Académique Documents
Professionnel Documents
Culture Documents
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
1
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
2
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
Red Cells
Granulocytes
Lymohocytes
Monocytes
3
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.
4
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
5
CSF
Gross Appearance
Color of Supernatant
6
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
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
7
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
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
8
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
Lymphocytes
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Monocytes
Neutrophil, Eosinophil,Basophil
Fungal
Neutrophilic pleocytosis
10
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)
11
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
12
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
13
CSF- Leukemia/Lymphoma
CSF-Malignant Lymphoma
14
CSF- Malignant Cells
Pleural Effusion
15
Paracentesis
INDICATIONS:
Differential diagnosis of ascites
respiratory distress
Differential diagnosis of acute peritonitis
Paracentesis
The procedure to
remove abnormal
collection of fluid
from the
peritoneal cavity.
Peritoneal Dialysis
16
Pericardial Fluid
17
CELL TYPES IN SEROUS FLUIDS
NORMAL AND ABNORMAL
DIFFERENTIALS IN ABNORMAL
PLEURAL FLUID
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.
18
Pleural Fluid- Pleomorphic
Lymphocytes
19
Pleomorphic Mesothelial Cells
Plasma Cells
20
Macrophage
21
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
22
Pleural Fluid- Malignant Cells
23
Synovial Fluid
24
CELL TYPES IN SYNOVIAL FLUIDS
NORMAL AND ABNORMAL
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%
*************************************************
Synovial Fluid
Monocyte
Lymphocyte
Synovial Lining Cell
26
Synovial Fluid- Neutrophils
27
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 Crystals
Needle-
Needle-shaped
monosodium
crystals seen by
light microscopy of
synovial fluid in a
patient with gout.
28
Synovial Fluid Crystals-
Monosodium Urate
Synovial fluid
with sodium urate
crystals, polarized
light with red
compensator
microscopic.
29