This document provides information on the examination of cerebrospinal fluid (CSF). CSF is produced by the choroid plexus and circulates in the subarachnoid space, providing mechanical protection and transport functions. Diagnosis of various central nervous system conditions can be made through CSF analysis, including meningitis, encephalitis, and malignancy. Proper specimen collection and examination of opening pressure, cell count, glucose, protein and microscopic analysis can help diagnose these conditions.

1. SPUTUM AND BODY FLUIDS
DR.PRAGATI SATHE

2. SPUTUM EXAMINATION
Specimen Collection –
•Ideal – should contain lower respiratory tract secretions
1st
morning sample is ideal ,contaminant may be prt.
•Needs patient co-operation and understanding.
•May be spontaneous/induced.
•In children – 3 methods –
Nasopharyngeal swab, Cough plate, Cough swab
•Collection in sterile,disposable container with a screw cap
•Delivered to the lab immediately

3. SPUTUM –GROSS EXAMINATION
Consistency and appearance – normal – viscoelastic
Opaqueness – due to pus,epithelial cells, Curshmann’s spirals,
caseous material,bronchial casts,food subs.
Colour – Yellow – pus,epithelial cells
Green – pseudomonas
Rust coloured – infarct,pneumonia
Bright red – acute LVF, TB,malignancy.
Odour – normal – no odour
putrid – lung abscess,cavities.
Others – foreign bodies,parasites,broncholiths

4. SPUTUM –MICROSCOPIC EXAMINATION
•All suspicious particles – transferred with sterile instrument
•Smears can be dried ,heat fixed to kill organisms
•Special stains to be applied – Grams,ZN,pap,Giemsa,H&E
•Presence of alveolar macrophage – sample is representative
•Other cells – bronchial epithelial cells
•Abnormal cells – Intact neutrophils – pyogenic infections
Eosinophils – in large nos– bronchial asthma
RBC’s in large nos. exudate, haemorrhage

5. SPUTUM –MICROSCOPIC EXAMINATION
•Organisms – Mycobacteria – ZN stain
Fungi – direct mount with 10% NaOH
Gram positive cocci
Gram negative bacilli

6. SPUTUM CULTURE
•Microscopic examination should preceede culture
•2 methods –
Classic technique – streaking on agar plate
should be corelated with Grams stained smears.
Quantitive analysis of the organisms

7. SPUTUM IN DISEASE
•Tuberculosis – 24 hr specimen,early morning,bronchial
• washings,transtracheal aspiration & gastric
•Pretreatment by digestion procedures facilitates demonstration
•Liquefying sputum, lowering specific gravity,
Decontamination of other organisms
•Staining with ZN stain
•Flourescent microscopy.

8. AFB bacilli – ZN stain

9. SPUTUM IN DISEASE
•Fungal Diseases –Histoplasmosis,Coccidioides,Blastomyces,
Cryptococci , Aspergillus,Candida
•Bronchial Asthma – Eosinophilia >80%
Charcot Leyden crystals
Bronchial epi cells- Creola bodies-50%
Curschmann’s spirals
•Bronchiectasis – putrid odor,greenish
epithelial cells,Dittrich’s plugs,bacteria
•Chronic bronchitis – copious,mucoid,tenacious
histiocytes and monocytes,organisms,PMN’s
•Lung Abscess – initially less
after rupture foul smelling,copious
multiple organisms identified.

10. SPUTUM IN DISEASE
•Pneumonia- blood tinged sputum
epithelial cells,leucocytes
organisms seen - streptococci,staph,Haemophilus
•Pneumoconiosis – quantity and color varies
macrophages
dumb-bell shaped needles of asbestosis
needle shaped elongated bodies of silicosis
•PCP – protozoan parasite,cysts
•Viral infections – inclusion bodies intranuclear,intracyto
•Malignancy – single,early morning deep cough sputum
look for malignant cells from lung cancers
pap and giemsa stains

11. Viral Infections
Adenovirus CMV

12. Fungal Infections
Aspergillus Cyrptococcus

13. Strongyloidosis stercoralis

14. Pneumocystis carinii organisms

15. Malignant cells – Pap Stain

16. • Pleural
• Peritoneal / Ascitic
• Pericardial
• Synovial
• Amniotic
• Gastric and duodenal
BODY FLUIDS

17. SEROUS FLUIDS
•Pleural
•Peritoneal
•Pericardial

18. TRANSUDATE EXUDATE
• Appearance clear/straw cloudy/purulent
• Coagulation absent freq present
• Sp. Gravity <1.018 >1.018
• Proteins <2 gm% >2 gm%
• Cells few many
mesothelial inflammatory
• Bacteria absent present
• Serous memb normal inflamed
FLUID - TYPES

19. SPECIMEN COLLECTION
•Done by attending physician/nurse
•Delivered to the lab immediately
•Should be examined fresh – as cells disintegrate
bacteria grow fast
altered chemical composition
•Need for another collection to be avoided
•Avoid traumatic tap
•Anticoagulant may be needed
•Collection in 3 bulbs – microbiology,cells & proteins,
biochemistry.

20. Pleural effusion
• Transudate – CCF, hepatic cirrhosis, hypoproteinemia,
• Exudate - neoplasm,infection, trauma,pulmonary infarct,
Rheumatoid disease,SLE, pancreatitis
• Chylous - trauma, TB, lymphoma, Ca,
CAUSES OF FLUID ACCUMULATION

21. Peritoneal effusion
• Transudate – CCF, hepatic cirrhosis,hypoproteinemia
• Exudate - neoplasm,infection, trauma,pancreatitis, bile
peritonitis
• Chylous - trauma, TB, lymphoma, Ca, parasitic
infestation
• Pseudochylous – due to long standing effusions –
TB,rheumatoid pleuritis.
CAUSES OF FLUID ACCUMULATION

22. CAUSES OF FLUID ACCUMULATION
Pericardial effusion
•Infections – bacterial,TB,Fungal,viral
•Neoplastic – Ca,Lymphoma
•Haemorrhagic – trauma,anticoagulant therapy,aortic aneurysm
•Metabolic – uremia
•Hypersensitivity – SLE,rheumatoid dis.

23. FLUID - INDICATIONS
1. Pleural tap –
• Effusion of unknown aetiology
• Dyspnoea and fluid accumulation
• Intrapleural instillation of drugs
• Haemothorax
• Empyema
2. Pericardial tap –
• Effusion of unknown aetiology
• Acute / Chronic cardiac tamponade

24. 3. Peritoneal tap –
• Ascites of unknown aetiology
• Dyspnoea and fluid accumulation
• Suspected intestinal gangrene
• Intestinal perforation
• Intra-abdominal haemorrhage-?traumatic
• Instillation of cytotoxic drugs
FLUID - INDICATIONS

25. •Physical Examination
•Chemical Examination
•Microscopic Examination
•Microbiologic study
•Immunologic Studies
LABORATORY INVESTIGATIONS

26. Whole fluid without centrifugation-
• Appearance – clear,cloudy,chylous
• Colour – milky,red,yellow etc.
Normal = clear/straw-coloured
Red = trauma/rupture/tumour/ bleeding disorder
Milky = chylous - filariasis
Mucinous = adenocarcinoma
Purulent = empyema/peritonitis
FLUID – PHYSICAL EXAMINATION

27. FLUID – PHYSICAL EXAMINATION
•Turbid fluids can be centrifuged-
If supernatent clear – turbidity due to cells
If supernatent turbid – chylous/pseudochylous
•Specific Gravity – to decide whether transudate/exudate

28. Cytocentrifuge / undil. Fluid – Neubaeur Chamber
Total counts - >1000cells/cmm – exudate
>100,000 RBC’s /cmm – malignancy
Differential Counts-
• Lymphocytes > 50% = TB,viral,rheumatoid,SLE
• Neutrophils > 50% =empyema, pneumonia,infarction
> 300 cells/cumm = SBP, cirrhosis
• Eosinophils - ascites = CCF, eosinophilic GE, drugs
lymphoma, ruptured hydatid cyst, chr. peritoneal dialysis
• Mesothelial cells – reactive,TB,rheumatoid
• Malignant cells –Ca,lymphomas,leukemias,
mesotheliomas
FLUID – CYTOLOGY

29. • Protein – Qualitative – acetic acid
pleural = > 3 gm/dl ie. exudate
peritoneal = > 2.5 gm/dl ie. exudate
• Glucose – with flouride
< 60 mg/dl
- seen in – TB, bacterial sepsis, neoplastic,
carcinomatosis , Rheumatoid
• Lactate - >90 mg/dl – infectious pleuritis
• Enzymes – amylase , adenosine deaminase
• Lipids – > 110mg/dl - chylous effusions
• Tumour markers - CEA
FLUID - BIOCHEMISTRY

30. FLUID- MICROBIOLOGY
•Smear Preparation – Gram stain
ZN stain
•Culture Examination – first tube to be used
centrifuged specimen – use sediment.

31. FLUID- IMMUNOLOGY
•RF – seropositive RA
•ANA titers – lupus pleuritis - 1:160
•Decreased complement levels – rheumatoid ,lupus pleuritis

32. Plasma ultrafiltrate with added hyaluronic acid.
• Indications
a. synovitis - acute suppurative
- gout / pseudogout
b. DD of arthritis
• Collection - sterile, disposable plastic heparinized
syringe (1 ml in each large joint)
- Despatch similar to the other fluids
SYNOVIAL FLUID

34. LABORATORY INVESTIGATION
•Physical Examination
•Chemical Examination
•Microscopic Examination
•Microbiologic study
•Unique tests – Viscosity
Mucin clot test
•Immunologic Studies

35. Physical Examination –
Appearance – Clear,cloudy,pale yellow,milky,xanthochromic
reddish,dark brown,greenish
•Normal – Clear and viscous , straw-coloured ,does not clot
•Cloudy – inflammatory,crystals,fibrin,amyloid,cartilage frag.
•Purulent – Acute septic arthritis
•Xanthochromia – hemophilia,tumours,trauma
•Haemorrhagic – traumatic/non traumatic
SYNOVIAL FLUID

36. SYNOVIAL FLUID
Tests for viscosity –
Principle – based on presence of hyaluronic acid
•String test -hold a drop of fluid between thumb &index finger
-pull them apart to form a string
-normal -a string can be formed – 4-6 cm long
-abnormal - < 3 cm long and breaks
•Mucin clot test – clot formation on addition of acetic acid
-grading of clots
-normal – firm clot
-abnormal – poor clot.

37. SYNOVIAL FLUID - BIOCHEMISTRY
•Glucose Examination-
-Must be taken from fasting patient and treated with flouride
-Reported as difference between blood-synovial fluid glucose
-Normal and Non inflammatory arthritis – diff =10mg/dl
-Infectious arthritis –diff=25-50 mg/dl
-Severe inflammatory – diff=increased.
•Protein Content –
increased in severe inflammatory conditions
•Lactate – more values – septic arthritis

38. SYNOVIAL FLUID - MICROSCOPY
•Total and differential count – EDTA sample
Normal – count<200 cells/cumm,upto 25% neutrophils
Non inflammatory – counts <5000/cmm ,upto 25%neutrophils
Mild inflammatory – counts <10,000/cmm,upto 50%neutrophils
Severe inflammatory – counts-500-50,000/cmm, upto 90%
Infectious – counts – 500-2,00,000/cmm,upto 100% neutrophils
•Crystals – Wet preparation and sealed ,polarised,no anticoag
Gout – needle shaped – urate
Pseudogout – rhomboid calcium pyrophosphate
Rheumatoid arthritis – cholesterol – flat clear

39. Sodium urate Sodium urate
Ca pyrophosphate Ca pyrophosphate

40. SYNOVIAL FLUID- MICROBIOLOGY
•Smear Preparation – Gram stain
ZN stain
•Culture Examination – first tube to be used
centrifuged specimen – use sediment.

41. •Rh factor - +ve in 60% of RA patients
•ANA - +ve in SLE
•C4 - ↓ed in RA, SLE, arthritis, gout, etc.
SYNOVIAL FLUID - IMMUNOLOGY

42. • Water(99%),hydrocloric acid,organic acids,various
enzymes, mucin and intrinsic factor.saliva and
regurgitated duodenal contents are also present.
• Psycological factors,systemic diseases,GITract ds,
hence co related with clinical and other findings.
• No defined normal values.
• Unpleasant for the pt, time consuming and
cumbersome procedure
GASTRIC AND DUODENAL CONTENTS`

43. • To determine gastric acid secretion. Macrocytic
anaemia, pernicious anaemia, peptic ulcerand other
ulcerative lesions
• To measure amount of acid production in peptic ulcer
and post operative stomach ulcer
• Diagnosis of Zollinger-Ellison syndrome
• To determine completeness of vagotomy by insulin
tests
• M. tuberculosis isolation in children
• Detection of malignancy by cytology
INDICATIONS

44. GASTRIC INTUBATION
• Overnight fasting
• 55cms mark- antrum
• Contraindicated in esophageal varices, diverticula,
stenosis,aortic aneurysms,recent gastric haemorrhage,CCF
and pregnancy.
• Volume in ml, titrable acidity(0.1 N NaOH and phenol red
as indicator), pH measured electrometrically.

45. GASTRIC JUICE - Physical examination
• Pale grey,transluscent,slightly viscid and pungent
• Fasting volume varies upto 50ml
• Food particles indicate delayed gastric emptying
• Bile is seen in regurgitation, excess seen in intestinal
obstruction beyond the ampulla of Vater
• Blood may be due to trauma. Coffee ground may be
due to gastritis,ulcer, carcinoma or blood swallowed
from mouth, nasopharynx or pulmonary sources

46. GASTRIC JUICE - MICROSCOPY
•Normal - RBC’s,WBC’s,epithelial cells,yeasts,bacteria,mucus
•Neutrophils – increased nos. - gastric mucosal inflammation
•Epithelial cells – increased in gastritis
•Parasites rarely found – giardia,ascaris,strongyloides etc.
•Not really useful

47. AMNIOTIC FLUID
Indications –
•Cytogenetic – increased maternal age,chromosomal abn,
previous history,exposure to irradiation,single gene
disorders
•AFP – neural tube defects
•Erythroblastosis fetalis – based on presence of products of
hemolysis
•Surfactant analysis – lung maturation
•Intrauterine infections –
TORCH,syphilis,TB,Salmonella,candida,HIV

48. Cerebro-spinal fluid examination
Dr.Chetan Chaudhari
Asst.Prof. Dept.of Pathology
LTMG Hospital,Sion

49. CONTENT
• Anatomy and Physiology
• Specimen Collection
• Opening Pressure
• Total cell count
• Differential cell count
• Glucose
• Protein

50. Anatomy and Physiology
Clear & Colourless fluid
Produced by choroid
plexus & ultrafiltration
 leaves ventricular system by
lateral & medial foramina
 flows within subarachnoid
space
 CSF resorption occurs at
arachnoid villi.
Volume: 120 – 180 ml in adultsVolume: 120 – 180 ml in adults
60 – 80 ml in babies60 – 80 ml in babies

51. Functions of CSF
• Mechanical protection
• Transport of biomolecules
• Clearance of catabolites
• Maintenance of constant ICT

52. Diagnosis by CSF
• High sensitivity, high specificity
– Bacterial, TB, and fungal meningitis
• High sensitivity, moderate specificity
– Viral meningitis, SAH, MS, CNS syphilis, abcess
• Moderate sensitivity, high specificity
– Meningeal malignancy
• Moderate sensitivity, moderate specificity
– Intracranial hemorrhage, viral encephalitis,
subdural hematoma

53. Indications
• Meningeal infection
• Subarachnoid hemorrhage (SAH)
• CNS malignancy
• Demyelinating diseases

54. Specimen Collection
• Lumbar puncture (LP) is the insertion of a needle
into the subarachnoid space (the area under the
membrane that surrounds the brain and spinal cord)
of the lumbar (lower back) region for diagnostic or
therapeutic purposes. This allows access to the
cerebrospinal fluid (CSF) in which the brain and
spinal cord float.
• Although the subarachnoid space can be accessed
from other levels, the lumbar region is most often
used as it allows the needle to be inserted below the
end of the spinal cord.

56. Routine Lab Tests
• Required
• Opening CSF pressure
• Total cell count and differential (stained)
• Glucose (CSF/plasma ratio)
• Protein
• Optional
• Cultures, gram stain, antigens, cytology
• Protein electrophoresis, VDRL, D-dimers

57. Opening Pressure
• Normal opening pressure in adults
is 90~180mmH2O, 10~100mmH2O
in children.

58. Elevated pressure
• Congestive heart failure
• Meningitis
• Superior vena cava syndrome
• Cerebral edema
• Mass lesion

59. Decreased pressure
• Spinal-subarachnoid block
• Dehydration
• Circulatory collapse
• CSF leakage

60. CSF specimen is usually devided into 3 serially collected sterile tubes
Tube-1 For chemistry & immunological studies
Tube-2 For microbiological examination
Tube-3 For cell counts
Specimen should be delivered to the lab & processesd within 1 hr.
Refrigeration is contraindicated for culture examination
Gross Examination:
Normal CSF is clear & colourless
Abnormal CSF may appear cloudy, frankly purulent or pigment tinged
Viscous CSF may be seen in metastatic mucin producing
adenocarcinoma, cryptococcal meningitis.

61. Xanthochromia
• Pink, orange, or yellow discoloration
• RBC lysis or hemoglobin breakdown
• May be seen within hours of LP
• Peak intensity at 24 - 36 hours
• RBC > 6000/uL (SAH, ICH, infarct,
traumatic)
• Oxyhemoglobin, bilirubin, increased
protein
• Carotinoids, melanin, rifampin
therapy
CSF supernatantCSF supernatant
colourcolour
Disease/DisordersDisease/Disorders
PinkPink RBC lysisRBC lysis
YellowYellow HyperbilirubenemiaHyperbilirubenemia
OrangeOrange Hypervitaminosis AHypervitaminosis A
Yellow-GreenYellow-Green HyperbilirubenemiaHyperbilirubenemia
BrownBrown Metastatic melanomaMetastatic melanoma

67. Differential Dx of Bloody CSF
• Traumatic tap - blood clears between tubes
• Xanthochromia - pink tinge, RBCs
• SAH - blood does not clear or clot

68. Microscopic examination
Done on undiluted CSF in a manual Neubauer’s counting chamber.
No RBCs should be present in normal CSF
If numerous (except a traumatic trap) indicates trauma or pathologic
processes like malignancy, infarct, hemorrhage

70. Increased Neutrophils in CSF
• Meningitis (bacterial, early TB, viral, fungal)
• Other infections
• Following seizures
• Following CNS hemorrhage
• Following CNS infarct
• Reaction to repeated LP
• Foreign materials
• Metastatic tumor

71. Increased Lymphocytes in CSF
• Meningitis (aseptic, L
monocytogenes,Viral,tuberculous,fun
gal,syphilitic)
• Parasitic infections
• Degenerative disorders
– SSPE, MS, encephalopathy due to
drugs, GBS
• Other inflammatory conditions
– Sarcoidosis, polyneuritis,
periarteritis involving the CNS

72. Eosinophilic pleocytosis in CSF
• Commonly associated with
• Parasitic infections
• Fungal infections
• Reaction to foreign material
• Infrequently associated with
• Bacterial or tuberculous
meningitis
• Viral, rickettsial infection,
lymphoma, sarcoidosis

73. Conditions Associated with Increased
CSF Total Protein
• Increased blood-CSF permeability
– Meningitis (bacterial, fungal, TB)
– Hemorrhage (SAH, ICH)
– Endocrine disorders
– Mechanical obstruction (tumor, disc, abcess)
– Neurosypilis, MS, SSPE, GBS, CVD

74. Bacterial Meningitis
• 0 - 1m: Group B strept & E. coli (GNR)
• 1m - 5y: H. influenzae
• 5 - 29y: N. meningitidis
• >29y: S. pneumoniae
• Listeria monocytogenes common in newborns,
elderly, and other immunocompromised hosts

75. Bacterial Meningitis
CSF Gram stain showingCSF Gram stain showing Gram-negativeGram-negative
diplococcidiplococci characteristic of N.meningitidis.characteristic of N.meningitidis.

76. Granulocytic type of reaction:
1) Purulen meningitis
2) Initial phase of
serous inflammation
Purulent Meningitis – Prevalence of Granulocytes

77. Typical CSF Findings in Meningitis
TestTest BacterialBacterial ViralViral FungalFungal TuberculouTuberculou
ss
OpeningOpening
pressurepressure
ElevatedElevated Usually normalUsually normal VariableVariable VariableVariable
LeukocyteLeukocyte
countcount
>1000/microL>1000/microL <100/microL<100/microL VariableVariable VariableVariable
Cell differentialCell differential MainlyMainly
neutrophilsneutrophils
MainlyMainly
lymphocyteslymphocytes
MainlyMainly
lymphocyteslymphocytes
MainlyMainly
lymphocyteslymphocytes
ProteinProtein MarkedlyMarkedly
increasedincreased
Normal to mildNormal to mild
increaseincrease
IncreasedIncreased IncreasedIncreased
GlucoseGlucose <40 mg/dl<40 mg/dl NormalNormal DecreasedDecreased DecreasedDecreased
CSF to serumCSF to serum
glucose ratioglucose ratio
MarkedlyMarkedly
decreasedecrease
Usually normalUsually normal LowLow LowLow
Lactic acidLactic acid MarkedlyMarkedly
increaseincrease
Normal to mildNormal to mild
increaseincrease
Mild to modMild to mod
increaseincrease
Mild to modMild to mod
increaseincrease

78. • Gradual onset of headache and decreased
consciousness
– Low grade evening fevers
– Night sweats
– Weight loss
– Neck stiffness and positive Kernig’s sign
– Cranial nerve palsies result from exudate
around base of the brain
Mycobacterial Infection: M. tuberculosis
(TB Meningitis)
Presenting Signs and Symptoms

79. Diagnostics
• CSF Values
– Normal: 5-10%
– Protein: High (40mg/dl-100 mg/dl)
– WBC: 5-2000 (average is 60-70%
lymphocytes)
– Glucose: low (<20 mg/dl)
– AFB smear pos: 20%

80. Fungal Infection: Cryptococcus neoformans
(cryptococcal meningitis)
Diagnostics
• CSF Values
– Normal 20%
– Protein 30-150/dl
– WBC: 0-100 (mainly lymphocytes)
– Glucose decreased: 50-70mg/dl
– Culture positive: 95-100%
– India ink positive: 60-80%
– Crypt Ag nearly 100% sensitive and specific

81. Cryptoccocal meningitis: CSF
Indian ink examination

82. Tumorous type of reaction:
1)Malignant meningeal infiltration
2) Localized tumours
3) Special staining procedures required

83. Hypopituitary Carcinoma Lung Carcinoma – Toluidine Blue
Non-Hodgkin Lymphoma

84. THANK YOU