CLINICAL SIGNIFICANCE OF

ANALYTES IN NON-STANDARD
BODY FLUIDS
Deanna Franke, PhD, MT(ASCP), DABCC
Clinical Scientist, Core Laboratory & Toxicology
Pathology Consultants of South Broward, LLP
dfranke@mhs.net
Deanna Franke, PhD,
DABCC has no relevant
financial relationships
to disclose.






This presentation does
NOT cover laboratory
validation studies for
non-standard body
fluid testing

Disclosures Limitations
OBJECTIVES
Define non-standard body fluid testing

Describe pathogenesis of fluid accumulation

Explain clinical significance of analyte
measurements
Standard vs. Non-standard

Identify continued challenges



REALITY CHECK #1 QUESTIONS TO ASK
Do clinicians send fluids to your laboratory for
testing?

Does the laboratory understand (or even know)
clinical indication(s) for measuring analytes in body
fluids?

Are these results meaningful to the clinician?

How do you know?
DEFINITIONS
Lab Medicine 2004; 35(12): 707-708.
ROUTINE test
NO method/instrument modifications
COMPLIANT with
Manufacturers instructions (FDA approved)
All regulatory requirements (CLIA 88)
Standard
Laboratory Test

REFERENCE package insert
NOT listed = NSBF
Non-standard
Body Fluid
(NSBF)
DEFINITIONS
Lab Medicine 2004; 35(12): 707-708.

MODIFICATIONS method/instrument
NON - COMPLIANCE with
Manufacturers instructions (FDA approved use)
Non-Standard
Laboratory Test

Blood
Urine
CSF
Standard Body
Fluid
REALITY CHECK #2 WHAT DO I DO?
By definition = Laboratory Developed Tests (LDT)
Require documented method validation
Recommend working with clinical and technical
staff to minimally validate:
Accuracy
Reproducibility
Reliability
Recommend starting with clinically significant
analytes in NSBF
Review following glossary and discussion
Ask the doctors who are ordering


NON-STANDARD BODY FLUID GLOSSARY
Serous
Amniotic
Cerebral
Spinal Fluid
Dialysate
Cyst
SEROUS FLUIDS
SEROUS FLUIDS - FUNCTION
Facilitates movement of
parietal and visceral
membranes

Plasma filtrate

Pericardial Sac
Pleural Cavity
Peritoneal Cavity
Source: The McGraw-Hill Companies

SEROUS FLUID PRODUCTION AND CIRCULATION


Parietal
membrane
S
T
O
M
A
T
A



Visceral
membrane
Systemic Circulation Pulmonary Vessels
Lymphatics
Normal Pleural Total Volume = 8.4 4.3 mL / cavity
PATHOGENESIS OF SEROUS FLUID ACCUMULATION


Parietal
membrane
S
T
O
M
A
T
A



Visceral
membrane
Systemic Circulation Pulmonary Vessels
Lymphatics
Effusion develops imbalance of fluid resorption/production
SEROUS FLUIDS NON-STANDARD TESTS
Clinical Significance
Differentiate Exudates vs. Transudates

Exudate Transudate
Increased permeability
OR
Decreased resorption
Increased capillary pressure
OR
Decreased plasma oncotic pressure
Unilateral Bilateral
Infectious/Pneumonia
Neoplasms
Inflammatory disease
Congestive heart failure
Hepatic cirrhosis
Nephrotic syndrome
Henrys Clinical Diagnosis and Management by Laboratory Methods. 21
st
ed.
Chapter 28.
SEROUS FLUIDS NON-STANDARD TESTS
Lights Criteria Exudative Effusions

Pleural and Pericardial fluids only
Fluid LDH & Total protein - LDT
Requires matched serum sample - Routine

Test Value
Fluid/Serum Total Protein > 0.5
Fluid/Serum LDH > 0.6
Fluid LDH > 2/3 serum upper
limit of normal
SEROUS FLUIDS OTHER NON-STANDARD TESTS
CHOICE TEST:
Pleural Fluid pH

Question:
How does your lab
measure pleural fluid
pH?
A: pH paper
B: POC analyzer
C: pH meter in Lab
D: Blood gas analyzer
Henrys Clinical Diagnosis and
Management by Laboratory Methods.
21
st
ed. Chapter 28.
CAP Survey FLDA 2009
CLINICAL SIGNIFICANCE PLEURAL FLUID PH
Diagnostic and prognostic
Drainage and chest tube insertion pH <7.2
Potential to report significant false elevations
Loss CO2 = Increases pH +0.1-0.2
Anaerobic collection
Precision counts!
Method Standard Deviations
(pH Units)
Litmus paper 0.20-0.40
pH meter 0.12-0.14
Blood gas analyzer 0.04-0.06
CAP Survey FLDA2009
SEROUS FLUIDS NON-STANDARD TESTS
N Engl J Med. 2002; 346(25): 1971 - 1977
Lights criteria + cholesterol 99 98
AMNIOTIC FLUID
AMNIOTIC FLUID - FUNCTION
Function
Protects fetus trauma
Cushions umbilical cord
Serves as a nutrient reservoir
Supports lung, Musculoskeletal, and GI
development
Space
Growth factors
Fluid
Antibacterial properties
Clin Obstet Gynecol. 1997;40(2):280.
AMNIOTIC FLUID PRODUCTION DURING
GESTATIONAL PHASES
UpToDatev19.1: Physiology of amniotic fluid volume regulation.
Early
Exocoelomic cavity = Coelomic Fluid
Amniotic sac = Amniotic Fluid
Mid
Urine
Lung secretions
Late
Urine and lung secretions
Minor: Oral-nasal cavity secretions
Notable:
DECREASE: Sodium and Osmolality
INCREASE: Urea Nitrogen, Creatinine, Uric acid
Reflective of changes in amniotic fluid during gestation
AMNIOTIC FLUID NORMAL COMPOSITION
AMNIOTIC FLUID STANDARD TESTS
Fetal Lung Maturity
Phosphatidylcholine
Phosphatidylglycerol
Fetal Stress
Meconium (visual assessment)
Hemolytic Disease of Newborn (HDN)
Bilirubin
Neural Tube Defects
AFP
Acetylcholinesterase

AMNIOTIC FLUID NON-STANDARD TESTS
Lamellar Bodies
Laminated storage granules of pulmonary surfactant
Size: 1-5 m
Phospholipids, cholesterol, protein
Exudation of pulmonary fluid and fetal breathing
LDT on automated cell counter platelet channel
Lab Invest 2000 80: 395-403
CLINICAL SIGNIFICANCE LAMELLAR BODY COUNT
Respiratory Distress Syndrome
Incidence: Increases with decreasing gestational age

Pulmonary surfactant deficiency
Decreased lung compliance
Low lung volume
Collapse of alveoli
Respiratory failure

Determination of fetal lung maturity
Antenatal prevention corticosteroid administration
Treatment- surfactant replacement
http://www.arup.utah.edu/media/FLM/FLM.html (May 2012)

AMNIOTIC FLUID NON-STANDARD TESTS
Enzymes
Gamma-Glutamyl-Transferase (GGT)
Aspartate-amino-transferase (AST)
Total Alkaline Phosphatase (t-ALP)
5Nucleotidase (5NUC)
Amylase (AMY)
Recall AF composition 2
nd
to 3
rd
trimester

GGT
Abnormal bile excretion
GGT & t-ALP
Slow intestinal transit
Congenital extrahepatic
biliary damage
(Muller F et al, 1991)
Cystic Fibrosis
Brock D et al, 1988
Dechecchi et al., 1989
CLINICAL SIGNIFICANCE AF ENZYMES 1
Clin Biochem 2001 34: 317-322
GGT
5NUC
N = 3 3 17 N = 3 3 17
CLINICAL SIGNIFICANCE AF ENZYMES 2
Clin Biochem 2001 34: 317-322
GGT
N = 2 9 3
Gastroschisis N = 5
5NUC
AMNIOTIC FLUID NON-STANDARD TESTS
ENZYME SUMMARY
AF Enzymes 1
Chromosomal abnormalities
Trisomy 13: Patau Syndrome
Trisomy 18: Edwards
Syndrome
Trisomy 21: Down Syndrome
Significant differences
between GGT and
5nucleotidase levels


AF Enzymes 2
GI defects suggested by
ultrasound confirmed by
GGT and 5nucleotidase
enzyme analysis.

First demonstration of AF
enzymes in gastroschisis
Prognostic implications

CEREBROSPINAL FLUID
CEREBROSPINAL FLUID - FUNCTION
Provides mechanical
support

Maintains stable
biochemical
environment
Drainage sink
Nutrient pool

Supports
communication

Neuroscience. 2004;129(4):95770
UpToDate19.1: Cerebrospinal fluid: Physiology and utility of an examination in disease states
Image: www.brainandspinalcord.org
CEREBROSPINAL FLUID STRUCTURAL SUPPORT
Arachnoid mater:
spiderweb like connection to pia mater
makes up the subarachnoid space = fluid filled = CSF
CEREBROSPINAL FLUID PRODUCTION
Choroid Plexus
Epithelium
UpToDate19.1: Cerebrospinal fluid: Physiology and utility of an examination in disease states
Blood-CSF
Barrier
CSF actively secreted
by choroid plexus and
composition depends
on filtration, secretion,
absorbtion.
CEREBROSPINAL FLUID
COMPOSITION
Transport system
H
+
K
+
Ca
2+
Mg
2+

Other ionic components
Diffusion
Glucose
Urea
Creatinine
Proteins
Adapted from: Henrys Clinical Diagnosis and Management by Laboratory
Methods. 21
st
ed. Chapter 28.
CLINICAL INDICATIONS
CEREBROSPINAL FLUID STANDARD TESTS
Henrys Clinical Diagnosis and Management by Laboratory Methods. 21
st
ed.
Chapter 28.
CEREBROSPINAL FLUID NON-STANDARD TESTS

Adapted from: Henrys Clinical Diagnosis and Management by Laboratory
Methods. 21
st
ed. Chapter 28.
CEREBROSPINAL FLUID NON-STANDARD TESTS
Lactate/Lactic acid
Intermediate product
carbohydrate
metabolism

Production
Muscle, skin, renal
medulla, erythrocytes

Elimination
Liver Cori Cycle
Kidney


Biochemistry. 5th edition. Figure 16.33
CLINICAL SIGNIFICANCE CSF LACTATE









Anaerobic metabolism due to CNS tissue hypoxia
Adjunct test viral vs. bacterial meningitis
CSF pH
decreased
bacterial
meningitis
Postulated due
to lactate
Confirmed 1925
1917
Lactate is
ionized at
physiologic pH
- BBB
CSF lactate
independent
of blood
lactate levels
1967
Postgrad Med 1990; 88(5): 217.
CLINICAL SIGNIFICANCE CSF LACTATE
Meningitis Mean
(mmol/L)
Control
(mmol/L)
Investigators
Viral 2.2-3.0 1.6-3.3
Levinson, Bland
et al, Cunha,
Ellis et al
Bacterial 6.0-9.7 1.6-3.3
Bacterial
Partially Rx
3.0-6.0 1.6-3.3
Tuberculous 7.0 1.3 Tang
Cryptococcal 1.5 1.4 Duinkerke et al
Cerebral Malaria
(P. falciparum)
Mild
Severe


3.4
9.0


<2.2
<2.2

White et al
Adapted from Postgrad Med 1990; 88(5): 217.
CLINICAL SIGNIFICANCE CSF LACTATE
Method: Gas-liquid
chromatography
N=97 patients
207 samples
Cutoff = 35 mg/dL
Normal range: 16-17
mg/dL)

J Infect Dis 1978; 137(4): 384.
CLINICAL SIGNIFICANCE CSF LACTATE
J Infect Dis 1978; 137(4): 384.
CSF Lactate is a meaningful marker for
bacterial meningitis.
CEREBROSPINAL FLUID NON-STANDARD TESTS
Tumor Markers
Alpha-fetoprotein (AFP)
-human chorionic
gonadotropin (-hCG)

Commonly measured in
blood

CNS Germ Cell Tumors
<3% up to 11%
Midline locations
Tumor
Marker
Malignancy

AFP
Germ Cell (GCT)
GI Tract
Hepatocellular (HCC)
Ovarian


-hCG
Gastrointestinal
Germ cell (GCT)
Lung
Melanoma
Ovarian
Trophoblastic
Source: www.arupconsult/TumorMarkers
UpToDatev19.1: Pediatric Intracranial Germ Cell Tumors
CLINICAL SIGNIFICANCE CSF TUMOR MARKERS
Secreting GCTs > aggressive:
CSF AFP > 10g/L
CSF -hCG > 50 IU/L
Proper classification is critical:
NGGCT require more intensive therapy
NGGCT have less favorable prognosis
CLINICAL SIGNIFICANCE CSF TUMOR MARKERS
Overall Survival Event Free Survival
Pediatr Blood Cancer 2008; 51: 768-773.
Hazard ratio of death for patients with elevated
markers was 1.9X as high as that for patients
with normal markers.
CEREBROSPINAL FLUID NON-STANDARD TESTS
Cystatin C
Cysteine protease
inhibitor
Nucleated cells
Marker of GFR
Routine: blood
(2004) J Mol Biol. 341: 151-160.
Disease/Condition Study Report
Traumatic Brain Injury
Hanrieder et al 2009
Gao et al 2007
Guillain-Barre Syndrome
Yang et al 2009
Creutzfeldt-Jakob
Disease
Piubelli et al 2006
Amyotrophic Lateral
Sclerosis
Pasinetti et al 2006
Ranganathan et al 2005


Alzheimers Disease
Vinters et al 1990
Levy et al 2001
Deng et al 2001
Kaeser et al 2007
Mi et al 2007
Sundelof et al 2008
CLINICAL SIGNIFICANCE CSF CYSTATIN C
Nat Genet 2007; 39: 1440. J Immunol Methods 2010; 360: 84-88.
Cystatin C inhibits amyloid-
deposition in AD mouse models
APP+/CysC- APP+/CysC+
Recent paper demonstrating 2 assay
methods to measure Cystatin C in CSF
DIALYSATE & PERITONEAL FLUIDS
DIALYSIS FLUID - FUNCTION
Peritoneal dialysate

Clinical indication: ESRD
estimated GFR < 30
mL/min/1.73 m2

Remove waste normally
excreted by kidneys

Dialysate: 2.0-2.5 L
DIALYSIS FLUID - COMPOSITION
BASE
Electrolytes:
Calcium, Sodium,
Potassium, Magnesium
Buffers:
Lactate, Acetate,
Bicarbonate
Osmotic Agents:
Glucose polymers
(icodextrin*), Glucose
solutions
Amino Acids
Other:
Insulin, Antibiotics,
Heparin
TARGETED WASTE
PRODUCTS REMOVED

Creatinine

Urea

Toxins

Drugs
*NOTE: Icodextrin shown to have
negative interference in amylase
determinations.
DIALYSIS FLUID STANDARD VS. NON-STANDARD
TESTS
STANDARD
Creatinine clearance

NON-STANDARD
Urea

Fractional urea
clearance = Kt/Vurea
24-hour dialysate
24-hour urine
Matched serum


K - dialyzer clearance
of urea
t - dialysis time
V - Vdist of urea
(~total body water)

CAP Survey FLDB2009
CASE KT/VUREA
Equation 2 parts:
Kt (L per week)= 7 x ([Urea]dialysate + [Urea]24Hurine)
[Urea]serum
Watson formula: V(L) = -2.097 + [0.1069 x Height(cm)]
+ [0.2466 x Weight(kg)]
69 year-old woman on PD 3 samples
[Urea]serum = 40 mg/dL 400 mg/L
[Urea]dialysate = 35 mg/dL Tvol = 92dL. 3220 mg
[Urea]24Hurine = 300 mg/dL Tvol = 1dL. 300 mg
Height(cm) = 155 cm
Weight(kg) = 50 kg

CAP Survey FLDB2009
CASE KT/VUREA
Equation 2 parts:
Kt (L per week)= 7 x (3220 mg + 300 mg) / 400 mg/L
= 61.6L per week
V(L) = -2.097 + [0.1069 x 155] + [0.2466 x 50]
= 26.8 L

69 year-old woman on PD Kt/V = 2.3

NKF Clinical Practice Guideline
Minimum weekly Kt/Vurea target of at least 1.7

PD for this patient is ADEQUATE

CAP Survey FLDB2009
DIALYSIS FLUID CLINICAL SIGNIFICANCE
Monitored quarterly

Determine adequacy of dialysis dose

Missed target (<1.7):
More frequent exchanges
Employ a larger volume of dialysate per exchange
Use a higher concentration of glucose or glucose
polymer in the dialysate
PERITONEAL FLUID NON-STANDARD TESTS
Creatinine

Albumin

Amylase and Lipase

Bilirubin


All tests considered to
be Lab Developed Tests
CLINICAL SIGNIFICANCE PERITONEAL FLUID
Creatinine
Cleared by kidney
Concentrated in urine

Indicative of presence of urine
Defect
Trauma
Surgery

Matched serum critical for comparison

CLSI C49-A
CLINICAL SIGNIFICANCE PERITONEAL FLUID
Albumin
High vs. low gradient - calculation
Hydrostatic pressure portal circulation
Serum-Ascites Albumin Gradient

Semin Liver Dis. 1997; 17:191-202.
CLINICAL SIGNIFICANCE PERITONEAL FLUID
Amylase and Lipase

Useful marker
pancreatic fluid
Acute pancreatitis
Lavage post-trauma
Dialysis complications
peritonitis
Amylase: 50 IU/mL
Lipase: 15 IU/mL

High concentration
secretion from
pancreas

Up to X000 U/mL

Matched serum critical
for comparison


CLSI C49-A
CLINICAL SIGNIFICANCE PERITONEAL FLUID
Bilirubin
Bile leakage
(conjugated)

Gall bladder perforation

Levels several fold
higher

Matched serum critical
for comparison
Nephrol Dial Transplant (2006) 21: 11211122
CYST FLUID
CYST FLUID - CASE
44yo F
H&P:
Dull epigastric pain x 1 month.
Possible alcohol abuse and bout of acute pancreatitis 6
months prior
Mild epigastric tenderness but no palpated masses
ABD CT scan
circumscribed 6.8 cm multiloculated, macrocystic mass in the
body of the pancreas.
Radiologic evaluation
No direct communication between the pancreatic cyst and
main pancreatic duct.
No other pancreatic, hepatic or biliary masses are noted.
CAP Survey FLDB2010
CYST FLUID - CASE
Endoscopic Ultrasound
aspirate 3.6 mL of
slightly thick fluid

CEA and Amylase
ordered

Supervisor calls PhD
Clinical Chemist to ask:
What do I do?
Is this useful or is Dr.
X just fishing?
CEA
AMYLASE
CAP Survey FLDB2010
PANCREATIC CYSTS
Malignancy risk:
LOW
Malignancy risk:
HIGH
CLINICAL SIGNIFICANCE - CYST FLUID
Tumor Markers: CEA*, CA-19.9, CA 125
Amylase*
In concert with imaging studies, cytology, and
viscosity:
Distinguish between mucinous and nonmucinous
pancreatic cysts
Determine the likely type of malignant pancreatic cyst

Ann Surg. 1993; 217: 41-47
CYST FLUID - CASE
Which type of
pancreatic cyst is most
likely to yield
significantly elevated
cyst fluid CEA levels?
A: Pseudocyst
B: Serous cyst
C: Mucinous cyst
Which type of
pancreatic cyst is most
likely to yield
significantly elevated
cyst fluid Amylase
activity?
A: Pseudocyst
B: Serous cyst
C: Mucinous cystic
neoplasm


CAP Survey FLDB2010
ANSWER: C
ANSWER: A
NON-STANDARD BODY FLUID GLOSSARY
Serous
Amniotic
Cerebral
Spinal Fluid
Dialysate
Cyst
This is not all encompassing others: IV fluid, saliva, JP
drains, synovial fluids, etc
CHALLENGES AND WHATS AHEAD ...
CHALLENGE REALITY CHECK #3
Review your menu
Understand and
communicate importance
of matched serum sample
Appreciate information or
lack thereof:
Case based reports
Normal reference range
Absence of method or
platform description

Develop validation
strategy to determine:
Accuracy
Reproducibility
Reliability

Collaboration is KEY
Physicians
Laboratory
Vendors/Reagent
Manufacturers

Clinical and Laboratory
Standards Institute.
Analysis of Body Fluids in
Clinical Chemistry;
Approved Guideline. CLSI
document C49-A [ISBN 1-
56238-638-7]. Clinical and
Laboratory Standards
Institute, 940 West Valley
Road, Suite 1400, Wayne,
Pennsylvania 19087-1898
USA, 2007.
Henrys Clinical Diagnosis
and Management by
Laboratory Methods. 21
st

ed. Chapter 28.
AACC for providing a
forum to start the
dialog on NSBF testing
Frank H. Wians, Jr.,
PhD, MT(ASCP),
DABCC, FACB
David Grenache, PhD,
MT(ASCP), DABCC,
FACB
REFERENCES ACKNOWLEDGEMENTS