E-Klinická biochemie Biochemical and morphological investigation of cerebrospinal fluid •MUDr. Zdeňka Čermáková E-Klinická biochemie Anatomy ØClear colorless liquid ØFormed and secreted by the choroid plexus, a special tissue that has many blood vessels and that lines in • the ventricles in the brain. P:\Technical Directors shared documents\Wikilite\Main page design\TBS logo black JPG.jpg Where is CSF? •Between arachnoid and pia mater meninges • • “Intrathecal” refers to under the arachnoid membrane i.e. in the CSF Meninges •On this diagram you can see the brain (white) covered by 3 protective membranes (meninges) the cranium (NB the skull is composed of the cranium and the mandible), the periosteum (a fibrous membrane that covers the outer surface of all bones, except at the joints of long bones) and lastly the skin. •CSF is located between arachnoid and pia mater meninges •The term “intrathecal” refers to the area under the archnoid membrane – ie in the CSF E-Klinická biochemie Fyziology •Formatio of cerebrospinal fluid: oActive secretion of chorioid plexus (50-70%) oUltrafiltration of blood plasma oTransfer from brain cells •Resorption: oLarge intracranial venous sinuses through the arachnoid villi and granulations Pacchions o Veins and dural sinuses allow passage of cerebrospinal fluid directly into the venous blood. o oTotal amount of CSF is an adult 120-180 mL oDaily production 500-600 mL Ø P:\Technical Directors shared documents\Wikilite\Main page design\TBS logo black JPG.jpg TIGHT JUNCTIONS “Blood-CSF barrier” Barriers (BBB) 280px-Hematolikvorov%C3%A1_bari%C3%A9ra Two largest interfaces between blood and brain extracellular fluids the epithelial layer has tight gap junctions between the cells on the side facing the ventricle (apical surface). These gap junctions prevent the majority of substances from crossing the cell layer into the CSF; thus the CP acts as a blood–CSF barrier. There are four choroid plexuses in the brain, one in each of the ventricles. CSF is formed as plasma is filtered from the blood through the epithelial cells. CP epithelial cells actively transport sodium, chloride and bicarbonate ions into the ventricles and water follows the resulting osmotic gradient In addition to CSF production, the CP act as a filtration system, removing metabolic waste, foreign substances, and excess neurotransmitters from the CSF. In this way the CP has a very important role in helping to maintain the delicate extracellular environment required by the brain to function optimally. CHoroid plexus- a rich network of blood vessels (derived from those of the pia mater, in each of the brains ventricles. It is responsible for the production of CSF While the largest interface between blood and brain is the BBB, this is also smaller less direct interface between blood and cerebrospinal fluid (CSF). Goldmann first demonstrated the existence of the blood-CSF barrier in 1913. Through the use of dyes with different properties it was found that the blood-CSF barrier was selectively permeable, rather than absolute (Bradbury, 1979).. Filtration is selective with low molecular mass proteins passing more readiy into CSF than larger ones. There is also much active transport of substances into, and out of, of the CSF as it is made. The choroid plexus and the arachnoid membrane act together at the barriers between the blood and CSF. On the external surface of the brain the ependymal cells fold over onto themselves to form a double layered structure, which lies between the dura and pia, this is called the arachnoid membrane. Within the double layer is the subarachnoid space, which participates in CSF drainage. Passage of substances from the blood through the arachnoid membrane is prevented by tight junctions (Nabeshima et al., 1975). The arachnoid membrane is generally impermeable to hydrophilic substances, and its role is forming the Blood-CSF barrier is largely passive. The choroid plexus forms the CSF and actively regulates the concentration of molecules in the CSF. The choroid plexus consist of highly vascularized, "cauliflower-like" masses of pia mater tissue that dip into pockets formed by ependymal cells. The preponderance of choroid plexus is distributed throughout the fourth ventricle near the base of the brain and in the lateral ventricles inside the right and left cerebral hemispheres. The cells of the choroidal epithelium are modified and have epithelial characteristics. These ependymal cells have microvilli on the CSF side, basolateral interdigitations, and abundant mitochondria (Segal, 1999). The ependymal cells, which line the ventricles, form a continuous sheet around the choroid plexus. While the capillaries of the choroid plexus are fenestrated, non-continuous and have gaps between the capillary endothelial cells allowing the free-movement of small molecules, the adjacent choroidal epithelial cells form tight junctions preventing most macromolecules from effectively passing into the CSF from the blood (Brightman, 1968). However, these epithelial-like cells have shown a low resistance as compared the cerebral endothelial cells, approximately 200 W ·cm2, between blood and CSF (Saito and Wright, 1983); Figure NB: The blood-brain barrier (BBB) is the specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. Unlike peripheral capillaries that allow relatively free exchange of substance across / between cells, the BBB strictly limits transport into the brain through both physical (tight junctions) and metabolic (enzymes) barriers. Thus the BBB is often the rate-limiting factor in determining permeation of therapeutic drugs into the brain. Additionally, BBB breakdown is theorized to be a key component in central nervous system (CNS) associated pathologies. BBB investigation is an ever growing and dynamic field studied by pharmacologists, neuroscientists, pathologists, physiologists, and clinical practitioners. P:\Technical Directors shared documents\Wikilite\Main page design\TBS logo black JPG.jpg Blood-brain barrier ENDOTHELIAL CELL ASTROCYTE FOOT TIGHT JUNCTIONS •Endothelial cells 280px-Hematoencefalick%C3%A1_bari%C3%A9ra E-Klinická biochemie Blood x CSF Blood CSF Na+ (mol/l) 140 140 Cl- (mmol/l) 100 125 Total protein 60-80 0,15 – 0,40 IgG 6-16 0,025 Glucose (mmol/l) 5,6 E-Klinická biochemie Function •Mechanic protection (shocks , changes in temperature and pressure) •Homeostasis - optimal environment for cells of CNS (constant ion composition , pH , osmolality ). •Ensures evacuation products of catabolism , such as lactate and CO2,variety of bioactive substances. •Protection against pathogenic microorganisms E-Klinická biochemie When is it ordered? Ø ØDiseases of CNS Ø oInfection inflammation Ø oAutoimmune diseases that affect the central nervous system(Guillain-Barré syndrome and multiple sclerosis) Ø oOncologic diseases • oIntracerebral bleeding E-Klinická biochemie Lumbar puncture • Soubor:Thisisspinaltap.jpg lumbalni_punkce_technika E-Klinická biochemie External ventricle drainage E-Klinická biochemie V-P shunt ventrikuloperitonealni_drenaz E-Klinická biochemie Complications of lumbar puncture ØDry puncture - incorrect needle position , arthritic changes Ø ØThere is the potential for the needle to contact a small vein on the way in. This can cause a "traumatic tap," which just means that a small amount of blood may leak into one or more of the samples collected. Soubor:Spinal needles.jpg E-Klinická biochemie Analysis of CSF – basic set Ø ØCSF color, clarity – ØCytology –CSF cell count (total number of cells present) –CSF differential cell count (numbers of different types of cells present) • ØCSF protein ,CSF glucose , lactate E-Klinická biochemie Cell count ØFuchs Rosenthal chamber (volume 3 ml) ØCell count /ml Ø Øref. range: 0-3(5) WBC/ml (newborn- 15/ml) Ø obrázky k přednášce 014 IMG_0271 IMG_0277 E-Klinická biochemie Qualitative cytology ØPermanent cytological preparation ØCytocentrifugation, cytosedimentation ØBasic hematology staining ØCytological diagnosis - assessment of the representation of individual cell types, functional state of cells • Ø IMG_0331 IMG_0332 IMG_0333 E-Klinická biochemie Physiological finding • •Oligocytosis 0-3cells/ul •Lymfocytes 50-80%, monocytes 20-50% • • • • • • 012 E-Klinická biochemie Aseptic meningitis Zvětšení 200x P7045314.JPG E-Klinická biochemie Aseptic meningitis F:\Rosík Filip\2.9.2013\P9045439.JPG Zvětšení 400x E-Klinická biochemie Aseptic meningitis Zvětšení 1000x E-Klinická biochemie Types of CSF cytological findings ØLymfocyte pleocytosis Ø non bacterial inflammatory diseases Ø(viral infection, infection caused by Borrelia, Leptospira or tuberculosis bacilli) Ø – – Ø Ø Ø ØLymfocyte oligocytosis ØNon bacterial inflammatory diseases Øearly stage of multiple sclerosis. . 002 E-Klinická biochemie Bacterial neuroinflammation F:\Novotný Pavel\14.7.2013\nález 14.7.2013.bmp F:\Novotný Pavel\14.7.2013\P7195382.JPG Soubor:Neurinom akustiku.png E-Klinická biochemie Bacterial neuroinflammation F:\Novotný Pavel\14.7.2013\P7195385.JPG Cytospin po naředění (zvětšení 400x) E-Klinická biochemie Types of CSF cytological findings ØPolynuclear pleocytosis (predominant polynucleares) • Occurs in purulent inflammations • Ø Ø Ø ØPolynuclear oligocytosis • is frequent in early stage nonbacterial inflammation or cerebral ischemia Ø • IMG_0186 E-Klinická biochemie Types of CSF cytological findings ØMonocyte pleocytosis or oligocytosis Øwith the presence of activated monocytes is a nonspecific finding Øcharacteristic of non-infectious diseases - compression syndrome, an autoimmune disease ØThe finding is also characteristic of the final stage of inflammation IMG_0003 IMG_0016 E-Klinická biochemie Types of CSF cytological findings ØTumorosis pleocytosis or oligocytosis ØMalignity CNS (metastasis or primary tu) Ø Ø IMG_0307 IMG_0342 E-Klinická biochemie Total protein Ø ØPhysiological value of 0.15 to 0.40 g / l Øalbumin, prealbumin, transferrin, immunoglobulines haptoglobin, C-reactive protein, C3 and C4 complement , antithrombin III, α1-antitrypsin,orosomucoid, ØIncrease of total protein inflammation (disorder BBB) CSF circulation disorder intrathecal synthesis of Ig ØMethod of measurement - photometry reaction with benzethonium chloride E-Klinická biochemie Glucose Ø ØThe basic energy source of nerve tissue ØDepends on the level of glucose (60% serum) ØRatio of CSF glucose / S-glucose ( 0.6) Ø ØDecrease: • bacterial meningitis tumors hemorrhage E-Klinická biochemie Lactate Ø ØPhysiological value of 1.2 to 2.1 mmol / l Ø ØNot depend on the plasma concentration, practically does not cross the BBB Ø ØIncrease: Inflammation - the resolution of viral and bacterial meningitis (produced mainly by bacteria in anaerobic glycolysis) Oxygen supply to the brain disorder - ischemia, hemorrhage Increase the intensity of metabolism - tumors Ø E-Klinická biochemie Albumin Ø Syntesis in the liver ØAlbumin in CSF only from peripheral blood ØReference values: CSF-Albumin: 120-300 mg / l oAlbumin quotient - Qalb = alb.CSF / alb.S (depends on age): under 15 years: ≤ 5 × 10-3 to 40 years: ≤ 6.5 × 10-3 to 60 years: ≤ 8 × 10-3 oAlbumin quotient is used for: to assess the degree of disability the blood-brain barrier – for the calculation of intrathecal synthesis of immunoglobulines Ø E-Klinická biochemie The albumin quotient (QAlb) CSF albumin Serum albumin = Normal individuals Albumin quotient (QAlb) [USEMAP] Alternatively, quantitative IgG analysis may be used as a measure of intrathecal immunoglobulin synthesis. To ensure that measurements represent local (intrathecal) synthesis and not IgG which has diffused from the blood, values are corrected using albumin measurements. This serves as a marker of the blood-CSF barrier function because albumin is never synthesised within the CNS. For example, the IgG index is calculated as follows: E-Klinická biochemie The albumin quotient (QAlb) CSF albumin Serum albumin = Impaired blood brain barrier Albumin quotient (QAlb) Alternatively, quantitative IgG analysis may be used as a measure of intrathecal immunoglobulin synthesis. To ensure that measurements represent local (intrathecal) synthesis and not IgG which has diffused from the blood, values are corrected using albumin measurements. This serves as a marker of the blood-CSF barrier function because albumin is never synthesised within the CNS. For example, the IgG index is calculated as follows: E-Klinická biochemie Immunoglobulines ØSource – serum ØLocal synthesis (intrathecal) –perivascular infiltrates of B lymphocytes, which proliferate locally and ripen in plasmocytes and produce antibodies • • oCSF-IgG: 12,0-40,0 mg/l oCSF-IgM: 0,2-1,2 mg/l oCSF-IgA: 0,2-2,1 mg/l – E-Klinická biochemie Intratecal synthesis IgG Reiberův_diagram.png (780×720) Ø1. Quantitative-calculation according to Reiber Area 1 - normal findings Area 2 - isolated BBB failure without local synthesis of Ig Area 3 - impaired BBB with intrathecal synthesis of Ig Area 4 - isolated intrathecal Ig synthesis without failure the BBB Area 5 - the area of analytical errors E-Klinická biochemie Oligoclonal bands Kvalitativní průkaz intratékální syntézy IgG ØElectrophoresis in a gradient of pH – dividing according to isoelectric point of proteins • C:\Users\3050\Desktop\IMG_2409.JPG E-Klinická biochemie E-Klinická biochemie C:\Users\3050\Desktop\IMG_2411.JPG E-Klinická biochemie E-Klinická biochemie C:\Users\3050\Desktop\IMG_2415.JPG E-Klinická biochemie E-Klinická biochemie E-Klinická biochemie E-Klinická biochemie \\fnbrno.cz\tree\Home\3050\My Pictures\2017-04-28\085.JPG \\fnbrno.cz\tree\Home\3050\My Pictures\2017-04-28\087.JPG E-Klinická biochemie Intratecal synthesis IgG Ø2. Qualititive conclusion by isoelectric focusation – oligoclonal bands E-Klinická biochemie Perspectives of development of cerebrospinal fluid examination ØDiagnosis of degenerative diseases and prion infections - determination of 14-3-3 protein in CSF. ØDiagnosis of Alzheimer's disease - determining β-amyloid, τ-protein and phospho-τ-protein. ØStructural protein S-100 belongs to a family of calcium-binding proteins. Increased levels of S100 may indicate failure of neuronal involvement. Øβ2 microglobulin among the proteins which are present in all body fluids. Increase of its concentration in CSF are found in conditions generally associated with lymphocyte activation and multiplication and macrophages elements. ØIn patients with multiple sclerosis was investigated neuronal presence of antibodies - were found in cerebrospinal fluid antibodies against myelin molecules pack. This is mainly of myelin basic protein (MBP) from the group of structural proteins, which is the basis of myelin. Antibodies against MBP reflect the degree of myelin destruction. E-Klinická biochemie Spectrophotometry of CSF Ø ØSuspection of intracerebral bleeding. Ø ØBenefit in the early stages (no changes in the cytological image) Ø ØSpectrophotometry is 10 times more sensitive than the human eye, we can get a positive result even in seemingly colorless cerebrospinal fluid. Ø ØRegistration absorbance in the visible light (380-700 nm), detects the presence of oxyhemoglobin and bilirubin. E-Klinická biochemie Spectrophotometry of CSF Ø Spektrofotometrie_oxy_a_bili E-Klinická biochemie Subarachnoidal bleeding -recent IMG_0162 IMG_0161 E-Klinická biochemie E-Klinická biochemie IMG_1881 E-Klinická biochemie IMG_1885 Subarachnoidal bleeding - elderly E-Klinická biochemie Rhinorhea, otorhea Ø ØDrainage of cerebrospinal fluid through the nose,ear Ø ØBeta trace protein Ø ØBeta-2 transferrin E-Klinická biochemie beta 2 transferin Ø Obrázek1 Obrázek2