Cerobro Spinal Fluid collection and Analysis Process

Cerobrospinal Fluid is the fluid that protect our brain and spinal cord. It uses as the sample for analysis of different diseases. Some characteristics of CSF and collection process is described in this slide.

1. Specimen collection By: Raju Yadav M.Sc. Medical Microbiology

2. Definition Specimen collection is the process of obtaining tissue or fluids for laboratory analysis .  It is often a first step in determining diagnosis and treatment . The specimen must be collected in following manner: Appropriate to the patient’s clinical presentation; Collected at the right time; Collected in a way that minimises contamination; Collected in a manner that reduces risk to all staff (including laboratory staff); Collected using the correct tools; Documented clearly, including the process; Stored/transported appropriately.

3. Continue T he types of biological samples accepted in most clinical laboratories are: serum samples, virology swab samples, biopsy and necropsy tissue, cerebrospinal fluid, whole blood for PCR, and urine samples. These are collected in specific containers for successful processing in the laboratory. Specimen from the patients is collected before the administration of antibiotics. General specimen selection and collection guidelines include proper labelling of the sample to include t o patient identifiers, the source of the sample, and information on who collected the sample and at what time collection occurred.

4. Transportation of samples Transportation of samples means transporting the samples of patients from the area of collection to the laboratory for the laboratory examination . For sample transportation sterile container with ice is used. Sample processing: Sam ple processing is a process done to a sample to prepare it for testing . The accuracy of the results depends upon the purity of the sample provided.

5. Cerebrospinal fluid(CSF) It is the clear, color less fluid formed in the ventricles of the brain mainly by choroid plexus (mesh work of tiny small blood vessels in the lateral third and fourth ventricles). It is mainly ultra filtration of plasma. CSF is contained within cerebral ventricles, spinal cord and subarachnoid space surrounding the brain and spinal cord. It cushions the brain and spinal cord from injury and also serves as a nutrient delivery and waste removal system for the brain. CSF is reabsorbed in the blood through arachoid villi of dural venous sinuses.

6. Composition of CSF Total volume: 100-150 ml (10-60 ml in newborn) Color: Colorless Appearances: clear Clot: Absent Viscosity: is similar to water. Glucose: 40-80 mg/dl Proteins: 15-45mg/dl. Bilirubin: Absent Chloride: 116-127 mmol /L

7. Continue Cells Adults: 0-5 cells/ cmm Infants: 0-30 cells/ cmm 1-4: 0-20 cells/ cmm 5-18: 0-10 cells/ cmm Fumction of CSF : It acts a shock absorber and protects the brain and spinal cord from injury Serve as medium between blood and brain for the supply of nutrients to and removal of waste products from the brain.

8. Collection of CSF For the collection of CSF from the subarachnoid space, spinal cord, LP( Lumbarl puncture) needle is passed between 3 rd and 4 th or 4 th and 5 th lumber vertebrae. LP is carried out at this level to prevent the injury of nerve cells of the spinal cords.

9. Continue The patient is in a side- lying position with his back absolutely vertical to the edge of the bed, with the knee drawn up and his head flexed onto his chest. This position increase the surface between the lumber vertebrae. Alternately, the patients may be in sitting position.

10. Continue The selected site is disinfected with chorhexidine containing solution or povidon iodine Covered with the sterile drapes and after injection of local anesthesia, a sterile lumber puncture needle, preferably 22guge is inserted slowly. An increase resistance is absorbed as the needle passes through the spinal ligaments and dura matter. As the needle enters the subarachnoid space loss of resistance is felt. The stylet is withstand slowly. When the drops of CSF appear preassembled manometer is attached to the needle inorder to record the opening pressure of CSF. The patient should be in the lateral position for measurement of opening pressure.

11. Continue CSF collection in the sterile tubes as follows: Tube 1:(1ml):Chemistry(for glucose, protein and other serological tests) Tube 2: (2ml):Bacterial culture(blood agar, chocolate agar), fungus culture( Sabouraud -dextrose agar), culture for tuberculosis. Tube 3 : (1ml): Hematology(total cell count, differential cell count) Tube 4: (1ml): Cytology, special studies, immunology. The needle is withdraw after replacing the stylet and sterile dressing is applied. A venous blood is also collected to measure the glucose level in the blood. Usually 3-5 ml CSF is collected. If the opening preeure was high, closing pressure is measured after collection.

12. Laboratory examination of CSF After collection Specimen of the CSF should transfer immediately to the laboratory & examined with out any delay because: Cell disintegrated rapidly Reduction of glucose level occurs due to glycolysis. CSF should be examined with in 1hr of collection and CSF cell count are always done with in 30-60 minutes of the collection. Glass tube should not be used for the collection since cells adherence to the glass reduce the cell count. Specimens for the bacterial culture should not be refrigerated because fastidious organisms( Haemophilus influenzae , Neisseria meningitidis ) do not survive in cold temperatures.

13. Continue CSF chemical examination results should always be compared with those in plasma since any change in plasma is reflected in CSF. CSF examination includes: Opening pressure 60-100 mm of water in adults 10-100 mm of water in children. Cause of increased CSF Pressure: Tension Meningitis Cardiac failure

14. Continue Cause of decrease CSF Pressure Lumbar puncture Leakage of spinal cord due to brain trauma Completely blockage of spinal cord Large difference between opening and closing pressure indicates postal or complete spinal block. If opening CSF preeure is >20 mm, no more than 1-2 ml of CSF should be collected. Gross appearance of CSF Normal CSF is clear, color less like distilled water and does not Clot. Abnormal CSF is turbid, blood mixed, xanthochromic or viscous.

15. Continue Clot formation in CSF is abnormal and indicates increased proteins present in CSF. Turbid CSF is mainly due to the presence of leukocyte >200 cells/mm and RBC>400 cells/mm. Microorganism like bacteria, fungi, amoeba raised proteins. Blood mixed CSF may results due to injury to choroid plexus in the spinal walls. Cell Count: Cell count in CSF is done mainly on the undiluted samples in the counting chamber. It is necessary to do microscopic examination to all CSF samples as WBC count 200/ cmm and RBC count 400/ cmm is observed with the clear appearance. An increase in cell count in CSF is called as pleocytosis .

16. Continue Chemical examination of CSF: It consist of estimation protein and glucose. Normal range of protein in adults 15-45 mg/dl but if blood is mixed with CSF the value of the chemical may varies. If the facilities for estimates of protein is not available in laboratory, then pandy’s test for globulins may be performed. In this test CSF is added to saturated solution of ethanol. If cloudiness is developed immediately, indicates presence of increased protein and the test is positive, if no then test is negative. Glucose estimation in csf : Normal glucose level in CSF is 2/3 rd of blood glucose. A sample for blood glucose is drawn 1hr befoe Lumbar puncture for comparison with CSF glucose. Normal range of glucose is 45-80 mg/dl and abnormal glucose level is <40mg/dl. Glucose is decrease due utilization by bacteria, leukocyte, cancer cells in CSF.

17. Continue Microbiology examination of CSF: Microbiology examination is carried out on CSF samples are Direct wet mount of CSF in suspected cases of cryptococcus , manigoencyphalities , Candida infections and trypanosomasis . Gram smear should be done if CSF is turbid and neutrophils are increased. Ziehl-Neelseen smear is done if tuberculosis Maningitis is suspected. Lattex agglutination test for the bacterial and Cryptococcal antigens. Culture for bacteria including Mycobacterium tuberculosis Serology test for syphilis and PCR for Mycobacterium and viruses.