Cerebrospinal Fluid Examination Procedures - Research Proposal Example

Name : xxxxxxxxxxx Institution : xxxxxxxxxxx Course : xxxxxxxxxxx Title : CSF Examination Procedures Manual Tutor : xxxxxxxxxxx @2010 Table of Contents Table of Contents 2 Latex agglutination testing for Neisseria menengitidis 11 Interprating Microscopic observations 11 Table 2 13 Table 3 14 Common microbial agents associated with CSF 14 Conclusion 15 Bibliography 16 CSF Examination Procedures Manual Introduction Cerebrospinal fluid (CSF) infections are caused by different things which include bacteria which are the most serious cause, viruses, fungi, drugs’ reaction as well as environmental toxins, for instance heavy metals (Milan 2001). Most of the bacteria and viruses causing meningitis are usually common and are allied to other routine diseases. Bacteria and viruses that normally infect the skin, urinary system, gastrointestinal in addition to respiratory tract can spread through the bloodstream to the meninges through cerebrospinal fluid, the fluid that circulates within and in the region of the spinal cord (Milan 2001). In some incidences of bacterial meningitis, the bacteria can spread to the meninges in case of a serious head trauma or severe local infection, like ear infection or sinusitis (Feldman 2004). The organisms causing CSF infections normally colonize within an individual’s respiratory track and can be transmitted through close contact with people who could be having the infections (Kee 2003). The organisms can also be transmitted by respiratory secretions. A CSF infection normally begins within the respiratory track then moves into the bloodstream where it can reach both the brain and spinal cord as well (Kee 2003). The most microorganisms involved in CSF infections include; Group B streptococcus, Escherichia coli, Listeria monocytogenes, Haemophilus influenzae type b or H. influenzae, Neisseria meningitides, Strepolioviruses, enteroviruses like coxsackie viruses and echoviruses, paramyxovirus, herpes simplex virus (HSV) and ptococcus pneumoniae (Kee 2003). Collecting CSF Rationale (i) Place the patient in a foetal lateral position and flex the back This is to widen the intervertebral spaces. (ii) Clean the skin area using povidone iodine, and after that clean using 70% alcohol. This is to destroy the normal flora of the skin to: •prevent contamination of the specimen •prevent inoculation of normal skin flora into the cerebrospinal fluid. The spinal cord typically terminates at the point of the second lumbar vertebra in adults. Therefore the sub-arachnoid space must be entered below the level of the second lumbar vertebra to avoid injury to the cord. (iii) Introduce a spinal needle into the sub-arachnoid gap between the 3rd and 4th or the 4th and 5th lumbar vertebrae. (iv) Remove obturator from the centre of the needle and let CSF drop out. Provided the needle is correctly positioned. (v) Attach a three-way stopcock and manometer to the needle to measure the spinal fluid pressure. CSF should not be collected if there is raised intracranial pressure, as this will cause the brain stem to cone, causing death. Normal pressure is 17–18cm of CSF. (vi) Collect 1 to 2 ml of aliquots of spinal fluid into three sterile screw topped tubes, marked 1, 2, 3 in order of the collection. For paediatric collections – collect as much CSF as possible, 0.5 ml to 1 ml aliquots into 3 sterile tubes. For collections from the elderly – collect as much CSF as possible – see adult collection. Separate procedures will be performed on each of the specimens and if during the procedure a blood vessel is hit then the first specimen will be the most blood stained. (vii) Withdraw the needle and place a dry sterile dressing over the puncture site.   (Milan 2001). Microscopic examination of CSF Add the CSF sample to the counting chamber of the Kova Slide while using a capillary tube (Steven1998). View the slide microscopically using the x 10 and/or x40 objectives. Count the number of red blood cells and leukocytes present in one large square or one small square of the chamber if possible. After counting, use a suitable conversion factor to calculate the number of cells per litre of CSF. Leukocyte counts up to 5 x 106/L should be normal; counts between 100–1000 x 106/L occur most regularly in cases of viral meningitis and higher counts typically indicate presence of bacterial meningitis (Pezzlo 1998). Culturing CSF specimens Centrifuge CSF specimen (Steven1998). Remove the supernatant using a sterile pipette and transfer it in a sterilized tube; this will be used in chemical tests. NB; leave two drops of the specimen in the bottom of the tube. Resuspend the sediment. Using a sterilized loop, inoculate Blood agar plate and Chocolate agar plate and streak (Evan 2001). Incubate the plates at 37°C overnight. Blood agar plate should be incubated aerobically while Chocolate agar plate should be incubated under carbon dioxide (Henry 2006). Staining of the CSF Prepare two smears on microscope slides from the resuspended CSF sediment (Steven1998). Perform Gram stain on the first smear to assist in the identification of the morphology of any bacterial pathogen that could be present in the CSF. On the second smear, perform a Giemsa stain in order to differentiate and quantify various leucocytes in the CSF (Henry 2006). Preparation of CSF smears Transfer the resuspended sediment into two clean microscope slides (Lessing 1998). Allow the smear to dry and add another loopful of sediment to every smear. Allow the smear to dry and add further loopful of sediment to every smear. The addition of further specimen sediments will help in concentrating the cells and organisms which could be present in the CSF (Lessing 1998). Fix the slides using heat (Lessing 1998). Gram stain one slide and stain the other slide using Giemsa stain. Since the nucleus of the cells present in the specimen are now stained, one can differentiate and quantitate the numbers of polymorphonuclear leukocytes and monocytes, from the Giemsa stain. In polymorphonuclear leukocytes, the cytoplasm will have a granular appearance while the nucleus will have several lobes since it will appear lumpy inside. The monocytes will have a granular cytoplasm as well as large nucleus which is kidney or horse shaped (Lessing 1998). Count the number of polymorphonuclear leukocytes present within a sample of 50 cells. Calculate the percentage present of these cells (Henry 2006). PART B Microscopy Total WBC and RBC counts should be performed on the specimen that is not centrifuged, rather the last specimen taken in, within a counting chamber. Cell count is not supposed to be done on specimens with a clot since this will invalidate the results (Zabransky 2005). Differential leucocyte count 1. Counting chamber procedure This method is recommended for lower WBC counts (Prober 2003). Non- or lightly blood stained specimens Stain the unspun CFC with 0.1 percent stain solution, like, toluidine, methylene or Nile blue. This lyses the RBCs and stains the leucocyte nuclei for differentiation to ensue. Count and record the definite numbers of every leucocyte type. Consider the dilution factor and express it as the number of cells per litre (Zabransky 2005). Heavily stained bloodstained specimens Dilute the specimen with WBC diluting fluid (Gregory 2005). Leave it for five minutes prior to loading the counting chamber. This lyses the RBCs and stains the leucocyte nuclei for differentiation to happen. Count and record the definite numbers of every leucocyte type. Consider the dilution factor and express it as the number of cells per litre (Zabransky 2005). 2. Stained method This method is recommended for very high WBC counts where differentiation within the counting chamber is not easy (Watt 2002). Prepare a slide from the CSF deposit which has been centrifuged as for the Gram stain. Allow it to air and dry as well. Fix within alcohol and stain with the appropriate stain for WBC morphology. NB. Heat fixation will distort cellular morphology. Count and record the definite numbers of every leucocyte type. Considering the dilution factor, express as the number of cells per litre (Watt 2002). NB. A cytocentrifugation deposit, for example Cytospin gives the most accurate cell differentiation. Care should be taken and a sterilized tube should be used if the deposit is to be utilized in Gram stain examination (Watt 2002). Gram stain Carry out Gram stain on all specimens apart from; clotted specimens (Gregory 2005). Centrifuge the specimen within a sterile, capped, conical-bottomed container at 1200 *g for five to ten minutes. NB. If examination of Mycobacterium species is necessary, centrifugation time can be increased to 15 to 20 minutes at 3000 * g58 and the same deposit can be used for this in addition to routine microscopy and culture. Transfer all apart from the last 0.5 ml of the supernatant using a sterile pipette to another sterilized container for further testing if necessary, for example protein or virology (Braunwald 2005). Resuspend the deposit within the remaining fluid. Place one drop of the centrifuged deposit using sterilized pipette on a clean microscope slide. Spread the deposit using a sterile loop to make a thin smear for Gram staining. NB. The sensitivity of the Gram stain can be improved through “building up” the serial drops on the slide after every drop has dried, to maximize the quantity examined. Care should be taken to make sure that the smear is not washed off during staining. Observe the specimen (Prober 2003). Table 1 Culture media, Conditions and Organisms for all Specimens: Clinical details/conditions Standard media Incubation Temp 0C, Atmos, Time A Cultures read Target organisms Meningitis Post neurosurgery Reservoirs Ventriculitis Immunocomprised Chocolate agar Blood agar 35-37 5-10% CO2 40-48 hrs 35-37 5-10% CO2 40-48 hrs Daily Daily Any organism (Wallach 2000). For these conditions, add the following; Clinical details/conditions Supplementary media Incubation Temp 0C Atmos Time Cultures read Target organisms Immunocompromised patients Sabourand plate 35-37 air 2-5days >40 hrs to 8 wks Fungi Brain abscess Ventriculitis Reservoirs Post neurosurgery Post otitis media with complications Fastidious Anaerobe agar 35-37 anaerobic 7 -14days 40hrs and at 5 days Anaerobes If mixed infection suggested by Gram-stained film Neomycin Fastidious Anaerobe agar 35-37 anaerobic 7 -14days 40hrs and at 5 days Anaerobes (Musher 2001) Latex agglutination testing for Neisseria meningitidis Neisseria meningitidis contains specific polysaccharide surface antigens that can be detected by Latex agglutination test (LAT) (Steven1998). CSF should be tested for Neisseria meningitidis with LAT using the BIO-RAD PASTOREX® MENINGITIS Kit. The kit should have sera to detect antigens of Neisseria meningitides (Steven1998). Perform the test was using the supernatant according to the manufacturer's instructions. A positive test confirms presence of Neisseria meningitidis while a negative test indicates absence of Neisseria meningitides (Steven1998). Interpreting microscopic observations Turbidity When the CSF appears turbid after collection, this indicates presence of leucocytes and bacteria in CSF; demonstrates bacterial meningitis. Still, in case of turbidity viral pathogens are absent since viral specimens are clear (Braunwald 2005). Colour If CSF is yellow in color it indicates presence of blood breakdown products and Hyperbilirubinemia. Orange color indicates presence of Blood breakdown products and High carotenoid ingestion within CSF. Pink color indicates Blood breakdown products while Green color indicates Hyperbilirubinemia or Purulent CSF. Finally, if CSF is brown in color it indicates presence of meningeal melanomatosis. White Blood Cells The normal number of WBC in CSF is very low. Augmented amount of White Blood cells in the CSF indicates an infection (Braunwald 2005). WBC differential assists in differentiating different types of infections. Increased number of lymphocytes indicates viral infection. Increased number of polymorphonuclear leukocytes (neutrophils) indicates bacterial or fungal infection (Braunwald 2005). Eosinophils indicate allergy and ventricular shunts. Macrophages with ingested bacteria indicate meningitis (Braunwald 2005). Table 2 Test Bacterial Viral Fungal Tubercular Opening pressure Elevated Usually normal Variable Variable White blood cell count ≥1,000 per mm3 Read More