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CSF and Other Body Fluids

specimens are maintained in the ff manner: Provides a physiologic system to supply Hematology tubes are nutrients to the nervous tissue refrigerated. Remove metabolic wastes Microbiology tubes remain at room Produce mechanical barrier to cushion temp. the brain and spinal cord against trauma Chemistry and serology tubes are Formation and Physiology frozen. Lined the meninges which consist of: Appearance Dura mater Normal CSF is crystal clear. Arachnoid The major terminology used to describe CSF appearance includes crystal clear, Pia mater cloudy or turbid, milky, xantochromic Produced in choroid plexuses of the two and hemolyzed/bloody. lumbar ventricles and the third and fourth Cloudy, turbid or milky specimen - can be ventricles the result of an increased protein or lipid The fluid flows to subarachnoid space concentration. It might also be indicative of located between arachnoid and pia mater infection with the cloudiness being cause by Blood-brain barrier- tight fitting structure of the presence of WBCs. the endothelial cells in the choroid plexuses. It Xanthochromia is a term used to described prevent the passage of many molecules into CSF supernatant that is pink orange, or the brain tissue. yellow. Specimen Collection and Handling This may be caused by a CSF is collected by lumbar puncture variety of factors but the most between the third, fourth, or fifth lumbar common is the presence of RBC vertebrae. degradation products. The volume of CSF that can be removed Depending on the amount is based on the volume available in the of blood and length of time it has patient (adult vs neonate) and the been present, the color wil vary opening pressure of the CSF taken when from pink (slight amount of the needle first enters the subarachnoid oxyhemoglobin) to orange (heavy space. hemolysis) to yellow (conversion Specimens are collected in 3 sterile of oxyhemoglobin to unconjugated tubes labeled 1, 2, 3 in the order in bilirubin). which they are withdrawn. Other causes of Tube 1 is used for chemical and xanthochromia include elevated serologic tests because these serum bilirubin, carotene, tests are least affected by blood or melanoma pigment and markedly bacteria as a result of the tap increased protein concentrations. procedure. Xanthochromia that is Tube 2 is for microbiology. caused by bilirubin due to Tube 3 is for cell count because it immature liver function is seen in is the least likely to contain cells infants, particularly premature introduced by the spinal tap infants. procedure. Traumatic Tap A fourth tube may be drawn for Three visual examinations are done to microbiology laboratory to provide determine whether grossly blood CSF is better exclusion of skin contamination the result of a hemorrhage or traumatic and other additional serologic tests tap. Ideally tests done on CSF are performed 1. Uneven Distribution of Blood in STAT basis but if not possible Blood from cerebral hemorrhage will be evenly distributed 1

CSF (Cerebrospinal Fluid)

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throughout the three CSF tubes while a traumatic tap will have the heaviest concentration of blood in tube 1, with gradually diminishing amounts in tubes 2 and 3. Streaks of blood may also be seen in specimens acquired through a traumatic tap. 2. Clot formation Fluid collected from a traumatic tap may form clots owing to the introduction of plasma fibrinogen into the specimen. Bloody CSF caused by intracranial hemorrhage does not contain enough fibrinogen to clot 3. Xanthochromic supernatant The result of blood that has been present longer than that introduced by the traumatic tap. To examine a bloody fluid for presence of xanthochromia the fluid should be centrifuged and the supernatant examined against a white background. Additional testing for differentiation includes microscopic examination and Ddimer test. The microscopic finding of macrophages containing ingested RBCs or hemosiderin granules is indicative of intracranial hemorrhage. Cell Count WBC count is the routinely performed cell count on CSF specimens RBC count is only done when traumatic tap has occurred and correction for WBCs or protein is desired Cell counts should be performed immediately because WBCs (particularly granulocytes) and RBCs begin to lyse within 1 hr, with 40% of the WBCs disintegrating after 2 hrs. Normal adult CSF contains: 0-5 WBCs/ul, higher in children and as many as 30 mononuclear cells/ul can be considered normal in newborns An improved Neubauer counting chamber is used for performing Cell Counts CSF cellular Constituents The cells found in normal CSF are primarily lymphocytes and monocytes. Adults usually have a predominance of monocytes (70:30) wheras the ratio is reversed in children. The presence of increased number of normal cells/pleocytosis, is considered abnormal, as is the finding of immature leukocytes, eosinophils, plasma cells, macrophages, increased tissue cells and malignant cells When pleocytosis involving neutrophils, lymphocytes or monocytes is present, CSF differential count is mostly associated with providing the type of cell that is causing an infection of meninges. A high CSF WBC count in which majority of the cells are neutrophils is indicative of bacterial meningitis. A moderately elevated CSF WBC count with high percentage of lymphocytes and monocytes suggests meningitis of viral, tubercular, fungal or parasitic origin. Chemistry Tests Protein Determination The most frequently performed chemical test on CSF Albumin makes up the majority of CSF protein Prealbumin-second most prevalent fraction in CSF Alpha globulins include primarily haptoglobin and ceruloplasmin Transferrin-major beta globulin present Tau- separate carbohydrate deficient fraction, seen in CSF but not in serum CSF gamma globulin: Ig G with only small amount of Ig A Not found in normal CSF: Ig M, fibrinogen, beta lipoprotein Clinical Significance of Elevated Protein Values Abnormally low values: present when fluid is leaking from the CNS Causes of elevation: damage to bloodbrain barrier, productions of immunoglobulins within the CNS, decreased clearance of normal protein from the fluid and degeneration of neural tissue Most common cause of elevation: meningitis and hemorrhage Methodology 2

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The most routinely used techniques for measuring total CSF protein use the principles of turbidity production or dye binding ability Nephelometry-adaptation of turbidity method in automated instrumentation Microbiology Tests The role of the microbiology laboratory in the analysis of CSF lies in the identification of the causative agent in meningitis. For positive identification, the microorganism must be recovered from the fluid by growing it on the appropriate culture medium. This can take anywhere from 24 hours in cases of bacterial meningitis to 6 weeks for tubercular meningitis. CSF culture is a confirmatory rather than a diagnostic procedure. Normal synovial fluid does not clot, however diseased joint may contain fibrinogen and will clot Arthritis- Damage to articular membranes produces pain and stiffness in the joint. Collection and Handling Synovial fluid is collected by needle aspiration called ARTHROCENTESIS Often collected in a syringe that have been moistened with heparin It should be distributed to following tubes based on the tests required: Sterile heparinized tube- gram stain and culture Heparin or EDTA tube- cell count Sodium fluoride tube- glucose analysis Nonanticoagulated tube for other tests Normal Synovial fluid Values Volume = less than3.5 ml SYNOVIAL FLUID Color = colorless to pale Referred to as joint fluid Clarity = clear Synovial fluid acts as a lubricant, Viscosity = able to string 4-6 cm long reducing the friction between articular Leukocyte count = <200cells/ul cartilage and other tissues in joints, and Neutrophils = <25% of the differential also as a shock absorber during Crystals = none present movement. Glucose: plasma difference = <10mg/dl It is secreted by synovial membranes. lower than the blood glucose The synovial membranes contain Total protein = <3g/dl specialized cells called synoviocytes. Crystal Identification It is formed as an ultrafiltrate of plasma Primary crystals seen in synovial fluid are: across the synovial membrane. Monosodium urate (uric acid) The fluid contains hyaluronic acid (MSU) found iin cases of gout secreted by fibroblast-like cells in the Calcium pyrophosphate (CPPD) synovial membrane and interstitial fluid seen in psuedogout. filtered from the blood plasma. Additional crystals may be present The large hyaluronate molecules include: contribute to the viscosity of the synovial Hydroxyapatite (basic calcium fluid. phosphate) associated with calcified Normal viscous fluid resemples egg cartilage degeneration. Osteoarthritis white. Cholesterol crystals- associated Normal amount of fluid in the adult knee with chronic inflammation cavity is less than 3.5 mL but increase to Corticosteroid- injections greater than 25ml of synovial fluid Calcium oxalate crystal in renal indicates an inflammation. dialysis patients Slide preparation 3

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May be examined as an unstained wet preparation Can be initially examined under low and high power using a regular light microscope MSU crystals routinely seen as needle-shaped crystals. Frequently seen sticking through the cytoplasm of the cell CPPD crystals appear rhombicshaped but may appear as short rods. Usually ;located within the vacuoles of the neutrophil

PLEURAL FLUID

Obtained from pleural cavity, located between the parietal pleural membrane lining the chest wall and the visceral pleural membrane covering the lungs. PF to serum chole ratio >0.3= exudate PF chole > 60mg/dl= exudate PF to serum TB ratio 0.6<= exudate Appearance Normal and trans= clear and pale yellow Turbid= presence of WBC or RBC Milky PF= chylous material Hematology tests PF neutrophils= bact inf SEROUS FLUID PF lymphocytes= TB, viral inf, etc. Pleural, pericardial and peritoneal Eosinophils= if greater than 10%, cavities are lined by 2 serous pleural cavity trauma membranes. One lines the cavity wall Chemistry tests (parietal membrane) and the other Most common chemical tests performed covers the organs within the cavity in pleural fluid are glucose, pH, (visceral membrane). The fluid in adenosine deaminase (ADA) and between membranes is called serous amylase fluid. glucose levels- Tuberculosis, It provides lubrication between parietal rheumatoid inflammation and purulent and visceral membranes infections. Formation lactate levels- bacterial infection Are formed as ultrafiltrates of the plasma amylase levels- pancreatitis Hydrostatic and colloidal pressures Micobiologic and Serologic tests Effusion- disruption of the mechanisms G/S, C/S, AFB of serous fluid formation and ANA and Rf reabsorption causes increase in fluid between the membranes Pericardial fluid Specimen collection and handling 10-50ml of fluid is found Needle aspiration Pericardial effusions are primary the Greater than 100 ml are collected result of changes in permeability of EDTA- for cell and differential counts membranes due to infection Green top- used for microbiology and (pericarditis), malignancy and trauma cytology producing exudates Maintained anearobically in ice Appearance The aspiration procedures are referred to as: Normal and transudate: clear and pale Thoracentisis- pleural yellow Pericardiocentesis- pericardial Turbid: infection and malignancy Paracentesis- peritoneal Blood streaked: malignant Transudates and Exudates Milky: chylons and pseudochylons Transudates- formed because of a Laboratory tests systemic disorder Neutrophil count- bacterial Exudates- formed by conditions directly endocarditis involved the membrane of a cavity 4

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Fluid to serum protein LDH Bacterial culture G/S and the absorption from fetal swallowing and intramembranous flow Increases throughout pregnancy, reaching a peak of approximately 1L during the third trimester and gradually decreases prior to delivery During each episode of fetal breathing movement, secreted lung liquid enters the amniotic fluid After the first trimester, fetal urine is a major contributor liquid enters the amniotic fluid After the first trimester, fetal urine is a major contributor to the amniotic fluid volume Failure of the fetus to begin swallowing the urine results in excessive accumulation of amniotic fluid (polyhydramnios) and is an indication of fetal distress often associated with neural tube disorders and may be secondarily associated with fetal structural anomalies, cardiac arrhythmias, congenital infections or chromosomal abnormalities Decreased amniotic fluid or oligohydramnios may be due to urinary tract deformities and membrane leakage. It may be associated with umbilical cord compression resulting in decelerated heart rate and fetal death Chemical Composition Placenta is the ultimate source of amniotic fluid water and solutes The composition of the amniotic fluid is similar to that of the maternal plasma Contains small amount of sloughed fetal cells from the ski, digestive system and urinary tract thus 5

PERITONEAL FLUID
Ascites- accumulation between peritoneal membranes Peritoneal lavage- sensitive test for detection of intra-abdominal bleeding in blunt trauma cases Appearance Nomal: clear and pale yellow Turbid: bact inf Blood streaked: intestinal disorders or malignancy Milky: blockage or lymphatic vessels Laboratory tests Normal WBC count: <500 cells/uL glucose level: tubercular peritonitis amylase level: gastrointestinal perforations ALP: intestinal perforation

AMNIOTIC FLUID
Testing for amniotic fluid is frequently associated with cytogenetic analysis Product of fetal metabolism. Constituents that are present in the fluid provides information about the metabolic process taking place during-as well as the progress of fetal maturation Primary functions are to provide cushion for the fetus, allow fetal movement, stabilize the temperature to protect the fetus from extreme temperature changes, and to permit proper lung development Where exchange of water and chemicals from the maternal circulation to the fetus takes place Regulated by a balance between the production of fetal urine and lung fluid

Function

Volume

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o Mothers age of 35 or more at delivery o Family history of chromosome abnormalities such as trisomy 21 (down syndrome) o Parents carry an abnormal chromosome rearrangement o Earlier pregnancy with a child with a birth defect\parent is a carrier of a metabolic disorder o History of genetic diseases o Elevated maternal serum alpha fetoprotein o Abnormal triple marker screening test o Previous child has a neural tube disorder o 3 or more miscarriages Evaluation of amniocentesis is indicated later in pregnancy (20-42 weeks) to evaluate: Differentiating Maternal Urine From o Fetal lung maturity Amniotic Fluid o HDN caused by Rh blood type Needed to determine incompatibility possible premature o Infection membrane rupture or Collection accidental puncture of the Via needle aspiration in the maternal bladder during amniotic sac specimen collection (amneocentesis) Chemical analysis of Usual procedure done is a creatinine, urea, glucose transabdominal and protein aids in the amniocentesis differentiation Uses the aid of an Specimen Collection ultrasound for guidance Amniocentesis- Recommended when screening Vaginal amniocentesis has blood tests such as maternal serum alpha greater risks of contracting protein test, triple screening test (AFP, hCG and an infection unconjugated estriol UE3) or quadruple test Performed during the 14th (AFP,hCG, UE3 and inhibin A) yield results that week of gestation are abnormal Fluid for chromosome Indications for Performing Amniocentesis analysis is usually collected May be indicated at 15-18 at approximately 16 weeks weeks of gestation for the gestation ff. conditions to determine Tests for fetal distress and early treatment: maturity are performed later in the third trimester Maternal Urine Amniotic Fluid Maximum of 30mL of Creatinin Higher (10 Lower (does not exceed e level mg/dl) 3.5 mg/dl) amniotic fluid is collected Urea 300 mg/dl Does not exceed 30 mg/dl Fluid for bilirubin and HDN analysis must be protected Glucose More likely to contain from light and glucose and CHON CHON Specimen Handling and Processing
Fern Test Presence of fern crystals

providing basis for cytogenetic analysis Contains biochemical substances that are produced by the fetus such as bilirubin, lipids, enzymes, electrolytes, nitrogenous compounds, and proteins that can be determined to assess the health of the fetus Concentration of creatinine, urea, and uric acid increases, whereas glucose and protein concentrations decreases when fetal urine production begins Fetal age could be determined through measurement of the amniotic fluid cratinine

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-Zone 1: mildly affected fetus FLM (fetal lung maturity) -Zone 2: careful monitoring should transported with ice and be done refrigerated up to 72 hours -Zone 3: severely affected fetus prior to testing or kept frozen. Neural Tube Defects -Filtration must be employed *Alpha-fetoprotein - major protein produced by the fetal in separating the fluid from cellular liver during early gestation elements in order to prevent the loss of - increased in concentration in cases of phospholipids anencephaly and spina bifida Specimens for cytogenic - after 12-15 weeks of gestation, AFP studies- maintained at room declines temp. or body temp(37C) -serum and amniotic AFP levels are Color and Appearance reported in terms of multiples of the Normal median(MoM) -colorless - the median is the laboratorys -slightly to moderately turbid reference level for a given week of gestation. A value 2x the median is Blood-streaked considered abnormal. -can be a result of traumatic *Amniotic Acetylcholinesterase tap,abdominal trauma or intra-amniotic - test more specific for neural tube hemorrhage defects -Kleihauer-Betke test: detects fetal - the specimen must not be hemoglobin contaminated with blood Yellow Tests for Fetal Maturity -due to the presence of bilirubin Fetal lung maturity -indicative of RBC destruction resulting *Respiratory Distress syndrome(RDS) fr. HDN - most frequent complication of early Dark green delivery -due to meconium, known as the -lack of lung surfactant newborns first bowel movement -surfactant: allows the alveoli to remain -due to fetal intestinal secretions open throughout the normal cycle of Very dark red-brown inhalation and exhalation. -may be associated with fetal death Lecithin-Sphingomyelin Tests for Fetal Distress Ratio Hemolytic Disease of the -reference method to which tests of FLM Newborn are compared -unconjugated bilirubin is present in the *Lecithin amniotic fluid due to destruction of fetal - primary component of surfactants red blood cells -produced at low and constant rate until * Spectrophotometric Analysis the 35th week of gestation - measures the amniotic fluid bilirubin *Sphingomyelin -Optical density in normal fluid is highest - produced at a constant rate after at 365 nm and decreases linearly to 550 about 26 weeks of gestation nm; when bilirubin is present a rise in OD - serve as a control on which to is seen at 450 nm because this is the base the rise in lecithin. wavelength of maximum bilirubin * Prior to 35th week of gestation, absorption. The difference between the OD of the theoretic baseline and the OD the L/S rati is < 1.6 because large at 450nm represents the amniotic amounts of lecithin are not being bilirubin concentration, This difference in produced at this time. It will rise to 2.0 or OD is referred to as the absorbance higher when lecithin production difference at 450nm is then plotted on a increases to prevent alveolar collapse. Liley graph to determine the severity of hemolytic disease. 7

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Therefore, when L:S reaches 2.0, a preterm delivery is considered. Amniostat-FLM it correlates with the amount of phospholipid present in the fetal lungs. -Lamellar body diameter: can be counted with the use of platelet channel of hematology analyzers. -can be counted using resistance-pulse counting.

*Phosphatidyl glycerol - a surfactant that is essential for lung maturity -parallels the production of lecithin but is delayed when there is maternal SWEAT FLUID diabetes Sweat formation * Amniostat FLM - uses an antisera specific for It is produced continuously by the sweat phospatidyl glycerol glands -not affected by specimen Types of Sweat formation: contamination with blood and meconium. 1. Insensible (no visible) perspiration Foam stability The amount is 300-700 mL/day -shake test 2. Sensible perspiration -bedside procedure - amniotic fluid is mixed with 95% The amount is 10-14 L/day. ethanol(antifoaming agent), shaken for This can create a severe problem in the 15 seconds and allowed to sit maintenance of water and electrolytes undisturbed for 15 minutes Sweat function -the presence of continous line of It plays an important role in body bubbles around the edge indicates that temperature regulation by cooling and there is sufficient amount of evaporation phospholipid - Values > 47 indicate fetal lung maturity Sweat Composition: Microviscosity: fluorescence 1. The major cations (Na+, K, Mg2+, etc) Polarization Assay and anions -presence of phospolipid decreases (Cl-, HCO3-, etc) are similar to that microviscosity which can be measured found in plasma using the principle of fluorescence 2. pH of sweat varies considerably usually polarization assay bet. 5.2-7.3 - This assay measures the polarization of a fluorescent dye that combines with 3. Glucose is present in very small amount both albumin and surfactants. Dye 4. Urea is 4-6 times that of plasma bound to surfactant has a longer 5. Proteins and lipids- only traces are fluorescence time and exhibits low present polarization 6. There are significant amounts of amino -albumin is used as an internal standard because it remains constant throughout acids 7. Lactic acid concentration is higher than gestation. - A value of 55mg surfactant per gram that in blood or urine albumin or greater provides a Sweat disorders: conservative indicator of FLM 1. Miners (Stokers) Craps Lamellar bodies and Optical The excessive loss of NaCl in perspiration density under extremely hot or humid conditions -represents a storage form of may result in miners craps surfactant - Type 2 pneumocytes: produce and secrete surfactants responsible for FLM -as the fetal lung matures, lamellar bodies increased in number,thus Treatment Small amount of salt (NaCl) should be added to the drinking water 8

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2. Pancreatic Cystic Fibrosis It is an autosomal recessive disease and generalized malabsorption due to lack of delivery of pancreatic digestive enzymes to small intestine. It is the most common, fatal, inherited disease of white men. This condition can lead to steatorrhea (presence of fat in stool) and also to fat-soluble vitamin deficiencies. Abnormalities affected are airways, lungs, pancreas,liver, intestine and sweat glands. There is an abnormal electrolytes transport, especially Cl- secretion from the epithelial cells of airways, pancreas, intestine and sweat glands. Defective Cl- secretion causes hyperactivity of Na+ absorption which causes the secreted mucus to become viscous and sticky. The causes of elevated of Cl- level in the sweat is due to failure of reabsorption in the sweat glands Determination of the sweat Cl- level is used as standard diagnostic test for cystic fibrosis