Hema I Chapter 14 CSF.ppt

1. Cell counts on CSF and other
body fluids
CHAPTER 14
Aschalew K. (MSc.)
7/22/2022
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2. Objectives
At the end of this chapter, students will be able to:
 Define terms related to body fluids
 Identify different types of body fluids
 Define CSF
 Define Serous fluids: pleural, pericardial, peritoneal (Ascitic),
and Synovial fluid
 Explain the analysis of CSF
 Discuss how to perform cell count on other body fluids
 Apply QC measures in body fluid examination
 Determine sources of error during CSF analysis
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3. Brainstorming questions
1. What are the common body fluids that used in the laboratory for
diagnosis of the patients?
2. What are serous fluids?
3. What is the difference between transudate and exudate effusions?
4. Would you explain about hydrostatic and colloid osmotic
pressures?
5. Where anatomical place of CSF in the body?
6. How to collect the specimen of CSF?
7. Would you remember method of CSF analysis?
8. What is the clinical significance of CSF analysis?

4. 14.1. Introduction to Body Fluids
Body fluids
 Are ultra-filtrates of plasma
 Fluids serve as lubricants as membranes move against each other
 Body fluids commonly analyzed in hematology lab include:
CSF
Serous fluid
Synovial fluid (fluid from the joints)
Semen
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5. Introduction cont’d
Serous fluids:
 Are fluids from closed body cavities such as pleural, pericardial,
peritoneal/ascitic cavities:
Pleural fluid from the pleural cavity of lungs
Pericardial fluid from around the heart
Peritoneal fluid from around the abdominal and pelvic
organs
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6. Definition of terms
 Effusion: an increase in volume of any serous fluid
 Transudates: effusion as a result of a mechanical disorder affecting
movement of fluid across a membrane
 Exudates: are effusions resulting from inflammatory responses that
directly affect the serous cavity (includes infections and malignancies)
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8. Characteristics of Serous Effusions: Transudate
versus Exudate
Observation / Test Transudes Exudates
Appearance Watery, clear, pale
yellow, does not clot
Cloudy, turbid, purulent, or bloody;
may clot (fibrinogen present)
WBC count Low, < 1,000/µL with
> 50% mononuclear
cells
>1,000 cells/µL, with increased PMNs,
increased lymphocytes with TB or
rheumatoid arthritis
Red cell count Low, unless from a
traumatic tap
> 100,000/µL, especially with a
malignancy, trauma, or pulmonary
infarction
Total protein Low >3g/dl (or > than half the serum level)
Lactate
dehydrogenase
Varies with serum
level
Increased (>60% of serum level
because of cellular debris)
Glucose Not applicable Lower than serum level with some
infections and high cell counts
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9. 14.2. Cerebrospinal fluid (CSF) analysis
I. About CSF
 Fluid in the space called sub-arachnoid space between the
arachnoid mater and pia mater
 Protects the underlying tissues of the central nervous system
(CNS)
 Serve as mechanical interface to:
prevent trauma
regulate the volume of intracranial pressure
circulate nutrients
remove metabolic waste products from the CNS
Act as lubricant
 Has composition similar to plasma except that it has less
protein, less glucose and more chloride ion
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11. CSF cont’d
 Maximum volume of CSF
 Adults 150 mL
 Neonates 60 mL
 Rate of formation in adult is 450-750 mL per day or 20 ml per
hour
 reabsorbed at the same rate to maintain constant volume
 Collection by lumbar puncture/tap done by experienced medical
personnel
 About 1-2ml of CSF is collected for examination
 lumbar puncture is made from the space between the 4th and 5th lumbar
vertebrae under sterile conditions.
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12. Fig. Collecting a CSF specimen
Location of CSF
 Collected in three sequentially
labeled tubes
 Tube 1 for chemical and
immunologic tests
 Tube 2 for Microbiology
 Tube 3 for Hematology
(gross examination, total
WBC & Diff)
 This is the list likely to
contain cells introduced
by the puncture
procedure
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13. 7/22/2022
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14. CSF ont’d
II. Clinical Significance
 Diagnosis of meningitis of bacterial, fungal, mycobacterial and
amoebic origin or differential diagnosis of other infectious
diseases
 Subarachnoid hemorrhage or intracerebral hemorrhage
III. Principle of CSF analysis
 CSF specimen examined visually and microscopically and total
number of cells can be counted and identified
 i.e. Routine:- Gross examination, Cell counts and diff count,
Glucose and Protein values. Further more:- Cultures, Stains
(Gram, Acid Fast), Cytology, electrophoresis…..etc.
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15. CSF ont’d
IV. Specimen: the third tube in the sequentially collected tubes*
 must be counted within 1 hour of collection (cells disintegrate
rapidly). If delay, store at 2-8oC.
 All specimens should be handled as biologically hazardous
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Composition of normal CSF
Protein 15-45mg/dl
Glucose 50-80mg/dl
Urea 6.0-16mg/dl
Uric acid 0.5-3.0mg/dl
Creatinine 0.6-1.2mg/dl
Cholesterol 0.2-0.6mg/dl
Ammonia 10-35mg/dl
Sodium 135-150mEq/L
Potassium 2.6-3.0mEq/L
Chloride 115-130mEq/L
Magnesium 2.4-3.0mEq/L

16. Lab analysis
V. Equipment and Reagents: same as for WBC counting on whole
blood
VI. Method
Gross appearance
 Is visual assessment of CSF for turbidity, color and viscosity
Normal CSF is clear in appearance with viscosity comparable to
water
Abnormal CSF may appear
 cloudy, smoky, hazy, turbid or grossly bloody
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17. Method: cont’d
1. Turbidity
Turbidity may be graded from 0 to 4+ as follows:
 0 = crystal clear fluid
 1+ = faintly cloudy, smoky or hazy with slight turbidity
 2+ = turbidity clearly present but news print easily read through tube.
 3+ = news print not easily read through tube
 4+ = grossly turbid, news print cannot be seen through tube.
Note:
 Slight haziness indicates WBC count of 200-500/uL
 Turbidity indicates WBC count of > 500/uL
 Turbidity in general could result from large number of leukocytes
or bacteria, or increase in proteins or lipids
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18. Method: cont’d
2. Bloody specimens
Can result from a traumatic spinal tap (often occur in children)
Grossly blood specimen: may indicate subarachnoid hemorrhage or
intra-cerebral hemorrhage
 If the specimen is bloody:
There is a need to differentiate between a traumatic tap and a
patient’s clinical condition
 If the specimen in the 1st tube is bloody and is clear in the last tube, it
indicates traumatic tap
 If the specimen has the same bloody color in all the three tubes, it
indicates clinical condition
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19. Method: cont’d
3. Color
 Any color should be reported (N.B. normal CSF is clear)
Xanthochromia: is yellow coloration of CSF
Yellow color could be due to:
 Result of release of hemoglobin from lysed red blood cells
increase in bile pigments
 Specimen collected 2 hours post arachnoid hemorrhage
Xanthochromia of the CSF refers to a pink, orange, or yellow
color of the supernatant after the CSF has been centrifuged.
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20. Method: cont’d
4. Viscosity
 Normal CSF has viscosity comparable to that of water
 Viscosity may be associated with:
clotting
increased fibrinogen
a traumatic tap
or rarely may be associated with meningitis or subarachnoid
block
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21. Method: cont`d
Microscopic cellular enumeration
 Cell count is performed by manual method
 Electronic methods should be used with care
RBC counts have limited value
WBC counts are useful in developing differential diagnosis
 NR:
 0-5 WBC/µL or 0-5 x 106/L
 Neonates have higher value of 0-30 cells/µL
 Low WBC with turbidity could indicate high concentration of bacteria
 WBC between 100-10,000 x 106/L could indicate acute untreated bacterial
meningitis
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22. Method: cont’d
 Differential count is performed when WBC >30 cells/ µL
 Smear is prepared from centrifuged CSF settlement
Total Leucocyte Count on CSF
 If CSF is clear
Mix well the undiluted CSF and properly charge the improved
Neubauer counting chamber
Count the cells in all 9mm2 area
Multiply the number by 10/9 to get the number of WBC/mm3
WBC/mm3 = Number of WBCs X 10
9
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23. Method: cont’d
 If CSF is slightly turbid
prepare a 1:10 dilution with 10% acetic acid (1 drop CSF and 9
drops 10% acetic acid)
count the cells in all 9mm2 area in the improved Neubauer
counting chamber
Multiply counted cells by 100/9
 If CSF is purulent, proceed with the dilution and counting as for a
blood sample
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24. Method: cont`d
Technique for Counting Mixture of WBC and RBC
 This is done to find the true WBC count when the CSF is bloody
due to
Traumatic tap
 Perform the WBC and RBC counts on the patient’s blood and CSF.
 Multiply the ratio of RBC count on CSF to RBC count on blood
by the blood leucocyte count and subtract this product from the
WBC count of CSF.
RBCCSF x WBCblood= WBCADDED
RBCBLOOD
Corrected WBCCSF = WBCCSF – WBCADDED
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25. Excercise
 Example:
RBCBLOOD = 5 x 106/mm3
RBCCSF = 2,500/mm3
WBCBLOOD = 12 x 103/mm3
WBCCSF = 70/mm3
WBCADDED = 2,500 x 12,000
5 x 106
= 60/mm3
There fore:- Corrected WBCCSF = 70 – 60 = 10/mm3
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26. Method: cont`d
Differential Leukocyte Count on CSF
 Centrifuge the CSF at 2500 rpm for 10 min
 Remove supernatant (can be saved for other analysis)
 Re-suspend the sediment
 Prepare a smear from the re-suspended sediment
 Stain using Wright stain
 Wash off stain with water and air-dry
 Identify the types of leucocytes (PMNs or mononuclear cells) and
their number may be expressed as percentage of the total count
 Count at least 100 cells using the oil immersion objective
 Artifacts due to distortion of cells can lead to misidentification
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27. Method: cont`d
Differential Leukocyte Cont`d
 Cells in the spinal fluid may include:
granulocytes
mature or reactive lymphocytes,
momonuclear phagocytes,
plasma cells, blast cells and
malignant cells (indicating primary tumors of brain and
spinal cord)
Others like nucleated red cells, and intracellular bacteria.
Other rare cells unique to spinal fluid (ependymal cells,
choroidal cells) may be found
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28. VII. Quality control
 Count both sides of hemocytometer (18 mm2 area) for the total
WBC
 Increasing the number of cells to be counted for differential count
(instead of 100 cells count 200 cells)
 Check staining quality (e.g. staining time, pH, filtering)
 Proper centrifugation (Speed and time)
 Properly follow SOP
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29. VIII. Sources of errors
 General sources of error in dilution, charging, counting ,
calculating ….etc that were discussed for WBC count also apply
here
 Delay in analysis
 Centrifugation time and speed during sediment preparation for
Diff count
 Staining time
 Improper handling of sample
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30. IX. Interpretation of CSF diff count
 Normal range <5 cells/mm3
 Increased Neutrophil indicate bacterial infection
 Meningitis
 Sub arachnoid hemorrhage
 metastasis
 Increased Eosinophils indicate:
 Systemic parasitic and fungal infection
 systemic drug reaction
 Idiopathic eosinophilic meningitis
 Increased basophils indicate
 Chronic basophilic leukemia
 Chronic granulocytic leukemia
 Purulent meningitis
 Inflammatory processes
 Parasitic infections 7/22/2022
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31. Interpretation of CSF diff cont’d
 Increased lymphocytes indicate:
 viral infections
 viral meningeal encephalitis
 Aseptic meningitis syndrome (majority
of the cases)
 Fungal meningitis
 Partially treated bacterial meningitis
 Syphilitic meningeal encephalitis
 Non-infectious cases of increased
lymphocytes may indicate multiple
sclerosis.
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32.  Increased monocytes (>2%)
indicate:
 Tuberculosis meningitis
 Syphilis and viral encephalitis
 Meningeal irritation
 Subarachnoid hemorrhage
 Leukemic infiltration of the
meninges and infectious state
 Increased macrophages
 Infectious diseases
 CNS leukemia
 Lymphoma
 Malignant myeloma, and other
metastatic tumors spreading to
the brain 7/22/2022
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Interpretation of CSF diff cont’d

33.  Plasma cells increased in:
 Viral disorders such as herpes
simplex infection
 Meningeal encephalitis
 Syphilitic involvement of the CNS
 Post subarachnoid hemorrhage
 Erythrocytes increased in:
 Traumatic tap specimens
 Patients with a bleeding
subarachnoid hemorrhage or
intracerebral hemorrhage
 Chronic myelogenous leukemia or
erythroleukoblastic conditions
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Interpretation of CSF diff cont’d

34. Associated findings
 Glucose and protein values should be correlated with macro and
microscopic hematological findings
Generally decreased glucose in CSF with normal blood
glucose indicates bacterial utilization correlates with
increased PMNs in the Diff
Elevated protein suggestive of inflammatory reaction or
bacterial infection
Viral infections will not have dramatic effect on either CSF
glucose or protein levels
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35. Other body fluids
 Pleural fluid, pericardial fluid, peritoneal/ascitic fluid, synovial
fluid, semen
Same protocol followed as with CSF
 Macroscopic examination for:
Turbidity
Color
Viscosity
 Microscopic
Total white cell count
Differential count using Wright`s stain
 Gram stain and culture is done in bacteriology laboratory
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36. Review Questions
1. What is the function of body fluids?
2. What is the role of CSF in our body?
3. Mention at least three different types of body fluids
4. What is the difference between transudates and exudates
5. Define serous fluids
6. Define CSF
7. How do you perform the total leukocyte count on a:
a) clear CSF b) slightly turbid CSF c) purulent CSF
8) How do you correct the total leukocyte count to a true value
when the count is performed on a sample of CSF that is slightly
turbid due to traumatic tap?
9) How is the differential leukocyte count performed on a sample
of CSF?
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