10

Pleural, Pericardial, and

Peritoneal Fluid Analysis

LEARNING OBJECTIVES
After studying this chapter, the student should be able to: 5. Compare and contrast chylous and pseudochylous
1. Describe the function of serous membranes as they ­effusions.
relate to the formation and absorption of serous fluid. 6. Correlate the microscopic examination and differential
2. Describe four pathologic changes that lead to the cell count of serous fluid analyses with diseases that
formation of an effusion. affect the serous membranes.
3. Discuss appropriate collection requirements for serous 7. Correlate the concentrations of selected chemical
fluid specimens. constituents of serous fluids with various disease states.
4. Classify a serous fluid effusion as a transudate or an 8. Discuss the microbiological examination of serous
exudate based on the examination of its physical, fluids and its importance in the diagnosis of infectious
microscopic, and chemical characteristics. diseases.

CHAPTER OUTLINE
Physiology and Composition, 267 Amylase, 278
Specimen Collection, 269 Lipids (Triglyceride and Cholesterol), 278
Transudates and Exudates, 270 pH, 279
Physical Examination, 271 Carcinoembryonic Antigen, 279
Microscopic Examination, 272 Microbiological Examination, 279
Total Cell Counts, 272 Staining Techniques, 279
Differential Cell Count, 272 Culture, 279
Cytologic Examination, 278 References, 279
Chemical Examination, 278 Bibliography, 280
Total Protein and Lactate Dehydrogenase Ratios, 278 Study Questions, 280
Glucose, 278

K E Y T E R M S1
ascites mesothelial cells
chyle paracentesis
chylous effusion pseudochylous effusion
effusion serous fluid
exudate transudate
Definitions are provided in the chapter and glossary.
1

is named for the organ or organs it encloses. The lungs are

PHYSIOLOGY AND COMPOSITION
individually surrounded by a pleural cavity, the heart by the
The lungs, heart, and abdominal organs are surrounded by a pericardial cavity, and the abdominal organs by the peritoneal
thin, continuous serous membrane, as well as by the internal cavity. The serous membranes that line these cavities consist
surfaces of the body cavity wall. A space or cavity filled with of a thin layer of connective tissue covered by a single layer
fluid lies between the membrane that covers the organ (vis- of flat mesothelial cells. Within the membrane is an intricate
ceral membrane) and the membrane that lines the body wall network of capillary and lymphatic vessels. Each membrane
(parietal membrane) (Fig. 10.1). Each cavity is separate and is attached firmly to the body wall and the organ it surrounds;
267
268 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis

Lung
Parietal membrane
(pericardium)
Visceral membrane
(pleura) Pericardial cavity

Pleural cavity
Visceral membrane
(pericardium)
Parietal membrane
(pleura)
Body wall

Parietal membrane Liver

(peritoneum)
Peritoneal cavity Stomach

Visceral membrane Intestine

(peritoneum)

FIG. 10.1 Parietal and visceral membranes of the pleural, pericardial, and peritoneal cavities. Parietal
membranes line the body wall, whereas visceral membranes enclose organs. The two membranes are
actually one continuous membrane. The space between opposing surfaces is identified as the body
­cavity (i.e., pleural cavity, pericardial cavity, peritoneal cavity).

however, the opposing surfaces of the membrane—despite the cavity; at the same time, plasma proteins in the capillaries
close contact—are not attached to each other. Instead, the produce a force (oncotic pressure) that opposes this filtration.
space between the opposing surfaces (i.e., between the vis- The permeability of the capillary endothelium regulates the
ceral and parietal membranes) is filled with a small amount rate of ultrafiltrate formation and its protein composition. For
of fluid that serves as a lubricant between the membranes, example, increased permeability of the endothelium will cause
which permits free movement of the enclosed organ. The cav- increased movement of protein from the blood into the cav-
ity fluid is created and maintained through plasma ultrafiltra- ity fluid. When this occurs, the now protein-rich fluid in the
tion in the parietal membrane and absorption by the visceral cavity further enhances the movement of more fluid into the
membrane. The name serous fluid is a general term used to cavity. Such an accumulation of fluid in a body cavity is termed
describe fluids that are an ultrafiltrate of plasma and therefore an effusion and indicates an abnormal or pathologic process.
have a composition similar to that of serum. The lymphatic system, or the fourth component in cavity fluid
The process of fluid formation and absorption in the pleu- formation, plays a primary role in removing fluid from a cav-
ral, pericardial, and peritoneal cavities is dynamic. Fluid ity by absorption. However, if the lymphatic vessels become
formation is controlled simultaneously by four factors: (1) obstructed or impaired, they cannot adequately remove the
permeability of the capillaries in the parietal membrane; (2) additional fluid, resulting in an effusion. Other mechanisms
hydrostatic pressure in these capillaries; (3) oncotic pressure can cause effusions, and they may occur with a variety of pri-
(or colloid osmotic pressure) produced by the presence of mary and secondary diseases, including conditions that cause
plasma proteins within the capillaries; and (4) absorption of a decrease in hydrostatic blood pressure (e.g., congestive heart
fluid by the lymphatic system (Box 10.1). Hydrostatic pressure failure, shock) and those characterized by a decrease in oncotic
(i.e., blood pressure) forces a plasma ultrafiltrate to form in pressure (i.e., disorders characterized by hypoproteinemia).
 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis 269

BOX 10.1 Forces Involved in Normal peritoneal cavity. The term ascites refers to an effusion spe-
Pleural Fluid Formation and Absorption cifically in the peritoneal cavity, and ascitic fluid is simply
another name for peritoneal fluid.
Forces Favoring Fluid Formation Collection of effusions from a body cavity is an invasive
Hydrostatic pressure (in systemic capillary) + 30 mm Hg surgical procedure performed by a physician using sterile
Oncotic pressure (in systemic capillary) − 26 mm Hg technique. Unlike cerebrospinal fluid and synovial fluid col-
Intrapleural pressure + 5 mm Hg lections, serous fluid collections from effusions in the pleural,
Net pressure favoring fluid formation in + 9 mm Hg pericardial, and peritoneal cavities often yield large volumes
pleural cavity of fluid. Consequently, the amount of fluid obtained often
exceeds that needed for diagnostic testing. Note that at times,
Forces Favoring Fluid Absorption additional or repeat puncture procedures are necessary to
Hydrostatic pressure (in pulmonary − 11 mm Hg remove a recurring effusion from a cavity for therapeutic pur-
capillary)
poses, such as when the effusion is compressing or inhibiting
Oncotic pressure (in pulmonary capillary) + 26 mm Hg the movement of vital organs.
Intrapleural pressure − 5 mm Hg Before serous fluid is collected from a body cavity, the lab-
Net pressure favoring fluid absorption out of + 10 mm Hg oratory should be consulted to ensure that appropriate col-
pleural cavity lection containers are used and suitable volumes are obtained
(Table 10.1). In microbiological studies, the percentage of
positive cultures obtained increases when a larger volume of
A pleural, pericardial, or peritoneal effusion is diagnosed specimen (10 to 20 mL) is used or when a concentrated sedi-
by a physical examination of the patient and on the basis ment from a centrifuged specimen (50 mL or more) is used to
of radiographic, ultrasound, or echocardiographic imag- inoculate cultures.
ing studies. The collection and clinical testing of pleural, Normally, serous fluids do not contain blood or fibrino-
pericardial, and peritoneal fluids play an important role in gen, but a traumatic puncture procedure, a hemorrhagic effu-
determining the type of effusion present and in identifying sion, or an active bleed (e.g., from a ruptured blood vessel)
its cause. can result in serous fluid that appears bloody and clots spon-
taneously. Therefore to prevent clot formation, which entraps
cells and microorganisms, sterile tubes coated with an anti-
SPECIMEN COLLECTION coagulant such as sodium heparin or liquid ethylenediami-
The term paracentesis refers to the percutaneous puncture netetraacetic acid (EDTA) are used to collect fluid specimens
of a body cavity for the aspiration of fluid. Other anatomi- for the microscopic examination and microbiological studies.
cally descriptive terms denote fluid collection from specific In contrast, serous fluid for chemical testing is placed into a
body cavities. Thoracentesis, for example, refers to the surgi- nonanticoagulant tube (red top), which will allow clot forma-
cal puncture of the chest wall into the pleural cavity to col- tion when fibrinogen or blood is present. Serous fluids should
lect pleural fluid, pericardiocentesis into the pericardial cavity, be maintained at room temperature and transported to the
and peritoneocentesis (or abdominal paracentesis) into the laboratory as soon as possible after collection to eliminate

TABLE 10.1 Suggested Serous Fluid Specimen Requirements

Physical Examination Volume Acceptable Containers
Color and clarity Recorded at bedside by physician and noted on test
request form
Microscopic Examination
Cell counts, differential 5–8 mL EDTA
Cytology study (PAP stain, cell block) 50 mL recommended; Plain tube/container, sodium heparin, EDTA
15–100 mLa
Chemical Examination
Glucose 3–5 mL Plain tube, sodium fluoride
Protein, lactate dehydrogenase, amylase, 5–10 mL Plain tube, sodium heparin
triglyceride, cholesterol, others
pH (pleural fluid) 1–3 mL Heparinized syringe; anaerobically maintained
Microbiological Studies
Gram stain, bacterial culture 10–20 mLb Sterile container; SPS, none, sodium heparin
Acid-fast stain and culture 15–50 mLb Sterile container; SPS, none, sodium heparin
EDTA, Ethylenediaminetetraacetic acid; PAP, Papanicolaou; SPS, sodium polyanetholsulfonate.
a
No upper limit to the amount of fluid that can be submitted; large volumes of fluid enhance the recovery of cellular elements.
b
Large fluid volumes may facilitate the recovery of viable microbial organisms.
270 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis

TABLE 10.2 Differentiation of Transudates and Exudates

Parameter Transudates Exudates
Causes Increased hydrostatic pressure Increased capillary permeability
Decreased oncotic pressure Decreased lymphatic absorption
Physical Examination

Clarity Clear Cloudy

Color Pale yellow Variable (yellow, greenish, pink, red)
Clots spontaneously No Variable; often yes
Microscopic Examination
WBC count <1000 cells/μL (pleural) Variable, usually
<300 cells/μL (peritoneal) >1000 cells/μL (pleural)
>500 cells/μL (peritoneal)
Differential count Mononuclear cells predominate Early, neutrophils predominate; late,
mononuclear cells
Chemical Examination
Bilirubin ratio (fluid-to-serum) ≤0.6 >0.6
Glucose Equal to serum level Less than or equal to serum level
TP concentration <50% of serum >50% of serum
TP ratio (fluid-to-serum) ≤0.5 >0.5
LD activity <60% of serum >60% of serum
LD ratio (fluid-to-serum) ≤0.6 >0.6
Cholesterol ratio (fluid-to-serum) ≤0.3 >0.3
LD, Lactate dehydrogenase; TP, total protein; WBC, white blood cell.

potential chemical changes, cellular degradation, and bacte- transudate from an exudate in all patients.1 Table 10.2 lists
rial proliferation. Note that refrigeration (4–8°C) adversely parameters and the values associated with transudates and
affects the viability of microorganisms and should not be used exudates.
for serous fluid specimens. However, serous fluid samples Classifying an effusion as a transudate or exudate is
intended for cytology examination are an exception and can important because this information assists the physician in
be refrigerated at 4°C when storage is necessary. identifying its cause. Transudates primarily result from a sys-
A blood sample must be collected shortly before or after temic disease that causes an increase in hydrostatic pressure
the paracentesis procedure to enable comparison studies of or a decrease in plasma oncotic pressure in the parietal mem-
the chemical composition of the effusion with that of the brane capillaries. These changes are noninflammatory and are
patient’s plasma. These studies enable classification of the frequently associated with congestive heart failure, hepatic
effusion (transudate or exudate, chylous or pseudochylous), cirrhosis, and nephrotic syndrome (i.e., hypoproteinemia).
which assists in diagnosis and treatment. Note that for chemi- Once an effusion has been identified as a transudate, further
cal analysis, the same type of specimen collection tube (non- laboratory testing usually is not necessary.
anticoagulant, sodium heparin) should be used for both the In contrast, exudates result from inflammatory processes
fluid specimen and the blood collection (serum or plasma). that increase the permeability of the capillary endothelium
In addition, specimen transport and handling conditions in the parietal membrane or decrease the absorption of
should be the same to eliminate result variations due to these fluid by the lymphatic system. Numerous disease processes
potential differences. such as infections, neoplasms, systemic disorders, trauma,
and inflammatory conditions may cause exudates. Addi­
tional laboratory testing is required with exudates, such as
TRANSUDATES AND EXUDATES microbiological studies to identify pathologic organ-
An effusion, particularly in the pleural or peritoneal cavity, isms or cytologic studies to evaluate suspected malignant
is classified as a transudate or an exudate. This classification neoplasms.
is based on several criteria, including appearance, leukocyte Table 10.3 summarizes various causes of pleural, pericar-
count, and total protein, lactate dehydrogenase, glucose, dial, and peritoneal effusions. Unlike pleural and peritoneal
and bilirubin concentrations; however, because of the over- effusions, pericardial effusions usually are not classified as a
lap among categories, no single parameter differentiates a transudate or an exudate. Most often, pericardial effusions
 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis 271

TABLE 10.3 Serous Effusions: Types, Mechanism of Formation, and Associated Conditions
Effusion Type Mechanism of Formation Conditions
Pleural and Transudates Decreased hydrostatic pressure Congestive heart failure
peritoneal Hepatic cirrhosis
Decreased oncotic pressure Nephrotic syndrome
Pleural and Exudates Increased capillary permeability Infection (e.g., bacterial, tuberculous, viral, fungal)
peritoneal Tumors/neoplasms
Pleural: lung and metastatic cancers
Peritoneal: hepatic and metastatic cancers
Systemic disease (e.g., rheumatoid arthritis,
systemic lupus erythematosus)
Gastrointestinal disease (e.g., pancreatitis)
Decreased lymphatic absorption Tumors/neoplasms (e.g., lymphoma, metastasis)
Trauma or surgery
Pericardial Not categorized as Increased capillary permeability Infections (e.g., bacterial, tuberculous, viral, fungal)
transudate or exudate due to changes in parietal Cardiovascular disease (e.g., myocardial infarction,
membrane aneurysms)
Tumors/neoplasms (e.g., metastatic cancers)
Hemorrhage
Systemic disease (e.g., rheumatoid arthritis,
systemic lupus erythematosus)

result from pathologic changes of the parietal membrane chylous effusion is caused by obstruction of or damage to the
(e.g., because of infection or damage) that cause an increase lymphatic system. In the pleural cavity, this can be caused by
in capillary permeability; hence the majority of pericardial tumors, often lymphoma, or by damage to the thoracic duct
effusions could be considered exudates. due to trauma or accidental damage during surgery. Chylous
effusions in the peritoneal cavity result from obstruction to
lymphatic fluid drainage, which can occur with hepatic cir-
PHYSICAL EXAMINATION rhosis and portal vein thrombosis. Note that chronic effu-
Reference values for the characteristics of normal serous fluid sions (as seen with rheumatoid arthritis, tuberculosis, and
in the pleural, pericardial, and peritoneal cavities are not avail- myxedema) can have a similar appearance, owing to the
able because in healthy individuals, the fluid volume in these breakdown of cellular components; they also have a charac-
cavities is small and the fluid is not normally collected. Only teristically high cholesterol content. Consequently because of
effusions are routinely collected and categorized as a transudate their visual similarity, chronic effusions are often called pseu-
or an exudate (see Table 10.2). Transudates are usually clear flu- dochylous effusions and are differentiated from true chylous
ids, pale yellow to yellow, that have a viscosity similar to that effusions by their lipid composition (i.e., triglycerides, chy-
of serum. Because transudates do not contain fibrinogen, they lomicron content). In a chylous effusion, lipoprotein analy-
do not spontaneously clot. In contrast, exudates are usually sis will show an elevated triglyceride level (i.e., greater than
cloudy; vary from yellow, green, or pink to red; and may have 110 mg/dL) and chylomicrons present, whereas a pseudo-
a shimmer or sheen to them. Because exudates often contain chylous effusion has a low triglyceride level (less than 50 mg/
fibrinogen, they can form clots, thus requiring an anticoagu- dL) and no chylomicrons present. Table 10.4 summarizes the
lant (e.g., EDTA, sodium heparin) in the collection tube. The characteristics that assist in differentiating chylous and pseu-
physical appearance of an effusion usually is recorded on the dochylous effusions.
patient’s chart by the physician after paracentesis and should be Blood can be present in transudates and exudates because
transcribed onto all test request forms. If this information is not of a traumatic paracentesis procedure. As with other body
provided, the laboratory performing the microscopic examina- fluids (e.g., cerebrospinal fluid, synovial fluid), the origin of
tion should document the physical characteristics of the fluid. the blood is determined by the distribution of blood during
A cloudy paracentesis fluid most often indicates the pres- paracentesis. If the amount of blood decreases during the col-
ence of large numbers of leukocytes, other cells, chyle, lipids, lection and small clots form, a traumatic tap is suspected. If
or a combination of these substances. In pleural or perito- the blood is homogeneously distributed in the fluid and the
neal fluid, a characteristic milky appearance that persists after fluid does not clot (indicating that the fluid has undergone
centrifugation usually indicates the presence of chyle (i.e., fibrinolytic changes in the body cavity—a process that takes
an emulsion of lymph and chylomicrons) in the effusion. A several hours), the patient has a hemorrhagic effusion.
272 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis

TABLE 10.4 Differentiation of Chylous and Pseudochylous Effusions

Parameter Chylous Effusion Pseudochylous Effusion
Physical Examination Milky Milky
Chemical Examination
Chylomicrons Present Absent
Triglycerides >110 mg/dL (1.2 mmol/L) <110 mg/dL (1.2 mmol/L)
Cholesterol Usually <200 mg/dL (5.2 mmol/L) Usually >200 mg/dL (5.2 mmol/L)
Microscopic Findings Lymphocytes Variety of cell types
Lipid-laden macrophages
Cholesterol crystalsa
Conditions Pleural effusions due to Chronic diseases such as
• Trauma or surgery (caused damage to thoracic duct) • Tuberculosis
• Obstruction of lymphatic system: tumors (lymphomas), • Rheumatoid arthritis
fibrosis • Collagen vascular disease
• Peritoneal effusions due to
• Hepatic cirrhosis
• Portal vein thrombosis
Presence confirms or establishes fluid as pseudochylous effusion.
a

MICROSCOPIC EXAMINATION hematocrit of the fluid can assist in identifying a hemorrhagic

effusion. With pleural fluid, RBC counts can also be used to
The microscopic examination of pleural, pericardial, and identify hemorrhagic effusions. However, high RBC counts
peritoneal fluids may include a total cell count of erythrocytes (greater than 10,000 cells/μL) are frequently associated with
(red blood cells, RBCs) and leukocytes (white blood cells, neoplasms or trauma of the pleura. With peritoneal fluid, a
WBCs), a nucleated cell differential count, cytology studies, WBC count exceeding 500 cells/μL with a predominance of
and, at times, identification of crystals. Note that in contrast neutrophils (greater than 50%) suggests bacterial peritonitis.
to cerebrospinal fluid, which is always present in the central However, the volume of peritoneal fluid (or ascites) can change
nervous system regardless of health status, serous fluids (i.e., significantly because of extracellular fluid shifts, and these
pleural, pericardial, peritoneal fluids) are normally not pres- fluid shifts can significantly change the cell count obtained.
ent in a sufficient amount in body cavities to be analyzed. In Hence a wide range of WBC counts can be encountered in
other words, their presence indicates a pathologic process; peritoneal effusions throughout the course of a disease.
hence, there are no “normal” reference values for serous fluids.
As with other body fluids, cloudy effusions must be diluted Differential Cell Count
for cell counting using normal saline or another suitable dilu- Microscope Slide Preparation
ent. (See Appendix D for acceptable diluents and their prepa- A cytocentrifuged-prepared smear of a body fluid is most often
ration.) Acetic acid diluents are avoided because they cause used to perform a differential cell count. Cytocentrifugation
cells to clump, which prevents accurate cell counting. Cell is easy and fast, and enables good cell recovery in a concen-
counts can be performed manually using a hemacyto­meter or trated area of the microscope slide. Despite minimal cell
an automated analyzer. For details, see Chapters 16 and 17. distortion, some recognizable artifacts associated with cyto-
In the microbiology laboratory, a Gram stain is performed centrifugation are well known and they are listed in Box 17.4.
on serous fluids when requested to aid in the microscopic For additional details on slide preparation, dilutions, and dilu-
identification of microbes (see subsection Microbiological ents, see Chapter 17, subsection “Body Fluid Analysis: Manual
Examination). Hemacytometer Counts and Differential Slide Preparation.”
Total Cell Counts Low-Power Examination
Total RBC and WBC counts have little differential diagnos- Each cytocentrifuged smear should be scanned in its entirety
tic value in the analysis of pleural, pericardial, and peritoneal using a low-power objective (10 ×) to detect abnormalities
fluids. No single value for a WBC count can be used reliably that may be few in number yet apparent at this magnification,
to differentiate transudates from exudates; hence these counts such as malignant cell clumps or other significant findings
have limited clinical use. However, WBC counts in transu- (see Fig. 9.4). In addition, this overview gives the microsco-
dates are usually less than 1000 cells/μL, whereas those in pist a glimpse of the fluid’s composition, that is, its cellularity,
exudates generally exceed 1000 cells/μL. density, and cell distribution on the slide (Fig. 10.2). Note that
With pericardial fluid, a WBC count of greater than 1000 it does not matter whether this evaluation is done before or
cells/μL is suggestive of pericarditis, whereas an RBC count or after the nucleated cell differential.
 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis 273

Nucleated Cell Differential and mesothelial cells. Table 10.5 is provided as a guide for dif-
A differential nucleated cell count is performed using a high- ferentiating monocytes, macrophages, and mesothelial cells.
power oil immersion objective (i.e., 50 × or 100 ×). The cell Monocytes move into body fluids from the bloodstream
types that can be present in pleural, pericardial, and perito- in response to inflammation and infection. These round or
neal fluids include granulocytes (i.e., neutrophils, eosino- slightly irregular-shaped mononuclear cells are typically 12 to
phils, basophils), lymphocytes, plasma cells, mononuclear 20 μm in diameter (Fig. 10.3). The smooth-bordered nucleus
phagocytes (i.e., monocytes, macrophages), mesothelial cells is kidney bean or horseshoe–shaped and it can have deli-
(lining cells of serous membranes), and malignant cells. Note cate, brain-like folds. The nuclear chromatin appears loose
that all nucleated cells are counted, including mesothelial with a lacy or streaked (i.e., raked, combed) appearance. The
cells and malignant cells. ­cytoplasm of monocytes is pale blue-gray and azurophilic
Monocytes, macrophages, and mesothelial cells. It is im- granules may be present, although few in number. Monocytes
portant to note that both monocytes and mesothelial cells can can demonstrate blebs or pseudopods on their cytoplasmic
transform into macrophages. During this process, the visual borders. They may also have a few vacuoles or phagocytic
appearance of intermediate “transitioning” cells can be very remnants, but they do not form cell clumps.
difficult to specifically identify (Figs. 10.3 and 10.4). Fortu- In serous body fluids, macrophages derive from either
nately, because there is usually no clinical value in differen- monocytes or mesothelial cells. They appear in a variety of
tiating these three cell types (monocytes, macrophages, and irregular shapes (i.e., pleomorphic) and their size is highly
mesothelial cells), they are often counted together in a single variable, ranging from 12 to 85 μm in diameter (Figs. 10.3 and
group or category.2 Despite this, when performing the nucle- 10.4). Their oval or round nucleus is eccentric with a smooth
ated cell count, some institutions choose to identify and report border; it can also be lobular or bean-shaped. The nuclear
these cell types in two categories: monocytes/macrophages chromatin can vary from fine to dense and may be coarsely
clumped. The pale blue-gray cytoplasm often appears cloudy
or foamy and azure granules can be present. These actively
phagocytic cells have numerous vacuoles of varying sizes, and
their cytoplasmic borders often have blebs or pseudopods
present. At times during cell transformation to a macrophage,
it is difficult to distinguish whether the cell is a monocyte or
a macrophage, in which case they are identified and enumer-
ated in a “monocyte-macrophage” category.
Macrophages can also be descriptively named based on
the type of inclusions within them (as discussed in Chapter 9,
section Cerebrospinal Fluid). Erythrophages (macrophages
with RBC inclusions) and siderophages (macrophages with
hemosiderin inclusions) are associated with hemorrhage into
a body cavity (see Figs. 9.10–9.12). When numerous neu-
trophils are present in a body fluid, macrophages can ingest
FIG. 10.2 Overview of a peritoneal (ascites) fluid. Mesothelial them. These neutrophages will contain one or several neutro-
cells, macrophages, neutrophils, and lymphocytes are appar-
phils in various stages of degeneration and are associated with
ent. Cytocentrifuged smear, Wright’s stain, ×200. (From
Rodak BF, Fritsma GA, Doig K: Hematology: clinical principles
acute inflammation. Although lipophages (macrophages with
and applications, ed 3, St. Louis, 2007, Saunders.) numerous small, clear, lipid-containing vacuoles) may be seen

FIG. 10.3 (A) Peritoneal (ascites) fluid. Cytocentrifuged smear, Wright’s stain, ×1000. (B) Cell identification: monocyte (A), mono-
cyte/macrophage (B, C), macrophage (D, E, F), mesothelial cell (G, H, I). Note that mesothelial cell “I” is binucleated.
274 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis

FIG. 10.4 Peritoneal (ascites) fluid. (A) Cytocentrifuged smear, Wright’s stain, ×1000. (B) Cell identification: monocyte-macro-
phage (A, B, C), macrophage (D–J), mast cell (K), and lymphocyte (L).

TABLE 10.5 Guide for Differentiation of Monocytes, Macrophages, and Mesothelial Cells2–4
Feature Monocytes Macrophages Mesothelial Cells

Size 12–20 μm 15–85 μm 12–30 μm

Cell Shape Round to irregular Pleomorphic; irregular Round to oval
N:C ratio 3:1 1:3 1:1 or less
Nucleus:
Shape Horseshoe-shaped, bean- Oval, round, lobular, or bean- Oval to round; nucleoli can be
shaped, or delicately folded; shaped; eccentric prominent; can be multinucleated
brain-like folds
Outline Smooth Smooth Smooth; well-defined membrane
Chromatin Loose or lacy with streaked Fine to dense and coarsely Evenly distributed; can be fine to
appearance; can have small clumped dense
clumps
Cytoplasm:
Appearance Pale blue-gray; can have few Pale blue-gray; often foamy Light to darkly basophilic; may show
azurophilic granules or cloudy; can have azure perinuclear halo; may have grainy
granules texture
Phagocytic Sometimes Almost always Rare
remnants present
Vacuoles None to few Often many; variable sizes Few, usually peripheral
Border Irregular; can have small blebs Often with blebs or Ruffled, fringed or lacey edges; can
or pseudopods pseudopods have blebs or pseudopods
Cell clumps or Not seen Rare Often present
clusters

in pleural and pericardial fluids, they are more often seen in their appearance, these cells are sometimes called “signet ring”
the cerebrospinal fluid (see Fig. 9.13) and synovial fluids. macrophages (Fig. 10.5). However, it is important that these
In a macrophage, several vacuoles can merge together to macrophages are not confused with signet ring carcinoma,
form a single large vacuole. When this occurs, the nucleus an adenocarcinoma of glandular epithelial cells most often of
becomes pressed against the cell membrane and because of the stomach. Therefore, to reduce confusion for clinicians, the
 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis 275

FIG. 10.5 Pleural fluid. A “signet ring” macrophage and red

blood cells. Cytocentrifuged smear, Wright’s stain, ×400.
(Courtesy Charlotte Janita.)

FIG. 10.7 Three plasma cells in pleural fluid (×1000). Other

nucleated cells present are: a small lymphocyte, a reactive
lymphocyte, and a macrophage (upper left edge). (From Carr
JH, Rodak BF: Clinical hematology atlas, ed 3, St. Louis, 2008,
Saunders.)

mesothelial cells can show a perinuclear halo, they sometimes

resemble plasma cells (Fig. 10.7), which can also be in pleural
and peritoneal fluids.
Mesothelial cells have ruffled or fringed cytoplasmic
borders that can form blebs or pseudopods (see Fig. 10.3).
They can have a few small vacuoles which, when present, are
usually located at the cell’s periphery. In serous body fluids,
mesothelial cells appear singly and as cell clumps. Therefore,
it is imperative that when cell clumps are present they are
correctly identified and distinguished from malignant cells.
Note that as mesothelial cells transform into macrophages,
FIG. 10.6 Pleural fluid. Wright’s stain, ×500. Mesothelial cells,
their cytoplasm becomes less basophilic and more vacuolated,
singly and in small clumps. Note the “windows” between
cells in the larger cluster. (From Carr JH, Rodak BF: Clinical giving it a foamy appearance.
hematology atlas, ed 3, St. Louis, 2008, Saunders.) Granulocytes. Conditions associated with an increase in
granulocytes, lymphocytes, and plasma cells in serous flu-
ids are varied and briefly listed in Table 10.6. Neutrophils,
when present in large numbers, suggest an infection but
term signet ring should not be used when reporting macro- they could originate from blood contamination. Infections
phages in serous fluids. include peritonitis, pericarditis, bacterial pneumonia, and
A detailed discussion of the formation of these various fungal infections. Although rare, lupus erythematosus (LE)
macrophages is provided in Chapter 9, section Macrophages. cells (i.e., neutrophils with a phagocytized homogeneous
Note that because macrophages are counted collectively in a nucleus) may be present in fluids from patients with sys-
monocyte/macrophage/mesothelial cell category, some insti- temic lupus erythematosus, a chronic autoimmune disorder
tutions append a comment to the report that indicates the (Fig. 10.8).
descriptive type of macrophages present, for example, “many Eosinophilia, when the eosinophil count is greater than
erythrophages present, rare siderophage present.” 10%, can accompany a variety of conditions, such as parasitic
Mesothelial cells are the lining cells of serous membranes infections, foreign body response (e.g., shunts, chest tubes,
and are routinely sloughed off. Hence, they are often seen in peritoneal dialysis), allergies, air in the body cavity (e.g., pneu-
effusions. These cells are 20 to 50 μm in diameter, with light mothorax), leukemias, lymphoma, and others (Fig. 10.9).
gray to deep blue staining cytoplasm that can appear grainy Basophils and mast cells can look similar but are actually
(Fig. 10.6). Their nuclei may be eccentric with smooth, regu- derived from different cell precursors. A mast cell is usually
lar borders and they can be multinucleated (see Fig. 10.3). larger than a basophil and its nucleus is round compared with
The nuclear chromatin is evenly distributed and can be fine the segmented nucleus of a basophil. Both cells have numer-
or dense; one to three nucleoli may be present. Because ous blue-black cytoplasmic granules that can fill the cell such
276 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis

TABLE 10.6 Cell Types and Associated Conditions in Serous Fluids

Basophils/
Neutrophils Eosinophils Mast Cells Lymphocytes Plasma Cells
Bacterial infection Parasitic infection Inflammatory Viral infections a
Chronic infections
conditions
Fungal infection Foreign body response Foreign body Chronic inflammatory Chronic inflammatory
response conditionsb conditionsb
Blood contamination Air in body cavity (e.g., Congestive heart failure Adenocarcinoma
pneumothorax)
Allergies Cirrhosis
Asthma Nephrotic syndrome
Hypersensitivity Malignancy
Leukemia/lymphoma Trauma or obstruction to
lymph
a
Viral infections include infectious mononucleosis, viral hepatitis, viral pneumonia, cytomegalovirus infection, etc.
b
Chronic inflammatory conditions include tuberculosis, rheumatoid arthritis, collagen vascular disease, etc.

FIG. 10.9 Peritoneal dialysate. Numerous eosinophils; also

FIG. 10.8 Pleural fluid. A lupus erythematosus cell. Note the present are small lymphocytes (2) and monocyte-macro-
engulfed homogeneous mass that pushes the nucleus of the phages (3). Cytocentrifuged smear, Wright’s stain, ×1000.
neutrophil to its periphery. Additional neutrophils, red blood
cells, and a macrophage also present. Cytocentrifuged smear,
Wright’s stain, ×1000. (From Carr JH, Rodak BF: Clinical to moderate amount of light blue cytoplasm; a round to oval
hematology atlas, ed 3, St. Louis, 2008, Saunders.) nucleus; and condensed, dark-staining chromatin (Figs. 10.2,
10.4, 10.7, and 10.9). A few small azurophilic granules may be
present in the cytoplasm. In comparison, large lymphocytes
that it is difficult to see the nucleus. There is a greater den- are 12 to 18 μm in diameter, primarily due to a larger amount
sity of these granules in a mast cell compared with a basophil. of cytoplasm. Reactive lymphocytes are usually the largest
The peritoneal fluid in Fig. 10.4 shows a mast cell based on lymphocytes, typically 10 to 30 μm in diameter, with mod-
its round, eccentric nucleus. It is unusual to see basophils or erate to abundant cytoplasm. The cytoplasm can be pale to
mast cells in serous fluids. However, they often accompany dark blue, and in some cells the cytoplasm stains darker blue
eosinophilia (e.g., parasitic infections), are associated with a at the periphery with a light area or zone near the nucleus,
foreign body response (e.g., peritoneal dialysis), and may be resembling a plasma cell. The nucleus of reactive lymphocytes
encountered in other inflammatory conditions. is larger and the nuclear chromatin less basophilic compared
Lymphocytes and plasma cells. Lymphocytes can be with smaller lymphocytes, and several nucleoli may be prom-
seen in all body fluids. They often vary both in the number inent. Reactive lymphocytes accompany other inflammatory
­present and in their size. Note that lymphocytes transform cells and are frequently observed molding around RBCs and
in response to various stimuli. Consequently, a spectrum of other cells in the cytocentrifuged smear.
­different types of lymphocytes can be encountered in a sin- Note that lymphocytes also transform under various
gle body fluid. Small lymphocytes (7–12 μm) have a small stimuli to form mature plasma cells. As this process occurs,
 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis 277

BOX 10.2 Cellular Features Associated

with Malignancy
• Nuclear-to-cytoplasmic ratio (N:C) is high
• Nuclear shape is irregular; may have folds or projections
• Nuclear membrane/border is indistinct or jagged
• Nuclear chromatin is unevenly distributed with prominent
parachromatin spaces
• Nucleoli are large, irregularly shaped; often increased in
number
• Multinucleated with nuclei of different sizes and shapes
• Prominent cytoplasmic granules
• Nuclear molding
• Cell clumps or clusters

single cell. Nuclear molding, where the nucleus of one cell

indents the nucleus of another cell, can produce unusual cell
FIG. 10.10 A “Mott” cell (arrow), that is, a plasma cell with
clusters that are usually detectable using low-power magnifi-
vacuoles containing immunoglobulin. Also present are a lym-
cation. Lastly, a feature of malignancy that may be observed
phocyte and red blood cells. Cytocentrifuged smear, Wright’s
stain, ×1000. is cytoplasmic granules, that is, the cells have large clumps of
prominent granules that are normally not present. It can be
very difficult to discern between normal and malignant cells;
various intermediate cell types are formed: reactive lympho- therefore always consult a pathologist whenever suspicious
cytes, plasmacytoid lymphocytes, and immunoblasts. When cells are observed in a body fluid. This professional interac-
mature, a plasma cell is 8 to 20 μm in diameter and has an tion provides an opportunity for learning and team building
eccentric round or oval nucleus with no nucleoli and coarse- in the laboratory. For example, reactive and malignant meso-
ly clumped chromatin. Sometimes the chromatin takes on thelial cells can be very difficult to differentiate in cases of
a “clock-face” pattern. Plasma cells can be binucleated and malignant mesothelioma.
rarely multinucleated. Their moderate to abundant cytoplasm Most malignant effusions are caused by metastatic adeno-
has no granulation and stains moderate to dark blue with carcinomas (Fig. 10.11). However, any of the blood cancers,
a light area or clear zone called a “hof ” next to the nucleus leukemias, or lymphomas can infiltrate body cavities to cause
(Fig. 10.7). Vacoules may or may not be present. an effusion. In these cases, the use of immunocytochemistry
In response to infections, inflammation, or malignancy, and flow cytometry is valuable in determining an accurate
plasma cells can produce antibodies. These plasma cells, also diagnosis. In summary, proper identification of malignant
called “Mott” cells, have vacuoles that contain immunoglobu- cells in body fluids by a skilled professional (e.g., pathologist,
lin proteins. Their cytoplasm is usually dark blue with a tinge cytologist) is crucial and is performed when a smear is for-
of pink (Fig. 10.10). warded for review or when a cytologic examination is specifi-
Malignant cells. Malignant cells are common in effusions cally requested.
from patients with neoplastic disease. However, the number
of malignant cells found in body fluids can vary dramatically,
which highlights the importance of the low-power examina-
tion of an entire cytocentrifuged smear to search for any rare
malignant cells or cell clumps.
The morphology of malignant cells can be divided into
two types: monomorphic (i.e., cells that show no variation in
morphology; all cells look alike) and pleomorphic (numer-
ous variations in cellular morphology). A variety of morpho-
logic features are associated with malignant cells, and the
most common are listed in Box 10.2. At times, the changes
observed can be obvious, with cells looking extremely bizarre
and atypical; other times, the changes may not be as recog-
nizable. Malignant characteristics include a high nucleus-
to-cytoplasmic (N:C) ratio and changes in the nucleus size,
shape, and appearance. Nucleoli are atypically large and FIG. 10.11 Peritoneal (ascites) fluid. Wright’s stain, ×400.
irregular in shape, and there are changes in the chromatin Adenocarcinoma in a cytocentrifuged smear. Note the numer-
density and distribution. Cells can be oddly multinucleated, ous features in Box 10.2 that apply to this prominent cell
with the various nuclei differing in size and shape within a clump. (Courtesy Charlotte Janita.)
278 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis

Clinical Value of the Nucleated Cell Differential specific gravity values that were equivalent to those of tran-
The clinical value of a nucleated cell differential varies with sudates and vice versa), a better discriminator was needed.
the origin of the paracentesis fluid. In pleural fluid, neutro- Useful tests for classifying a serous fluid as a transudate or
phils predominate in about 90% of effusions caused by acute an exudate are simultaneous determinations of the serum
inflammation (i.e., exudates). Lymphocytes predominate and serous fluid TP concentration and lactate dehydrogenase
in 90% of effusions caused by tuberculosis, neoplasms, and (LD) activity. From these values, the fluid-to-serum TP ratio
systemic diseases. Similarly, in peritoneal fluid, neutrophils and the fluid-to-serum LD ratio can be determined as follows:
predominate (greater than 25%) in most exudates, suggesting
TPfluid
bacterial infection. In peritoneal transudates and in exudates TP ratio = Equation 10.1
caused by decreased lymphatic absorption (e.g., tuberculosis, TPserum
neoplasms, lymphatic obstruction), lymphocytes predomi-
nate. Pericardial fluid differential counts are often not per- LDfluid
LD ratio =
Equation 10.2
formed because a variety of conditions (e.g., bacterial and LDserum
viral pericarditis, postmyocardial infarction) can produce the
same cell differential; hence, a pericardial fluid differential These ratios together provide a reliable means to distin-
count provides little diagnostic information. guish a transudate from an exudate. If the TP ratio is less than
0.5 and the LD ratio is less than 0.6, the fluid is classified as
Cytologic Examination a transudate. In contrast, exudates are those fluids with a TP
When malignant disease is suspected, large volumes (10– ratio greater than 0.5, a LD ratio greater than 0.6, or both.
200 mL) of the pleural, pericardial, or peritoneal effusion
should be submitted for cytologic examination. The fluid Glucose
should be concentrated to increase the yield of cells, and a cell Once a fluid has been classified as an exudate, several chemi-
block and cytocentrifuged smears can be prepared. Cytologic cal tests can be used for further evaluation. The tests selected
examination is an important, sensitive, and specific proce- and their usefulness vary with the origin of the fluid. The
dure in the diagnosis of primary and metastatic neoplasms simultaneous measurement of serum and serous fluid glucose
and is performed by a cytologist or a pathologist. concentrations has limited value. If the serous fluid glucose is
less than 60 mg/dL or if the glucose difference between serum
and fluid is greater than 30 mg/dL, an exudative process is
CHEMICAL EXAMINATION identified. Only low fluid glucose levels are clinically signifi-
The chemistry tests selected to evaluate pleural, pericardial, cant, and a variety of disease processes are associated with
and peritoneal fluids assist the physician in establishing or them, particularly rheumatoid arthritis. Other conditions
confirming a diagnosis for the cause of an effusion. Once a such as bacterial infection, tuberculosis, and malignant neo-
diagnosis has been established, appropriate treatment can plasm may also present with decreased fluid glucose levels;
be initiated, and further testing usually is not required. A however, a normal serous fluid glucose value does not rule
specific diagnosis based on laboratory findings from serous out these disorders.
fluids is limited to (1) malignancy, when malignant cells are
recovered and identified; (2) systemic lupus erythematosus, Amylase
when characteristic lupus erythematosus cells are found The determination of simultaneous serum and fluid amylase
during the microscopic examination; and (3) infectious dis- levels, particularly in pleural and peritoneal fluids, is clini-
ease, when microorganisms (e.g., bacteria, fungi) are identi- cally useful and has become routine in many laboratories. A
fied by Gram stain or culture. Several disease processes can serous fluid amylase value that exceeds the established upper
occur simultaneously, each contributing to the development limit of normal (for serum specimens) or is 1.5 to 2 times the
of an effusion. Therefore chemistry tests initially classify the serum value is considered abnormally increased.5 These high
effusion as a transudate or an exudate. Transudates usually fluid amylase levels most often occur in effusions caused by
require no further chemical analysis, whereas exudates are pancreatitis, esophageal rupture (salivary amylase), gastro-
tested further to identify their causative agents or cause. A duodenal perforation, and metastatic disease.
systematic approach in serous fluid testing greatly facilitates
the diagnostic process. Lipids (Triglyceride and Cholesterol)
Because identification of a chylous effusion is clinically sig-
Total Protein and Lactate Dehydrogenase Ratios nificant, determining the triglyceride level of a fluid is an
No single test can identify specifically the disease process important adjunct when evaluating serous fluids. The milky
causing effusions in the pleural, pericardial, and peritoneal appearance of an effusion does not identify it specifically as a
cavities. Historically, transudates and exudates were classi- chylous effusion because pseudochylous effusions can have a
fied by the total protein (TP) content or specific gravity of similar appearance. Therefore fluid triglyceride levels are used
the fluid alone. Because of the significant overlap noted when as an additional determining factor. A serous fluid triglyceride
these criteria were used (i.e., exudates with protein content or value that is greater than 110 mg/dL (1.2 mmol/L) indicates
 CHAPTER 10 Pleural, Pericardial, and Peritoneal Fluid Analysis 279

a chylous effusion, whereas a triglyceride value of less than for immediate identification of microorganisms. Depending
50 mg/dL rules it out. If the triglyceride level is between 50 on the suspected diagnosis, this may include Gram stain, an
and 110 mg/dL, lipoprotein electrophoresis can be performed; acid-fast stain, and other staining techniques. The sensitivity
the presence of chylomicrons identifies a chylous effusion, of these techniques depends on two factors: (1) the appro-
whereas the absence of chylomicrons indicates a pseudochy- priate collection, processing, and handling of the fluid speci-
lous effusion. Chylous effusions are associated with obstruc- men, and (2) the technical competence of the microscopist
tion or damage to the lymphatic system, which can occur with reading the smears. If either aspect is substandard, optimal
neoplastic disease (e.g., lymphoma), trauma, tuberculosis, and results will not be obtained. In fluid specimens that have been
surgical procedures. Pseudochylous effusions are most often allowed to clot, microorganisms can be caught in the clot
encountered with chronic inflammatory conditions (e.g., rheu- matrix and obstructed from view; similarly, contamination of
matoid arthritis). Note that the presence of cholesterol crystals the specimen during its collection or delays in handling and
in a serous fluid is diagnostic of a pseudochylous effusion.6 processing can yield false-positive results from in vitro bacte-
The cholesterol level of pleural fluid can be useful for dif- rial proliferation. Because of the potential presence of stain
ferentiating between a chylous and a pseudochylous effusion. precipitates, cellular components, and other debris, smears
A fluid-to-serum cholesterol ratio of greater than 1.0 indi- must be evaluated by appropriately trained and experienced
cates a pseudochylous effusion. The cholesterol ratio can also laboratorians. Under the best conditions, a Gram stain is
be helpful in identifying a pleural effusion as a transudate or positive in about 30% to 50% of bacterial effusions, whereas
exudate when other chemical results (TP ratio, LD ratio) are acid-fast stains are positive in only 10% to 30% of tubercu-
equivocal. In these cases, a fluid-to-serum cholesterol ratio of lous effusions.
greater than 0.3 indicates an exudate.6,7
Culture
pH As with smear preparations, the larger the volume of pleural,
With pleural fluid, an abnormally low pH value can help pericardial, or peritoneal fluid used or the more concentrated
identify patients with parapneumonic effusions (i.e., exudates the inoculum used, the greater the chances of obtaining a
caused by pneumonia or lung abscess) that require aggres- positive culture. Both aerobic and anaerobic cultures should
sive treatment. Parapneumonic effusions can involve the be performed. The sensitivity of a positive culture varies with
parietal and visceral membranes, produce pus, and loculate the origin of the fluid and the organism present. Positive
in the pleural cavity. Studies show that if the pleural fluid bacterial cultures are obtained in approximately 80% of all
pH is less than 7.30, despite appropriate antibiotic therapy, bacterial effusions. In contrast, peritoneal tuberculous (or
the placement of drainage tubes is necessary for resolution mycobacterial) effusions culture positive in 50% to 70% of
of the effusion. In contrast, if the pleural fluid pH exceeds cases, pericardial effusions culture positive in about 50% of
7.30, the effusion completely resolves after antibiotic treat- cases, and pleural tuberculous effusions culture positive in
ment alone. An important note is that the collection of pleu- only about 30% of cases.
ral fluid specimens for pH measurement requires the same
rigorous sampling protocol as the collection of arterial blood
gas specimens (i.e., an anaerobic sampling technique using a
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Pericardial and peritoneal fluid pH measurements cur- Laboratory Methods. Philadelphia: WB Saunders; 1991.
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Carcinoembryonic Antigen Kjeldsberg’s Body Fluid Analysis. Chicago: American Society of
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smears using a concentrated or cytocentrifuged specimen and transudates. Chest. 1999;99:1097–1102.