Lecture 7: Introduction to Body Fluid and CSF

INTRODUCTION TO BODY FLUID AND CSF 1  BODY FLUIDS ANALYSIS  Analysis of each type of body fluid involves multiple departments of the laboratory:  Hematology:  Examinination of the cells and crystals found.  Clinical chemistry:  To assess significant physiologic...

INTRODUCTION TO BODY FLUID AND CSF 1  BODY FLUIDS ANALYSIS  Analysis of each type of body fluid involves multiple departments of the laboratory:  Hematology:  Examinination of the cells and crystals found.  Clinical chemistry:  To assess significant physiologic changes in the patient.  Microbiology:  Detection of infectious agents in a nearby body cavity or membrane.  Immunology and other miscellaneous tests:  Provide the physician with critical information.  Further consultation with pathology may be required for the2 identification of tumor cells and other abnormal cells.  BODY FLUIDS ANALYSIS SIGNIFICANCE  In general, studies of body fluids are most helpful to assess:  Inflammation  Infection  Malignancy 3  Hemorrhage  BODY FLUID COMPOSITION  Body fluids vary in composition but the important determinants are water and electrolytes.  Water enters the system through:  Consumption of either water or food  Cellular metabolic processes (the metabolic oxidation yield about 300 mL of water per day).  Fluids (water) of the body can be:  Intracellular fluids (55%)  Extracellular fluids (45%) that divided into:  Interstitial fluid 4  Transcellular fluids in various body cavities (Body Fluids)  Plasma  TYPES OF BODY FLUIDS  Body fluids can be divided into categories such as: 1) Cerebrospinal fluid (around the brain and spinal cord) 2) Synovial (around the joints) 3) Peritoneal (around the abdominal and pelvic cavities) 4) Pericardial (around the heart) 5) Pleural (around the lungs) 6) Semen fluid (secreted by the gonads) 7) Amniotic fluids (surrounds the unborn fetus during pregnancy) 8) Other fluids such as (sweat, gastric, nasal, saliva & tears) 5  CEREBROSPINAL ANATOMY  The brain and spinal cord are lined by the meninges which consists of three layers (membranes) that envelope the brain and the spinal cord:  The dura mater:  The outer layer that located next to the skull bone (lines the skull and vertebral canal).  The arachnoid mater:  The middle layer is a filamentous (spiderlike) inner membrane.  The pia mater:  The thin membrane lining the surfaces of the brain and spinal cord (adhere to the brain). 6  The dura mater layer contains sinuses are called the arachnoid villi.  CEREBROSPINAL ANATOMY  The ventricular system of the brain and the central canal of the spinal cord are lined with lined by special epithelial cells is called ependymal cells which involved in the production of cerebrospinal fluid (CSF) 30%.  In the choroid plexuses, the endothelial cells (that line the choroid plexus are called choroidal cells instead of ependymal cells).  Together with the endothelium of capillaries, choroidal cells form the blood–brain barrier (have very tight-fitting junctures termed the blood-brain barrier).  Disruption of the blood-brain barrier by diseases such as meningitis 7 and multiple sclerosis allows leukocytes, proteins, and additional chemicals to enter the CSF.  CEREBROSPINAL ANATOMY  In the choroid plexuses, the endothelial cells have very tight-fitting junctures, this structure is termed the blood-brain barrier.  Any structures lined by epithelial cells is called ependymal cells include the cerebral ventricle.  Disruption of the blood-brain barrier by diseases such as meningitis and multiple sclerosis allows leukocytes, proteins, and additional chemicals to 8 enter the CSF.  BLOOD BRAIN BARRIER  Occurs due to tight fitting endothelial cells that prevent filtration of larger molecules.  Essential to protect the brain.  Blocks chemicals, harmful substances.  Antibodies and medications also blocked.  Restricts entry of large molecules, cells, etc.  Therefore CSF composition is unlike the blood (CSF is not an ultrafiltrate).  About 70% of the CSF is formed by a combined process of active secretion and ultrafiltration from plasma.  The rest 30% is formed by the ependymal lining cells of the ventricles and the cerebral/subarachnoid space.  https://www.youtube.com/watch?v=sqLb-lzbbWA 9  https://www.youtube.com/watch?v=azy41OJ2n5s 10 11 12  FORMATION OF CEREBROSPINAL FLUID  CSF is produced in the choroid plexuses of the two lumbar ventricles and the third and fourth venticles (Choroid plexus of the 4 ventricles by modified Ependymal cells).  About 20 mL/hour of fluid is produced (500 mL/day) in adult.  CSF flows through the Subarachnoid space:  Adult volume 90-150 mL  Neonate volume 10-60 mL  To maintain that normal volume, the CSF is reabsorbed back into the blood capillaries in the arachnoid 13 granulations/villae at a rate equal to its production.  to be eventually reabsorbed into the blood  CEREBROSPINAL FUNCTIONS 1) CSF provides a physiologic system to supply nutrients to the nervous tissue 2) Remove metabolic wastes 3) Produce a mechanical barrier to cushion the brain and spinal cord against trauma. 4) regulate the volume of the intracranial contents (adjust its volume in response to 14 changes in cerebral vessel changes)  INDICATIONS FOR CSF EXAMINATION  Indications for performing a lumbar puncture and CSF examination include:  Suspicions of encephalitis, meningitis, multiple sclerosis, and neurosyphilis.  Evaluate for intracranial or subarachnoid hemorrhage.  Patients with unexplained seizures.  Diagnose malignancies, leukemia 15  Patients who have fever of unknown origin. COMPOSITION OF THE CSF  CSF contains water and water-soluble substances such as (chloride, CO2, creatinine, glucose) and urea diffuse rapidly across the blood–brain barrier. 16  SPECIMEN COLLECTION AND HANDLING  The procedure to remove CSF is called a lumber puncture or spinal tap.  Routinely lumbar puncture done between 3rd& 4th, or 4th& 5th lumbar vertebrae under sterile conditions.  Intracranial pressure measurement taken before fluid is withdrawn and careful technique to prevent the introduction of infection or the damaging of neural tissue. 17 18  SPECIMEN COLLECTION AND HANDLING  The volume of CSF removed is based on the volume available in the patient (adult vs. neonate).  Typically, 10–20 mL of CSF is slowly removed into three or four sterile tubes that are numbered sequentially 1, 2, 3, and 4. 19 20  SPECIMEN COLLECTION AND HANDLING  Specimen collection:  Tube 1 –chemistries and serology  Tube 2 –microbiology cultures  Tube 3 –hematology  Testing considered STAT and the specimen potentially infectious  If immediate processing not possible:  Tube 1 (chem-sero) frozen  Tube 2 (micro) room temp 21  Tube 3 (hemo) refrigerated  CEREBROSPINAL FLUID APPEARANCE  Normal -Crystal clear, colorless  Descriptive Terms –hazy, cloudy, turbid, milky, bloody, xanthochromic  Often are quantitated–slight, moderate, marked, or grossly.  Unclear specimens may contain increased lipids, proteins, cells or bacteria.  Clots indicate traumatic tap  Milky –increased lipids 22  Oily –contaminated with x-ray media 23  CEREBROSPINAL FLUID APPEARANCE  Xanthochromic:  Yellowing discoloration of supernatant (may be pinkish, or orange).  Most commonly due to presence of (old blood).  Other causes include increased bilirubin, carotene, proteins, melanoma. 24 25 26 27  CEREBROSPINAL FLUID APPEARANCE  Clots –indicates increased fibrinogen & usually due to traumatic tap, but may indicate damage to blood-brain barrier. (see below)  Pellicle formation in refrigerated specimen associated with tubercular meningitis. 28  Normal CSF Red CSF from fresh hemorrhage xanthochromic CSF from old hemorrhage 29 CSF from a traumatic tap 30  ROUTINE TESTS OF CSF  The routine tests should include: 1) Cell count, differential count 2) Glucose level 3) Protein level 31  CEREBROSPINAL FLUID (CSF) -PROCEDURES  All specimens should be examined microscopically (hematology)  Stat priority (RBC lyse in 1 hour, WBC in 2 hrs).  Refrigerate if not able to process immediately.  Electronic counters generally unusable. Manual count.  No dilution usually required (use saline if needed)  Use standard Neubauer hemacytometer 32 counting chamber 33 34 35 36  FORMULA FOR CALCULATIONS  Results in number of cells (# cells) / µL  Count and record cells from both sides of the chamber.  Average the two sides  Multiply by dilution factor (if no dilution is made, this number is 1)  Divide by number of squares counted X volume of each square  Large squares, such as # 1-9 in the next slide have volume of 0.1  Small squares – in center # 5 have volume of 0.004 37 ave. # cells counted x dilution # squares counted x volume of each square 38  EXPECTED RESULTS  Normally 0 RBCs/µL regardless of age  WBCs:  Adult – up to 5 mononuclear WBCs/uL  Newborn – up to 30 mononuclear WBCs/uL  Children (1-4) - up to 20 mononuclear /uL  Children (5+) – up to 10 mononuclear / uL  Increased numbers = Pleocytosis  Pleocytosis: is an increased amount of white 39 blood cell (WBC) in a body fluid.  WBC COUNTS  3% acetic acid can be used to lyse RBC  Methylene blue staining will improve visibility  WBCs are counted in the four corner squares, and the center square on both sides of the hemocytometer and the number is multiplied by the dilution factor to obtain the 40 number of WBCs per microliter  DIFFERENTIAL COUNT  Usually performed on cytocentrifuged preparations  Stained with Wright stain  Few cells normally present in CSF  Lymphocytes and monocytes are predominant  Neutrophils are not a common finding in CSF  In adults, normal proportions of cells in CSF:  28–96% lymphocytes  16–56% monocytes  0–7% neutrophils  Eosinophils, ependymal cells, and histocytes are only rarely seen  Adults usually have a predominance of lymphocytes to monocytes (70:30), whereas the ratio is essentially reversed41in children  DIFFERENTIAL COUNT  Most laboratories that do not have a cytocentrifuge:  Concentrate specimens with routine centrifugation:  Spin for 5 to 10 minutes  Supernatant fluid is removed and saved for additional tests  Slides made from the suspended sediment  Allowed to air dry  Stained with Wright’s stain. 42  CSF SLIDE DIFFERENTIAL BY CYTOCENTRIFUGATION  Wrights stained smear of concentrated sediment.  Cytocentrifuge: places cells on the microscopic slide. Increases number of cells to evaluate, however, risk of cell distortion from the centrifugation process.  Use of albumin reduces cell distortion 43 44 45  CEREBROSPINAL FLUID (CSF)  Entire smear should be evaluated for:  abnormal cells, inclusions within cells, Clusters, Presence of intracellular organisms  Normal differential values:  Adults: 70% lymps, 30% monos.  Children / newborns: monocyte  Types of cells:  Neutrophils – occasionally (with normal count)  Macrophages – increase following CVA 46  Ependymal cells, and normal lining cells can also be seen. 47  OTHER CELLS  Eosinophils:  Often associated with parasitic / fungal infections.  Ependymal cells:  Normal cell, unique to CSF (line the ventricles, produce CSF fluid).  Suspicious / unclassified or malignant cells are reported as “other” or “unclassified”:  Sent to pathology (always consult with hematology 48 specialist / pathologist).  CHEMISTRY TESTS: CEREBROSPINAL PROTEIN  Normal CSF contains a very small amount of protein (N.R 15 –45 mg/dL)  CSF contains protein fractions similar to those found in serum:  Albumin makes up the majority of CSF protein followed by prealbumin.  The alpha globulins:  Primarily haptoglobin and ceruloplasmin.  Transferrin is the major beta globulin present  Gamma globulin is primarily IgG, with only a small amount of 49 IgA.  IgM, fibrinogen, and beta lipoprotein are not found in normal CSF CLINICAL SIGNIFICANCE OF ELEVATED PROTEIN VALUES  Decreased levels not significant  The causes of increased CSF protein include:  damage to the blood-brain barrier  as in meningitis or hemorrhage  Production of immunoglobulins within the CNS  Multiple sclerosis  Degeneration of neural tissue 50  METHODOLOGY  The two most routinely used techniques for measuring total CSF protein use the principles of: 1) Turbidity production methods:  Adapted to automated instrumentation in the form of nephelometry  Dyebinding ability methods –preferred:  Alkaline biuret  Coomassie brilliant blue -a blue color produced is proportional to the amount of protein present (Beers Law)  CSF Protein electrophoresis  Looking for oligoclonal bands (MS)  Myelin Basic Protein:  Abnormal protein that indicates demyelination of neuron axons 51  Measurement used to monitor course of disease (MS) and effectiveness of treatment  CEREBROSPINAL FLUID GLUCOSE  Selective transport across the blood brain barrier  Results in a normal value that is 60% to 70% of the plasma glucose.  Blood glucose test must be run for comparison (the blood glucose should be drawn about 2 hours prior to the spinal tap)  STAT procedure, glycolysis reduces level quickly.  Decreased levels seen in:  Bacterial & fungal meningitis  Hypoglycemia  Brain tumors  Leukemias  Other disorders producing damage to the CNS 52  CSF LACTATE  Normal values = 11-22 mg/dL  Valuable aid in the diagnosis and management of meningitis cases  Levels greater than 35 mg/dL (bacterial meningitis)  Viral meningitis (lower than 25 mg/dL)  Increase as result of hypoxia  Used to monitor severe head injuries  Falsely elevated results may be obtained on 53 xanthochromic or hemolyzed fluid (RBCs)  CSF GLUTAMINE:  Normal range is 8-18 mg/dL  Increased levels associated with increases in ammonia (toxin)  CSF Enzymes:  Lactate dehydrogenase(LDH or LD):  5 isoenzyme types; LD1&LD2 are in brain tissue  Creatinekinase(CPK or CK): 54  Isoenzyme CK3/ CK-BB from brain tissue 55 56  MICROBIOLOGY TESTS  Gram stain: Extremely important for early diagnosis of bacterial meningitis  Even when well performed, 10% false negatives occur  Use of Cytospin to concentrate specimen increases sensitivity  Cultures: Aerobic & Anaerobic. Culture blood at same time  Organisms:  Newborns:  E. coli & group B Strep.  Children:  Streptococcus pneumonia  Hemophilus influenzae 57  Neisseria meningitidis  MICROBIOLOGY TESTS  Organisms in adults:  Neisseria meningitidis  Streptococcus pneumoniae  Staph aureus (if a shunt is present)  Immunocompromised:  Cryptococcus neoformans,  Candida albicans, Coccidioides, or  any opportunistic organism  India-ink / nigrosine preparation: 58  Negative stain to view the encapsulated Cryptococcus neoformans (often AIDs /immunocompromised)  SEROLOGIC TESTING  VDRL (Veneral Disease Research Laboratory)  For detection of neurosyphilis  On CSF test low sensitivity, but great specificity  Fluorescent treponemal antibody absorption (FTA-ABS) test:  More sensitive than VDRL 59 SUB ARA HEM 60 61