in patients with CNS germ cell tumors (Seregni et al,
2002; Ferguson et al, 2008).
Elevation of CSF ferritin is a sensitive indicator of CNS malignancy
but has very low specificity because it is also increased in patients with
inflammatory neurologic diseases (Zandman-Goddard et al, 1986). This
assay has greater diagnostic utility in detecting subarachnoid hemorrhage
in patients presenting late (Petzold et al, 2011).
MICROBIOLOGICAL EXAMINATION
A thorough and prompt examination of cerebrospinal fluid is essential for
the diagnosis of CNS infection because an inaccurate or delayed report
may result in significant mortality or morbidity. Although changes in
opening pressure, total cell and differential counts, total protein, and
glucose suggest an infectious origin (Table 29-6), Gram stain, culture and
other relevant studies are critical for a definitive diagnosis.
Bacterial Meningitis
The most common agents of bacterial meningitis are group B streptococ-
cus (neonates), Neisseria meningitidis (3 months and older) (Fig. 29-9),
Streptococcus pneumoniae (3 months and older), Escherichia coli and other
gram-negative bacilli (newborn to 1 month), Haemophilus influenzae (3
months to 18 years), and Listeria monocytogenes (neonates, older adults,
alcoholics, and immunosuppressed) (Graves, 1989; Wenger et al, 1990).
H. influenzae, once the most common bacterial cause of meningitis in
Figure 29-9  Cerebrospinal fluid Gram stain showing gram-negative diplo-
cocci characteristic of N. meningitidis.
491
 TABLE 29-6 
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
Typical Lumbar CSF Findings in Meningitis
Test Bacterial Viral Fungal Tuberculous

Opening pressure Elevated Usually normal Variable Variable

Leukocyte count ≥1000/µL <100/µL Variable Variable
Cell differential Mainly neutrophils* Mainly lymphocytes† Mainly lymphocytes Mainly lymphocytes
Protein Mild to marked increase Normal to mild increase Increased Increased
Glucose Usually ≤40 mg/dL Normal Decreased Decreased: may be <45 mg/dL
CSF/serum glucose ratio Normal to marked decrease Usually normal Low Low
Lactic acid Mild to marked increase Normal to mild increase Mild to moderate increase Mild to moderate increase

Data from Body BA, Oneson RH, Herold DA: Use of cerebrospinal fluid lactic acid concentration in the diagnosis of fungal meningitis, Ann Clin Lab Sci 17:429, 1987; Tang
LM: Serial lactate determinations in tuberculous meningitis, Scand J Infect Dis 20:81, 1988; Arevalo CE, Barnes PF, Duda M, Leedom JM: Cerebrospinal fluid cell counts
and chemistries in bacterial meningitis, South Med J 82:1122, 1989; Fishman RA: Cerebrospinal fluid in diseases of the nervous system, ed 2, Philadelphia, 1992, Saunders;
Wubbel L, McCracken GH Jr: Management of bacterial meningitis, Pediatr Rev 19:78, 1998; Zunt JR, Marra CM: Cerebrospinal fluid testing for the diagnosis of central
nervous system infection, Neurol Clin 17:675, 1999.
CSF, Cerebrospinal fluid.
*Lymphocytosis present in about 10% of cases.
†
Neutrophils may predominate early in disease.

young children, has decreased dramatically from widespread use of H.
influenzae type b vaccine. Cerebrospinal fluid shunts, head trauma, and
neurosurgery place patients at risk for CNS infections from Staphylococcus
species, gram-negative bacilli, and Propionibacterium species.
The Gram stain remains an accurate, rapid method by which to diag-
nose CNS infection. All specimens should be concentrated by centrifuga-
tion before Gram stain and culture. Depending upon the type of infecting
microorganism and its concentration in the cerebrospinal fluid, Gram stain
sensitivity ranges from 60% to 90%, with the greatest sensitivity corre-
sponding to higher concentrations of bacteria (about 105 colony-forming
units/mL). For example, the sensitivity of the Gram stain for detecting
Listeria monocytogenes and gram-negative bacilli is 50% or less (Greenlee,
1990). For patients with many polymorphonuclear leukocytes but no
organisms seen on Gram stain, the more sensitive acridine orange stain
may be helpful. Cultures have a sensitivity of 80% to 90%, but are about
30% less sensitive in partially treated cases (Greenlee, 1990).
Although standard culture-based methods remain the mainstay for
diagnosis, the BinaxNOW Streptococcus pneumoniae antigen test, an immu-
nochromatographic membrane assay that detects the presence of the C
polysaccharide cell wall antigen common to all pneumococcal serotypes, has
been proven to be a valuable tool for the rapid diagnosis of pneumococcal
meningitis from CSF. Latex agglutination bacterial antigen tests performed
on CSF to detect H. influenzae, N. meningitidis, S. pneumoniae, and
β-hemolytic group B streptococci historically are used as adjuncts to Gram
stain and culture; however, the sensitivity is approximately the same as the
Gram stain, and a negative test does not rule out the diagnosis of bacterial
meningitis. Perhaps the best application of latex agglutination antigen tests
is seen in partially treated, community-acquired meningitis that is negative
for microorganisms by Gram stain (Perkins et al, 1995; Wilson, 1997).
The polymerase chain reaction (PCR) and sequencing of 16S ribosomal
RNA in CSF have been shown to be very useful in the diagnosis of bacterial
meningitis (Schuurman et al, 2004). Compared with bacterial culture, the
assay shows a sensitivity of 86%, a specificity of 97%, a positive predictive
value of 80%, and a negative predictive value of 98%. Nucleic acid ampli-
fication tests may also be helpful in patients already receiving antimicrobial
therapy and in detecting more fastidious pathogens such as N. meningitidis
(Porritt et al, 2000; Seward & Towner, 2000; Baethgen et al, 2003).
Spirochetal Meningitis
The incidence of neurosyphilis has increased in recent years, primarily in
patients with HIV infection. In one report, 44% of patients with neuro-
syphilis had AIDS (Flood et al, 1998). Unfortunately, the remaining
patients, who may have had HIV infection without AIDS, were not studied.
The diagnosis of CNS infection in patients with syphilis relies primarily
on CSF parameters and serologic testing, although molecular DNA testing
may now represent another approach. Abnormalities in CSF protein and
cell counts are common in syphilitic meningitis, but are nonspecific. CSF
serologic testing to diagnose neurosyphilis is difficult. The standard non-
treponemal test performed on CSF is the Venereal Disease Research Labo-
ratory (VDRL). If few erythrocytes are contaminating the CSF, the VDRL
specificity is high, but its sensitivity is only 50% to 60% (Davis & Schmitt,
1989). Treponemal tests, such as the treponemal antibody absorption
(FTA-ABS), are both sensitive and specific for syphilis; however, their use
in CSF for neurosyphilis is controversial. The CSF FTA-ABS is highly
sensitive, but false-positive results may occur. In the absence of CSF
abnormalities or clinical suspicion, it should not be used as a screening
test. Thus, the following generalizations have been proposed (Davis &
Schmitt, 1989): (1) A nonreactive serum FTA-ABS test rules out neuro-
syphilis; (2) a reactive serum FTA-ABS test with a nonreactive CSF
FTA-ABS test essentially rules out neurosyphilis; (3) a reactive CSF VDRL
test makes a diagnosis of neurosyphilis likely; and (4) a reactive CSF
FTA-ABS test may indicate active neurosyphilis, asymptomatic neuro-
syphilis, treated neurosyphilis, or a false-positive reaction. Other studies
have demonstrated the utility of a PCR-based assay as a sensitive and
specific means of diagnosing neurosyphilis (Leslie et al, 2007).
Diagnosis of Lyme meningitis represents another challenge for the
laboratory. The mainstay of diagnosis remains serologic analysis per-
formed on a serum specimen, using an enzyme-linked immunosorbent
assay (ELISA)-based screening assay followed by confirmation with
Western blot. PCR-based analysis may be performed on CSF specimens;
however, the sensitivity of this approach may be low, particularly in patients
with chronic neuroborreliosis (Steere, 2010).
Rapid diagnosis of leptospiral and other bacterial infections is now
possible using genomic methods, including next-generation DNA sequenc-
ing analysis. In a recent study, a patient with neuroleptospirosis in whom
a diagnosis could not be established by conventional methods was success-
fully diagnosed using next-generation sequencing analysis of CSF (Wilson
et al, 2014). This methodology offers the hope of increasingly rapid diag-
nosis of a range of infections, as well as a way to rapidly track infection
outbreaks (Sherry et al, 2013).
Viral Meningitis
Enteroviruses (echoviruses, coxsackieviruses, polioviruses) and arboviruses
are responsible for the majority of meningitis cases; a seasonal peak in
spring to autumn has been noted for these agents. As an example, echovi-
ruses 9 (E9) and 30 (E30) were found to be mainly responsible for an
increase in cases of aseptic meningitis (Morbidity and Mortality Weekly
Report, 2003). Most patients present with a CSF pleocytosis, and although
neutrophils may be observed early in the infection, patients soon develop
a predominance of lymphocytes.
Before molecular diagnostic testing, viral meningitis was a diagnosis of
exclusion because the sensitivity of viral cultures can be low. Thus, in
an early study, a specific etiologic diagnosis by viral cultures varied from
72% for enteroviruses to 5% for herpes simplex virus (HSV) (Marton &
Gean, 1986).
Reverse transcriptase polymerase chain reaction (RT-PCR) is signifi-
cantly more sensitive than cell culture (Dumler & Valsamakis, 1999; Haus-
fater et al, 2004). Arguably, it has evolved as the “gold standard” for the
diagnosis of viral meningitis secondary to enterovirus, herpes simplex
virus, cytomegalovirus, varicella zoster, and JC virus. The use of RT-PCR
may result in significant cost savings by shortening hospital stays and
eliminating unnecessary diagnostic and therapeutic interventions (Ramers
et al, 2000). For patients with arbovirus-associated meningitis, acute and
convalescent serologic testing in serum remains the cornerstone for diag-
nosis, with CSF testing a possible adjunct. Presumptive diagnosis of West
Nile viral meningitis may be made on a CSF specimen using an IgM

492
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antibody-capture ELISA assay. PCR testing for West Nile virus and other
arboviruses may also be done on CSF specimens; however, these
assays have low sensitivity because of the short-lived nature of the
viremia in these diseases and are not recommended for routine use
(Vaughn et al, 2010).
PCR amplification of HSV DNA has revolutionized the testing for
HSV infection in CSF and has replaced brain biopsy as the primary
method used in the early diagnosis of HSV meningoencephalitis (Tunkel,
2008). False negatives might occur in very early infections and with bloody
taps, necessitating analysis of a second CSF specimen (Tyler, 2004).
Human Immunodeficiency Virus
A wide variety of CSF abnormalities may be found in HIV-positive patients
with or without neurologic disease, including lymphocytic pleocytosis,
elevated IgG indexes, and oligoclonal bands (Chalmers et al, 1990; Hall &
Snyder, 1992). Identifying opportunistic infection is the most important
indication for examining the CSF. Serious fungal infections may exist in
the presence of few or no CSF parameter abnormalities.
Fungal Meningitis
Cryptococcus neoformans is the most frequently isolated fungal pathogen
from CSF. India ink or nigrosin stains for cryptococcus capsular halos have
a sensitivity of about 25%, increasing to 53% with multiple lumbar punc-
tures, and to greater than 90% in untreated HIV-infected patients (Marton
& Gean, 1986). Detection of cryptococcal antigen from sera or CSF using
latex agglutination has higher sensitivity, ranging from 60% to 95%. False-
negatives due to a prozone effect or low concentration of polysaccharide
antigen may occur. Early disease, intraparenchymal infection, infection
with nonencapsulated C. neoformans variants, and immune complexes (cor-
rected with pronase treatment) may also produce false-negatives. Con-
versely, sera or CSF from patients with rheumatoid factor or Trichosporon
asahii infection may be falsely positive. If clinical suspicion for dimorphic
or filamentous fungi is high, large volumes of CSF (approximately 15 to
20 mL) are optimal for culture to improve the recovery of fungal
organisms.
Tuberculous Meningitis
Abnormal CSF with elevated protein and lymphocytic predominance are
the hallmark features of tuberculous meningitis. The sensitivity of CSF
acid-fast stains for the diagnosis of tuberculous meningitis is highly vari-
able, ranging from 10% to 12% (Greenlee, 1990) to greater than 50%
(Thwaites et al, 2004). Large volumes of CSF, often obtained from mul-
tiple lumbar punctures with the use of concentration techniques, are rec-
ommended to improve the sensitivity of both acid-fast stain and culture
(Marton & Gean, 1986).
PCR nucleic acid amplification for detecting Mycobacterium tuberculosis
DNA-specific sequences have shown great promise in the rapid and accu-
rate diagnosis of tuberculous meningitis (Lin et al, 1995; Desai & Pal,
2002), but PCR methods using a single primer are associated with rela-
tively low sensitivity (Pai et al, 2003). Multiplex PCR analysis, using
primers to multiple relevant tubercular genes, appears to be a significantly
more sensitive method in the rapid diagnosis of tuberculous meningitis
(Kusum et al, 2011).
TABLE 29-7 
Synovial Fluid Findings by Disease Category
Group I
Finding Normal Noninflammatory
Clarity Transparent Transparent
Color Clear to pale yellow Xanthochromic
WBCs/mL 0-150 <3000
PMNs, % <25 <30
RBCs No No
Glucose (blood/SF 0-10 0-10
difference, mg/dL) (0-0.56 mmol/L) (0-0.56 mmol/L)
Modified from Kjeldsberg CR, Knight JA: Body fluids: laboratory examination of amniotic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993, © American
Society for Clinical Pathology, with permission.
PMNs, Polymorphonuclear cells, neutrophils; RBCs, red blood cells; SF, synovial fluid; WBCs, white blood cells.
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DOT ELISA has been standardized to detect tuberculosis antigens and
antibodies against M. tuberculosis in CSF. Using this technique, a positive
reaction was present in 86% of cases with suspected tuberculous meningitis
(Kashyap et al, 2003). Only 5% of patients with other disorders, mainly
pyogenic meningitis, were positive.
Other tests have been shown to be useful in some cases of tuberculous
meningitis. ADA levels are significantly higher in tuberculous meningitis
than in other types of meningitis and CNS disorders (Pettersson et al, 1991;
Choi et al, 2002; Solari et al, 2013), although the degree of increase indica-
tive of tuberculous infection varies depending on the specific assay used.
Primary Amebic Meningoencephalitis
PART 3
This rare disease is caused by the free-living ameba Naegleria fowleri,
Acanthamoeba species, and Balamuthia mandrillaris. Naegleria and Bala-
muthia are more likely to cause an acute inflammatory response with a
neutrophilic pleocytosis, a decreased glucose level, an elevated protein
concentration, and the presence of erythrocytes. Gram stain is always
negative. Acanthamoeba more often produces a granulomatous meningitis.
Motile Naegleria trophozoites may be visualized by light or phase-contrast
microscopy in direct wet mounts, allowing rapid diagnosis. Intact and
degenerating organisms may be identified on Wright’s- or Giemsa-stained
cytospins but must be distinguished from macrophages (dos Santos, 1970;
Benson et al, 1985). Acridine orange stain is useful in differentiating
amebas (brick red) from leukocytes (bright green). Immunocytochemical
staining of CSF specimens using an antibody to Naegleria fowleri has been
shown to improve microscopic detection of this organism (Visvesvara,
2010). Multiplex PCR-based methods are an even more sensitive modality
for detecting multiple amebic organisms in CSF (Qvarnstrom et al, 2006).
SYNOVIAL FLUID
Synovium refers to the tissue lining synovial tendon sheaths, bursae, and
diarthrodial joints, except for the articular surface. It is composed of one
to three cell layers that form a discontinuous surface overlying fatty,
fibrous, or periosteal joint tissue.
Synovial fluid (SF) is an imperfect ultrafiltrate of blood plasma com-
bined with hyaluronic acid produced by the synovial cells. Small ions and
molecules (e.g., Na+, K+, glucose, urea) readily pass into the joint space
and therefore are similar in concentration to plasma; large molecules are
absent or present in trace amounts. Resorption of synovial molecules is by
lymphatics and is not size dependent. SF acts as a lubricant and adhesive
and provides nutrients for the avascular articular cartilage.
Examination of the SF is essential to distinguish infectious from non-
infectious arthritis. Results from gross and microscopic examination of
SF have traditionally been divided into “reaction types,” as depicted in
Table 29-7. These groupings are largely descriptive, and considerable
overlap among them is evident. Except for Gram stain, culture, and crystal
examination, SF parameters can be nonspecific and must be integrated into
the clinical context.
Noninflammatory effusions (Group I) typically have leukocyte counts
less than 3000/µL, with a minority of neutrophils. Osteoarthritis,
traumatic arthritis, neuropathic osteoarthropathy, pigmented villonodular
synovitis, and early rheumatic fever usually present with little
CATEGORY
Group II Group III
Inflammatory Infectious Group IV Hemorrhagic
Transparent/opaque Opaque Opaque
Xanthochromic to White Red-brown or xanthochromic
white/bloody
3000-75,000 50,000-200,000 50-10,000
>50 >90 <50
No Yes Yes
0-40 20-100 0-20
(0-2.2 mmol/L) (1.11-5.5 mmol/L) (0-1.11 mmol/L)
493
 inflammatory response. Early rheumatoid arthritis, early bacterial infec-
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
tion, and viral arthritis may also present as noninflammatory effusions.
Inflammatory effusions (Group II) have leukocyte counts between
3000 and 75,000/µL, with neutrophils accounting for more than 50% of
the population. Rheumatoid arthritis, systemic lupus erythematosus (SLE),
Reiter’s syndrome, rheumatic fever, acute crystal-induced arthritis, arthri-
tis associated with inflammatory bowel disease, psoriatic arthritis, and fat
droplet synovitis are examples of this reaction group.
Purulent (infectious) effusions (Group III) typically have leukocyte
counts greater than 50,000/µL, of which 90% or more are neutrophils.
Bacterial, fungal, and tuberculous joint infections constitute this group.
Hemorrhagic effusions (Group IV) may be seen in association with
traumatic arthritis, pigmented villonodular synovitis, synovial hemangi-
oma, neuropathic osteoarthropathy, joint prostheses, and hematologic
disorders (hemophilia, thrombocytopenia, anticoagulant therapy, sickle
cell disease or trait, myeloproliferative syndrome).
SPECIMEN COLLECTION
Joint fluid aspiration (arthrocentesis) should be confined to patients with
an undiagnosed effusion or a significant clinical change related to a known
effusion. It should be performed by an experienced operator using good
sterile technique. Caution is necessary to avoid aspirating a sterile joint in
someone with bacteremia or through a cutaneous or periarticular soft
tissue infection into a sterile joint. Large joints such as the knee normally
contain no more than 4.0 mL of SF, so small sample size is common unless
an effusion is present.
Synovial fluid must be collected with sterile, disposable needles and
plastic syringes to avoid contamination by birefringent particulates. The
syringe may be heparinized with 25 U of sodium heparin/mL of SF in
routine arthrocentesis. Oxalate, lithium heparin, and powdered ethylene-
diaminetetraacetic acid (EDTA) anticoagulants should be avoided because
they form crystal artifacts that may be misleading during the microscopic
examination. Before aspiration, the joint should be turned or otherwise
manipulated to ensure mixing of its contents.
Ideally, the specimen should be separated into three parts: 3 to 10 mL
into a sterile heparinized tube or syringe for microbiological studies; 2 to
5 mL in an anticoagulant tube (sodium heparin or liquid EDTA) for
microscopic examination; and about 5 mL into a plain (no anticoagulant)
tube for chemical analysis (normal SF does not clot because fibrinogen is
absent). Heparin concentrations greater than 125 U/mL have an inhibi-
tory effect on some pathogenic bacteria (Rosett & Hodges, 1980). Speci-
mens for culture, therefore, should be at least 1 to 2 mL in volume if they
are submitted in green-top heparin tubes (Becton Dickinson, Rutherford,
N.J.) containing 143 U/tube of heparin, or submitted in recapped syringes
after removal of the needle and excess air.
Dry taps may still have fluid within the needle, which may be sufficient
for the most critical tests. Such specimens should be submitted with the
needle still on the syringe and its tip stuck into a sterile cork. Good com-
munication with the laboratory is crucial to the appropriate processing of
such specimens.
RECOMMENDED TESTS
Laboratory examination of SF is of major importance in the differential
diagnosis of joint disease, especially in crystal-induced and infectious
arthritis. When either is suspected, arthrocentesis and a systematic exami-
nation of the SF are imperative, and when examination is carried out
properly, it is usually diagnostic. In other joint diseases, a specific diagnosis
may not be possible. Nevertheless, fluid examination is still important, if
only to rule out infectious arthritis, which is a critical diagnosis to make
in that a joint may be irreversibly damaged within 2 to 3 days if not prop-
erly treated. This is especially true when Staphylococcus aureus is the infec-
tious organism. Therefore, routine tests should be directed toward the
diagnosis of these two disorders (Box 29-8). Although other tests are not
of practical value for routine use, they may provide important diagnostic
information under certain circumstances.
It is critical that these tests be performed well because they can provide
highly specific diagnostic information. Quality performance may be com-
promised in smaller laboratories that examine only a small number of SF
specimens (Hasselbacher, 1987; Rabinovitch & Cornbleet, 1994).
GROSS EXAMINATION
Total volume should be recorded at the bedside, especially if the sample
is to be divided for submission to different laboratory sections.
494
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BOX 29-8 
Recommended Synovial Fluid Tests
Routine tests
Gross examination (color, clarity)
Total and differential leukocyte counts
Gram stain and bacterial culture (aerobic and anaerobic)
Crystal examination with polarizing microscope and compensator
Useful tests in certain circumstances
Fungal and acid-fast stains and cultures
PCR for bacterial and mycobacterial DNA
Serum–synovial fluid glucose differential
Lactate and other organic acids
Complement
Enzymes
Uric acid
Modified from Kjeldsberg CR, Knight JA: Body fluids: laboratory examination of amni-
otic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993, ©
American Society for Clinical Pathology, with permission.
DNA, Deoxyribonucleic acid; PCR, polymerase chain reaction.
Color should be evaluated in a clear glass tube against a white back-
ground. Normal SF is colorless but is often pale yellow because of diape-
desis of a few RBCs associated with even mild trauma. Noninflammatory
and inflammatory disorders impart a straw to yellow color (xanthochro-
mia). Septic fluid may be yellow, brown, or green depending on the chro-
mogens produced by the offending organism and the host response,
including the presence of WBCs and RBCs.
A traumatic tap produces an uneven distribution of blood during
arthrocentesis or streaking in the syringe. Although pale yellow xantho-
chromia is difficult to distinguish from normal, a red-brown color follow-
ing centrifugation is good evidence of pathologic hemarthrosis.
Clarity relates to the number and type of particles within the SF.
Because normal SF is transparent, newsprint is easily read through the
tube. Although translucent fluid obscures details, black and white areas can
be distinguished, but opaque fluid completely obscures the background.
Leukocytes are most commonly responsible for changes in clarity.
However, very large numbers of crystals may produce an opaque, milky,
opalescent fluid without leukocytes. A shimmering, oily-appearing speci-
men suggests an abundance of cholesterol crystals, which may grossly
resemble pus.
Increased turbidity is less often due to concentrations of fibrin, free-
floating rice bodies (fragments of degenerating proliferative synovial cells
or microinfarcted synovium), metal and plastic particles from patients with
joint prostheses, or cartilage fragments in osteoarthritis. A ground pepper
appearance from pigmented cartilage fragments may be the result of a
metabolic disorder (i.e., ochronosis).
MICROSCOPIC EXAMINATION
Total Cell Count
Total leukocyte counts should be performed promptly to avoid degenera-
tive cell loss, which begins as soon as 1 hour following arthrocentesis. Cell
counts are usually performed in a standard hemocytometer. A wet-prep
slide count of 0 to 2 leukocytes per high-power field (hpf) (averaged over
10 fields) predicts fewer than 1300 WBCs by cell count (Clayburne et al,
1992). Leukocyte counts over 50,000/µL require dilution, which should
be done with saline, not acetic acid, to avoid mucin clot formation and cell
clumping. Automated cell counters may be used, but their use risks clog-
ging the machine aperture or obtaining spuriously high cell counts from
non-WBC particles (e.g., crystals, fat globules), especially in multichannel
and flow cell–based machines. Pretreatment of highly viscous SF samples
with hyaluronidase improves automated cell counting using these instru-
ments (Aulesa et al, 2003). Automated cell counting in SF samples using
digital image-based technology avoids the clogging and spurious count
problems and now offers a reliable method for these counts (Walker et al,
2009; Scott, 2014).
Leukocyte counts greater than 10,000/µL, and often greater than
50,000/µL, are characteristic of crystal-induced arthritis (e.g., gout, pseu-
dogout), chronic inflammatory arthritis (e.g., rheumatoid arthritis, sys-
temic lupus erythematosus, ankylosing spondylitis), and septic arthritis
(Kjeldsberg, 1993). Osteoarthritis, osteochondritis dissicans, trauma, and
synovioma usually have total WBC counts less than 10,000/µL.
Erythrocytes should be routinely counted unless it is an obvious trau-
matic tap. If a large number of RBCs interferes with the leukocyte count,
they may be lysed by dilution with 0.3 normal saline or 1% saponin in
saline.
The upper reference level for SF leukocytes is 150 to 200/µL (Kjelds-
berg, 1993). Elevated cell counts are used to help divide findings into
different disease categories but are nonspecific for any particular disease
because of extensive overlap.
Differential Leukocyte Count
Cytospin preparations are preferred over smears from centrifuged SF
because the cell morphology is significantly better preserved. Treatment
with hyaluronidase may be necessary to produce thin smears in viscous
specimens. Liquid-based thin-layer processing instruments may also be
used effectively to produce good-quality preparations of SF for micro-
scopic examination (Policarpio-Nicolas & Valente, 2013). Automated leu-
kocyte differential counts utilizing flow cytometers may be reliably
performed on SF samples (Aulesa et al, 2003), with even better perfor-
mance, including detection of crystals, using digital image-based instru-
ments (Walker et al, 2009; Scott, 2014).
Neutrophils normally account for about 20% of SF leukocytes. Neu-
trophils generally exceed 50% in urate gout, pseudogout, and rheumatoid
arthritis (RA); they most often exceed 75% in acute bacterial arthritis.
When 75% is used as a cutoff, the sensitivity for an inflammatory process
is about 75%, and the specificity is 92% (Shmerling et al, 1990). These
cells frequently exhibit degenerative changes and may contain bacteria,
crystals, lipid droplets, vacuoles, or dark blue to black granular inclusions
thought to consist of immune complexes (ragocytes, RA cells). The pres-
ence of ragocytes in patients with RA may indicate a poorer outcome
(Davis et al, 1988).
Lupus erythematosus (LE) cells, sometimes present in patients with
lupus arthritis, are most often neutrophils that have phagocytosed the
nuclei of degenerating cells (Fig. 29-10). However, LE cells are not
pathognomonic for SLE because they have also been identified in the SF
of RA patients (Hunder & Pierre, 1970).
Lymphocytes, normally constituting about 15% of SF cells, are prom-
inent in early RA and other autoimmune disorders, as well as in chronic
infections. Reactive forms, including immunoblasts, are occasionally
present.
Monocytes and macrophages are the most common cells present in
normal SF, accounting for approximately 65% of the cell count. Monocy-
tosis may be self-limited in viral arthritis or serum sickness, or more
chronic in SLE or undifferentiated connective tissue disorders. Reiter’s
cells, originally believed to be specific for Reiter’s syndrome, are macro-
phages containing degenerating neutrophils (Fig. 29-11).
Eosinophilia, defined as more than 2% of the leukocyte count, has
been reported in Lyme disease, rheumatoid arthritis, rheumatic fever,
metastatic carcinoma, parasitic infection, chronic urticaria, and angio-
edema, and following arthrography (allergic reaction to dye) and irradia-
tion (Podell et al, 1980; Kay et al, 1988).
Synovial cells have no pathologic significance. They appear similar to
mesothelial cells and may be difficult to distinguish from monocytes and
macrophages.
Lipid bodies are associated with trauma, aseptic necrosis, and RA.
These droplets often form Maltese crosses under polarized light, can be
Figure 29-10  Lupus erythematosus cell in the synovial fluid from a patient
with systemic lupus erythematosus.
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associated with a leukocyte response, and may cause spurious elevations of
the automated WBC count (Wise et al, 1987).
Crystal Examination
Crystals in SF lead to acute inflammation with increased WBC counts and
a neutrophil-predominant infiltrate. Crystal identification, especially if
intracellularly in neutrophils or macrophages, is pathognomonic for a
crystal-induced arthritis (Judkins & Cornbleet, 1997).
Gout refers to the process of crystal deposition in articular tissue.
Common usage typically implies urate gout, and an inflammatory response
to crystal deposition is referred to as gouty arthritis. The most common
PART 3
types of endogenous crystals responsible for gouty arthritis are monoso-
dium urate monohydrate (urate gout), calcium pyrophosphate dihy-
drate (pyrophosphate gout, chondrocalcinosis, or “pseudogout”), apatite
and other basic calcium phosphates (BCP; apatite gout), calcium oxalate
(oxalate gout), and lipids (lipid gout).
Except for BCP, all of the above crystals can be detected by polarized
light microscopy. A high-quality polarizing microscope with a first-order
red plate compensator should be used. The polarizer filter is placed
directly above the light source. The analyzer (another polarizing filter) is
placed between the specimen slide and the microscope oculars, oriented
90 degrees from the polarizer to produce a dark background. The com-
pensator is placed between the polarizer and the analyzer, usually oriented
45 degrees (halfway) between the planes of the two polarizing filters.
Initial examination should be performed on a wet preparation using
polarized light. Phase-contrast microscopy enhances crystal detection.
The slide and coverslip must be cleaned and carefully dried immediately
before use to avoid birefringent dust particulate artifacts. The coverslip
edges are sealed with nail polish or other sealant, which retards but does
not prevent evaporation. The coverslip edge is used to find the proper
plane of focus; however, crystals in this location should be ignored because
they are most likely artifacts. Most crystals are scanned with a 10× objective
and are evaluated with at least a 40× objective, concentrating especially on
cellular areas. Complete examination requires 100× oil immersion,
however, because apparently negative fluids on scanning may contain a
large population of small crystals (Gatter & Schumacher, 1991). Aligning
the crystals’ orientation to the compensator by rotating the microscope
stage or the compensator facilitates recognition and identification. Crystal
morphology, the extinction angle, strength, and signs of any birefringence
(i.e., the ability to refract light and split the incident light into two rays: a
fast ray and a slow ray) are noted. The sensitivity and specificity of polar-
ized microscopy for crystals are 78% and 79%, respectively, for monoso-
dium urate (Hasselbacher, 1987), and 12% and 67% for calcium
pyrophosphate dihydrate (McGill & York, 1991). Repeat examination fol-
lowing 24 hours of refrigeration at 4° C may result in a significant increase
in the number of crystal-positive fluids (Yuan et al, 2003). Cytospin prepa-
rations are also used effectively in the detection of crystals in SF samples
(Theiler et al, 2014).
The Diff-Quik staining method may be a reliable alternative to polar-
ized microscopy. Overall specificity, sensitivity, and accuracy are 87.5%,
94.4%, and 91.9%, respectively. The overall positive predictive value is
92.7%; the negative predictive value is 90.3% (Selvi et al, 2001). Other
more sophisticated and reliable methods, such as X-ray crystallography
Figure 29-11  Reiter’s cells in the synovial fluid from a patient with Reiter’s
syndrome.
495
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
Figure 29-12  Monosodium urate crystals under polarized light from a patient
with urate gout.
Figure 29-13  Monosodium urate crystals in synovial fluid. Compensated
polarized light. (From Kjeldsberg CR, Knight JA: Body fluids: laboratory examination
of amniotic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993,
© American Society for Clinical Pathology, with permission.)
and Fourier transform infrared spectroscopy, have been described for iden-
tifying and characterizing crystals in biological specimens (Rosenthal &
Mandel, 2001).
Monosodium urate (MSU) crystals appear as needle-shaped rods 5 to
20 µm long, but they may be only 1 to 2 µm in length or, rarely, may
appear as rounded spherolites. They are strongly birefringent (Fig. 29-12):
yellow when oriented parallel to the compensator and blue with perpen-
dicular orientation (negative birefringence or elongation) (Fig. 29-13). A
control slide of MSU crystals should always be used for comparison.
Alternatively, betamethasone, a steroid that appears as a strongly negative
birefringent rod, can be used to prepare a reference slide for the polarizing
microscope (Judkins & Cornbleet, 1997).
MSU crystals are found in 90% of acute urate gout and in about 75%
of patients between attacks. Intracellular MSU crystals are characteristic
of acute urate gout. They may also occasionally be observed as a result of
inflammation in septic arthritis (McCarty, 1988).
Calcium pyrophosphate dihydrate (CPPD) crystals are found in a
group of conditions collectively known as CPPD crystal deposition disease.
These crystals appear as rhomboids, rods, or rectangles 1 to 20 µm in
length. CPPD crystals are weakly birefringent with positive elongation
(blue when aligned with the compensator axis; Fig. 29-14). Many are too
small to polarize the light, making them difficult to detect without phase-
contrast microscopy. Extinction is incomplete and occurs between 20 and
30 degrees from the angle of the polarizer and analyzer (oblique or inclined
extinction).
CPPD crystals are associated with degenerative arthritis and are seen
in arthritides associated with hypomagnesemia, hemochromatosis, hyper-
parathyroidism, and hypothyroidism (Jones et al, 1992).
Calcium hydroxyapatite and the other basic calcium phosphate (BCP)
crystals are typically too small and nonbirefringent (isotropic) to see with
496
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Figure 29-14  Calcium pyrophosphate dihydrate crystals in synovial fluid from
a patient with pseudogout. Compensated polarized light. (From Kjeldsberg CR,
Knight JA: Body fluids: laboratory examination of amniotic, cerebrospinal, seminal,
serous and synovial fluids, ed 3, Chicago: © American Society for Clinical Pathology;
1993, with permission.)
Figure 29-15  Cholesterol crystals in synovial fluid. Polarized light.
light microscopy, unless they are clumped into 1- to 50-µm spherical
microaggregates. Alizarin red S dye may be used to stain these and other
calcium-containing crystals (Lazcano et al, 1993). At present, identifica-
tion of BCP crystals is not important for diagnosis or prognosis, or as a
guide in treatment.
Calcium oxalate dihydrate crystals are 5- to 30-µm bipyramidal octa-
hedral “envelopes” with variable birefringence and positive elongation.
They are seen in arthropathy associated with chronic renal dialysis and
primary oxalosis, a rare inborn error of metabolism. The monohydrate
form is birefringent but nondescript in shape.
Lipid crystals are 1- to 20-µm spheres with a Maltese cross appearance
and positive birefringence under compensated polarized light. They have
been implicated as a cause of acute arthritis (McCarty, 1988).
Crystalline corticosteroids from intraarticular injection may have an
appearance similar to MSU or CPPD crystals and persist up to 1 month
following injection. Most often, they have blunt, jagged edges without
clear crystal structure because they are prepared by grinding up larger
crystalline forms. Triamcinolone hexacetonide is negatively birefringent,
but most others show positive birefringence.
Cholesterol crystals typically appear as irregular birefringent plates,
often with notched corners (Fig. 29-15). In chronic effusions (e.g., tuber-
culous arthritis, RA, SLE), needle- or rhomboid-shaped crystals similar to
MSU or CPPD may be present (Ettlinger & Hunder, 1979). Very small
(1 to 5 microns) irregular, rod- and needle-shaped cholesterol crystals have
been identified by X-ray diffraction analysis and by ultrastructural studies
in osteoarthritis effusions (Fam et al, 1981). Cholesterol crystals are
ethanol and ether soluble and are not phagocytosed by leukocytes. If
cholesterol crystals are detected, quantitative analysis should show that the
SF cholesterol level exceeds the plasma level.
TABLE 29-8  dehydrogenase is elevated in RA, gout, failed arthroplasties, and infec-
Reference Intervals for Synovial Fluid Constituents
Constituent Synovial Fluid Plasma
Total protein 1-3 g/dL 6-8 g/dL
Albumin 55%-70% 50%-65%
α1-Globulin 6%-8% 3%-5%
α2-Globulin 5%-7% 7%-13%
β-Globulin 8%-10% 8%-14%
γ-Globulin 10%-14% 12%-22%
Hyaluronic acid 0.3-0.4 g/dL
Glucose 70-110 mg/dL 70-110 mg/dL
Uric acid 2-8 mg/dL 2-8 mg/dL
Lactate 9-29 mg/dL 9-29 mg/dL
Modified from Kjeldsberg CR, Knight JA: Body fluids: laboratory examination of amni-
otic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993, ©
American Society for Clinical Pathology, with permission.
Glove powder (modified cornstarch) introduced during joint surgery
appears as round, strongly birefringent particles 5 to 30 µm in diameter,
with a central notch and a Maltese cross appearance when polarized.
A variety of other crystals or particulates may be present in SF. These
include monoclonal Ig crystals or cryoglobulins, Charcot-Leyden crystals,
amyloid fragments, cartilage fragments, collagen fibrils and fibrin strands,
hematoidin crystals from prior hemorrhage, crystals from certain antico-
agulants, nail polish, prosthetic fragments, and dust particles (Gatter &
Schumacher, 1991).
CHEMICAL ANALYSIS
Chemical analyses of SF generally offer only supportive information to the
routine tests. High viscosity may be remedied by dilution with normal
saline, sonication, or hyaluronidase treatment. Reference intervals for the
more important chemical analytes are shown in Table 29-8.
Mucin Clot Test
Addition of acetic acid to SF precipitates hyaluronate into a mucin clot,
which may be graded as good, fair, or poor. A fair to poor mucin clot test
reflects dilution and depolymerization of hyaluronic acid—a nonspecific
finding of several inflammatory arthritides. Although of historic interest,
the mucin clot test has minimal clinical utility today (Baker, 1991).
Glucose
Proper interpretation of SF glucose values requires comparison with serum
levels, ideally preceded by a fast of 8 hours to allow glucose to equilibrate
across the synovial membrane. The serum-synovial differential is less than
10 mg/dL in normal and many noninflammatory conditions. In septic
arthritis, this difference ranges from 20 to 60 mg/dL but overlaps signifi-
cantly with other inflammatory conditions, thereby limiting its clinical
usefulness. When a cutoff value of 75 mg/dL is used, the sensitivity of low
glucose for detecting an inflammatory joint disease is only 20%; the speci-
ficity is 84% (Shmerling et al, 1990).
In vitro glycolysis by large numbers of leukocytes may falsely reduce
SF glucose values unless testing is performed within 1 hour of collection.
Tubes containing sodium fluoride, an inhibitor of glycolysis, prevent the
loss of glucose.
Protein
The mean normal protein concentration is 1.38 g/dL in living volunteers
(Weinburger & Simkin, 1989). A reliable reference interval is 1.0 to 3.0 g/
dL. With increasing inflammation, larger proteins (e.g., fibrinogen) enter
the synovial space. Spontaneous clot formation may be detected in non-
anticoagulated specimen tubes (fibrin clot test). Although elevation of the
SF protein level may be associated with inflammatory and infectious condi-
tions, measurement of SF protein is highly nonspecific; the sensitivity is
about 52% and the specificity 56% for inflammatory disorders (Shmerling
et al, 1990).
Enzymes
Numerous enzymes have been studied in SF, including lactate dehydroge-
nase, aspartate aminotransferase, adenosine deaminase, acid and alkaline
phosphatase, and lysozyme, among others (Kjeldsberg, 1993). Lactate
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tious arthritis. This increase most likely reflects the neutrophil infiltrate.
Elevated acid phosphatase may have negative prognostic value in RA but
is not specific (Luukkainen et al, 1989). Although enzyme analysis of SF
is currently not clinically relevant, the measurement of various hydrolases
may have significant predictive value in joint prognosis, especially RA.
Organic Acids
When compared with nonseptic monoarticular arthritis, SF lactic acid
levels are usually increased in patients with septic arthritis (Kjeldsberg,
1993). Levels significantly greater than 30 mg/dL (3.7 mmol/L) are com-
PART 3
monly associated with septic arthritis due to gram-positive cocci and gram-
negative bacilli. Measurement of D-lactate levels in SF appears to be a
useful method for rapid diagnosis of bacterial synovitis (Gratacos et al,
1995). In other studies, however, normal or intermediate lactate levels
neither rule in nor rule out infection. Moreover, gonococcal arthritis is
notorious for having normal SF lactate levels (Curtis et al, 1983).
When gas-liquid chromatography is used, the presence of other organic
acids not normally present in SF (e.g., n-valeric, n-hexanoic, and suc-
cinic acids) may be very helpful in differentiating septic from nonseptic
arthritis (Borenstein et al, 1982).
Uric Acid
Synovial fluid uric acid levels generally, although not always, parallel serum
levels in gout and noninflammatory arthropathies. This assay provides
little clinical value in SF analysis except in some cases where gout is sus-
pected but crystals are not identified (Reeves, 1965). Increased SF uric acid
levels in these cases support a diagnosis of gout.
Lipids
In contrast to plasma, normal SF contains extremely low concentrations
of lipids. Synovial fluid lipid abnormalities include (1) rare cholesterol-rich
pseudochylous effusions typically associated with chronic RA; (2) lipid
droplets, usually the result of trauma; and (3) extremely rare chylous effu-
sions seen in association with RA, SLE, filariasis, pancreatitis, and trauma
(Wise et al, 1987). These diseases can usually be differentiated clinically
and by gross and microscopic examination; quantification of lipids cur-
rently has no clinical value in joint fluid analysis, except in cases in which
cholesterol crystals may resemble MSU or CPPD. In these cases, a cho-
lesterol level that exceeds the plasma level supports the presence of cho-
lesterol crystals.
IMMUNOLOGIC STUDIES
Rheumatoid factor (RF) is found in the SF of about 60% of RA patients,
usually at a titer equal to or slightly lower than the serum titer. Antinu-
clear antibodies (ANA) are found in the SF of about 70% of patients with
SLE and 20% of patients with RA. Neither is specific enough for practical
use. SF complement levels, normally about 10% of serum levels, increase
to 40% to 70% of serum activity with inflammation, proportional to the
increase in protein exudation. Complement consumption in SLE and RA,
in particular, results in levels less than 30% of serum complement. Com-
plement is also decreased in some cases of bacterial and crystal-induced
arthritis, so measurement is impractical for routine diagnosis.
MICROBIOLOGICAL EXAMINATION
Immediate transportation of joint fluid and good communication of clini-
cal suspicions to the laboratory are extremely important in the rapid iden-
tification of an infectious agent.
Septic arthritis may be acute or chronic, and Gram stain and culture
should be performed as part of the routine SF evaluation. Gram stain
sensitivity varies from about 75% for staphylococcal infections and 50%
for most gram-negative organisms, to less than 25% for gonococcal infec-
tions (Goldenberg & Reed, 1985). Concentration methods, including
cytocentrifugation, may increase the sensitivity of the Gram stain.
Culture sensitivity ranges from 75% to 95% for nongonococcal joint
infections in patients who have not received antibiotics. For patients with
gonorrhea, the sensitivity is only 10% to 50% (Shmerling, 1994). In par-
tially treated patients, the use of resin-containing blood culture bottles for
culturing SF may improve isolation and identification of the responsible
organism.
Although not yet in routine practice, the use of polymerase chain
reaction (PCR) with primers to detect bacterial DNA is helpful, particu-
larly for the more fastidious, uncultivable pathogens (e.g., Borrelia burg-
dorferi, Chlamydia spp., Mycoplasma spp.) (Nocton et al, 1994; Li et al,
497
 1996; Jalava et al, 2001). Viruses are often associated with acute infectious BOX 29-9 
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
arthritis, and, depending on the putative virus, serology, viral culture, Classification of Pleural Effusions
and detection of viral DNA by nucleic acid amplification should be
performed. Transudates: Increased Hydrostatic Pressure or Decreased  
Depending on the clinical history, infectious arthritis may be associated Plasma Oncotic Pressure
with particular exposures and their associated pathogens. Arthritis devel- Congestive heart failure
ops in approximately 60% of patients with Lyme disease resulting from Hepatic cirrhosis
exposure to ticks infected with Borrelia burgdorferi (Golightly, 1993). The Hypoproteinemia (e.g., nephrotic syndrome)
PCR test for detecting B. burgdorferi DNA in SF is positive in 96% of Exudates: Increased Capillary Permeability or Decreased  
untreated cases (Nocton et al, 1994; Exner & Lewinski, 2003). Lymphatic Resorption
In patients with a travel history or outdoor occupations, SF/tissue Infections
should be examined for fungal pathogens by KOH/calcofluor white stain Bacterial pneumonia
and cultured on selective fungal media. For example, a patient with a recent Tuberculosis, other granulomatous diseases (e.g., sarcoidosis,
travel history to Arizona may present with a monoarticular arthritis sec- histoplasmosis)
ondary to Coccidioides immitis. Viral or mycoplasma pneumonia
Patients with chronic arthritis and risk factors for Mycobacterium tuber- Neoplasms
culosis or nontuberculous infections should undergo a synovial biopsy (Ver- Bronchogenic carcinoma
ettas et al, 2003; Titov et al, 2004). Ziehl-Neelsen or Kinyoun stains for Metastatic carcinoma
acid-fast organisms have a sensitivity of about 20%. Cultures for M. tuber- Lymphoma
culosis are positive in about 80% of proven cases. Because conventional Mesothelioma (increased hyaluronate content of effusion fluid)
culture methods for M. tuberculosis are often very time-consuming, apply- Noninfectious inflammatory disease involving pleura
ing PCR in SF for the detection of M. tuberculosis is a useful technique for Rheumatoid disease (low pleural fluid glucose in most cases)
more rapid diagnosis (Fujimoto et al, 2010). Systemic lupus erythematosus (LE cells are occasionally present)
Pulmonary infarct (may be associated with hemorrhagic effusion)
Fluid from Extrapleural Sources
PLEURAL FLUID
Pancreatitis (elevated amylase activity in effusion fluid)
The pleural cavity is a potential space lined by mesothelium of the visceral Ruptured esophagus (elevated amylase activity and low pH)
and parietal pleurae. The pleural cavity normally contains a small Urinothorax (elevated creatinine and low pH)
amount of fluid that facilitates movement of the two membranes against
each other. This fluid is a plasma filtrate derived from capillaries of the LE, Lupus erythematosus.
parietal pleura. It is produced continuously at a rate dependent on capillary
hydrostatic pressure, plasma oncotic pressure, and capillary permeability. TABLE 29-9 
Pleural fluid is reabsorbed through the lymphatics and venules of the
visceral pleura. Laboratory Criteria for Pleural Fluid Exudate
An accumulation of fluid, called an effusion, results from an imbal­ Pleural fluid/serum protein ratio ≥0.50
ance of fluid production and reabsorption. This fluid accumulation in the
Pleural fluid/serum LD ratio ≥0.60
pleural, pericardial, and peritoneal cavities is known as a serous
effusion. Pleural fluid LD ≥2 3 upper limit of normal
serum LD
Pleural fluid cholesterol >45 mg/dL
SPECIMEN COLLECTION
Pleural fluid/serum cholesterol ratio ≥0.30
Thoracentesis is indicated for any undiagnosed pleural effusion or for Serum–pleural fluid albumin gradient ≤1.2 g/dL
therapeutic purposes in patients with massive symptomatic effusions.
Pleural fluid/serum bilirubin ratio ≥0.60
Pleural fluid samples are frequently collected, handled, and/or analyzed in
a less than satisfactory manner. Indeed, improper collection/handling and LD, Lactate dehydrogenase.
undertesting or inappropriate testing are more common than with other
body fluids. The laboratory often receives a large syringe or vacuum bottle,
which must be circulated through the various laboratory sections. More- RECOMMENDED TESTS
over, a large blood or fibrin clot may be present as the result of inadequate
anticoagulation or mixing. The evaluation of serous body fluids (pleural, pericardial, peritoneal) is
Except for an EDTA tube for total and differential cell counts, the directed first toward differentiating transudative from exudative effusions.
specimen should be collected in heparinized tubes to avoid clotting. Ali- Transudates generally require no further workup. However, the fluid
quots for aerobic and anaerobic bacterial cultures are best inoculated into should be retained for 7 to 10 days in case further testing is needed. To
blood culture media at the bedside. If malignancy, fungal infection, or separate the two, several chemical parameters have been proposed,
mycobacterial infection is suspected, all remaining fluid (≥100 mL) should although none is 100% accurate (Table 29-9).
be submitted to maximize the yield of stains and culture. Because serous Classical teaching stressed that exudates and transudates can be distin-
effusions are more forgiving than CSF in maintaining cellular integrity, guished on the basis of total protein concentrations above (exudates) or
fresh specimens for cytology may be stored for up to 48 hours in the below (transudates) 3.0 g/dL. However, using total protein alone misclas-
refrigerator with satisfactory results. For pH measurements, the fluid sifies both exudates and transudates by about 30% (Melsom, 1979). It is
should be collected anaerobically in a heparinized syringe and submitted now well accepted that test combinations increase sensitivity, improve
to the laboratory on ice. Grossly purulent specimens do not require pH accuracy, and serve as the basis for the well-established Light criteria
measurement and may clog the analyzer. (Light et al, 1972). An exudate meets one or more of the following criteria:
(1) Pleural fluid/serum protein ratio greater than 0.5; (2) pleural fluid/
serum LD ratio greater than 0.6; and (3) pleural fluid LD level greater
TRANSUDATES AND EXUDATES than two thirds of the serum upper limit of normal. Using these criteria,
It has long been recognized that the initial classification of a pleural fluid the sensitivity and specificity are about 98% and 80%, respectively.
as a transudate or an exudate greatly simplifies the process of arriving at Several alternative measurements have been proposed to differentiate
a correct final diagnosis. Moreover, it determines whether further testing exudates from transudates. Testing for total cholesterol, the albumin gradi-
is needed. ent, or a combination of LD and total cholesterol may discriminate effu-
Transudates are usually bilateral owing to systemic conditions leading sions with equivocal Light’s criteria results. For example, the albumin
to increased capillary hydrostatic pressure or decreased plasma oncotic gradient is recommended to confirm a clinical transudate misclassified as
pressure (Box 29-9). Malignant effusions may infrequently be transudative an exudate by Light’s criteria (Light, 1997), that is, a serum albumin level
as the result of a simultaneous confounding clinical condition such as greater than 1.2 g/dL higher than the pleural fluid level indicates that the
congestive heart failure (Ashchi et al, 1998). Exudates are more often fluid is a transudate (Burgess et al, 1995). In many such cases, the patient
unilateral, associated with localized disorders that increase vascular perme- is being diuresed. Other test combinations have equaled but not surpassed
ability or interfere with lymphatic resorption (see Box 29-9). the performance of Light’s criteria. In most cases, the same categorization

498
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BOX 29-10  TABLE 29-10
Pleural Effusion: Recommended Tests
Routine Tests
Gross examination
Pleural fluid/serum protein ratio
Pleural fluid/serum LD ratio
Examination of Romanowski-stained smear (malignant cells, LE cells)
Useful Tests in Most Patients
Stains and cultures for microorganisms
Cytology
Useful Tests in Selected Cases
Pleural fluid cholesterol
Pleural fluid/serum cholesterol ratio
Albumin gradient
pH
Lactate
Enzymes (ADA, amylase, LD)
Interferon-γ
C-reactive protein
Lipid analysis
Tumor markers
Immunologic studies
Tuberculostearic acid
Pleural biopsy
Modified from Kjeldsberg CR, Knight JA: Body fluids: laboratory examination of amni-
otic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993, ©
American Society for Clinical Pathology, with permission.
ADA, Adenosine deaminase; LD, lactate dehydrogenase; LE, lupus erythematosus.
of pleural fluids as exudates or transudates may be achieved using measure-
ments of protein and LD alone without comparison with a blood sample,
as was accomplished with application of Light’s criteria (Murphy & Jen-
kinson, 2008).
Some tests, such as the combination of pleural fluid LD and choles-
terol, may be more convenient and cost-effective by avoiding the need for
simultaneous blood tests (Costa et al, 1995). Bilirubin measurement is not
a strong discriminator of effusions (Heffner et al, 1997).
Further analysis of exudates is directed toward ruling out malignancy
and infection. Cytology and appropriate bacterial stains and cultures are
the most useful tests in this regard. Moreover, given that pleural fluid DNA
levels are significantly increased in exudates, quantitative analysis may be
an effective method to evaluate the causes of serous effusion (Chan et al,
2003). Recommended tests for the evaluation of pleural effusions are sum-
marized in Box 29-10. The types of tests ordered and the interpretation
of test results should always be correlated with clinical findings and dif-
ferential diagnosis. Total leukocyte, differential, and RBC counts are of
limited use in the evaluation of serous effusions.
GROSS EXAMINATION
Transudates are typically clear, pale yellow to straw-colored, and odorless,
and do not clot. Approximately 15% of transudates are blood tinged. A
bloody pleural effusion (hematocrit >1%) suggests trauma, malignancy, or
pulmonary infarction (Jay, 1986). A traumatic tap is suggested by uneven
blood distribution, fluid clearing with continued aspiration, or formation
of small blood clots. A pleural fluid hematocrit greater than 50% of the
blood hematocrit is good evidence for a hemothorax (Light, 1995).
Exudates may grossly resemble transudates, but most show variable
degrees of cloudiness or turbidity, and they often clot if not heparinized.
A feculent odor may be detected in anaerobic infections. Turbid, milky,
and/or bloody specimens should be centrifuged and the supernatant exam-
ined. If the supernatant is clear, the turbidity is most likely due to cellular
elements or debris. If the turbidity persists after centrifugation, a chylous
or pseudochylous effusion is likely.
True chylous effusions are produced by leakage from the thoracic duct
resulting from obstruction by lymphoma, carcinoma, or traumatic disrup-
tion. A creamy top layer of chylomicrons may form in the specimen on
standing. Idiopathic congenital chylothorax is the most common form of
pleural effusion in the newborn.
Pseudochylous or chyliform effusions may have a milky, greenish, or
“gold paint” appearance. They accumulate gradually through the break-
down of cellular lipids in long-standing effusions such as rheumatoid
pleuritis, tuberculosis, or myxedema. Features that distinguish true chylous
from pseudochylous effusions are summarized in Table 29-10.
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Characteristic Features of Chylous and Pseudochylous Effusions
Feature Chylous Pseudochylous
Onset Sudden Gradual
Appearance Milky-white, or Milky or greenish,
yellow to bloody metallic sheen
Microscopic Lymphocytosis Mixed cellular reaction,
examination cholesterol crystals
Triglycerides*† ≥110 mg/dL <50 mg/dL
PART 3
(≥1.24 mmol/L) (<0.56 mol/L)
Lipoprotein Chylomicrons present Chylomicrons absent
electrophoresis
Modified from Kjeldsberg CR, Knight JA: Body fluids: laboratory examination of amni-
otic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993, ©
American Society for Clinical Pathology, with permission.
*Values in parentheses are SI units.
†
Triglyceride levels between 50 mg/dL and 110 mg/dL are equivocal and require
electrophoresis to confirm chylothorax.
MICROSCOPIC EXAMINATION
Cell Counts
Total cell counts may be performed using manual hemocytometer methods;
however, automated cell counts are increasingly used with pleural and
other serous fluid specimens (Aulesa et al, 2003; Conner et al, 2003; Yang
et al, 2013). Leukocyte counts have limited utility in separating transudates
(<1000/µL) from exudates (>1000/µL). Although RBC counts above
100,000/µL are highly suggestive of malignancy, trauma, or pulmonary
infarction, they are not specific for these conditions.
Differential Leukocyte Count and Cytology
Examination is commonly performed on a stained smear, preferably pre-
pared by cytocentrifugation and with the air-dried smear stained with a
Romanowski stain. Indeed, examination by the hematology laboratory can
be highly effective in the detection of malignant cells, especially hemato-
logic malignancies (Kendall et al, 1997). Filtration or automated concen-
tration methods with Papanicolaou stain may also be used, especially if cell
loss is a matter of concern. Automated WBC differential counts may be
done on pleural fluid samples; however, some variation in results from
those of manual methods may be seen (Conner et al, 2003). Liquid-based
thin-layer methods are often used to prepare pleural and other serous fluid
specimens for cytopathologic examination and show good performance in
the detection of malignant cells (Moriarity et al, 2008).
Cytologic analysis will establish the diagnosis of metastatic carcinoma
in 70% or more of cases when both smears and cell blocks are examined
(Light, 2002). The sensitivity is significantly lower if the patient has meso-
thelioma (10%), squamous cell carcinoma (20%), lymphoma (25% to
50%), or sarcoma (25%). Preparation of cell blocks is unnecessary, except
for effusions in which malignancy is an important consideration (Jonasson
et al, 1990).
Mesothelial cells are common in pleural fluids from inflammatory
processes (Fig. 29-16). They are, however, conspicuously scarce in patients
with tuberculous pleurisy and rheumatoid pleuritis, and in patients who
have had pleurodesis. Fibrin deposition and fibrosis occurring in these
conditions prevent mesothelial cell exfoliation. Well-differentiated carci-
noma cells may be easily recognized (Fig. 29-17) or may be highly undif-
ferentiated (Fig. 29-18). A panel of immunocytochemical stains may be
necessary for confirmation, and these studies are enhanced by the use of
liquid-based preparation methods (Sioutopoulou et al, 2008).
Neutrophils predominate in pleural fluid from patients with pleural
inflammation (Box 29-11). More than 10% of transudates will also have a
predominance of neutrophils, but this has limited clinical significance.
Lymphocytes predominate in the disorders summarized in Box 29-11.
Most are small, but medium, large, and reactive (transformed) variants
may be seen. Nucleoli and nuclear cleaving are more prominent in effu-
sions than in the peripheral blood, and these features may be particularly
prominent in cytocentrifuge preparations. Plasma cells may also be
observed. Lymphocytosis associated with transudates is of limited clinical
significance.
Low-grade non-Hodgkin’s lymphoma and chronic lymphocytic leuke-
mia (CLL) may be difficult to distinguish from benign lymphocyte-rich
serous effusion (Fig. 29-19). Immunophenotyping by flow cytometry or
immunocytochemistry, in conjunction with cellular morphology, is usually
499
 BOX 29-11 
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
Cellular Differential of Pleural Effusions
Neutrophilia (>50%) Uremic effusions
Bacterial pneumonia Transudates (≈30%)
(parapneumonic effusion) Eosinophilia (>10%)
Pulmonary infarction Pneumothorax (air in pleural
Pancreatitis space)
Subphrenic abscess Trauma
Early tuberculosis Pulmonary infarction
Transudates (>10%) Congestive heart failure
Lymphocytosis (>50%) Infection (especially parasitic,
Tuberculosis fungal)
Viral infection Hypersensitivity syndromes
Malignancy (lymphoma, other Drug reaction
neoplasms) Rheumatologic diseases
True chylothorax Hodgkin’s disease
Rheumatoid pleuritis Idiopathic
Systemic lupus erythematosus
Figure 29-16  Mesothelial cells in pleural fluid.

Figure 29-17  Well-differentiated breast carcinoma cells in pleural fluid.

Figure 29-19  Pleural effusion in patient with non-Hodgkin’s lymphoma, small
lymphocytic type. The cells are small round forms, difficult to distinguish from
benign lymphocytes. (From Kjeldsberg CR, Knight JA: Body fluids: Laboratory exami-
nation of amniotic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago,
1993, © American Society for Clinical Pathology, with permission.)

(Karcher & Alkan, 1997). The diagnosis is usually confirmed by demon-

strating in the neoplastic cells the presence of human herpesvirus-8
(HHV-8)–associated antigens and/or DNA by immunophenotypic and/or
molecular analysis.
An eosinophilic effusion is one that has 10% or more eosinophils. The
most common causes are related to the presence of air or blood in the
pleural cavity (see Box 29-11). Most of these are exudates; however, in
about 35% of patients, the cause is unknown (Adelman et al, 1984).
Although not of much assistance in diagnosing the cause of an effusion,
eosinophilia appears to be independently associated with longer survival
(Rubins & Rubins, 1996). A small number of mast cells or basophils often
accompany eosinophils. Eosinophil-derived Charcot-Leyden crystals
may also be seen.

Figure 29-18  Small cell carcinoma of the lung showing typical molding of
nuclei.
helpful in making a correct diagnosis. The relative proportions of T and
B cells are, by themselves, not definitive for separating benign from malig-
nant exudates (Ibrahim et al, 1989); however, the pattern of expression of
Ig light chains and/or other distinctive cell marker combinations may allow
specific diagnosis of lymphoproliferative disorders. Molecular DNA
analysis may be another useful adjunct to morphologic analysis. An
unusual form of high-grade B-cell non-Hodgkin’s lymphoma, called
primary effusion lymphoma, may be seen in pleural, peritoneal, and/or
pericardial fluid specimens, typically from immunocompromised patients,
with a characteristic highly anaplastic and immunoblastic morphologic
appearance and without expression of most B cell–associated antigens
CHEMICAL ANALYSIS
Protein
The measurement of pleural fluid total protein or albumin has little clinical
value except when combined with other parameters to differentiate exu-
dates from transudates. Protein electrophoresis shows a pattern similar to
serum, except for a higher proportion of albumin; it has little value for
differential diagnosis (Light, 1995).
Glucose
The glucose level of normal pleural fluid, transudates, and most exudates
is similar to serum levels. Decreased pleural fluid glucose, accepted as a
level below 60 mg/dL (3.33 mmol/L) or a pleural fluid/serum glucose ratio
less than 0.5, is most consistent and dramatic in rheumatoid pleuritis and
grossly purulent parapneumonic exudates (Sahn, 1982). Low pleural fluid

500
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glucose may also be present in malignancy, tuberculosis, nonpurulent bac-
terial infection, lupus pleuritis, and esophageal rupture.
Lactate
Pleural fluid lactate levels can be a useful adjunct in the rapid diagnosis of
infectious pleuritis. Levels are significantly higher in bacterial and tuber-
culous pleural infections than in other pleural effusions. Values greater
than 90 mg/dL (10 mmol/L) have a positive predictive value for infectious
pleuritis of 94% and a negative predictive value of 100% (Gastrin &
Lovestad, 1988).
Enzymes
Amylase elevations above the serum level (usually 1.5 to 2.0 or more times
greater) indicate the presence of pancreatitis, esophageal rupture, or malig-
nant effusion (Light & Ball, 1973). Elevated amylase derived from esopha-
geal rupture or malignancy is the salivary isoform, which differentiates it
from pancreatic amylase (Kramer et al, 1989).
Pleural fluid lactate dehydrogenase (LD) levels rise in proportion to the
degree of inflammation. In addition to their use in separating exudates from
transudates, declining LD levels during the course of an effusion indicate
that the inflammatory process is resolving. Conversely, increasing levels
indicate a worsening condition requiring aggressive workup or treatment.
ADA, which is particularly rich in T lymphocytes, is significantly
increased in tuberculous pleuritis. At a level of 50 U/L, the sensitivity,
specificity, positive predictive value, negative predictive value, and effi-
ciency for tuberculosis are 91%, 81%, 84%, 89%, and 86%, respectively
(Burgess et al, 1996). When the lymphocyte/neutrophil ratio is 0.75 or
greater, the percentages are 88%, 95%, 95%, 88%, and 92%, respectively.
ADA levels of 40 U/L or greater are present in about 99.6% of patients
with verified tuberculous pleuritis (Lee et al, 2001); however, in patients
with lymphocyte-rich pleural fluids from nontuberculous causes, ADA
levels less than 40 U/L are present in 97.1% of cases.
Interferon-γ
Pleural fluid interferon (IFN)-γ levels are significantly increased in the pleural
fluid of patients with tuberculous pleuritis. The sensitivity of levels of
3.7 IU/L or greater is 99%, and the specificity is 98% (Villena et al, 1996a).
Test sensitivity does not differ in HIV-positive and HIV-negative patients.
Only about 20% of patients with effusions due to hematologic malignancies
have IFN-γ levels slightly above 3.7 IU/L (Villena et al, 2003a).
pH
Pleural fluid pH measurement has the highest diagnostic accuracy in
assessing the prognosis of parapneumonic (pneumonia-related) effusions
(Heffner et al, 1995). A parapneumonic exudate with a pH greater than
7.30 generally resolves with medical therapy alone. A pH less than 7.20
indicates a complicated parapneumonic effusion (loculated or associated
with empyema), requiring surgical drainage.
Patients with borderline complicated exudates (pH 7.20 to 7.30) may
be closely watched with repeat measurements. A concomitant pleural
glucose level below 60 mg/dL (3.33 mmol/L), however, strongly suggests
impending empyema. Rheumatoid pleuritis and malignant effusions with
a poor response to pleurodesis also have pH values below 7.20 and a low
glucose level (Rodriquez-Panadero & Mejias, 1989). A pH below 6.0 is
characteristic of esophageal rupture, although the pH in severe empyema
may be 6.0 or less (Good et al, 1980).
Urinothorax, a collection of urine presumably produced by lymphatic
drainage of perirenal accumulations into the pleural cavity, is also associ-
ated with a pleural fluid pH less than 7.30. These effusions are transuda-
tive, because of their low protein content, and smell of urine. They have
a creatinine level greater than in simultaneously drawn serum (Miller et al,
1988).
Lipids
Some serous effusions appear to be chylous (i.e., a milky appearance) but
are not (pseudochylous), whereas others may not look chylous but are
(Maldonado et al, 2009). Although pseudochylous fluids may be partially
due to increased leukocytes and necrotic debris, they are primarily due to
the presence of increased lecithin-globulin complexes. A true chylous effu-
sion has chylomicrons on lipoprotein electrophoresis. Lipid measurements
are also helpful in identifying chylous effusions (Staats et al, 1980). Thus,
pleural fluid triglyceride levels above 110 mg/dL indicate a chylous effu-
sion; values from 60 to 110 mg/dL (0.68 to 1.24 mmol/L) are less certain
and require lipoprotein electrophoresis to confirm a chylothorax, particu-
larly in fasting and postoperative patients (Maldonado et al, 2009). Non-
chylous and pseudochylous effusions generally have triglyceride levels
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below 50 mg/dL (0.56 mmol/L) and no chylomicrons on electrophoresis
(see Table 29-10).
Cholesterol measurements may be useful in separating transudates
from exudates, especially when there is a question regarding Light’s crite-
ria. A total cholesterol value of 54 mg/dL or more and a pleural fluid/
serum cholesterol ratio of 0.32 or higher have sensitivity and specificity
values similar to Light’s criteria (Suay et al, 1995). Elevated levels and the
presence of cholesterol crystals may be seen with pleural effusions that
have been present for several years.
C-Reactive Protein
PART 3
Pleural fluid CRP is a clinically useful screening test for organ disease,
index of disease activity, and measure of response to therapy (Castano &
Amores, 1992). Pleural fluid CRP levels >30 mg/L reportedly have a
sensitivity of 93.7%, a specificity of 76.5%, and a positive predictive
value of 98.4% in parapneumonic infections (Turay et al, 2000). Mean
CRP values are about 90 mg/L in parapneumonic infections compared
with 26 mg/L and 23 mg/L for tuberculous and malignant effusions,
respectively. Measurement of pleural fluid CRP contributes to both the
diagnosis and assessment of severity of parapneumonic effusions (Porcel
et al, 2012).
Tuberculostearic Acid (10-Methyloctadecanoic Acid)
Tuberculostearic acid (TSA) was first isolated from the bacillus Mycobac-
terium tuberculosis. This fatty acid is a structural component of mycobac-
teria and is not normally present in human tissue. Using gas
chromatography–mass spectroscopy, TSA was measured in sputum,
bronchial washings, and pleural fluid from patients with pulmonary
tuberculosis (Muranishi et al, 1990). Here, pleural fluid TSA was identi-
fied in 24 of 32 (75%) patients with active tuberculosis; bronchial wash-
ings were positive for TSA in 15 of 22 cases. In patients with other
pulmonary disorders, only 4 of 46 pleural fluids and 3 of 69 bronchial
washings had detectable levels. A later smaller study reported the follow-
ing for pleural fluid TSA: Sensitivity 54%, specificity 80%, positive pre-
dictive value 75%, negative predictive value 61%, and efficacy 66%
(Yorgancioglu et al, 1996). Combining TSA and adenosine deaminase
(ADA) analysis in pleural fluid samples appears to increase the diagnostic
sensitivity for tuberculous pleuritis (Muranishi et al, 1992).
Tumor Markers
Although not recommended as a routine test, various tumor markers are
often a useful adjunct in enigmatic noninflammatory exudates with nega-
tive cytology. Several tumor markers, especially CEA, CA 15-3, CA 549,
CA 72-4, and CYFRA 21-1, among others, have been studied in pleural
fluids. CEA is probably the most useful single marker for adenocarcino-
mas, but reported cutoff values vary considerably. The sensitivity of CEA
for malignant effusions varies depending on tumor origin and is about 50%
overall. Although complicated parapneumonic effusions may result in
elevated CEA levels (Garcia-Pachon et al, 1997), they are usually not a
problem to distinguish clinically.
A combination of tumor markers increases the accuracy of diagnosis of
malignant effusions. Thus, a combination of CEA, CA 15-3, and CA 72-4
had an accuracy of 90% with 78% sensitivity, 95% specificity, 88% positive
predictive value, and 91% negative predictive value (Villena et al, 1996b).
Similarly, CA 15-3 and CEA combined had an accuracy of 87% (Romero
et al, 1996); a combination of CA 549, CEA, and CA 15-3 had a sensitivity
of 65%, specificity 99%, and accuracy 85% (Villena et al, 2003b). The use
of the cytokeratin 19 fragment (CYFRA 21-1) may also be useful in com-
bination with other tumor markers. Complicating the use of these tests,
different tumor markers may be positive in both malignant and inflamma-
tory pleural effusions (Topolcan et al, 2007).
Other tumor markers may also be useful in the diagnosis of unexplained
effusions. For example, a marked increase in pleural fluid prostate-specific
antigen (PSA) can lead to the correct diagnosis of metastatic prostate
cancer in pleural and pericardial effusions, even when negative by cytologic
examination (Chin et al, 1999).
IMMUNOLOGIC STUDIES
Approximately 5% of patients with RA and 50% with SLE develop pleural
effusions sometime during the course of their disease.
RF is commonly present in pleural effusions associated with seroposi-
tive RA. Although a pleural fluid titer of 1 : 320 or greater in a patient with
known RA is reasonable evidence of rheumatic pleuritis (Halla et al, 1980),
elevated RF titers up to 1 : 1280 have been identified in 41% of patients
with bacterial pneumonia, 20% of patients with malignant effusions, and
501
 14% of patients with tuberculosis, making this test of little value (Levine
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
et al, 1968).
ANA titers once appeared to be useful in the diagnosis of effusion due
to lupus pleuritis; (Good et al, 1983); however, later studies have indicated
no benefit of this testing in pleural fluid specimens over and above serum
testing (Porcel et al, 2007).
Decreased complement levels (CH50 <10 U/mL or C4 level below
10 × 10–5  U/g protein) are present in most patients with rheumatoid or
lupus pleuritis (Hunder et al, 1972; Halla et al, 1980); however, decreased
complement is not highly specific for these diseases and is of little value
for routine diagnosis, although it may be helpful in the diagnosis of oth-
erwise enigmatic effusions.
MICROBIOLOGICAL EXAMINATION
Bacteria most commonly associated with parapneumonic effusions are
Staphylococcus aureus, Streptococcus pneumoniae, β-hemolytic group A strep-
tococci, enterococci, and some gram-negative bacilli. Anaerobic bacteria
are isolated in a significant proportion of cases, so both anaerobic and
aerobic cultures should be performed. The sensitivity of the Gram stain is
approximately 50% (Ferrer et al, 1999), and concentration methods, such
as cytocentrifugation, may increase the sensitivity. Use of resin-containing
blood culture bottles may improve the isolation of certain bacteria in
partially treated patients.
For patients with suspected M. tuberculosis, direct staining of tubercu-
lous effusions for acid-fast bacteria has a sensitivity of 20% to 30%, and
positive cultures are found in 50% to 70% of cases (Baer & Smith, 2001).
Pleural biopsy yields the highest culture sensitivity (50% to 75%) and may
provide a rapid presumptive diagnosis of tuberculosis by histopathologic
demonstration of granulomas or acid-fast bacteria. Combining culture and
acid-fast stains with pleural biopsy increases the sensitivity to about 95%
(Jay, 1986). Real-time PCR analysis of pleural fluid specimens shows good
sensitivity and specificity for the diagnosis of M. tuberculosis and may
provide an effective less invasive alternative method for rapid diagnosis
(Rosso et al, 2011).
ADA can provide rapid chemical evidence for tuberculous effusions
independent of HIV status (Burgess et al, 1996; Riantawan et al, 1999; Lee
et al, 2001). Although the ADA-2 isoenzyme form is elaborated by acti-
vated lymphocytes in tuberculosis, only mild elevations occur in
lymphocyte-rich pleural effusions from nontuberculous causes. However,
the relatively low prevalence of tuberculous pleurisy in North America
anticipates a lower positive predictive value rate compared with the excel-
lent results reported in the Asian and European literature, where tubercu-
losis is more common.
Pleural fluid interferon-γ is significantly increased in tuberculous pleu-
ritis and may be helpful in some cases because it is independent of HIV
status and is only modestly increased in about 20% of hematologic malig-
nancies (Villena et al, 2003a).
PERICARDIAL FLUID
From 10 to 50 mL of fluid is normally present in the pericardial space,
produced by a transudative process similar to pleural fluid. Pericardial
effusions are most often caused by viral infection, and enterovirus is the
most common etiologic agent. They may also develop as a result of bacte-
rial, tuberculous, or fungal infection, or in association with autoimmune
disorders, renal failure, myocardial infarction, mediastinal injury, or the
effects of various drugs, or they may be idiopathic (Box 29-12). HIV-
infected patients commonly have asymptomatic pericardial effusions,
which may become large in more advanced disease (Silva-Cardosa et al,
1999), or they may be associated with primary effusion lymphoma (Karcher
& Alkan, 1997). Many of the recommended laboratory tests described for
pleural fluid also pertain to pericardial effusions (see Box 29-10).
The postpericardiotomy syndrome is a fairly common but nonspe-
cific complication of cardiac surgery (or other cardiac damage) that devel-
ops days to weeks following the initial injury. It is hallmarked by the
development of fever, pleuritic chest pain, and other signs of pleural,
pericardial, and, less often, lung inflammation. Exudative pleural effusions
develop in more than 80% of cases. These are often serosanguineous to
hemorrhagic and typically have a pH greater than 7.4 and a normal glucose
level (Stelzner et al, 1983). No specific tests are available for diagnosing
this syndrome. Diagnosis, therefore, remains one of clinical exclusion.
Although the cause is uncertain, the time course, presence of antimyocar-
dial antibodies, and response to antiinflammatory therapy suggest an
immune-mediated process. Increased levels of antimyocardial antibodies
relative to serum, decreased complement levels, and immune complexes
502
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BOX 29-12 
Causes of Pericardial Effusions
Idiopathic (most often viral) Renal failure
Infection Hemorrhage
Bacteria Trauma
Tuberculosis Anticoagulant therapy
Fungi Leakage of aortic aneurysm
Viruses Autoimmune disorders
AIDS-related (usually viral) Hypothyroidism
Neoplasm Rheumatoid arthritis
Metastatic carcinoma Systemic lupus erythematosus
Lymphoma Inflammatory bowel disease
Drugs Wegener’s granulomatosis
Hydralazine Acute myocardial infarction
Procainamide Radiation therapy
Phenytoin
AIDS, Acquired immunodeficiency syndrome.
have been documented in pleural fluid from a single patient with the
syndrome (Kim & Sahn, 1996).
SPECIMEN COLLECTION
Pericardial effusions of unknown origin or large effusions with signs of
cardiac tamponade are generally submitted for laboratory examination.
Fluid may be obtained by pericardiotomy following limited thoracotomy,
or by pericardiocentesis (sterile needle aspiration).
GROSS EXAMINATION
Normal pericardial fluid is pale yellow and clear. Large effusions (>350 mL)
are most often caused by malignancy or uremia, or they may be idiopathic.
In HIV-associated cardiac tamponade, 45% of cases are idiopathic, and
tuberculous and bacterial effusions each account for about 20% of cases
(Chen et al, 1999). Infection or malignancy typically produces turbid effu-
sions, whereas effusions due to uremia are usually clear and straw-colored.
These and several other disorders may produce hemorrhagic effusions.
Blood-like fluid obtained by pericardiocentesis might represent a hem-
orrhagic effusion or inadvertent aspiration of blood from the heart. Blood
obtained from the heart chamber will have a hematocrit comparable with
that of peripheral blood, and blood gas analysis yields results similar to
venous or arterial blood. In contrast, the hematocrit of a hemorrhagic
effusion is usually lower than that of peripheral blood. Blood from a cardiac
puncture clots, but a hemorrhagic effusion usually does not.
A milky appearance suggests the presence of a chylous or pseudochy-
lous effusion. Identification and differentiation of these effusions are dis-
cussed in the Pleural Fluid section earlier in the chapter.
EXUDATES AND TRANSUDATES
According to Light’s criteria, a pleural exudate has one or more of the
following: pleural fluid/serum protein ratio >0.5; pleural fluid/serum LD
ratio >0.6; and pleural fluid LD level >200 U/L. Light’s criteria have also
been shown to be the most reliable diagnostic tool for identifying pericar-
dial exudates and transudates (Burgess et al, 2002a).
Routine testing of pericardial effusions should probably be limited to
cell count, glucose, total protein, LD, bacterial culture, and cytology
(Meyers et al, 1997). Other, more specific tests are appropriate only when
there is a high clinical suspicion of unusual causes of pericardial effusion.
MICROSCOPIC EXAMINATION
The hematocrit and RBC count document the presence of a hemorrhagic
effusion, but are of limited value for differential diagnosis. Total leukocyte
counts over 10,000/µL suggest bacterial, tuberculous, or malignant peri-
carditis; however, low counts may be encountered in these conditions,
limiting the value of this measurement (Agner & Gallis, 1979). Leukocyte
differential counts may be helpful in determining the cause of certain
pericardial effusions, particularly when combined with other pericardial
fluid findings (Reuter et al, 2006). Even when the differential count isn’t
helpful, examination of a stained smear should always be performed to
evaluate for atypical or malignant cells.
Cytologic identification of malignant cells is usually not difficult. Meta-
static carcinomas of the lung and breast are most frequently observed in
malignant pericardial effusion. Cytology has a sensitivity of 95% and a
specificity of 100% (Meyers et al, 1997).
CHEMICAL ANALYSIS
Chemical parameters for the diagnosis of pericardial effusions have not
been studied to the same extent as in other body fluids. Although pericar-
dial effusions are very similar to pleural fluids, routine application of these
tests requires additional studies to fully appreciate their diagnostic
importance.
Protein
A value greater than 3.0 g/dL has a sensitivity of 97% for exudative effu-
sions, but a specificity of only 22%, which significantly limits its usefulness.
Thus, total protein has no discriminating power in pericardial diagnosis
(Meyers et al, 1997).
Glucose
Pericardial glucose levels less than 60 mg/dL have a diagnostic accuracy
of only 36% in identifying pericardial exudates (Meyers et al, 1997). Values
less than 40 mg/dL (<2.22 mmol/L) are common in bacterial, tuberculous,
rheumatic, or malignant effusions.
pH
Pericardial fluid pH may be markedly decreased (<7.10) in rheumatic or
purulent pericarditis. Malignancy, uremia, tuberculosis, and idiopathic dis-
orders may have moderate decreases in the range of 7.20 to 7.30 (Kindig
& Goodman, 1983).
Lipids
Separation of true chylous from pseudochylous effusions may be facilitated
by triglyceride and cholesterol measurements, as well as by lipoprotein
electrophoresis for chylomicrons (see Table 29-10). See Lipids in the
Pleural Fluid section earlier in the chapter for further details on the diag-
nosis of chylous effusions.
Enzymes
A pericardial fluid LD level greater than 200 U/L has been suggested as
a cutoff for pericardial exudates (Burgess et al, 2002a). Moreover, the
measurement of LD and creatine kinase in postmortem pericardial fluid
within 48 hours of death may be useful in establishing acute myocardial
injury when such injury is suspected but cannot be established by the usual
histologic methods (Luna et al, 1982; Stewart et al, 1984). Pericardial fluid
levels of CK-MB, myoglobin, and troponin I in postmortem pericardial
fluid are significantly increased in patients with myocardial injury (Perez-
Carceles et al, 2004).
ADA activity is a useful adjunctive test for tuberculous pericarditis in
suspicious cases with negative acid-fast stains. The median ADA level in
tuberculous pericarditis is significantly higher than in other pathologic
effusions (Burgess et al, 2002b). Using a cutoff of 30 U/L, the sensitivity
is 94%, specificity 68%, and positive predictive value 80%. Using a cutoff
of 40 U/L, the sensitivity and specificity are 93% and 97%, respectively
(Koh et al, 1994). Combining pericardial ADA levels with other findings
adds to the diagnostic utility of measuring this analyte (Reuter et al, 2006).
Interferon-γ
Increased IFN-γ levels have been reported in tuberculous serous effusions,
including tuberculous pericarditis (Burgess et al, 2002b). Here, the IFN-γ
level was greater than 1000 pg/L, which was significantly higher than in
effusions from other pathologic conditions. A cutoff value of 200 pg/L
results in a sensitivity and specificity of 100% for the diagnosis of tuber-
culous pericarditis. As with ADA, combining IFN-γ levels with other
pericardial fluid findings improves the diagnostic utility of this assay
(Reuter et al, 2006).
Polymerase Chain Reaction
PCR is a sensitive technique and may be more specific than adenosine
deaminase in the diagnosis of tuberculous pericarditis (Lee et al, 2002);
however, a negative test does not rule out tuberculous pericarditis because
some pericardial fluids from patients with large tuberculous effusions may
not contain M. tuberculosis.
IMMUNOLOGIC STUDIES
A negative ANA test makes the diagnosis of lupus serositis highly unlikely.
Conversely, high ANA titers in pericardial effusions lack specificity, even
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when they are as high as 1 : 5120 (Leventhal et al, 1990; Wang et al, 2000).
If a high ANA titer is unexplained, malignancy should be considered.
MICROBIOLOGICAL EXAMINATION
The sensitivity of the Gram stain and culture for bacterial pericarditis is
similar to other serous body fluids (i.e., about 50% and 80%, respectively).
Important aerobic bacteria include S. aureus, S. pneumoniae, S. pyogenes, and
gram-negative bacilli. Although infectious pericarditis due to anaerobic
bacteria may be encountered rarely, the bacteria are sometimes not recog-
nized because of inconsistent methods used for their isolation and identi-
PART 3
fication (Brook, 2002). For this reason, proper laboratory technique is of
particular importance when infection with an anaerobic organism is sus-
pected. The major anaerobic organisms are the Bacteroides fragilis group,
anaerobic streptococci, Clostridium species, Fusobacterium species, and Bifi-
dobacterium species.
Identification of a specific etiologic agent in viral pericarditis is difficult
because the viruses (e.g., coxsackieviruses, influenza virus, mumps) are
rarely isolated from pericardial fluid. Obtaining acute and convalescent
sera for antibody response to suspected viral pathogens may help support
the diagnosis (Bellinger & Vacek, 1987). Viral infection probably accounts
for most idiopathic HIV-associated pericardial effusions.
The sensitivity of acid-fast stains and culture for tuberculous pericar-
ditis is about 50% (Agner & Gallis, 1979). PCR is a sensitive technique
and may be more specific than the use of adenosine deaminase in the
diagnosis of tuberculous pericarditis (Lee et al, 2002). However, a negative
test does not exclude the diagnosis of tuberculous pericarditis.
PCR-based assays may aid in the rapid diagnosis of a variety of bacterial
and other infections in the pericardial space (Tenenbaum et al, 2005; Levy
et al, 2006).
PERITONEAL FLUID
Ascites is the pathologic accumulation of excess fluid in the peritoneal
cavity. Up to 50 mL of fluid is normally present in this mesothelial-lined
space. As with pleural and pericardial fluids, it is produced as an ultrafiltrate
of plasma dependent on vascular permeability and on hydrostatic and
oncotic Starling forces.
TRANSUDATES AND EXUDATES
Common causes of peritoneal effusions are listed in Box 29-13. The labo-
ratory criteria for classifying ascitic fluid as a transudate or an exudate are
not as well defined as they are for pleural and pericardial fluids. For
example, infected or malignancy-related samples are not uncommonly
BOX 29-13 
Causes of Peritoneal Effusions
Transudates: Increased Hydrostatic Pressure or  
Decreased Plasma Oncotic Pressure
Congestive heart failure
Hepatic cirrhosis
Hypoproteinemia (e.g., nephrotic syndrome)
Exudates: Increased Capillary Permeability or  
Decreased Lymphatic Resorption
Infections
Primary bacterial peritonitis
Secondary bacterial peritonitis (e.g., appendicitis, bowel rupture)
Tuberculosis
Neoplasms
Hepatoma
Lymphoma
Mesothelioma
Metastatic carcinoma
Ovarian carcinoma
Prostate cancer
Trauma
Pancreatitis
Bile peritonitis (e.g., ruptured gallbladder)
Chylous Effusion
Damage to or obstruction of thoracic duct (e.g., trauma, lymphoma,
carcinoma, tuberculosis and other granulomas [e.g., sarcoidosis,
histoplasmosis], parasitic infestation)
503
 reported with protein concentrations in the transudative range (i.e., <3.0 g/ BOX 29-14 
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
dL), and many patients with cirrhotic or heart failure ascites have protein Criteria for Evaluation of Peritoneal Lavage
values in the exudative range (>3.0 g/dL) (Runyon et al, 1992).
The serum-ascites albumin gradient, defined as the serum albumin Positive Result
concentration minus the ascitic fluid albumin concentration, is widely Aspiration of >15 mL gross blood on catheter placement
considered as the most reliable method to differentiate peritoneal transu- Grossly bloody lavage fluid
dates from exudates (Runyon et al, 1992). Ascites caused by portal hyper- RBC >100,000/µL after blunt trauma
tension has a gradient of at least 1.1 g/dL (>11 g/L; transudate), whereas RBC >50,000/µL after penetrating trauma
ascites produced by other causes has a gradient less than 1.1 g/dL (exudate) WBC >500/µL
(Runyon et al, 1992). The diagnostic accuracy is 98% for the serum-ascites Amylase >110 U/dL
albumin gradient compared with only 52% to 80% for four other markers: Indeterminate Result
Ascitic fluid total protein; ascites/serum total protein ratio; ascitic fluid LD Small amount of gross blood on catheter placement
concentration; and ascites/serum LD ratio (Akriviadis et al, 1996). RBC 50,000-100,000/µL after blunt trauma
An ascitic fluid/serum bilirubin ratio of 0.6 or greater is also signifi- RBC 1000-50,000/µL after penetrating trauma
cantly associated with exudates (Elis et al, 1998). Indeed, the accuracies of WBC 100-500/µL
the bilirubin ratio, the serum-ascites albumin gradient, and Light’s criteria Negative Result
were 81.5%, 84%, and 80.2%, respectively. Others have suggested that if
RBC <50,000/µL after blunt trauma
the ascitic fluid LD is >130 U/L and the ascitic fluid/serum total protein
RBC <1000/µL after penetrating trauma
ratio is >0.4, the fluid should be regarded as an exudate (Paramothayan &
WBC <100/µL
Barron, 2002).
Although the serum-ascites albumin gradient is probably the best single Modified from Feied CF: Diagnostic peritoneal lavage, Postgrad Med 85:40, 1989,
method to differentiate an ascitic exudate from a transudate, other methods with permission.
compare favorably. Nevertheless, no ideal biochemical markers allow com- RBC, Red blood cells; WBC, white blood cells.
plete discrimination between ascitic fluid exudates and transudates.

SPECIMEN COLLECTION BOX 29-15 

Recommended Tests in Peritoneal Effusions
Paracentesis
Diagnostic paracentesis is performed in most patients with new ascites, or Useful in Most Patients
if there is a change in the clinical picture of a patient with ascites, such as Gross examination
rapid fluid accumulation or fever development. A minimum of 30 mL is Cytology
needed for complete evaluation. If possible, at least 100 mL should be Stains and culture for microorganisms
provided for cytologic examination. Samples for cell counts should be Serum-ascites albumin concentration gradient
placed in an EDTA-anticoagulated venipuncture tube. Culture specimens Useful in Selected Disorders
should include blood culture bottles that have been inoculated at the Total leukocyte and differential cell counts
bedside with ascitic fluid (10 mL per culture bottle). RBC count (lavage)
Bilirubin
Diagnostic Peritoneal Lavage Creatinine/urea nitrogen
This procedure is no longer recommended as a routine technique for the Enzymes (ADA, ALP, amylase, LD, telomerase)
evaluation of abdominal trauma. Concerns of oversensitivity and nonspeci- Lactate
ficity, and improvements in noninvasive diagnostic procedures such as Cholesterol (malignant ascites)
computed tomography and ultrasound have limited its common use to (1) Fibronectin
rapid screening for significant abdominal hemorrhage in hemodynamically Tumor markers (CEA, PSA, CA 19-9, CA 15-3, CA-125)
unstable patients, and (2) evaluation of hollow viscous injuries. Immunocytology/flow cytometry
A catheter is placed through a small incision into the abdominal cavity. Tuberculostearic acid
If less than 15 mL of gross blood can be aspirated, diagnostic peritoneal Modified from Kjeldsberg CR, Knight JA: Body fluids: Laboratory examination of
lavage (DPL) is performed by infusing 1.0 L of saline or Ringer’s solution amniotic, cerebrospinal, seminal, serous and synovial fluids, ed 3, Chicago, 1993, ©
(20 mg/kg in children) and retrieving the fluid by gravity drainage. At least American Society for Clinical Pathology, with permission.
600 mL should be recovered to avoid falsely low counts (Sullivan et al, ADA, Adenosine deaminase; ALP, alkaline phosphatase; CEA, carcinoembryonic antigen;
1997). The catheter is sometimes left in place so that DPL may be repeated LD, lactate dehydrogenase; PSA, prostate-specific antigen; RBC, red blood cell.
in 2 to 3 hours if initial results are negative or indeterminate.
Commonly accepted criteria for DPL interpretation after trauma are
shown in Box 29-14. The positive predictive value is only 23% for an Peritoneal Washings
isolated (no other abnormal criteria) leukocyte count of 500/µL or greater This procedure is performed intraoperatively to document early intraab-
(Soyka et al, 1990). dominal spread of gynecologic and gastric carcinomas. Samples are gener-
The conventional DPL criteria may be unreliable in detecting hollow ally sent for cytologic examination only.
viscous injury when blood is present from a simultaneous solid organ injury
not requiring surgical repair, resulting in unnecessary exploratory lapa-
rotomies. Suggested modifications of DPL criteria to adjust for this source
RECOMMENDED TESTS
of bleeding include either of the following: (1) WBC count greater than The most important tests for the evaluation of ascitic fluid are listed in
or equal to RBC count divided by 150, where RBC is 10 × 104/mm3 or Box 29-15. Relative importance varies depending on the type of sample
greater (Otomo et al, 1998); or (2) cell count ratio greater than 1.0 (Fang and the clinical findings. For example, RBC and WBC counts are more
et al, 1998). The cell count ratio is defined as the ratio between WBC and important than cytology or the serum-ascites albumin gradient in the
RBC counts in the lavage fluid divided by the WBC/RBC ratio in the evaluation of abdominal effects of trauma. Gross examination may provide
peripheral blood. These criteria have a reported specificity of 97% for immediate information in the clinical and laboratory triage.
hollow organ injury, especially if DPL is performed at least 3 hours fol-
lowing injury.
Other applications of DPL include the evaluation of patients with
GROSS EXAMINATION
suspected acute peritonitis or pancreatitis. A WBC count in the lavage Whereas transudates are generally pale yellow and clear, exudates are
fluid of 200 cells/mm3 is associated with a 99% probability of acute peri- cloudy or turbid because of the presence of leukocytes, tumor cells, or
tonitis (Larson et al, 1992). increased protein levels. The presence of food particles, foreign material,
or green-yellow bile staining in a DPL specimen suggests perforation of
Peritoneal Dialysis the gastrointestinal or biliary tract. Acute pancreatitis and cholecystitis
Dialysate fluid from renal patients undergoing chronic ambulatory perito- may also cause greenish discoloration.
neal dialysis should be submitted to the laboratory to check for Blood-tinged or grossly bloody fluid must be distinguished from a
infection. traumatic tap in which the blood usually clears with continued

504
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Figure 29-20  Neutrophils in a patient with bacterial peritonitis.
paracentesis. As little as 15 mL of blood per liter of fluid produces a bright
red opaque color such that newsprint cannot be read through the lavage
tubing. In most cases, the ability to read newsprint through the tubing
results in a negative DPL. Opaque specimens require cell counts because
newsprint readability is lost well below the 100,000 RBCs/µL criterion for
a positive DPL (Bellows et al, 1998). Bloody ascites is also seen in malig-
nancy and tuberculosis.
Milky fluid that does not clear with centrifugation suggests a chylous
or pseudochylous effusion. True chylous peritoneal effusions are signifi-
cantly less common than chylous pleural effusions. They are caused by
disruption or blockage of lymphatic flow by trauma, lymphoma, carci-
noma, tuberculosis, other granulomatous diseases (e.g., sarcoidosis),
hepatic cirrhosis, adhesions, or parasitic infestation. Differentiation of true
chylous and pseudochylous effusions is discussed in the Gross Examination
section in the Pleural Fluid section later in the chapter.
MICROSCOPIC EXAMINATION
The total leukocyte count is useful in distinguishing ascites due to uncom-
plicated cirrhosis from spontaneous bacterial peritonitis (SBP), which is
caused by migration of bacteria from the intestine into the ascitic fluid.
Approximately 90% of patients with SBP will have leukocyte counts
greater than 500/µL, more than 50% of which are neutrophils (Fig. 29-20)
(Runyon & Hoefs, 1984; Stewart et al, 1986).
The ascitic fluid total neutrophil count is the preferred method for the
diagnosis of SBP. Cutoff values of 250 and 500 neutrophils/µL have been
recommended, with a diagnostic accuracy of about 94% for 500
neutrophils/µL and about 90% for 250 neutrophils/µL (Stassen et al,
1986; Albillos et al, 1990).
Cell counts, total protein, and albumin gradient values vary with fluid
shifts associated with ascites formation and resolution. For example, diure-
sis may cause the WBC count to increase from 300/µL to 1000/µL or
more. When obtained by DPL, a leukocyte count of 200/µL or more is
reported to be associated with a 99% probability of acute peritonitis
(Alverdy et al, 1988; Larson et al, 1992).
Eosinophilia (>10%) is most commonly associated with the chronic
inflammatory process associated with chronic peritoneal dialysis. It has also
been reported in congestive heart failure, vasculitis, lymphoma, and rup-
tured hydatid cyst.
Cytology has an overall sensitivity of 40% to 65% for malignant ascites.
Peritoneal carcinomatosis accounts for about two thirds of malignant effu-
sions, and cytology has a sensitivity of over 95% when confined to these
cases (Runyon et al, 1988). Liquid-based thin-layer preparations of peri-
toneal effusions and pelvic washings have been shown to be superior to
standard cytologic preparations in the detection of carcinoma (Moriarity
et al, 2008). Immunocytochemical stains are useful in characterizing atypi-
cal cells in equivocal cases.
CHEMICAL ANALYSIS
Protein
The serum-ascites albumin gradient is superior to total protein content
in differentiating cirrhosis from other causes of peritoneal effusions
(Runyon et al, 1992). Spontaneous bacterial peritonitis (SBP) is commonly
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associated with low total protein (<3.0 g/dL) and a high serum-ascites
albumin gradient (>1.1 g/dL), making total protein measurements of little
value in this disorder. Extracellular fluid shifts associated with ascites for-
mation and resorption also cause variations in protein content.
Glucose
Early reports indicated that peritoneal fluid glucose levels of 50 mg/dL or
less are present in 30% to 60% of cases of tuberculous peritonitis and in
about 50% of patients with abdominal carcinomatosis (Polak & Torres da
Costa, 1973; Brown & An, 1976). Another study found decreased glucose
levels in most cases of tuberculous ascites (Bansal et al, 1998). Still, glucose
PART 3
measurements are of little value because the sensitivity and specificity are
generally too low to be of practical value.
Enzymes
Amylase activity in normal peritoneal fluid is similar to plasma levels. A
level greater than three times the plasma value is good evidence of
pancreas-related ascites, including acute pancreatitis and pancreatic pseu-
docyst (Runyon, 1987a). Amylase is not recommended in the routine
evaluation of ascites, however, because the prevalence of pancreatic ascites
is low. Retrospective amylase measurement on a stored specimen is indi-
cated if initial studies are not diagnostic. Amylase levels in peritoneal
lavage fluid may be valuable in patients following blunt and penetrating
abdominal trauma (McAnena et al, 1991). Here, amylase levels greater
than or equal to 20 U/L have a sensitivity of 87%, a specificity of
75%, and a positive predictive value of 46% for significant intraabdominal
injury. In these cases, laparotomy should be considered. Gastroduodenal
perforation, acute mesenteric vein thrombosis, intestinal strangulation, or
necrosis may also produce elevated amylase levels. Although various non-
pancreatic malignancies may rarely produce amylase elevations, isoenzyme
evaluation usually identifies the salivary isoform in these latter cases
(Kosches et al, 1989).
Elevated alkaline phosphatase (ALP) levels greater than 10 U/L in
diagnostic peritoneal lavage fluid are useful in predicting hollow visceral
injury in patients who would otherwise not undergo laparotomy (specific-
ity 99.8%, sensitivity 94.7%) (Jaffin et al, 1993). Ascitic fluid ALP mea-
surements may also be helpful in the differentiation of primary bacterial
peritonitis from secondary bacterial peritonitis due to bowel perforation.
Secondary peritonitis has significantly higher mean ALP levels than spon-
taneous bacterial peritonitis (SBP). ALP levels >240 U/L were present in
92% of patients with secondary peritonitis versus 12% with SBP (Wu et al,
2001). The sensitivity and specificity for differentiating secondary perito-
nitis from SBP were 92% and 88%, respectively.
LD activity is often increased in malignant effusions (Gerbes et al,
1991). An ascitic fluid/serum LD ratio greater than 0.6 has a reported
sensitivity of 80% (Boyer et al, 1978). Combined measurement of ascitic
fluid LD and cholesterol totally discriminated peritoneal carcinomatosis
from cirrhosis and hepatocarcinoma-related ascites (Castaldo et al, 1994;
Halperin et al, 1999). Although both serum and peritoneal fluid LD levels
are significantly higher in patients with ovarian cancer than in those with
benign ovarian tumors or other gynecologic malignancies, peritoneal fluid
LD has higher diagnostic sensitivity (87%) and diagnostic accuracy (90%)
than serum LD (60% and 77%, respectively) (Schneider et al, 1997). LD
has also been used for the early diagnosis of spontaneous bacterial perito-
nitis (SBP), in which it has a diagnostic accuracy of about 74% using an
ascitic fluid/serum ratio cutoff of 0.4 (Lee et al, 1987).
The presence of telomerase is a specific discriminatory marker in
malignant ascites (Tangkijvanich et al, 1999). Telomerase activity was
detected in 81% of malignant peritoneal effusions with a sensitivity of 76%
and a specificity of 95.7%.
ADA is commonly used in endemic areas to identify patients with
tuberculous peritonitis (Burgess et al, 2001). Using receiver operating
characteristic curves and a cutoff value of 30 U/L, the sensitivity and
specificity were 94% and 92%, respectively. Using a cutoff value of
33 U/L, the sensitivity, specificity, positive and negative predictive values,
and overall diagnostic accuracy for diagnosing tuberculous peritonitis were
100%, 96.6%, 95%, 100%, and 98%, respectively (Dwivedi et al, 1990).
Fibronectin
Using a cutoff value of 85 µg/mL (85 mg/L), fibronectin was more reliable
in differentiating malignant from sterile ascites (diagnostic accuracy, 79%)
than were total protein, LD, γ-glutamyltransferase, pH, amylase, triglyc-
erides, leukocyte count, and cytologic examination (Colli et al, 1986).
In a subsequent study using a cutoff of 94.6 µg/mL, the sensitivity, specific-
ity, positive accuracy, negative accuracy, and overall diagnostic accuracy in
the diagnosis of malignant ascites were 100%, 95%, 93.8%, 100%, and
505
 97.1%, respectively (Sood et al, 1997). Further studies have confirmed the
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
utility of fibronectin measurement in the diagnosis of malignant ascites
(Lee et al, 2006).
Lactate
Ascitic fluid lactate has been used with pH measurements to differentiate
spontaneous bacterial peritonitis (SBP) from uncomplicated ascites. Sen-
sitivity and specificity for lactate levels are approximately 90% using a
cutoff of 40 mg/dL (4.44 mmol/L), with a positive predictive value of 62%
(Stassen et al, 1986). Although not as accurate as leukocyte counts, the
high specificity of lactate in hepatic ascites suggests that it has some value
in the diagnosis of SBP in otherwise equivocal cases. Malignant and tuber-
culous ascites are also associated with elevated lactate levels.
Patients with hollow viscous perforation, gangrenous intestine, perito-
nitis, or intraabdominal abscess have a peritoneal fluid minus plasma
lactate level of at least 13.5 mg/dL (1.5 mmol/L), which reportedly sepa-
rates these patients completely from those with other conditions producing
acute abdominal problems (DeLaurier et al, 1994). Still, the clinical utility
of measuring ascitic fluid lactate in surgical decision making remains
unclear.
Creatinine and Urea
Measurement of creatinine and urea nitrogen is useful to differentiate
between peritoneal fluid and urine. Elevated peritoneal fluid urea
nitrogen and creatinine, in association with elevated serum urea but
normal serum creatinine (due to back-diffusion of urea), suggest urinary
bladder rupture.
Bilirubin
The mean (±SD) ascitic fluid bilirubin concentration in various types of
ascites has been reported as 0.7 ± 0.8 mg/dL, and the mean ascitic fluid/
serum bilirubin ratio as 0.38 ± 0.44 (Runyon, 1987b). An ascitic fluid bili-
rubin greater than 6.0 mg/dL and an ascitic fluid/serum bilirubin ratio
over 1.0 suggest choleperitoneum from a ruptured gallbladder. A ratio of
0.6 or greater has been advocated as an additional marker for an exudative
process, although its accuracy is not as high as that of the serum-ascites
albumin gradient (Elis et al, 1998).
pH
Ascitic fluid pH may be helpful in the diagnosis of spontaneous bacterial
peritonitis (SBP) in patients with cirrhotic ascites, especially if it is used in
conjunction with the leukocyte count (Attali et al, 1986; Stassen et al,
1986). A pH less than 7.32 or a blood–ascitic fluid pH difference of more
than 0.1 has a reported sensitivity and specificity of about 90% for SBP,
with the pH differential being slightly more accurate. Peritoneal fluid pH
appears useless in detecting SBP, however, in the absence of neutrophils
(Runyon & Antillon, 1991). Patients with an ascitic fluid pH of less than
7.15 have a poor prognosis (Attali et al, 1986). Low pH is also found in
patients with malignant and pancreatic ascites and tuberculous
peritonitis.
Cholesterol
The ascitic fluid cholesterol level is a moderately useful index in separating
malignant ascites (>45 to 48 mg/dL) from cirrhotic ascites (Mortensen
et al, 1988; Castaldo et al, 1994). The sensitivity and specificity average
just over 90% using a cutoff value of 45 to 48 mg/dL (1.2 mmol/L). Thus,
using a cutoff value of 48 mg/dL, the sensitivity, specificity, positive and
negative predictive value, and overall diagnostic accuracy for differentiat-
ing malignant from nonmalignant ascites were reported as 96.5%, 96.6%,
93.3%, 98.3%, and 96.6%, respectively (Garg et al, 1993).
Interleukin-8
Interleukin-8, a cytokine produced by a variety of cells in response to
stimuli such as bacterial lipopolysaccharide, is significantly higher in spon-
taneous bacterial peritonitis (SBP) compared with sterile ascites (Martinez-
Bru et al, 1999). Using a cutoff value of 100 ng/L, the sensitivity and
specificity were both 100% in cirrhotic patients.
Tuberculostearic Acid (10-Methyloctadecanoic Acid)
As noted in the Pleural Fluid section, TSA was detected in pleural fluid in
75% of patients with pulmonary tuberculosis using gas chromatography–
mass spectroscopy (Muranishi et al, 1990). Using quantitative chemical
ionization gas chromatography–mass spectrometry, the measurement of
TSA is a valuable technique to identify tuberculous peritonitis, as well as
tuberculous meningitis (spinal fluid) and pneumonia (pleural fluid) (Brooks
et al, 1998).
506
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Tumor Markers
Because of their reportedly low sensitivity and specificity, the measure-
ment of tumor markers in peritoneal fluid is generally considered to be
of little value. They are often useful, however, in selected cases, such as
in following a patient’s response to therapy and in the early detection of
tumor recurrence. They may also be very useful in cases in which cytol-
ogy is negative but suspicion of malignant ascites is high. In one
study, cytologic examination was positive in only 40% (35 of 89
patients) of malignant cases, while tumor markers were positive in 80%
(Cascinu et al, 1997). Moreover, excluding small cell lung and renal
cancers, for which specific tumor markers are lacking, tumor markers
(i.e., CEA, CA 19-9, CA 15-3, PSA) in ascitic fluid for other carcinomas
were positive in 97% of cases. These tumor markers, as well as
α-fetoprotein, were also found to be highly specific (over 90%) for
serous fluid malignancies, although their sensitivities were low (19% to
38%) (Sari et al, 2001). The measurement of PSA may also be a valu-
able marker for the diagnosis of malignant effusions due to prostate
cancer (Appalaneni et al, 2004).
CEA has a sensitivity of 40% to 50% and a specificity of about 90%
for malignant effusions, using a cutoff value of 3.0 ng/mL (Mezger et al,
1988). In a similar study using a 5-mg/mL cutoff, the specificity was
about 97% (Gulyas et al, 2001). Elevated CEA levels in peritoneal wash-
ings suggest a poor prognosis in gastric carcinoma (Irinoda et al, 1998).
Ascitic fluid CA-125 is elevated to some degree in a variety of nonma-
lignant conditions. Indeed, cardiovascular and chronic liver disease may be
the most frequent diagnoses in patients with increased CA-125 levels
(Miralles et al, 2003), thereby supporting the general opinion that CA-125
in ascitic fluid lacks adequate specificity as a marker for malignancy.
Extremely high levels are likely to be caused by epithelial carcinomas of
the ovary, fallopian tubes, or endometrium. The sensitivity for ovarian
carcinoma depends on the tumor’s stage (range, 40% to 95%) and histo-
logic subtype (mucinous adenocarcinomas have lower values) (Molina
et al, 1998).
DNA ploidy analysis by flow cytometry or image analysis may provide
useful complementary diagnostic information in cases with equivocal
cytology results when the malignant cells carry an aneuploid karyotype,
although the added value of DNA ploidy analysis over cytology is small
(Bisht et al, 2014). Image analysis appears to be more practical than flow
cytometry when the tumor cells are scarce (Rijken et al, 1991).
MICROBIOLOGICAL EXAMINATION
Primary peritonitis occurs at any age and is seen in children with
nephrotic syndrome and in adults with cirrhotic liver disease. Spontane-
ous bacterial peritonitis (SBP) occurs in patients with ascites in the
absence of recognized secondary causes such as bowel perforation or
intraabdominal abscess. The bacteria in SBP are most often normal intes-
tinal flora, and more than 92% are monomicrobial. Aerobic gram-
negative bacilli (e.g., E. coli, Klebsiella pneumoniae) are responsible for two
thirds or more of all cases (Gilbert & Kamath, 1995), followed by S. pneu-
moniae, Enterococcus spp., and, rarely, anaerobes. The Gram stain has a
sensitivity of 25% in SBP (Lee et al, 1987), and routine cultures are posi-
tive in only about 50% of cases (Castellote et al, 1990). Inoculation of
blood culture bottles at the bedside and concentration of large volumes of
fluid can improve sensitivity, but up to 35% of infected patients may still
have negative ascitic fluid cultures (Marshall, 1988). Use of resin-
containing blood culture bottles may improve the isolation of certain
bacteria in partially treated patients.
Ascitic fluid total neutrophil count is the preferred method for the
diagnosis of SBP (see the Microscopic Examination section earlier in the
chapter). As noted earlier, in difficult cases, several analytes may be useful
in differentiating SBP from secondary bacterial or tuberculous peritonitis.
Because of the low number of bacteria in many cases of SBP, sensitive
methods for detection of bacteria in these specimens are often needed.
PCR has been successfully used in the detection of bacterial DNA in
culture-negative ascitic fluid (Such et al, 2002; Soriano et al, 2011), and
more recently in situ hybridization performed on leukocytes suspended in
peritoneal fluid has been used to detect bacteria in SBP (Enomoto et al,
2014).
The sensitivity of acid-fast stains for M. tuberculosis is no more than
20% to 30%, and cultures have a sensitivity of only 50% to 70% (Reimer,
1985). Application of PCR to detect M. tuberculosis DNA may be used, but
a negative result does not exclude the diagnosis (Schwake et al, 2003). In
a patient with a high clinical suspicion for tuberculous peritonitis, laparo-
scopic examination with biopsy may be indicated.
ALTERNATIVE SPECIMENS
SALIVA
Saliva is generally easily collected by noninvasive means, and so its
acquisition is well accepted by patients. Because it is a filtrate of plasma,
saliva has concentrations of some small molecules that are in equilibrium
with the free (unbound) active fractions of those substances in plasma.
This property has been especially useful for measuring free cortisol,
which is the physiologically important fraction that reflects the secretion
rate of cortisol and so is important for diagnosis. Salivary cortisol
measurements have been used to assess adrenal function in critically ill
subjects (Arafah et al, 2007) and in those with Cushing’s syndrome
and adrenal insufficiency (Raff, 2009), with late-night (or midnight)
salivary cortisol being an effective screening test for Cushing’s syndrome
(Raff et al, 1998). Unfortunately, salivary concentrations of several
other steroid hormones are not reliable measures of plasma free levels
because of rapid fluctuations in their salivary concentrations (e.g., estra-
diol, progesterone, testosterone, dehydroepiandrosterone, aldosterone)
(Wood, 2009).
Saliva also contains some antibody molecules that apparently derive
from plasma. Thus measurements are sometimes performed on saliva to
detect antibodies against infectious agents in circumstances when collec-
tion of saliva is much more convenient or acceptable to patients. Testing
of saliva for antibodies against human immunodeficiency virus has been
practiced widely in sexually transmitted disease clinics, although episodes
of false-positive results have diminished confidence in these point-of-care
tests (Cummiskey et al, 2008).
Genetic testing requires DNA from the patient that can be obtained
conveniently from leukocytes in blood specimens, or even more conve-
niently from buccal cells in saliva or from swabs of the interior of the
mouth.
Drug testing has also been performed in saliva to provide evidence of
ingestion of illicit substances. Although drugs such as amphetamines,
cocaine, and opioids are present in oral fluid at concentrations similar to
those in plasma, local absorption of these drugs in the mouth can increase
their concentration in saliva after use (Drummer, 2006).
MECONIUM
The analysis of body fluids can provide unique information based on
anatomic location (often remote or not readily accessible) or sequences of
metabolic processes and normal clearance of chemical constituents from
the body. An example of this clinical utility is the examination of meconium
from newborns for the detection of illicit drugs such as cocaine and
amphetamines that the mother might have abused while pregnant; the
drugs cross the placenta from maternal circulation into that of the fetus,
which excretes the drug or its metabolites in bile that remains in the
meconium until birth (Concheiro et al, 2013). This window of drug detec-
tion in meconium is much broader than that for testing of urine or blood
by several weeks and covers second and third trimesters of pregnancy. Such
testing is considered more reliable than self-reporting by mothers due to
fear of reprisal and possible loss of custody. These results can be used both
for medical management of the mother and the baby as well as for medical-
legal purposes. Recently, measurement of ethyl glucuronide in meconium
was found to be an objective measure of maternal alcohol use (Himes et al,
2015).
HAIR AND NAILS
Drug testing has time limitations of detection in blood for periods of
minutes to hours after ingestion. The need to detect illicit drug use over
longer periods of time has prompted analysis of other body sources. Con-
centrations of drugs in saliva follow the same time course as blood of
minutes to hours after ingestion. Detection in urine can be as soon as
minutes up to many days after drug use. Sweat concentrations also rise in
minutes but may persist for weeks. True long-term detection is possible in
hair and nails, where drugs of abuse can be detected days to even years
after ingestion. Although numerous companies offer drug testing of hair
or nail specimens, there has not been official adoption of this strategy by
government agencies, largely because of issues of uncertainty as to
whether the substances detected from hair or nail specimens were actually
“in” the hair following ingestion and incorporation into that tissue, or
whether the detected drug was simply “on” the surface of the specimen
because of incidental contact that did not involve ingestion (Curtis &
Greenberg, 2008).
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BREATH TESTING
Exhaled Nitric Oxide and Exhaled Breath Condensate
Inflammatory disorders of the lungs, including asthma, chronic obstructive
pulmonary disease, and chronic cough, have largely been evaluated in the
past by bronchoscopy, lavage, biopsy, and analysis of induced sputum.
Noninvasive techniques for monitoring these disorders are now available
as detection of chemical constituents in exhaled breath. Exhaled nitric
oxide (NO) is an inflammatory mediator that is unstable in tissues but is
much more stable in the gas phase, where it can be measured in single
breath exhalations by specialized devices where NO content is expressed
PART 3
in parts per billion. This use is currently considered investigational but
theoretically has great potential for monitoring pulmonary inflammation
by noninvasive means with much greater frequency than by invasive pro-
cedures (Dweik et al, 2011).
A second approach to monitoring pulmonary disease is to measure
nonvolatile inflammatory markers in exhaled breath condensate (EBC)
that is collected from exhaled breath that is condensed by passage through
a cooling device. Such specimens of respiratory fluid are scant, but they
contain a rich assortment of substances such as cytokines, leukotrienes,
oxidants, antioxidants, and so on that can reveal inflammatory activity by
noninvasive means (Horvath et al, 2005). Even pH of EBC has been found
useful in assessing lung disease; EBC tends to be slightly alkaline (mean
pH 7.7 ± 0.49) in health, but it becomes more acidic with disease changes
in the lower airways (Vaughan et al, 2003). EBC analysis is currently con-
sidered investigational.
Carbon-13-Urea Breath Test
Diagnosis of infection with Helicobacter pylori may be aided by the carbon-
13-urea breath test in which the patient drinks a solution of 13C-urea. H.
pylori has the enzyme urease, which breaks down urea releasing 13CO2 into
the patient’s breath. Samples of breath taken in a balloon or other special-
ized collection devices are analyzed by mass spectrometry (Patel et al,
2014) (see Chapters 4 and 23). Detection of 13CO2 indicates infection with
H. pylori. An alternative procedure utilizes 14C in place of 13C, although
14
C detection is based on low-level radioactivity. 13C has the advantage of
not being radioactive but the disadvantage of not being measured in most
local laboratory facilities. The 13C-urea breath test has special utility for
pediatric patients (Frenck et al, 2006).
Opportunities for analysis of additional body fluid types will increase as
collection methodologies expand, particularly in collaboration with invasive
radiologic approaches. Some problems expected with these new unique
specimens include scant specimen volumes, unknown protein and other
chemical composition that might adversely affect measurements, and lack of
reference ranges to distinguish normal healthy results from those in disease.
TISSUE ASPIRATES
Breast nipple aspirate fluid can be obtained by noninvasive means such as
massage or with automated collection devices to yield material suitable for
cytologic examination for early detection of breast cancer. This new col-
lection approach may lead to other effective applications of cancer detec-
tion using modalities such as proteomics or still to be discovered biomarkers
(Alexander et al, 2004).
Fine-needle aspiration for cytologic examination is frequently used to
evaluate head and neck masses. Rapid identification of a mass of parathy-
roid tissue has been done by measuring parathyroid hormone (PTH) in
saline, into which the needle aspirate is flushed (Conrad et al, 2006). This
method was shown to be 99% accurate for diagnosis of parathyroid tissue
because the measured values of PTH are orders of magnitude higher in
parathyroid aspirates than in those from other tissues such as thyroid,
adipose, or lymphatic. This type of specimen is truly unusual and would
not likely be included in any manufacturer’s claims for assay performance;
however, this practice is well recognized by endocrine surgeons who now
consider it a standard of practice.
Another diagnostic application of tumor markers is for aspirates col-
lected from pancreatic cysts during surgery, although such fluids might
also be collected by direct aspiration with a needle guided through ultra-
sound. High levels of CA19-9 in pancreatic cyst fluid suggest malignancy,
whereas low levels are more consistent with benign processes (Khalid &
Brugge, 2007). CA19-9 measurement in pancreatic cyst fluid is an aid to
diagnosing malignancy in conjunction with cytology and other studies; the
advantage of CA19-9 in this context is its relatively fast turnaround time
by immunoassay that might be completed during the time period of surgi-
cal procedure. CEA has also been used for this purpose (Rockacy & Khalid,
2013).
507
 BILLING FOR TESTS IN NONSTANDARD
29  Cerebrospinal, Synovial, Serous Body Fluids, and Alternative Specimens
SPECIMENS
Although considerations of medical necessity provide strong motivation
for performing tests in unusual specimen types to provide unique informa-
tion that could not be obtained by other means, reporting of the results
and subsequent billing may lead to confusion. Most laboratory computer
systems have strictly defined specimen types, such as blood, serum, urine,
and CSF. When dealing with an unusual specimen type that is not indi-
vidually coded or recognized in a computer system, laboratories should
take care not to enter these results as though they were serum or other
commonly tested fluid. For both medical needs and reimbursement, some
disclaimer should be included to indicate the specimen type, so that it is
not confused with serum or other conventional fluid.
CHEMICAL MEASUREMENTS
IN BODY FLUIDS
Plasma, serum, cerebrospinal fluid, and urine are standard fluids that are
often submitted for chemical analysis. Manufacturers generally provide
product claims for their assays in one or more of these body fluids, but
they have not established assay behavior in other specimen types such as
pleural fluid, peritoneal fluid, SF, and other accumulations of abnormal
fluids such as drainage or lavage fluids. These types of fluids are not nor-
mally present in health but rather can form as the result of various patho-
logic processes, such as hemodynamic imbalance, infection, inflammation,
malignancy, or other organ dysfunction. Consequently, the composition of
these fluids can range widely, thereby leading to unpredictable effects on
laboratory measurements. Matrix effects from variations in protein con-
centration can alter fluid surface tension, viscosity, and miscibility in a
reaction mixture. All of these variations potentially might cause differences
in measurements because of errors in pipetting fixed volumes or in speed
and completeness of mixing with reagents in an assay. Other constituents
present in these pathologic fluids (e.g., hyaluronic acid in SF) but not in
serum also have the potential to alter measurements in assays intended for
use in serum. This potential for erroneous measurements in body fluids
due to matrix effects should be recognized when such requests for analysis
are received.
A second issue concerning chemical measurements in pathologic fluids
is what reference range to use for comparison in an interpretive report.
Because abnormal fluid accumulations do not exist in a state of health, they
cannot, of course, be collected from a normal healthy population to estab-
lish reference ranges.
These two issues of potential interferences and lack of reference ranges
for analysis of body fluids fly in the face of Clinical Laboratory Improve-
ment Act requirements for assay validation. In fact, the lack of manufactur-
ers’ claims for body fluid testing might actually place a greater burden on
a laboratory to validate these assays than for use in serum for which manu-
facturer claims are typically cleared by the U.S. Food and Drug Adminis-
tration. Without such validation, measuring chemical constituents in body
fluids could be considered an off-label use of commercial assays.
In recognition of these difficulties faced by laboratories, the Clinical
and Laboratory Standards Institute (CLSI) has developed the document
C49A, Analysis of Body Fluids in Clinical Chemistry, to provide guidance
in this situation (CLSI, 2007). The specific recommendations from CLSI
are as follows:
1. Review performance claims of the manufacturer for the possibility of
extending a commercial assay to body fluids with special attention to
accuracy, precision, analytic measurement range, reference interval
(usually in comparison with simultaneous measurement in serum, in
addition to the patient’s body fluid), and interferences from substances
in the body fluid.
2. Existing methods may be suitably modified for body fluid analysis when
necessary to obtain medically relevant information. Matrix effects that
could alter the accuracy of measurement must be recognized. Further-
more, lack of a reference range for the analyte in body fluids should be
compensated for by interpretation of results in comparison with a
simultaneously collected serum specimen.
3. The preconditions for using a routine assay for an alternate body fluid
are that the measurement system in another specimen type (serum
plasma, urine)
a. Has acceptable test characteristics.
b. Has a reference method by which to ascertain bias.
c. Has calibrators and controls.
d. External proficiency testing should be done whenever available.
4. Specimen collection of body fluid and its handling, processing, and
storage should follow guidelines for specimens of plasma or serum col-
lected for that measurement. Special attention should be directed to
the possibility of interference from anticoagulants into which a body
fluid might be collected (e.g., heparin, EDTA, citrate).
5. Unusual properties of a body fluid such as high viscosity should be given
consideration if they have the potential to alter the analyte concentra-
tion in the final solution of the reaction mixture (e.g., inaccurate pipet-
ting, inadequate mixing).
6. The presence of an interferent in a body fluid can be assessed by testing
the fluid neat and at 1 : 2 and 1 : 4 dilutions. Recovery of similar con-
centrations suggests lack of interference. Low concentrations can be
checked for interference by mixing with a routine (serum) sample with
a high measurable value to measure recoverability.
7. Result reporting should include the measured value and the type of
fluid analyzed plus a statement that accuracy might be affected by
sample type, and that results should be interpreted in the clinical
context. The laboratory is urged to contact ordering physicians to
explain these limitations.
A key point is that to be useful clinically, measurements in body fluids
need not necessarily be highly accurate but instead must be within a clini-
cally acceptable range of the true value. This CLSI document provides an
extensive list of applications of many different chemical analytes that may
provide medically unique information about the source of a body fluid
(e.g., creatinine in peritoneal fluid to evaluate urinary tract injury; amylase
in peritoneal fluid to evaluate for pancreatitis; triglycerides in pleural fluid
as an indicator of chylous effusion from lymphatic damage).
REFERENCES
Access the complete reference list online at ExpertConsult.com.

SELECTED REFERENCES
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