CEREBROSPINAL FLUID BY Hossam HASSAN

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1 CEREBROSPINAL FLUID BY Hossam HASSAN
Welcome to first session of Unit 4 - Cerebrospinal Fluid Please carefully follow your course objectives as you move through this unit.

2 Overview of Body Fluid Analysis
Laboratory exam of body fluids Physical characteristics Chemical constituents Morphologic elements Culture for microorganisms Ancillary studies Body fluids, other than urine are not easily obtained – usually requiring the insertion of a needle deeply into a normally sterile cavity . There are inherit risks involved. The sample obtained is usually of a small volume and is very fragile. The results of the tests often have significant impact on the treatment and prognosis for the patient. Therefore Laboratories that offer tests on these body fluids must be able to provide Accurate & timely results as well as be a Source of information for , normal values, reliability of the results, and Proper specimen collection and handling. Depending on the physicians reason for drawing the body fluid, the examination involves evaluation of the Physical characteristics, Chemical analysis, microscopic review of the cells and other Morphologic elements as well as culturing for microorganisms. Ancillary or other special studies may be done in-house, or sent to a reference lab.

3 Cerebrospinal Fluid (CSF)
Composition and formation CSF is the 3rd major fluid of the body Adult volume mL Neonate volume mL Composition and formation CSF is the 3rd major fluid of the body after blood and urine. Adult volume mL Neonate volume mL

4 Cerebrospinal Fluid (CSF)
Produced at the Choroid plexus of the 4 ventricles by modified Ependymal cells At ml/day is produced CSF flows through the Subarachnoid space Where a volume of 90 – 150 ml is maintained (adults) Reabsorbed at the Arachnoid villus / granulation to be eventually reabsorbed into the blood CSF is Produced at the Choroid plexus of the 4 ventricles by modified Ependymal cells At ml / hr (adults) The CSF flows through the Subarachnoid space where the appropirate volume of the fluid is maintained (adults) The CSF is then Reabsorbed at the Arachnoid villus / granulation and is eventually reabsorbed into the blood This pattern of flow is shown in the drawing from the Strassinger reference textbook Med training 150 ml/day is produced.

5 Cerebrospinal Fluid (CSF)
Blood Brain Barrier Occurs due to tight fitting endothelial cells that prevent filtration of larger molecules. Controls / restricts / filters blood components Restricts entry of large molecules, cells, etc. Therefore CSF composition is unlike blood’s ** CSF is NOT an ultrafiltrate There is a secure barrier between the blood and the csf fluids that surround the brain called the Blood Brain Barrier This barrier is due to the tight fitting endothelial cells that prevent filtration of larger molecules Due to the blood brain barrier, CSF composition is very different from that of plasma. And therefore CSF is NOT considered an ultrafiltrate of plasma. Tjherefor Controls / restricts / filters blood components Restricts entry of large molecules, cells, etc. Therefore CSF composition is unlike blood’s **

6 Cerebrospinal Fluid (CSF)
Blood Brain Barrier Essential to protect the brain Blocks chemicals, harmful substances Antibodies and medications also blocked Tests for those substances normally blocked can indicate level of disruption by diseases: ie meningitis and multiple sclerosis. The purpose of the Blood Brain Barrier is to protect the brain from chemicals, and other harmful substances . In this process, Antibodies and medications also blocked Testing for those substances normally blocked - can indicate the level of disruption by diseases: ie meningitis and multiple sclerosis.

7 Cerebrospinal Fluid (CSF)
CSF functions Supplies nutrients to nervous tissues Removes metabolic wastes Protects / cushions against trauma There are 3 major functions of CSF . It Supplies nutrients to nervous tissues Removes metabolic wastes Protects / cushions against trauma

8 Cerebrospinal Fluid (CSF)
Four major categories of disease Meningeal infections Subarachnoid hemorrhage CNS malignancy Demyelinating disease There are Four major categories of disease Meningeal infections – such as bacterial or viral meningitis Subarachnoid hemorrhage – commonly refereed to as a stroke. Central Nervous System malignancy Demyelinating disease - such a multiple sclerosis.

9 Cerebrospinal Fluid (CSF)
Indications for analysis To confirm diagnosis of meningitis Evaluate for intracranial hemorrhage Diagnose malignancies, leukemia Investigate central nervous system disorders Four categories of disease lead to 4 Indications for analysis CSF us analyzed To confirm diagnosis of meningitis , Evaluate for intracranial hemorrhage, to Diagnose malignancies, and leukemia and to Investigate central nervous system disorders

10 Cerebrospinal Fluid (CSF)
Specimen collection and handling Routinely collected via lumbar puncture between 3rd & 4th, or 4th & 5th lumbar vertebrae under sterile conditions Intracranial pressure measurement taken before fluid is withdrawn. Specimen collection and handling CSF is Routinely collected via lumbar puncture between 3rd & 4th, or 4th & 5th lumbar vertebrae under sterile conditions Once the puncture has been made, the first step is to measure the Intracranial pressure . This must be done before the fluid is removed.. A low opening pressure is seen in trauma where there has been a loss of the csf fluid. and an increased pressure is diagnostic of intracranial hypertension – which is present in many pathological states including tumors, intracranial bleeding and meningitis.

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12 Cerebrospinal Fluid (CSF)
This slide from the web Adam series included only as a pictorial representation of the spinal puncture process.

13 Cerebrospinal Fluid (CSF)
Specimen collection and handling Tube 1 – chemistries and serology Tube 2 – microbiology cultures Tube 3 – hematology Testing considered STAT Specimen potentially infectious As the spinal fluid is removed it is collected into a series of tubes. The tubes are numbered to indicate how the fluid was removed. In other words, The first fluid that is removed will go into tube 1, the next amout of fluid will be in tube 2 and so forth. Usually 3 tubes are collected, but sometimes you will see four tubes. Tube 1 – chemistries and serology Tube 2 – microbiology cultures Tube 3 – hematology If there was a 4 th tube, usually the first tube is set aside – tube number 2 then becomes tube 1, tube 3 becomes number 2. Sounds confusing, but every thing just slides down a number. Keep in mind that the goal is to have the hematology tube be the last sample removed, so any cells present can be definitely said to have come from the spinal cannal and not a result of a traumic stick. All csf Testing considered STAT and always keep in mind that csf samples are potentially infectious

14 Cerebrospinal Fluid (CSF)
Specimen collection and handling If immediate processing not possible Tube 1 (chem-sero) frozen Tube 2 (micro) room temp Tube 3 (hemo) refrigerated I am a firm believer that CSF testing must be performed Stat. However, if immediate processing and testing is not possible Tube 1 (chem-sero) frozen Tube 2 (micro) room temp Tube 3 (hemo) refrigerated

15 Cerebrospinal Fluid (CSF)
Appearance Normal - Crystal clear, colorless Descriptive Terms – hazy, cloudy, turbid, milky, bloody, xanthrochromic Often are quantitated – slight, moderate, marked, or grossly. Unclear specimens may contain increased lipids, proteins, cells or bacteria. Use precautions. Clots indicate traumatic tap Milky – increased lipids Oily – contaminated with x-ray media CSF is Normally - Crystal clear, colorless When its transparency is not clear - Appropriate descriptive Terms include – hazy, cloudy, turbid, milky, bloody, When the non-clear terms are used, they are Often quantitated – so you may see a designation such as slightly hazy, moderately cloudy, or grossly bloody. Etc/ . Unclear specimens CSF specimens may contain increased lipids, proteins, cells or bacteria. Use precautions.\ xanthrochromic is a term used to describe a yellow / yellow-orangeish colored csf/ The picture in the slide shows how putting a newspaper behind the CSF sample can be used as aid in evaluating its clarity. The sample on the right is normal and the one on the left is moderately cloudy. – The cloudy sample is often an indication that it contains WBCs as would be seen in meningitis. Other reasons that the csf is not clear and colorless are also indicated on the slide.

16 Cerebrospinal Fluid (CSF)
Appearance Xanthrochromic – Yellowing discoloration of supernatent (may be pinkish, or orange). Most commonly due to presence of ‘old’ blood. Other causes include increased bilirubin, carotene, proteins, melanoma Xanthrochromia is a – Yellowing discoloration of the csf supernatent (may be pinkish, or orange).This term is unique to CSF samples. Xanthrochromia is Most commonly due to presence of ‘old’ blood. However it may be the result of increased bilirubin, or carotene, Other reasons may include proteins, melanoma Again, xanthrochromia – usually indicates old blood or bilirubin

17 Cerebrospinal Fluid (CSF)
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. Pellicle formation - picture at right (pellicle in L. tube, R is normal) Milky – increased lipids Oily – contaminated with x-ray media 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. An example of Pellicle formation is seen in the lower right picture. The tube at the far right is normal and pellicle webbing that is associated with tubularcar meningitis is seen in the left tube. A Milky appearing CSF likely contains increased lipids An Oily appearing CSF is sometime seen in samples that are – contaminated with x-ray /dye or contrast media

18 Traumatic collection vs cerebral hemorrhage
Even distribution of blood in the numbered tubes Clot formation possible Xanthrochromic supernatent – RBCs must have been in 2+ hours - D-dimer, fibrin degradation product from hemorrhage site Microscopic presence of erythrophages, or siderophages, Hemosiderin granules When blood is found in the csf sample there is need to determine whether the blood’s presence is due to A bleed or Trauma during the collection or due to a cerebral hemorrhage The characteristics of the Cerebral hemorrhage include Even distribution of blood in the numbered tubes – the picture below left represents two consecutive csf tubes. As you can see, They both appear to have a similar amount of blood in them- as would be seen in the cerebral bleed. If there is sufficient blood and fibrinogen present, clots may be seen. After spinning the csf sample in the centriguge, a Xanthrochromic / yellow supernatent will be seen in the patients with cerebral bleed, if enough time has gone by. – The authors note that the RBCs must have been in 2+ hours before this process results in the xanthrocromia. Serological test for - D-dimer, - which is a fibrin degradation product may also be helpful information from hemorrhage site On the The Microscopic differential you may find erythrophages, and siderophages, as well as Hemosiderin granules and hemotoidion bars

19 Cerebrospinal Fluid (CSF)
Expected results Normally 0 RBCs/uL 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 Normally there are no RBCs found in the CSF. The normal value and type of WBCs depend on the patients age Adults – up to 5 mononuclear WBCs/uL Newborns– up to 30 mononuclear WBCs/uL And the values for children are provided on the slids Children (1-4) - up to 20 mononuclear /uL Children (5+) – up to 10 mononuclear / uL Pleocytosis is the term used to indicate an increased number of cells. You can have a neutrophilic pleocytosis, lymphocytic pleocytosis, mixed cell pleocytosis, etc.

20 Cerebrospinal Fluid (CSF)
WBC counts 3% acetic acid can be used to lyse RBC Methylene blue staining will improve visibility If there are too many rbcs present to get an accurate wbc count, you can lyse the RBCs with 3% acetic acid. Using methylene blue stain will also help improve the visibility. If using a stain or lysing the rbcs with the acid, be sure you keep track of the dilution you are making and account for it in your calculation.

21 Cerebrospinal Fluid (CSF)
Correction of WBC count for traumatic tap contamination. Uses ratio of WBCs to RBCs in blood and compares it to same ratio (WBC/RBC) in CSF If patient’s peripheral cell counts are normal, can subtract 1 WBC for each 700 RBCs counted in CSF. Great chance for considerable error, makes this of little value. On occasion, it may be necessary to Correct the WBC count for traumatic tap contamination. Although I include the needed information on the slide, be warned that there are too many variables and assumptions that have to be made that would result in the possibility for considerable error. .Before doing such a correction, consult with the department head or pathologist. He or she may want to consult with the attending physician to make them aware of the error that is possible. As the more appropriate process would be to to have the sample recollected Uses ratio of WBCs to RBCs in blood and compares it to same ratio (WBC/RBC) in CSF If patient’s peripheral cell counts are normal, can subtract 1 WBC for each 700 RBCs counted in CSF. Great chance for considerable error, makes this of little value.

22 Differential Diagnosis of Meningitis by Laboratory Results
Bacterial Viral Tubercular Fungal Increased WBC count Neutrophils Lymphs Lymps & Monos Lymphs & Monos Marked ↑ protein Mod. ↑ protein Mod-Marked ↑ protein Marked ↓ glucose ↔ normal glucose ↓ glucose Normal to ↓ glucose Lactate > 35 mg/dL Lactate normal Lactate > 25 mg/dL + gram stains Pellicle formation + India ink with Cryptococcus neoformans + bacterial antigen tests + immunological test for C. neo. I have consolatated I strongly encourage you to spend some time evaluating this chart that compares the various lab results seen in bacterial, viral tubuarlular and fungal meningitis.

23 THANK YOU