Body Fluid Analysis

Marian J. Cavagnaro, MS, MT(ASCP)DLM

Director, Laboratory Services
Memorial Hospital West
Pembroke Pines, Florida

PARTICIPANTS (LEARNERS)
OBJECTIVES
The participant will learn about methods and techniques
for preparing body fluid cytospin smears.
The participant will recognize normal and abnormal cells
in CSF, synovial, and serous fluids on cytospin prepared
Wright-
Wright-Giemsa and Wright
Wrights stained smears.
The participant will be able to recognize differentials that
are abnormal in CSF, synovial, and serous fluids and that
correlate to different clinical conditions

BODY FLUID ANALYSIS

Physical (volume, color, clarity, viscosity)

viscosity)
Microscopic (total cell count and differential)
Chemical (protein, glucose, enzymes, etc.)
Microbiologic (bacteria, parasites, yeast/fungi)
Immunologic examination (not routine)
Cytologic examination (not routine)

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 BODY FLUID DIFFERENTIALS
(CYTOSPIN)
Ratio of cells counted on the hemacytometer
chamber to cells seen on cytospin preparation is
approximately 1:5 to 1:10
For any differential that does not reach 100 cells,
indicate number of WBC
WBCs counted
Differentials should still be reported on fluids that
present with clots
Cytocentrifuge artifacts (nucleus & cytoplasm)
Albumin enhances morphology

Cytocentrifuge
Manufacturers-
Manufacturers-(examples)-
(examples)- Wescor, Shandon
Lipshaw
Fluid vs. Drops/Slide-
Drops/Slide- (saline diluent)
Clear and colorless-
colorless- 10 drops
Slt. Cloudy-
Cloudy- 6-9 drops
Cloudy-
Cloudy-4-5 drops
Grossly Bloody/Cloudy-
Bloody/Cloudy- 1-2 drops
Synovial fluid-
fluid-push smears

Cytocentrifuge

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Cytocentrifuge
Speed/Time-
Speed/Time-
(examples)-
(examples)- 600 RPM
for 10 minutes; 800
RPM for 10 minutes;
1200 RPM for 5
minutes

CYTOCENTRIFUGE ARTIFACTS
NUCLEUS
Accentuation of nucleoli
Blebs and accentuation of lobulation
Denser chromatin in cells in center of slide
Peripheral localization of nuclear lobes
Vacuolization
CYTOPLASM
Clear or granular paranuclear area in mononuclear
cells
Localization of cytoplasmic granules
Irregular blebs and processes
Peripheral vacuolization

Cells in Body Fluids

Red Cells
Granulocytes
Lymohocytes
Monocytes

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CSF-Anatomy &Physiology
The cerebrospinal
fluid (CSF) bathes
the brain and spinal
cord. Most of the
CSF is in the
ventricles of the
brain, which are
large cavities within
the brain which
produce and
reabsorb the CSF.

CSF- Anatomy and Physiology

CSF- Specimen Collection

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 CSF-Specimen Collection
Collection- lumbar puncture between 3rd and 4th lumbar
Collection-
vertebrae
Specimen -divided into 3(or sometimes 4) samples and
placed into 3 sterile sequentially labeled tubes (1-
(1-4 mL
in each)
Tube #1-
#1- chemical and immunologic tests
Tube #2-
#2- microbiologic examination
Tube#3-
Tube#3- hematologic/cytologic
examination
cells counts and differential

ABNORMAL FINDINGS IN CSF

XANTHOCHROMIA (see notes **)
Hemorrhage
Severe and chronic jaundice
CLOTS
Paresis many small clots
Tuberculosis meningitis weblike clot
Blockage of spinal fluid circulation large clot
**NOTES:
1. Fluid from a subarachnoid hemorrhage has a pale
orange color supernatant if RBC
RBCs present within 2-
2-
4 hours; within 24 hours, hemoglobin is converted to
bilirubin and supernatant is yellowish color
2. In a bloody tap, lysis of RBC
RBCs occurs within 4 hrs
- process quickly to prevent a false +. xanthochromia

CSF TRAUMATIC TAP VS. SUBARACHNOID

HEMORRHAGE

Presence of blood in the tubes (varied vs.

no variation)
variation)
Supernatant (clear vs. xanthochromic)
xanthochromic)
Siderophage/erythrophages (absent vs.
present)
present)
Clot Formation (clot vs. no clot)
clot)
Repeat puncture (clear vs. not clear)
clear)

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CSF
Gross Appearance
Color of Supernatant

APPROACH TO CEREBROSPINAL FLUID

LABORATORY STUDIES
ROUTINE INITIAL STUDIES
Cell count/differential,
count/differential, Glucose, Total
Protein, Gram stain, Aerobic culture)
INITIAL SUTDIES (When indicated)
Cytology, Fungal culture, India ink
preparation, Cryptococcal Ag. (Latex
agglut.), AFB Culture, AFB Smear, Bacterial
Ag. (Latex agglut.), Viral cultures
RETROSPECTIVE STUDIES
VDRL, Oligoclonal band analysis,
Immunoglobulin studies, Viral antibody
titers, Tumor markers

CELL TYPES IN CSFS

NORMAL AND ABNORMAL

Lymphocyte Ventricular Lining Cells (ependymal

Monocyte or choroid plexus)
Segmented Neutrophil Chondrocyte (cartillage cell)
Band/Metamyelocyte Bacteria-
Bacteria-cocci or rods
Eosinophil Yeast/fungi
Basophil Macrophage
Promyelocyte Neutrophil macrophage with
Blast phagocytized fungi/bacteria
NRBC Erythrophage(containing RBC
RBCs)
Lymphocyte (reactive/atypical) Siderophage(containing hemosiderin)
Transformed Lymph (immunoblast) Hematin Crystals
Plasma cell Signet ring macrophage
Lymphoma cell Lipophage(containing lipid)
Malignant Cell Multinucleated histiocytic giant cell
Bone marrow cells

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 CSF- Bone marrow
contamination
Occurs because needle was inadvertently pushed to far anteriorly,
anteriorly, into
the marrow cavity of a vertebral body forcing bone-
bone-marrow cells into
the needle. After needle was pulled out and repositioned in the
subarachnoid space, adherent marrow cells were flushed out by the the
flow of CSF into the specimen
WBC may be falsely increased and differential may be uninterpretable
uninterpretable
because some or all of the cells (including mature cells) are of marrow
origin, making recognition of endogenous fluid cells difficult.
Finding of CSF pleocytosis in an infant ; or in an elderly woman who
has vertebral-
vertebral- bone abnormalities including osteoporosis, and
metastatic involvement by cancer should warn the physician to
consider bone marrow contamination.
A new specimen of CSF may be necessary

Blood Cell Maturation

Predominant Cells in CSF

Lymphocyte
Adult Normal-
Normal- 40-
40-80%
Children and Infants-
Infants- 5-35%

Monocyte
Adult Normal-
Normal- 15%-
15%-45%
Children and Infants -50%-
50%-90%

Neutrophil
Adult Normal less than 6%
Children and Infants less than 8%
Terry, 2004

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CSF- ventricular lining cells
Low ratio of nuclear to cytoplasmic cell
material
Round to oval nuclei with smooth nuclear
contours, evenly distributed nuclear
chromatin and inconspicuous nuclei
Sheets or clusters with minimal nuclear
molding

MONONUCLEAR PHAGOCYTIC SERIES

Monocyte/Macrophage
Erythrophage (macrophage containing
erythrocyte(s)
Lipophage (macrophage containing abundant
small lipid vacuoles)
Neutrophage (macrophage containing
neutrophil(s)
Siderophage (macrophage containing
hemosiderin)
With or without hematin (enzymatic
degredation of hemoglobin)

Monocyte/Macrophage

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 DIFFERENTIALS IN ABNORMAL CSF

Inc. PMN
PMNS Bacterial meningitis, early viral tuberculosis and
mycotic meningitis, cerebral abscess, CNS hemorrhage,
cerebral infarct, malignancies, CML in CNS

Inc. LYMPHS Viral meningitis, tuberculous meningitis, multiple

sclerosis, Guillain-
Guillain-Barre Syndrome, lymphoma and
leukemia

Inc. MONOS Chronic bacterial meningitis, partially treated bacterial

bacterial
meningitis, syphilitic meningitis, CNS malignancies

Inc. EOS Parasitic infections, fungal infections, reaction to foreign

material CNS (shunts, dyes), drug reactions

Neutrophils- PMN & Band

Lymphocytes

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Monocytes

Neutrophil, Eosinophil,Basophil

Cell Types seen in Meningitis

Bacterial
Neutrophilic pleocytosis -Increased
neutrophils(acute)
Viral
Lymphocytic pleocytosis-
pleocytosis-Predominance of
reactive lymphocytes
Small to medium to large lymphs with
plasmacytoid appearance
Neutrophilic pleocytosis (early)

Fungal
Neutrophilic pleocytosis

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Causes of Neutrophilic Pleocytosis
Bacterial Meningitis
Early Viral Meningitis (first 6-
6-8 hrs)
Cerebral abscess
CNS Hemorrhage
Trauma
Post-
Post-myelogram
Primary brain tumor or Metastatic tumor
Intrathecal injection of drugs
Previous lumbar puncture (8-(8-12 hrs before)

CSF- Bacterial Infection

Gram stain of
cerebrospinal
fluid showing
B. anthracis

CSF- Bacterial Meningitis

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Causes of Lymphocytic Pleocytosis
Viral Meningitis
TB Meningitis
Resolving Bacterial Meningitis (mature plasma
cells frequent)
CNS Syphilis
Multiple Sclerosis (plasmacytoid reactive forms)
CLL, Lymphoma
Disseminated Carcinoma

CSF-Viral Meningitis

CSF- Fungal Meningitis

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Cell types - in subarachnoid
hemorrhage
2- 24 hours:
Erythrocytes; Neutrophilic granulocytes
(30%-
(30%-60%); Lymphocytes;
Monocytes/Macrophages
12-
12-48 hours:
Monocytes/Macrophages;
Lymphocytes;Erythrophagocytosis
48 hours:
Monocytes/Macrophages;
Erythrophagocytosis; Siderophages and or
Hematin crystals
Kjeldsburg and Knight, 1993

CSF lymphoid cells,leukemic

lymphoblasts, lymphoma cells
Lymphoid cells
Mixture of small, large and transformed
lymphocytes
Leukemic lymphoblasts
Delicate dispersed chromatin nucleus;
nucleoli present
Lymphoma cells
Distinct nuclear clefts or irregularities

CSF- Leukemic Cells

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CSF- Leukemia/Lymphoma

CSF-Malignant Lymphoma

CSF- carcinoma (malignant) cells

High ratio of nuclear to cytoplasmic cell
material
Pleomorphic nuclei with irregularly
distributed chromatin and prominent
nucleoli
Clusters of cell with nuclear molding

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CSF- Malignant Cells

CSF- Malignant Cells

Pleural Effusion

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Paracentesis
INDICATIONS:
Differential diagnosis of ascites

Intraabdominal pressure causing

respiratory distress
Differential diagnosis of acute peritonitis

Paracentesis
The procedure to
remove abnormal
collection of fluid
from the
peritoneal cavity.

Peritoneal Dialysis

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Pericardial Fluid

APPROACH TO SEROUS FLUID

LABORATORY STUDIES
ROUTINE INITIAL STUDIES
Cell count/differential,
count/differential, Aerobic culture, Gram stain,
Albumin & Serum albumin (Ascites only), Protein &
Serum protein (Pleural effusion only), LDH & Serum
LDH
INITIAL STUDIES (When indicated)
Cytology,
Cytology, Anaerobic cultures, Fungal cultures, India
ink smear, AFB culture, AFB smear, pH
RETROSPECTIVE STUDIES
Glucose, Total protein (Ascites only), Amylase,
Lipid studies, Tumor markers, Immunologic stains

Pleural Fluids: Color/Turbidity

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 CELL TYPES IN SEROUS FLUIDS
NORMAL AND ABNORMAL

Lymphocyte Malignant cell

Monocyte
Segmented neutrophil
Band/Metamyelocyte
Eosinophil
Basophil & Mast cells
Myelocyte/Promyelocyte
Blast
Lymphocyte (reactive/atypical)
Transformed lymph (immunoblast)
Plasma cell
LE Cell
Degenerating cell, NOS
Parasites
Mesothelial cell
Reactive mesothelial cell
Macrophage
Lipid laden macrophage (Lipophage)
Neutrophil laden macrophage
(Neutrophage)
Erythrocyte laden macrophage
(Erythrophage)
Hemosiderin granules
Bacteria or Fungi
Cholesterol crystals
Uric acid crystals

DIFFERENTIALS IN ABNORMAL
PLEURAL FLUID

Inc. PMNS - Pneumonia, pancreatitis,

pulmonary infarction,
malignancy,CML
Inc. LYMPHS - Viral pneumonia, tuberculosis,
lymphoproliferative disorders
Inc. EOS - Pneumothorax, parasites, pulmonary
infarction, Hodgkin
Hodgkins disease,
eosinohilic leukemia, dermatologic
conditions.

Peritoneal Fluid-Transudate
Cytocentrifuged
smear contains 54%
macrophages, 43%
neutrophils, 3%
lymphocytes,
occasional reactive
mesothelial cells, and
moderate numbers of
red blood cells.
Infectious agents and
atypical cells are not
detected.

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Pleural Fluid- Pleomorphic
Lymphocytes

Pleural Fluid-Mesothelial Cell

( Multi-Nucleated)

Pleural Fluid-Mesothelial Cells

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Pleomorphic Mesothelial Cells

Mesothelial cell hyperplasia

Plasma Cells

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Macrophage

Pleural Fluid- Macrophage

Macrophages engulf
invaders and destroy
them with powerful
enzymes
Macrophage attacking
streptococcus bacteria
that cause pneumonia

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 Pleural effusion- Adult T-cell
Leukemia/Lymphoma

MORPHOLOGIC CHARACTERISTICS
BENIGN MESOTHELIAL VS. MALIGNANT CELLS

MORPHOLOGIC BENIGN
CHARACTERISTICS MESOTHELIAL MALIGNANT
NUCLEUS
Shape Round, oval, uniform Large, pleomorphic
Nuclear membrane Even Irregular
Chromatin Even Uneven
Multinucleated Yes, uniform size Yes, dissimilar size
Nucleoli
Small Large
Low High
N-C ratio
Absent In some carcinomas
Nuclear molding

CYTOPLASM
Uniform Non-
Non-uniform
Staining
Large, multiple Single or multiple
Vacuoles Single or mixed clusters Cohesive clusters
Signet ring cells

Pleural Fluid
Malignant cells
Reactive Mesothelial
cells

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Pleural Fluid- Malignant Cells

Pleural Fluid- Adenocarcinoma

Metastatic Pleural Effusion

(Primary in Breast)

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 Synovial Fluid

Synovial Fluid-Rheumatoid Arthritis

APPROACH TO SYNOVIAL FLUID -

LABORATORY STUDIES
ROUTINE INITIAL STUDIES
Cell count/differential,
count/differential, Glucose, Enzymes, Total protein, Gram
stain, Aerobic culture
INITIAL STUDIES (When indicated)
Mucin clot*, Cytology, Fungal culture, AFB culture, AFB
smear, Viral culture, Crystal identification
RETROSPECTIVE STUDIES
Countercurrent immunoelectrophoresis for microbial antigens,
Hemolytic complement titration, complement components
* measures hyaluronic acid-
acid- poor clot that fragments results
from inflammatory effusions

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 CELL TYPES IN SYNOVIAL FLUIDS
NORMAL AND ABNORMAL

Lymphocyte Synovial lining cell

Monocyte Mutinucleated synovial cell
Segmented Neutrophil Bacteria - cocci or rods
Band/Metamayelocyte Acid fast bacilli
Eosinophil Yeast/fungi
Basophil Macrophage
Myelocyte/Promyelocyte Neutrophil macrophage with or
Lymphocyte (reactive/atypical) without crystals
Transformed Lumph (immunoblast) Lipophage
Plasma cell Cholesterol crystals
Malignant cell Monosodium urate crystals
Degenerating neutrophils Calcium Purophosphate crystals
Reiter cell

DIFFERENTIALS IN ABNORMAL PERITONEAL

AND PERICARDIAL FLUIDS

Inc. PMN
PMNS - Peritonitis, malignancy
Inc. LYMPHS - Tuberculosis, chylous ascitis,
lymphoproliferative disorders
Inc. EOS
EOSS - Eosinophilic gastroenteritis, chronic
peritoneal dialysis, abdominal
lymphoma
*****************
Inc. PMN
PMNS - Bacterial pericarditis
Inc. LYMPHS - Viral pericarditis, tuberculosis,
lymphoproliferative disorders

CLINICAL CORRELATIONS
IN ABNORMAL SYNOVIAL FLUIDS
(CASE STUDIES)
GROUP I (NON-
(NON-INFLAMMATORY)
Degenerative joint disease, Traumatic arthritis, Osteochondritis
dissecans
GROUP II (INFLAMMATORY)
Rheumatoid arthritis, Reiter
Reiters syndrome, Ankylosing spondylitis
GROUP III (INFECTIONS)
Rheumatoid arthritis, Reiter
Reiters syndrome, Ankylosing spondylitis
GROUP IV (CRYSTAL-
(CRYSTAL-INDUCED)
Gout, Pseudogout
GROUP V (HEMORRHAGIC)
Hemorrhagic, Traumatic arthritis, Synoviomas

Classification of Arthritide

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 DIFFERENTIALS IN ABNORMAL
SYNOVIAL FLUID

GROUP I - Non-
Non-Inflammatory - PMN
PMNs = < 25%
GROUP II - Inflammatory - PMN
PMNs = 25 - 50%
GROUP III - Septic Reactions - PMN
PMNs = >75%
GROUP IV Crystal -Induced - PMN
PMNS = > 50%
GROUP V - Hemorrhagic Reactions - PMN
PMNS = > 25%
*************************************************

Increased neutrophils indicates a septic condition; whereas, an elevated

cell count with a predominance of lymphocytes suggests nonseptic
inflammation.
Other abnormal cells: LE cells, Reiter cells, and RA cells or ragocytes.
ragocytes.

Synovial Fluid
Monocyte
Lymphocyte
Synovial Lining Cell

Synovial Lining Cell

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Synovial Fluid- Neutrophils

Synovial Fluid- Neutrophils

SYNOVIAL FLUID CRYSTALS

Monosodium Urate (MSU) /Tophi-/Tophi-large crystal deposits in
joints, tendons, and soft tissue
Gout
Calcium Pyrophosphate Dihydrate (CPPD)
Pseudogout,degenerative or metabolic arthritis
Cholesterol
Chronic synovial effusions, rheumatoid arthritis
Calcium oxalate
Renal dialysis patients
Corticosteroid crystals/steroids
Drug injection for joint inflammation

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Synovial Fluid Crystal
Identification
Birefringence-
Birefringence- certain structures have the ability
to rotate or polarize light-
light-known as birefringence
(weakly/calcium pyrophosphate or
strongly/monosodium urate)
Polarizing filter-
filter- insert a polarizing filter between
light source and object; and then another
polarizing filter(this is analyzer) between
eyepiece and specimen

Synovial Fluid Crystal

Identification (cont.)
Polarizing filter with compensation-
compensation- using a polarizer
and and analyzer with a first order red compensator. The
red compensator is a retardation plate that alters the
passage of light into slow and first components when the
compensator is inserted between the polarizer and
analyzer, it retards the lights so that the field background
becomes red instead of black.
Monosodium urate crystals-
crystals- appear yellow when
longitudinal axis is parallel to the slow component of
the compensator and they appear blue when the axis
is perpendicular
Calcium pyrophosphate crystals -appear blue when
parallel to compensator and yellow when
perpendicular

Synovial Crystals
Needle-
Needle-shaped
monosodium
crystals seen by
light microscopy of
synovial fluid in a
patient with gout.

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Synovial Fluid Crystals-
Monosodium Urate
Synovial fluid
with sodium urate
crystals, polarized
light with red
compensator
microscopic.

Questions?.Thank you!Last one

in.

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