BODY FLUIDS

Mr. Rajesh Kumar Gupta PG Cl. Biochemistry

class ppt

Body Fluids

Extracellular fluid (ECF) Intracellular fluid (ICF)

• plasma, 1/4th • Interstitial fluid, 3/4th

liquid found between the cells or tissue fluidEg. lymph

• Transcellular fluidA body fluid that is not inside cells but is separated from plasma and interstitial fluid by cellular barriers.• CSF• pleural fluid• synovial fluid• Peritoneal fluid, etc

Cerebrospinal fluid (CSF)

Cerebrospinal Fluid (CSF)

• Produced at the Choroid plexus of the 4 ventricles by modified Ependymal cells • At rate @20 ml / hr (adults)

• CSF flows through the Subarachnoid space• Where a volume of 90 – 150 ml is

maintained (adults)• Neonate volume 10-60 mL

• Reabsorbed at the Arachnoid villus / granulation • to be eventually reabsorbed into the

blood

FUNCTIONS OF CSF

▪As shock absorber▪ As mechanical buffer▪ Act as cushion between the brain and cranium▪ Act as a reservoir and regulates the contents of the cranium▪ Serves as a medium for nutritional exchange in CNS▪ Transport hormones and hormone releasing factors▪ Removes the metabolic waste products through absorption

CSF Evaluation

• Tube 1- for cell count and differential• Tube 2- for glucose, protein & enzymes• Tube 3- for culture, Gram stain, AFB stain,

India ink etc• Tube 4- for cytology

Composition of CSF

Appearance Cells DLC Protein glucose

Clear/colorless

0-5/ul(lymphocytes predominant)

∙Adults: 70% lymps, 30% monos.∙Children / newborns: monocyte

15-45 mg/dl 45-75 mg/dl

Typical Viral Meningitis

• CSF WBC elevated, but < 250 (PMNs in early disease, then lymphocytes)

• CSF protein elevated, but < 150 mg/dl• Glucose > 25 mg/dlof serum concentration

Typical Bacterial Meningitis

• CSF WBC > 1000, PMN predominance• CSF protein > 500mg/dl• CSF glucose < 25 mg/dl

Cerebrospinal fluid (CSF)

Biochemical constituents:– Sp. Gravity – 1.003-1.008– CSF pressure – 60-100 mm H2O– pH – 7.28-7.32

Proteins:– 15-45mg/dl– Newborn (0-1mth) – 60-120mg/dl– Albumin/globulin – 3.1

Cerebrospinal fluid (CSF)

Mechanism of increased CSF protein:– increased permeability of the blood brain barrier d/t damage– Decreased reabsorption at the arachnoid villi– Mechanical obstruction of CSF flow due to spinal block above

the puncture site – Increased in inthrathecal immunoglobulin synthesis

• inflammatory meningitis - ↑ to about 125mg-1gm/dl• Neurosyphilis, encephalitis, abscess, tumor - ↑ to 20-

300mg/dl• Spinal cord tumor – 100 – 2000mg/dl

Cerebrospinal fluid (CSF)

Low CSF protein:• May normally occur in young children between 6 months to

12 years• Patient with increased CSF turnover– removal of large volume of CSF– CSF leak induced by trauma or lumbar puncture– increased intracranial pressure, probably due to an

increased rate of protein reabsorption by the arachnoid vili

Cerebrospinal fluid (CSF)

CSF protein estimation:1) Turbidimetric method:– Uses trichloroacetic acid (TCA) or sulfosalicylic acid (SSA) and

sodium sulphate for protein precipitation– Benzethonium chloride or benzalkonium chloride

2) Colorimetric method:– Uses Lowry method (Folin phenol reagent) or– Dye binding method using Coomassie brilliant blue (CBB) or

Ponceau S and– Biuret method

Cerebrospinal fluid (CSF)

Pyrogallol red technique:• Protein present in CSF will quantitatively bind with pyrogallol

red molybdate reagent dye at pH 2.5 violet colored complex

• intensity of this colored complex is measured at 600 nm in a spectrophotometer

• higher the concentration of protein, more intense or darker will be the color of the CSF solution

Cerebrospinal fluid (CSF)

Serum and CSF Albumin &IgG ratio:• assess permeability of blood brain barrier

CSF/Se. albumin index = CSF albumin (mg/dl) Se. albumin (g/dl)• Normal ratio: 1: 230

Cerebrospinal fluid (CSF)

CSF IgG:• 3-5%• MS - ↑ to about 15-18%• Increased intrathecal IgG assessed by

CSF/Se. IgG ratio = CSF IgG (mg/dl) Se. IgG (g/dl)

• Normal ratio: 1:390

Cerebrospinal fluid (CSF)

Cerebrospinal fluid (CSF)Electrophoresis of CSF:• Using cellulose acetate or agarose• Pre-albumin, albumin, alpha1, alpha2, beta1, beta2 and

gammaglobulins• CSF always contain pre-albumin and plasma does not• Pre-albumin or Transthyretin is thyroxine (T4) and retinol-

binding protein

Cerebrospinal fluid (CSF)

• Pattern is abnormal when IgG synthesis increases

• MS – gammaglobulin fraction↑(ologoclonal bands seen)• Multiple sclerosis(MS): is an inflammatory disease in

which the insulating covers of nerve cells in the brain and spinal cord are damaged

• CSF total protein/Gammaglobulin fraction exceeds 0.12 in about 65% of cases of MS

Cerebrospinal fluid (CSF)

High resolution agarose gel electrophoresis:

• Shows discrete patterns of IgG, oligoclonal bands

• Two or more bands necessary for diagnosis

• A highly sensitive stain like silver stain or Coomassie Brilliant Blue is required to identify the proteins in the gel

Cerebrospinal fluid (CSF)

• detection of oligoclonal bands is performed if there is suspicion of an inflammatory or demyelinating condition

• Concomitant serum sample for elecrophoresis and protein estimation is mandatory

• presence of oligoclonal bands in CSF combined with their absence in blood serum often indicates that immunoglobulins are produced in CNS

• Oligoclonal bands are detected in upto 90% of MS

Cerebrospinal fluid (CSF)

• oligoclonal bands are also seen in:

– Panencephilitis - Various viral CNS infection– Neurosyphilis - Neurobrucellosis– Cryptococcal meningitis - Guillian-Barre syndrome– Transverse myelitis - Meningial carcinomatosis– Burkitt’s lymphoma - Chronic relapsing

polyneuropathy– Cysticercosis - Trypanosomasis

Cerebrospinal fluid (CSF)

Glucose:– 50-85mg/dl (2.8-4.4mmol/L), about 2/3rd of plasma value

– normal CSF/plasma glucose ratio = 0.3-0.9

– Hypoglycorrhachia = < 35mg/dl (characteristic of bacterial, tuberculous and fungal meningitis)

– Some viral meningoencephalitis have low CSF glucose but not to that extent as in bacterial meningoencephalitis

Cerebrospinal fluid (CSF)

Decreased level of CSF glucose:– Meningeal involvement in malignant tumor– Sarcoidosis– Cysticercosis– Trichinosis– Amoeba– Acute syphilic meningitis– Intrathecial administration of radoiodinated serum albumin– Subarachonoid haemorrhage– Symptomatic hypoglycemia– Rheumatoid meningitis

Cerebrospinal fluid (CSF)

• Decreased CSF glucose results from increased anaerobic glycolysis in brain tissue and leucocytes and impaired transport to CSF

• CSF glucose normalize before protein levels and cells count during recovery of meningitis, making it a useful parameter in assessing response to treatment

Cerebrospinal fluid (CSF)

Lactate:• 9.0-26mg/dl (1.0-2.9mmol/L)• Elevated CSF lactate reflects CNS anaerobic metabolism due

to tissue hypoxia• Persistently increased – poor prognosis in patient with head

injury• Usually done to differentiate viral from bacterial,

mycoplasma, fungal and tuberculous meningitis where routine parameters yield equivocal results

• Viral meningitis - always <35mg/dl• Bacterial meningitis - >35mg/dl

Cerebrospinal fluid (CSF)

F2 isoprostanes:• Increased in Alzheimer’s disease

Urea:• Level is slightly lower than in blood• In uremia, urea conc. in CSF rises in parallel with that in

blood

Cerebrospinal fluid (CSF)

Enzymes:

Reference range

ADA ( pleural fluid)

Normal: <40 mg/dlSuspect: 40—50 Strong suspect: >50-60Positive: > 60

ADA(CSF) <10 mg/dlADA(Serum)

<15 mg/dl

ADA∙ADA involved in purine metabolism∙It converts Adenosine to inosine∙found mostly in lymphocytes and macrophages

1. Tuberculosis (cut off value > 60 U/L for pleural fluid)

2. ADA is also increased in various infectious disease like

• infectious mononucleosis• Typhoid• Viral hepatitis• Initial stage of HIV• Incase of malignant tumors • SLE

Cerebrospinal fluid (CSF)

Creatine kinase (CK):• Increased CSF CK activity are seen in numerous CSF disorders:– Hydrocephalus, cerebral infraction, primary brain tumors and

subarachnoid hemorrhage

• In patient with head trauma, CSF CK levels correlate directly with the severity of the Concussion

• CK-BB isoenzyme – better than CK-total

Cerebrospinal fluid (CSF)

• CK-BB isoenzyme increases about 6 hour following an ischemic or anoxic insult

CK-BB: < 5U/L - minimum neurological damage 5-20U/L - mild to moderate neurological

damage 21-50U/L - commonly correlated with death

Cerebrospinal fluid (CSF)

LDH:• < 40 U/L• used as the marker in estimating the potential outcome during the

early stages of ischemic brain injury• Also elevated in bacterial meningitis but not in aseptic or viral

meningitis

Ammonia:• Increased levels are generally proportional to the degree of

existing hepatic encephalopathy• Generally correlates with blood value• Also increases in Reye’s syndrome, inherited hyperammonemias

Cerebrospinal fluid (CSF)

Catecholamines:• Homovanillic acid (HVA), the major catabolite of dopamine

and 5-hydroxyindoleacetic acid (5-HIAA), the major catabolite of serotonin, are normally present in CSF

• The levels of both catabolites are reduced in patients with idiopathic or drug induced parkinsonism

Cerebrospinal fluid (CSF)

Tumor marker• Various tumor markers have been seen increased in CSF

of patients with both primary and metastatic tumors• eg. CEA, HCG, ALP

PLEURAL FLUID

Pleural Fluid

• pleural cavity normally contains small amount of fluid that facilitates movement of two membranes against each other

• Plasma filtrate derived from capillaries of the parietal pleura

• Produced continuously at the rate dependent on capillary hydrostatic pressure, plasma oncotic pressure and capillary permeability

• Reabsorbed – lymphatics and venules of visceral pleura

• Volume – about 10ml each

Pleural Fluid

• Accumulation of fluid – an effusion, results from imbalance between the fluid production and reabsorption

• Fluid accumulation in pleural, pericardial and peritoneal cavities serous effusion

Transudate

• Clear, pale yellow, watery substance• Influenced by systemic factors that alter the

formation or absorption of fluid• Increase in hydrostatic pressure • Decrease in plasma oncotic pressure • Contains few protein cells• Common causes: CHF and liver or kidney disease

Exudate• Pale yellow and cloudy substance• Influenced by local factors where fluid absorption is altered

(inflammation, infection, cancer) • Rich in protein (serum protein greater than 0.5)• Ratio of pleural fluid LDH and serum LDH is >0.6• Pleural fluid LDH is more the two-thirds normal upper limit

for serum• Rich in white blood cells and immune cells• Always has a low pH• Common causes: pneumonia, cancer, and trauma

Pleural Fluid

According to Light’s criteria exudate meets one or more of following criteria:

1. Pleural fluid protein/serum protein > 0.52. Pleural fluid LDH/serum LDH > 0.63. Pleural fluid LDH more than two-thirds normal upper

limit for serum

Pleural Fluid

Biochemical constituents:Protein:– contain < 50% of serum protein level– Estimation helps in differentiating transudate or exudate – Protein electrophoresis shows pattern similar to serum

except for higher proportion of albumin

Glucose:– Similar to serum glucose level– Low pleural fluid glucose – malignancy, TB, rheumatoid

pleuritis, non purulent bacterial infections, lupus pleuritis, etc

Pleural Fluid

Lactate:– Useful adjunct - rapid diagnosis of infectious pleuritis

(>90mg/dl)– Levels are significantly high in bacterial and tuberculous

pleural infections

Enzymes:Adenosine Deaminase:– Normal – about 36U/L– Significantly increased in tuberculous pleuritis

Pleural Fluid

Amylase:– Elevations above the serum level (usually 1.5-2 or more

times greater) – pancreatitis, esophageal rupture or malignant effusion

LDH:– Levels rise in proportion to degree of inflammation– Declining LDH level in course of an effusion – resolving of

inflammatory process

Pleural Fluid

Interferon – gamma (INF-gamma):– Useful diagnostic modality for TB pleural effusion– Levels increases significantly in pleural fluid of patient

with tuberculous pleuritis (>136pg/ml)

Lipids:– Pleural fluid Tg level > 110mg/dl – a chylous effusion– Pleural fluid/Se. cholesterol ≥ 0.32 – exudate

Pleural FluidTuberculostearic acid (TSA):– Structural component of Mycobacteriun tuberculosis, not

present normally in human tissue– Using gas chromatography or mass spectroscopy TSA is

measured in sputum, bronchial aspirates, washings or pleural fluid

Tumor markers:– Not recommended routinely– May be useful in diagnosing cases with negative cytology or

unexplained effusions

PERITONEAL FLUID

Peritoneal fluid

• Ascites – pathologic accumulation of excess fluid in the peritoneal cavity

• Normal volume – 50ml• Produced as an ultrafiltrate of plasma dependent on

vascular permeability, hydrostatic and oncotic pressure

Peritoneal fluid

Biochemical constituents:Protein:• serum-ascites albumin gradient or gap (SAAG) SAAG = albumin conc. of serum - albumin conc. of ascitic fluid

• high gradient: – > 1.1 g/dL – due to portal hypertension

• Important causes of high SAAG (> 1.1 g/dL) include:– high protein : heart failure, Budd Chiari syndrome– low protein : cirrhosis of the liver

Peritoneal fluid

• Low gradient:– < 1.1 g/dL - causes of ascites not associated with

increased portal pressure such as tuberculosis, pancreatitis, nephrotic syndrome and various types of peritoneal cancer

Glucose:– Decreased level in tuberculous ascites (< 50mg/dl)– Ascites glucose estimation are of little value

Peritoneal fluid

Enzymes:Amylase:– Amylase activity in normal peritoneal fluid is similar to

plasma level– Level greater than three times the plasma value is

good evidence of pancreas related ascites– Also increases in gastroduodenal perforation, acute

mesenteric vein thrombosis, intestinal strangulation or necrosis

Peritoneal fluid

ALP:– levels >10U/L – predicts hollow visceral injury

LDH:– increased in malignant effusions

Telomerase:– Increased in malignant ascites

Peritoneal fluid

ADA:– Increases in tuberculous peritonitis

Lactate:– Increased in malignant and tuberculous ascites

Creatinine and urea:– Essential in differentiating peritoneal fluid from urine– Increased peritoneal fluid urea and creatinine along with

increased serum urea but normal serum creatinine urinary bladder rupture

Peritoneal fluid

Bilirubin:– Ascitic fluid bilirubin > 6mg/dl and ascitic fluid/serum

bilirubin > 1.0 choleperitoneum

Tuberculostearic acid: helpful Tumor markers: – little value, however, CEA, PSA, α – fetoprotein found to

be very specific for serous fluid malignancies

PERICARDIAL FLUID

Pericardial fluid

• Normal volume: 10-50ml

• Pericardial effusion – excess accumulation

• Often caused by viral infection, most common by enterovirus

Pericardial fluid

• It may be:– transudative (congestive heart failure, myxoedema, nephrotic

syndrome),– exudative (tuberculosis, spread from empyema)– haemorrhagic (trauma, rupture of aneurysms, malignant

effusion).– malignant (due to fluid accumulation caused by metastasis)

• Light’s criteria - reliable diagnostic tool for identifying pericardial exudates and transudates

• Other indicators suggestive of exudate - Specific gravity >1.015, total protein >3.0 mg/dL, LDH >300 U/dL, glucose fluid-to-serum ratio < 1

Pericardial fluid

Biochemical constituents:• Biochemical parameters for the diagnosis of pericardial

effusions have not been studied to the same extent as in other body fluids

Glucose:– Value < 40mg/dl (2.22mmol/L) – bacterial, tuberculous,

rheumatic or malignant effusion

Pericardial fluid

Enzymes:LDH:– Level > 200U/L suggests pericardial exudate

• Significantly increased pericardial fluid levels of CK-MB, myoglobin and Troponin I in postmortem pericardial fluid – myocardial injury

ADA :– Useful adjunctive test for tuberculous pericardits

Pericardial fluid

Interferon-gamma:– Increased in tuberculous pericarditis– Cutoff value – 200pg/L

PCR:– More specific than ADA in diagnosing tuberculous

pericarditis but– Negative test does not rule out tuberculous pericarditis

since some pericardial fluids from patients with large tuberculous effusions may not contain M. tuberculosis

SYNOVIAL FLUID

SYNOVIAL FLUID

• Ultrafiltrate of blood plasma combined with hyaluronic acid produced in the joints space by the synovial cells lining synovial tendon sheaths, joints, etc

• Composition similar to plasma as small ions and molecules readily pass into the joint space

• Reabsorption – lymphatics

• Acts as a lubricant and adhesive, and provides nutrients for the avascular articular cartilage

SYNOVIAL FLUID

• Examination of synovial fluid is essential to differentiate infectious from non-infectious arthritis

Biochemical parameters:• Adds only supportive information to the routine test

Mucin clot test:• Add acetic acid to SF precipitates hyaluronate into a mucin clot

which may be graded as good, fair or poor• Fair to poor mucin clot reflects dilution and depolymerization of

hyaluronic acid, a non-specific finding of several inflammatory arthrites

SYNOVIAL FLUID

Glucose:• Proper interpretation of SF glucose values requires comparison

with serum levels, ideally preceded by eight hours fast to allow glucose to equilibrate across the synovial membrane

• Normally, Serum – synovial = < 10mg/dl, also in many non-inflammatory conditions

• In septic arthritis, this difference increases from 20-60mg/dl

SYNOVIAL FLUID

Protein:• Mean normal – 1.0-3.0 g/dl• Total protein estimation is not generally useful• With increasing inflammation, larger proteins enter the synovial

space

Enzymes:LDH:• Increased in RA, gout, failed arthroplasties and infectious arthritis

reflecting neutrophilic infiltration

SYNOVIAL FLUID

Acid phosphatase:• Elevated acid phosphatase may have negative prognostic

value in RA but is non-specific

Organic acids:Lactic acid:• increased in septic arthritis• Increased > 30mg/dl septic arthritis due to gram +ve cocci

and gram –ve bacilli

SYNOVIAL FLUID

• Using gas-liquid chromatography, presence of other organic acids not normally present in SF (eg. n-valeric, n-hexanoic and succinic acids) may be helpful in differentiating septic from non-septic arthritis

Uric acid:• Increased SF uric acid level supports a diagnosis of gout

Lipids:• Contains extremely low conc. of lipids than in plasma• Helps when cholesterol crystals of SF resemble MSU or CPPD

Reference intervals for synovial fluid constituents

Constituents Synovial fluid Plasma

Total protein 1 - 3 g/dl 6 – 8 g/dl

• albumin 55 – 70 % 50 – 65 %

• α1- globulin 6 - 8 % 3 – 5 %

• α2- globulin 5 – 7 % 7 – 13 %

• β – globulin 8 - 10 % 8 – 14 %

• γ – globulin 10 - 14 % 12 – 22 %

Hyaluronic acid 0.3 - 0.4 g/dl -

Glucose 70 - 110 mg/dl 70 – 110 mg/dl

Uric acid 2 – 8 mg/dl 2 – 8 mg/dl

Lactate 9 - 29 mg/dl 9 – 29 mg/dl

AMNIOTIC FLUID

AMNIOTIC FLUID

• fluid surrounds, protects, and nourishes a growing fetus during pregnancy

• allows the baby to move relatively freely and helps maintain a stable temperature

• increases in volume as fetus grows

• Highest – 34wks

• At first, it is mainly water with electrolytes, by about 12-14th week - proteins, carbohydrates, lipids and phospholipids and urea(all of which aid in the growth of the fetus)

AMNIOTIC FLUID

• detect and diagnose some birth defects, genetic diseases, and chromosome abnormalities in a fetus, especially if pregnancy screening tests are abnormal

• to evaluate fetal lung maturity

• obtained through a procedure - amniocentesis

AMNIOTIC FLUID

• 15 and 20 weeks - for genetic diseases, chromosome abnormalities and open neural tube defects

• after 32 weeks - to evaluate fetal lung maturity, when there is an increased risk of premature delivery

AMNIOTIC FLUID

• For genetic testing and chromosome analysis, fetal cells in the amniotic fluid are cultured and grown for several days in the laboratory, then are analyzed

• Biochemical tests, such as bilirubin and alpha-fetoprotein, and sometimes genetic tests can be performed directly on the amniotic fluid

AMNIOTIC FLUID

• Chromosome analysis, a cytogenetics test that may also be called karyotyping - detect chromosome abnormalities associated with a variety of disorders. (It evaluates the 22 paired chromosomes and the sex chromosomes (XY) in the nucleus of cells cultured from those collected in the sample of amniotic fluid and can be used to diagnose a variety of chromosomal disorders)(down’s, klinefelter, edward, patau’s, turner)

• Genetic testing, also called molecular testing.( It looks at fetal DNA to identify specific gene mutations and diagnose a variety of inherited diseases)(cystic fibrosis, tay sach’s disease, sickle cell anemia, thalessemia)

AMNIOTIC FLUID

• AFP (alpha-fetoprotein)—increased with neural tube defects

• Acetylcholinesterase—increased with neural tube defects and also other anatomic abnormalities

• Testing to evaluate fetal lung maturity- (tests are based upon the presence of adequate protective liquid substances called surfactants in the lungs, which are necessary for proper lung function)

• Tests for bilirubin may be performed on a regular basis, starting at about 25 weeks of pregnancy, to detect, evaluate and monitor the severity of the hemolytic anemia in the fetus

SEMINAL FLUID

SEMINAL FLUID

• Semi-gelatinous or liquid suspension containing spermatozoa and secretions from male accessory organ

• Net fluid formed by mixing of the testicular fluid, prostatic fluid and secretion from the seminal vesicles

• Composition is most suitable for the maintenance and survival of

spermatozoa

SEMINAL FLUID

• Has same pH as blood plasma

• Conc. of lactate, phosphate and citrate is higher than in blood

• Chloride and cholesterol are lower

• Sugar content is high –fructose

• If fructose is low – infertility (spermatozoa can’t survive)

SEMINAL FLUID

• semen analysis is used to determine whether a man might be infertile

Components Reference rangeVolume (ml) 2.3 – 2.99pH 7.19 – 8.47Osmolality (mosm) 254 – 423Fructose (mg/dl) 136 – 628Glucose (mg/dl) 5 – 295Total protein (mg/dl) 3700 – 7460Albumin (mg/dl) 1100 – 2000Urea (mg/dl) 13 – 98Lactic acid (mg/dl) 22 – 136Citrate (mg/dl) 304 – 751Ca (mg/dl) 16 – 53Cl (mg/dl) 130 – 158K (mg/dl) 50 – 248Mg (mg/dl) 7.89 – 31.8Na (mg/dl) 236 – 512Zn (mg/dl) 6.78 – 69.29

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