Body Fluid reporting & interpretation has many inherent challenges right from sample collection, selection of tests, test method validation, appropriate reference intervals & clinical decision limits. Recent published articles & guidelines clarify most of the above mentioned concerns for all laboratory to adopt & implement.

1. Body Fluids & Interpretation
Dr Rajesh V Bendre
Chief Pathologist & Head of Laboratory &
Blood Bank Services,
Jaslok Hospital & Research Centre, Mumbai

2. Body Fluid Challenges
Challenge 1: Pre-Analytical
Laboratories do not control collection of body fluids,
and this has several consequences. Possibility that
the volume of fluid and container submitted to the
laboratory is inadequate and/or inappropriate. The
source and site of fluid collection is often not clear
Challenge 2: Pre-Analytical
Which tests are actually useful and need to be
ordered to aid in patient management?
Challenge 3: Analytical
Body fluid matrix is not like serum or plasma need
test validation to rule out matrix interference. How
can laboratory validate these tests using serum kits?
Challenge 4: Post-Analytical
Scarcity of medical literature for reference intervals &
clinical decision limits to be applied for tests
performed on body fluids?
Introduction- Body Fluids & Interpretation

3. Body Fluids
 CSF
 Pleural Fluid
 Peritoneal Fluid
 Pericardial Fluid
 Synovial Fluid
Other Body fluids – with certain disease
specific Tests-
 Dialysis Fluid
 Drain Fluid
 Cyst Fluid
 Nasal/Ear secretion to rule out CSF leak
Introduction- Body Fluids & Interpretation
Clinician Expectation-
These are precious, critical samples- laboratory needs to prioritize, utilize
them optimally & give maximum information possible.

4. CSF Evaluation
Sample Collection-
 Tube 1-cell count and
differential
 Tube 2-glucose, protein
 Tube 3-cultures, gram
stain, cytology, “other”
studies (HSV PCR, India
ink, Crypto Antigen,
VDRL,AFB, etc…)
 Tube 4-cell count and
differential

5. cobweb
CSF Evaluation

7. CSF Serology

8.  Quantitative determination of immunoglobulin-(IgG) and albumin in cerebrospinal fluid
(CSF) is an important diagnostic test in the study of CNS inflammatory & infectious
diseases.
 Hence it is necessary to clearly differentiate & identify CSF proteins as CNS derived
proteins.
 This can be achieved by analysis of different proteins in CSF and Serum allowing the
calculation of different CSF/serum quotients.
 Low protein concentrations in normal CSF result from the known blood-to-CSF protein
gradients as per law of diffusion as 500:1 for IgG or 3000:1, for 1gM, for albumin as
200:1.
 The Quantitative indices for estimating the intrathecal IgG production-
- Link’ Index – CSF/serum IgG quotient calculated as linear relationship
- Tourtellotte Formula- calculation of IgG synthesis rate
- Reiber Formula - CSF/serum IgG quotient calculated as non-linear (hyperbolic
function) relationship- Currently recommended by IFCC
 All include correction for permeability changes across blood-CSF barrier & are calculated
using serum & CSF IgG & albumin values.
Neuroimmunonology- CSF Immunoglobulins

9. CSF Protein Electrophoresis with
Isoelectric focusing of Oligoclonal Bands
Case patient samples are in lanes 4 (CSF) and 4’ (serum). The CSF
sample is positive for oligoclonal bands (arrows). The IgG index was
0.7 (reference interval 0.3–0.8) and did not suggest increased
intrathecal synthesis. The serum/CSF albumin ratio was <9 indicating
normal permeability of the blood brain barrier. Controls are
indicated on the gel. Other patients are negative for oligoclonal
banding shown in lanes 2, 3, and 5.
Pattern Bands Observed Associated Diseases
1
Polyclonal pattern (no
discrete bands) in both
serum & CSF
Rare Multiple sclerosis
Myelitis
CNS vasculitis
Paraneoplastic syndromes
Systemic lupus erythematosis
2 Same number of OCB in
serum & CSF
Myelitis
CNS vasculitits
Paraneoplastic syndromes
Systemic infections
Systemic autoimmune
disease
Lymphoproliferative disorders
3
OCB in both serum &
CSF CSF has at least 2
more bands than serum
Multiple sclerosis
Most CNS infections
4
More than 2 OCB in
CSF & polyclonal
pattern in serum
Most Multiple sclerosis
5 Monoclonal band in both
serum & CSF
Multiple myeloma
MGUS, lymphoma

10. AUTOIMMUNE ENCEPHALITIDES-
Autoantibody IIF Disease
association
GAD Cerebellar neuronal
cytoplasm, granular
positivity
Stiff syndrome
NMDA
(NR1)
(NR2 & 3)
Mosaic with rat
hippocampus & cerebellum,
transfected HEK cells with
NMDA-NR1 & non-
transfected HEK cells
Limbic
encephalitis
Psychiatric
lupus
VGKC
(caspr2,Ligi1)
Mosaic similar to NMDA in
process - under validation
Limbic
encephaltis
There is evidence that specific autoantibodies directed
against neuronal proteins crucial to the control of
neurotransmission are responsible for a proportion (~8%
in one series ) of such cases.

11.  Guidelines on routine cerebrospinal fluid analysis. Report from an EFNS task force F. Deisenhammera, A. Bartosb, R. Egga, N. E.
Gilhusc, G. Giovannonid, S. Rauere and F. Sellebjergf. European Journal of Neurology 2006, 13: 913–922
CNS viral Infections - CSF Serology

12. Role of PCR of CSF – CNS viral infections
Though CSF PCR is the gold standard for the diagnosis of CNS viral infections, has limitations due to low viral
particles in CSF, as the major replication occurs intra-cellularly.
LIMITATIONS –
 Viral culture and/or PCR are unlikely to be positive in cases of Encephalopathy.
 Recent studies have found that CSF PCR may be negative when performed during the first 72 h. It would seem
prudent to interpret negative CSF PCRs with caution when they are obtained within 72 h of symptom
 An additional concern with viruses which associate with peripheral blood mononuclear cells (PBMCs) eg. CMV,
EBV might be carried into CSF in a setting of inflammation, be detected by PCR (“bystander”), and lead to a spurious
viral diagnosis.
ADVATAGES –
 A positive CSF PCR for viral nucleic acid strongly suggestive of direct infection rather than postinfectious immune-
mediated disease
 RT-PCR on CSF samples for certain infections eg HSV,CMV are useful in diagnosing & monitoring. The nucleic acid
copy number (viral load) may be a marker for the severity of disease or may also help to predict outcome, particularly
in immunocompromised patients.
Virus Sensitivity and specificity (at 3-5days of symptoms)
HSV-1 and 2 >95% sensitivity and specificity; quantitative PCR available; useful especially in neonatal disease
VZV Sensitivity and specificity >95%
CMV Sensitivity nearly 100% in immunosuppressed patients with neurological symptoms; can be
quantitated (range:10–104 copies/ml); useful to monitor therapy. Positive results seen in 60% of
affected infants.

13. Multiplex Nested PCR CSF –
Syndromic Approach to CNS infections
All Results should be reviewed in correlation with other serum results and clinical
evidence.

14. Intrathecal synthesis of specific antibodies as a marker of Herpes simplex encephalitis in patients with negative PCR
Eric Denesa, Catherine Labachb, Hélène Duroxa, Thierry Adoukonoub, Pierre Weinbrecka, Laurent Magyb, Sylvie Ranger-Rogezc
Department of Infectious Diseases, CHU Dupuytren, Limoges, France, Swiss Med Wkly. 2010;140:w13107
Case Studies

15. Case
 A 47 year-old male K/C/O NHL
on chemotherapy, noncompliant
with meds, presents with altered
mental status. He has had
progressively worsening neck
stiffness, photophobia, and
confusion over the past several
weeks. On exam he appears
somnolent and confused. LP,
opening pressure is 290 mm Hg,
and the following slide in the
micro lab.
CSF Evaluation

16. Thoracentesis
Indications
 Diagnostic - All NEW effusions (except if clearly due
to heart failure)
 Suspected parapneumonic effusions must be tapped
ASAP (“Don’t let the sun set on a new pleural effusion”)
 Therapeutic – Respiratory distress
Contraindications-
 Coagulopathy (INR > 2, platelets < 25,000)
 Severe lung disease on contralateral side (risk
of PTX – then what do you have left?)
 Mechanical ventilation (not due to risk of PTX
from PEEP, but due to decreased re-sealing)

17. What tests to order?
Serum LDH, total protein
Pleural fluid:
Total Protein, LDH
Glucose, cell count and diff, pH (on ice)
Gram stain, culture, fungal stain and culture, AFB
Cytology
Other: triglyceride level to r/o chylothorax;
amylase to r/o pancreatitis, esoph perf; Adenosine
deaminase (ADA) to eval for Tb pleuritis
Pleural Fluid Evaluation

18. Fluid- Exudate vs
Transudate
Sensitivity
%
Specificity
%
Light’s criteria 98 83
Protein/serum protein ratio
>0.5
85 84
LDH/serum LDH ratio >0.6 90 82
LDH >2/3 upper limits of serum
normal level
82 89
Others
Pleural-fluid cholesterol level
>60 mg/dL
54 92
Pleural-fluid cholesterol level
>43 mg/dL
75 80
Pleural-fluid/serum cholesterol
ratio >0.3
89 81
Serum/pleural-fluid albumin
level ≤1.2 g/dL
87 92
TRANSUDATE
 Result from imbalances in oncotic and
hydrostatic pressure
 Usually low serum oncotic pressure +/- high
serum hydrostatic pressure
 Pulm Edema/CHF
 Cirrhosis with ascites
 Hypoalbuminemia/Nephrotic syndrome, ESLD
 Fluid overload s/p aggressive IVF
 Peritoneal dialysis
EXUDATE
Caused by local, not systemic, factors
 Infection
 Neoplasm
 Pancreatitis
 Esoph perf
 RA
 SLE
 Sarcoid, Wegeners, PE, Meig’s, chylothorax
Pleural Fluid Evaluation

19. Pleural Fluid Evaluation- Approach
Pleural, peritoneal and pericardial effusions – a biochemical approach. Biochemia Medica 2014;24(1):123–37

20. Pleural Fluid eosinophilia
Eosinophilic pleural effusion (EPE) is defined by an eosinophil count
of ≧10% in the pleural fluid and often caused by air or blood in the
pleural space.
Other Causes
 Pulmonary infarct
 Drugs
 Malignancy
 Asbestos
 Parasitic/ Fungal infection
Pleural Fluid Evaluation- Approach

21. Pleural Fluid Evaluation- Approach
TB Diagnosis
Trajman A, Pai M, Dheda K & Menzies D ERJ 2008
PCR in tuberculosis: An extremely useful tool?. International Journal of Medical Microbiology and
Tropical Diseases, April-June, 2018;4(2):60-63

22. Pleural Fluid Evaluation- Cytology

23.  A 59-year old man with HIV and
Hepatitis C develops progressive
Breathlessness and presents to
the ER with 90% sats on RA. On
CXR, he has a large right-sided
pleural effusion.
 Serum LDH=200, serum protein =
5.6.
 Pleural fluid: LDH 100, protein
2700, WBC 400, pH 7.35,
glucose=85
 Exudate or transudate? Retap?
Abx? Chest drain?
Pleural Fluid Evaluation- Case
 Pleural fluid LDH/serum
LDH=100/200= 0.5
 needs to be > 0.6 to be exudate
 Pleural fluid protein/serum
protein=2700/5600= 0.4
 needs to be > 0.5 to be exudate
 Pleural fluid LDH is < 2/3 x (ULN
serum LDH)
 Transudate
 Cause is cirrhosis/ascites
 Presents w/right sided pleural
effusion
 No Abx or need to retap
 Treat the underlying problem
(ascites) w/ diuretics, aldactone;
optimize treatment for Hep C, HIV

24.  A 24 yr. woman with cystic fibrosis presents to the ER with fever, cough and
night sweats for 10 days. CXR shows LLL consolidation and surrounding free-
flowing effusion.
 Pleural fluid: cloudy, LDH=1360, RBC- 12100/cmm, WBC- 40000/cmm,
Neutrophils- 60%, lymphocytes- 40%, glucose = 36 mg/dl, gram stain- Neg
 Does she need a chest drain? Fibrinolytics?
Pleural Fluid Evaluation- Case
Exudate
 because LDH > 2/3 upper limits of normal serum LDH
Parapneumonic effusion- Category 3 (Moderate risk of poor outcome)
 Glucose < 60 mg/dL and/or Gram stain/culture positive and/or large (≥ ½
hemithorax if free-flowing) and/or loculated
 No frank pus
 Chest drain and Abx, consider fibrinolytics or surgical intervention

25. Parapneumonic Effusions
•Alternate to pH is pleural fluid glucose (< 60 mg/dl),
•ACCP recommendations, from Chest, 2000
Pleural Fluid Evaluation- Case

26. Paracentesis
Indications for paracentesis
 A febrile pt with ascites is assumed to
have SBP until proven otherwise
 New onset ascites—etiology?
 Increasing abdominal pain/discomfort
 Respiratory compromise
 Unexplained leukocytosis, acidemia, renal
failure
Contraindications
 Coagulopathy is NOT a contraindication, But no if
pt is in DIC
 Must be careful if minimal fluid visualized on U/S
 If peritoneal carcinomatosis, do not do this
procedure yourself

27. Ascitic Fluid Evaluation-
What tests to order?

28. Serum-to-ascites albumin gradient (SAAG)
= Serum albumin – ascitic fluid albumin
If the gradient is > 1.1:
Portal HTN (drives
fluids into peritoneum)
cirrhosis, alcoholic
hepatitis, CHF,
massive liver mets
Ascitic Fluid Evaluation- Approach
If the gradient is < 1.1:
(protein leaks into
peritoneum and fluid
follows)
Peritoneal
carcinomatosis, peritoneal
TB, pancreatitis, nephrotic
syndrome, peritonitis

29. Spontaneous Bacterial Peritonitis
SAAG > 1.1
Suspect if > 250 PMNs (> 100 PMNs in pt with
peritoneal dialysis catheter)
70% GNR (E.coli, Klebsiella)
30% GPC (S. pneumo, Enterococcus)
“Culture negative SBP” if > 250 PMNs but cx
neg; treat the same
Often Treated with ceftriaxone, cefotaxime,
cipro/flagyl
Ascitic Fluid Evaluation- Approach

30. Ascitic Fluid Evaluation- Approach
Pleural, peritoneal and pericardial effusions – a biochemical approach. Biochemia Medica 2014;24(1):123–37

31. Pericardial Fluid Evaluation- Approach
Pleural, peritoneal and pericardial effusions – a biochemical approach. Biochemia Medica 2014;24(1):123–37

32. Synovial Fluid Evaluation- Approach

33. Synovial Fluid Evaluation- Approach
 Samples with very high viscosity- how to perform cell count? - Use of pinch of hyalurinodase (bromelain) or
1-2 drops of 0.05% solution made in phosphate buffer – to be added in 0.5ml of synovial fluid
 RF has been detected in synovial fluid in only ~60% of patients with rheumatoid arthritis, and typically in
titers lower than those observed in serum
 Synovial fluid uric acid concentrations significantly greater than the upper reference limit for serum uric acid
may support such a diagnosis.

34. Biliary/Hepatic CSF Dialysate Drain Pancreatic Pericardial
Peritoneal/
Ascites Pleural Synovial
Alpha-Fetoprotein
(AFP) • • •
Amylase • • • • •
Bilirubin (Total) • • • •
CA 19-9 • • • • •
Carcinoembryonic
Antigen (CEA) • • • • •
Cholesterol • • • •
Chloride (Cl⁻) • • • • • •
Creatinine • • •
Glucose • • • • • •
Lactate
Dehydrogenase
(LDH) • • • • •
Lipase • • • • • • •
Potassium (K⁺) • • • • • •
Protein (Total) • • • • •
Rheumatoid
Factor (RF) • • • •
Sodium (Na⁺) • • • • • •
Triglycerides • • • •
Uric Acid • • • •
Certain Tests Requested on Body Fluids

35. Body Fluid Tests-
Recommendations for Laboratory

36. Body Fluid- Automated Cell counts Verification –
Recommendations for Laboratory

37. Body Fluid Tests- Validation - Recommendations for Laboratory

38. Body Fluid Tests- Validation - Recommendations for Laboratory
NOTE- Include following Remarks for all body fluid test reports-
1. Test performed using kit method approved for serum/plasma and inhouse validated for
body fluid as the sample type
2. All Results should be reviewed in correlation with cytology, serum results and other
clinical evidence.

39. BODY FLUID CHALLENGES RECOMMENDATION
Pre-Analytical
Laboratories do not control collection of body
fluids, possibility of fluid volume and container
submitted to the laboratory is inadequate and/or
inappropriate. The source and site of fluid
collection is often not clear.
Which tests are actually useful and need to be
ordered to aid in patient management?
• Creation & inclusion of laboratory offered tests on
body fluids along with required sample volume &
container as part of lab service manual
• Creation of separate body fluid test codes in HIS/LIS
& tag them as critical/stat samples
• Creation of test requisition forms to capture all
required details of clinical history, site & source of fluid
• Creation of disease specific body fluid packages after
discussing with clinicians
Analytical
Body fluid matrix is not like serum or plasma
need test validation to rule out matrix
interference. How can laboratory validate these
tests using serum kits?
• All body fluid tests offered by laboratory should use
fluid specific kits (if available) or in-house validate the
serum kit used for off-label application
Post-Analytical
Scarcity of medical literature for reference
intervals & clinical decision limits to be applied
for tests performed on body fluids?
• Test performed using kit method approved for
serum/plasma and in-house validated for body fluid as
the sample type
• All Results should be reviewed in correlation with
cytology, serum results and other clinical evidence.
• Include appropriate interpretation notes as applicable
based on available medical literature
CONCLUSION

40. Thank You