14_Hema_I_Chapter_14_CSF-1.ppt hematology

Hematology

1. CHAPTER 14
Cell counts on CSF and
other body fluids

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 semen analysis that can be done in hematology
Perform cell counts on Serous fluids: pleural, pericardial,
peritoneal (Ascitic)
Perform cell count on synovial fluid
Apply QC measures in body fluid examination

3. Outline

4. 14.1. introduction to Body Fluids
Are ultrafiltrates 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

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 from around the heart
Peritoneal from around the abdominal and pelvic organs

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
memebrane
Exudates: are effusions resulting from
inflammatory responses that directly affect the
serous cavity (includes infections and
malignancies)
More terms???

7. Characteristics of Serous Effusions:
Transudate versus Exudate
Observation / Test Transudate Exudate
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
500-1,000 cells/µL or more, with
increased PMNs, increased
lymphocytes with TB or rheumatoid
arthritis
Red cell countLow, unless from a
traumatic tap
> 100,000/µL, especially with a
malignancy
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

8. 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

9. CSF cont’d
Maximum volume of CSF
Adults 150 mL
Neonates60 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 4
th

and 5
th
lumbar vertebrae under sterile conditions.

10. 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

11. 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

12. 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 is unavoidable store 2-
8
o
C.
All specimens should be handled as biologically
hazardous

13. 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 crystal clear in appearance with
viscosity comparable to water
Abnormal CSF may appear
cloudy, smoky, hazy, opalescent, turbid or grossly
bloody

14. Method: Gross appearance cont’d
Turbidity may be graded from 0 to 4+ as follows:
 0 = crystal clear fluid
1+ = faintly cloudy, smoky or hazy with slight (barely visible)
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

15. Gross appearance cont’d
Bloody specimens
Can result from a traumatic a spinal tap (often occur in
children)
Grossly blood specimen: may indicate subarachnoid
hemorrhage or intracerebral 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 1
st
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

16. Gross appearance cont’d
2. Color
Any color should be reported (N.B. normal CSF is crystal
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

17. Gross appearance cont’d
3. Viscosity
Normal CSF has viscosity comparable to that of water
Clotting may occur
from increased fibrinogen
resulting from a traumatic tap
or rarely may be associated with meningitis or
subarachnoid block

18. Microscopic cellular enumeration
Cell count is performed by manual method
Electronic methods should be used with care
RBC counts are of limited value
WBC counts are useful in developing differential
diagnosis
NR:
0-5 WBC/µL or 0-5 x 10
6
/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 10
6
/L could indicate acute
untreated bacterial meningitis
WBC >50,000 x 10
6
/L are unusual and suggest
intraventricular rupture of a brain abscess

19. Microscopic examination 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 9mm
2
area
Multiply the number by 10/9 to get the number of
WBC/mm
3
 

20. Total Leucocyte Count on CSF 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 9mm
2
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

21. 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.
RBC
CSF x WBC
blood= WBC
ADDED
RBC
BLOOD
 
Corrected WBC
CSF = WBC
CSF – WBC
ADDED
 

22. Excercise
  Example:
RBC
BLOOD = 5 x 10
6
/mm
3
RBC
CSF
= 2,500/mm
3
WBC
BLOOD = 12 x 10
3
/mm
3
WBC
CSF
= 70/mm
3
 
WBC
ADDED
= 2,500 x 12,000
5 x 10
6

= 60/mm
3
 
Corrected WBC
CSF = 70 – 60 = 10/mm
3

23. 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

24. CSF cont’d
vii. Quality control:
count both sides of hemocytometer (18 mm
2
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

25. 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

26. Differential Leukocyte Count on CSF
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.
Lupus erythematosus (LE) cells are rarely found
Other rare cells unique to spinal fluid (ependymal
cells, choroidal cells) may be found

27. ix. Interpretation of CSF diff count
Normal range <5 cells/mm
3
Increased PMN indicate bacterial infection
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

28. 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 memningitis
Syphilitic meningeal encephalitis
Non-infectious cases of increased lymphocytes may
indicate multiple sclerosis

29. Interpretation of CSF diff cont’d
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

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

31. 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

32. 14.3. 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
Gram stain and culture is done in bacteriology laboratory

33. 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?