Presentation on theme: "Urinalysis and Body Fluids CRg"— Presentation transcript:
1 Urinalysis and Body Fluids CRg Unit 4Cerebrospinal FluidWelcome to first session of Unit 4 - Cerebrospinal FluidPlease carefully follow your course objectives as you move through this unit.
2 Overview of Body Fluid Analysis Laboratory responsibilitiesAccurate & timely resultsSource of informationNormal valuesReliability of results, effects of medication, etc.Proper specimen collection and handlingLaboratory exam of body fluidsPhysical characteristicsChemical constituentsMorphologic elementsCulture for microorganismsAncillary studiesBody 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 formationCSF is the 3rd major fluid of the bodyAdult volume mLNeonate volume mLComposition and formationCSF is the 3rd major fluid of the body after blood and urine.Adult volume mLNeonate volume mL
4 Cerebrospinal Fluid (CSF) Produced at the Choroid plexus of the 4 ventricles by modified Ependymal cellsAt ml / hr (adults)Med training 150 ml/day is producedCSF flows through the Subarachnoid spaceWhere a volume of 90 – 150 ml is maintained (adults)Reabsorbed at the Arachnoid villus / granulationto be eventually reabsorbed into the bloodCSF 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 bloodThis pattern of flow is shown in the drawing from the Strassinger reference textbookMed training 150 ml/day is produced.
5 Cerebrospinal Fluid (CSF) Blood Brain BarrierOccurs due to tight fitting endothelial cells that prevent filtration of larger molecules.Controls / restricts / filters blood componentsRestricts entry of large molecules, cells, etc.Therefore CSF composition is unlike blood’s** CSF is NOT an ultrafiltrateThere is a secure barrier between the blood and the csf fluids that surround the brain called the Blood Brain BarrierThis barrier is due to the tight fitting endothelial cells that prevent filtration of larger moleculesDue to the blood brain barrier, CSF composition is very different from that of plasma.And therefore CSF is NOT considered an ultrafiltrate of plasma.TjhereforControls / restricts / filters blood componentsRestricts entry of large molecules, cells, etc.Therefore CSF composition is unlike blood’s**
6 Cerebrospinal Fluid (CSF) Blood Brain BarrierEssential to protect the brainBlocks chemicals, harmful substancesAntibodies and medications also blockedTests 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 blockedTesting for those substances normally blocked - can indicate the level of disruption by diseases: ie meningitis and multiple sclerosis.
7 Cerebrospinal Fluid (CSF) CSF functionsSupplies nutrients to nervous tissuesRemoves metabolic wastesProtects / cushions against traumaThere are 3 major functions of CSF . ItSupplies nutrients to nervous tissuesRemoves metabolic wastesProtects / cushions against trauma
8 Cerebrospinal Fluid (CSF) Four major categories of diseaseMeningeal infectionsSubarachnoid hemorrhageCNS malignancyDemyelinating diseaseThere are Four major categories of diseaseMeningeal infections – such as bacterial or viral meningitisSubarachnoid hemorrhage – commonly refereed to as a stroke.Central Nervous System malignancyDemyelinating disease - such a multiple sclerosis.
9 Cerebrospinal Fluid (CSF) Indications for analysisTo confirm diagnosis of meningitisEvaluate for intracranial hemorrhageDiagnose malignancies, leukemiaInvestigate central nervous system disordersFour categories of disease lead to 4 Indications for analysisCSF us analyzed To confirm diagnosis of meningitis , Evaluate for intracranial hemorrhage, toDiagnose malignancies, and leukemia and toInvestigate central nervous system disorders
10 Cerebrospinal Fluid (CSF) Specimen collection and handlingRoutinely collected via lumbar puncture between 3rd & 4th, or 4th & 5th lumbar vertebrae under sterile conditionsIntracranial pressure measurement taken before fluid is withdrawn.Specimen collection and handlingCSF is Routinely collected via lumbar puncture between 3rd & 4th, or 4th & 5th lumbar vertebrae under sterile conditionsOnce 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.
11 Cerebrospinal Fluid (CSF) This slide from the web Adam series included only as a pictorial representation of the spinal puncture process.
12 Cerebrospinal Fluid (CSF) Specimen collection and handlingTube 1 – chemistries and serologyTube 2 – microbiology culturesTube 3 – hematologyTesting considered STATSpecimen potentially infectiousAs 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 serologyTube 2 – microbiology culturesTube 3 – hematologyIf 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
13 Cerebrospinal Fluid (CSF) Specimen collection and handlingIf immediate processing not possibleTube 1 (chem-sero) frozenTube 2 (micro) room tempTube 3 (hemo) refrigeratedI am a firm believer that CSF testing must be performed Stat. However, if immediate processing and testing is not possibleTube 1 (chem-sero) frozenTube 2 (micro) room tempTube 3 (hemo) refrigerated
14 Cerebrospinal Fluid (CSF) AppearanceNormal - Crystal clear, colorlessDescriptive Terms – hazy, cloudy, turbid, milky, bloody, xanthrochromicOften are quantitated – slight, moderate, marked, or grossly.Unclear specimens may contain increased lipids, proteins, cells or bacteria. Use precautions.Clots indicate traumatic tapMilky – increased lipidsOily – contaminated with x-raymediaCSF is Normally - Crystal clear, colorlessWhen 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.
15 Cerebrospinal Fluid (CSF) AppearanceXanthrochromic – Yellowing discoloration of supernatent (may be pinkish, or orange).Most commonly due to presence of ‘old’ blood.Other causes include increased bilirubin, carotene, proteins, melanomaXanthrochromia 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, melanomaAgain, xanthrochromia – usually indicates old blood or bilirubin
16 Cerebrospinal Fluid (CSF) AppearanceClots – 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 lipidsOily – contaminated with x-ray mediaClots – 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 lipidsAn Oily appearing CSF is sometime seen in samples that are – contaminated with x-ray /dye or contrast media
17 Traumatic collection vs cerebral hemorrhage Even distribution of blood in the numbered tubesClot formation possibleXanthrochromic supernatent– RBCs must have been in 2+ hours- D-dimer, fibrin degradation product from hemorrhage siteMicroscopic presence of erythrophages, or siderophages, Hemosiderin granulesWhen 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 hemorrhageThe characteristics of the Cerebral hemorrhage includeEven 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 siteOn the The Microscopic differential you may find erythrophages, and siderophages, as well as Hemosiderin granules and hemotoidion bars
18 Cerebrospinal Fluid (CSF) - procedures All specimens should be examined microscopically – hematologyStat priority, RBC lyse in 1 hour, WBC in 2 hrs. Refrigerate if not able to process immediately.Electronic counters generally unusable. Manual countNo dilution usually required (use saline if needed)Standard Neubauer hemacytometer counting chamberAll CSF specimens should be examined microscopically – this is usually perfomed in the hematology department.CSF samples must have stat priority, RBC lyse in 1 hour, WBC in 2 hrs.The hematology sample should be Refrigerated if not able to process immediately.Manual cell counts are needed because the numbers of cells present are to low to be counted accurately using automated cell counters.Rarely is a dilution needed but if a dilution is needed it should be normal salineThe counting is performed in a Standard Neubauer hemacytometer counting chamber as shown.
19 Neubauer hemacytometer / counting chamber Formula for calculations – results in # cells / uLCount and record cells from both sides of the chamber.Average the two sidesMultiply by dilution factor (if no dilution is made, this number is 1)Divide by number of squares counted X volume of each squareLarge squares, such as # 1-9 below have volume of 0.1Small squares – in center # 5 have volume of 0.004RBC and WBC counts are reported as # cells / uLTo obtain this you must Count and record cells from both sides of the chamber.Average the two sidesAnd enter thi information into the formula shownThe average number of cells from both sides of the chamber are multiplied by the dilution factor . This number will be 1 ,unless you needed to make a dilution. (On the denominator of the calculatiojn, you enter the number of squares you counted each side of the chamber. Times ths volume of each square.Large squares, such as # 1-9 seen on the picture below have volume of 0.1Small squares – in center # 5 have volume of 0.004
20 Cerebrospinal Fluid (CSF) Expected resultsNormally 0 RBCs/uL regardless of ageWBCsAdult – up to 5 mononuclear WBCs/uLNewborn – up to 30 mononuclear WBCs/uLChildren (1-4) - up to 20 mononuclear /uLChildren (5+) – up to 10 mononuclear / uLIncreased numbers = PleocytosisNormally there are no RBCs found in the CSF.The normal value and type of WBCs depend on the patients ageAdults – up to 5 mononuclear WBCs/uLNewborns– up to 30 mononuclear WBCs/uLAnd the values for children are provided on the slidsChildren (1-4) - up to 20 mononuclear /uLChildren (5+) – up to 10 mononuclear / uLPleocytosis is the term used to indicate an increased number of cells.You can have a neutrophilic pleocytosis, lymphocytic pleocytosis, mixed cell pleocytosis, etc.
21 Cerebrospinal Fluid (CSF) WBC counts3% acetic acid can be used to lyse RBCMethylene blue staining will improve visibilityIf 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.
22 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 CSFIf 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 recollectedUses ratio of WBCs to RBCs in blood and compares it to same ratio (WBC/RBC) in CSFIf 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.
23 Cerebrospinal Fluid (CSF) QCCSF controlsCheck techniquesCheck of reagentsCheck of centrifugesDecontaminate all counting chambers in bleach water 15 minutes. Rinse in water and cleaned again with alcohol.There are Quality control samples producedCSF controls should be used regularly to verify the accuracy of the techniques used by the laboratory staff who perform the cell counts. The controls also check the acceptability of the reagents and the centrifuges,A special safety reminder. Since CSF can transmit verulant pathogenic organisms, it is necessary to Decontaminate all counting chambers in bleach water 15 minutes. Rinse in water and cleaned again with alcohol - dried carefully and stored in a proper container that will prevent the chamber from being scratched.
24 Cerebrospinal Fluid (CSF) CSF Slide DifferentialWrights stained smear of concentrated sediment.Cytocentrifuge - places cells on filter/ membrane. Increases number of cells to evaluate, however, risk of cell distortion from the centrifugation process.Use of albumin reduces cell distortionMoving on to the CSF Slide DifferentialBecause of the low number of cells, it is necessary to concentrate the sediment by spinning it down in a special centrifugation – called a CytocentrifugeDuring the cerfugation process, the csf is filtered through a membrane which captures the cells.While the centrifugyation process concentrates and Increases number of cells to evaluate, the process can easily cause distortion of the cells. Most procedures add a small amount of albumin to help reduces cell damage and distortionThe membrane is then mounded on a microscope slide and stained with wrights stain.d sediment.Cytocentrifuge - places cells on filter/ membrane. Increases number of cells to evaluate, however, risk of cell distortion from the centrifugation process.Use of albumin reduces cell distortion
25 Cerebrospinal Fluid (CSF) Count and differentiate 100 nucleated cells.Any cell found in peripheral blood may be seen in CSF, other nucleated cells and malignant cells can also be found.Entire smear should be evaluated forabnormal cells, inclusions within cells, Clusters, Presence of intracellular organismsNormal differential valuesAdults: 70% lymps, 30% monos.Children / newborns: monocyteTypes of cellsNeutrophils – occasionally (with normal count)Macrophages – increase following CVAEpendymal cells, and normal lining cells can also be seen.After staining the slide with Wrights stain, you attempt to Count and differentiate 100 nucleated cells.Keep in mind that 0-5 WBC / uL is the normal for adults and 0-3 for children. So some times finding 100 nucleated cells is difficult, unless the patient has a pleocytosis.Any cell found in peripheral blood may be seen in CSF as well as cells from the tissues forming the membranes.However, you are going to find lymphocytes, perhaps a few monocytes / macrophages, and a rare neutrophile in normal patients.The published Normal differential values for Adults: 70% lymps, 30% monos. While Children / newborns should only have monocytes.If the patient has a central nervous system malignancy or a leukemia, these abnormal cells may be discovered in the CSF as well.Ependymal cells, and normal lining cells can also be seen.
26 Cerebrospinal Fluid (CSF) Entire smear should be evaluated forabnormal cellsinclusions within cellsClustersPresence of intracellular organismsAfter obtaining the 100 cell differential,Entire smear should be evaluated forabnormal cells, inclusions within cells, or Presence of intracellular organisms such as can be seen in the microphotograph below.
27 Cerebrospinal Fluid (CSF) Lymphocytes & monocytes / macrophagesMono / macro, segs and lymphL – lymphocytes & macrophagesTake a few minutes to examine this and the next couple of slides that contain microphotographs of examples of cells seen in CSF fluid.
28 Cerebrospinal Fluid (CSF) EosinophilsOften associated with parasitic / fungal infections, allergic reactions including reaction to shunts and other foreign objects.Eosinophils are occasionally seen in CSF and are Often associated with parasitic / fungal infections, allergic reactions including reaction to shunts and other foreign objects.
29 Cerebrospinal Fluid (CSF) Ependymal cellsNormal cell, unique to CSFLine the ventricles, produce CSF fluidLarge cell with distinct round/oval nucleus, sometimes found in sheetsEpendymal cells are unique to CSF.They are a Normal cell but not often found,. These cells Line the ventricles, and are responsible for producing the CSF fluidEpendymal cells are one of the larger cells you may find in the CSF and are identified byt their distinct round/oval nucleus and plentiful cytoplasm, sometimes found in sheets
30 Cerebrospinal Fluid (CSF) Suspicious / unclassified or malignant cells are reported as “other” or “unclassified” AND are sent to pathology. (as seen below)Cytology – send unstained slide to cytology / pathology1986 CAP CM10 CSF – blasts (appearance similar to peripheral blood, always consult with hematology specialist / pathologist) ( see below right)The cells on this and the next slide are the best examples I can provide you of suspicious or malignant cells as can sometimes be found in the CSF. Most of these pictures are from older CAP surveys.This course is not designed to give you the knowledge and skills of a cytologist, however the MLT would be expected to recognize the abnormal cells that do not fit the pattern or characteristics of the cells we have discussed previously.
31 Cerebrospinal Fluid (CSF) left is CAP CM 25 – CSF 250x malignant cellsRemember – we classify them as ‘other’ or ‘unclassified’ and take the slide to the cytologist / pathologistRight is leukemic cells found in CSFAccording to the protocol of your clinical facility or future work place, you would classify / call these cells suspicious, or unclassified. The slide must then be brought to the immediate attention of the department supervisor, a cytologist or pathologist. Again, follow the protocol of the clinical facility.
32 Cerebrospinal Fluid (CSF) Cellular inclusionsErythrophageSiderophageHematoidin crystals (see below)ASCP 21 CSF erythrophage, with few iron granules forming1991 CAP 30 CSF hematoidin crystal / bilirubin crystalASCP 6 macrophage, lymphocyte, siderophageMacrophages by definition are ‘big eaters’. They are produced by differentiation of monocytes. – that is to say, they are the result of a changed monocyte.both monocytes and macrophages are phagocytes.As such, Their role is to engulf and then digest cellular debris, foreign objects.A macrophage that has engulfed RBCs is more properly named an erythrophage.As the digestion of the RBCs takes place, the erythrophage becomes a siderophage.When the digestion process has continued along further, all that remains is the indigestible bilirubin crystal – also called hematoidin crystal.Review the microphotographs showing macrophages in each of these various phases. Again these are all macrophages, but be sure you can identify them by the more proper name or terminology.
33 Cerebrospinal Fluid (CSF) CSF Quality ControlCommercial quality control samples available>>>>>>>>>>>>>>>>>>>>>>>>>>>ChemistryBlood – brain barrier causes selective filtrationAbnormal valuesfrom altered permeabilityIncreased productionIncreased metabolismAs we move our discussion of analysis of CSF, I want to first inform you that there are commercially prepared quality control samples that should be analyzed in parallel with the patient CSF sample.As covered previously, the blood – brain barrier causes a selective filtration of plasma, giving the csf fluid a unique set of values for many chemical analytes and therefore the CSF normal values for the chemical analytes is different than for those of plasma.Abnormal values of CSF analytes are the result of an altered permeability of the blood- brain barrier or the result of an Increased production or am increased metabolism
34 Cerebrospinal Fluid (CSF) - protein Normal 15 – 45 mg/dL .Albumin fraction. If IgG – from damaged B-B, or CNS produced? Can electrophoresis to evaluate oligoclonal / malignant bands.Decreased levels not significantIncreases levelsDamaged B-B (as in meningitis or hemorrhage)Production of immunoglobulins within CNS (MS)Degeneration of neural tissueDye-binding methods – preferredAlkaline biuretCoomassie brilliant blue - a blue color produced is proportional to the amount of protein present (Beers Law)Cerebrospinal Fluid (CSF) – proteinThe first of the CSF chemistriys to discuss is the proteinCSF protein’s normal value is very low, as compared to serum or plasma. The csf protein normal range is 15 – 45 mg/dL .In normal situations, albumin is the only protein fraction found.The presence of the IgG gobulin fraction brings about the concern of whether it came from a damage in the blood- brain barrier or whether it was produced within the central nervous system it self.Protein electrophoresis is one method used to identify the presence of an oligoclonal – meaning few or small clone or / malignant bands.Decreased levels of csf protein are not significant, the pathology lies finding an abnormally increased protein level.CSF protein is best measured by a Dye-binding methods where the protein will couple with a dye or other molecule that can be easily measured in a spectrphotometer.The Alkaline biuret procedure has been used, but Coomassie brilliant blue procedure is preferred. In this procedure - a blue color produced is proportional to the amount of protein present ( and follows Beers Law)
35 Cerebrospinal Fluid –MS Panel Multiple SclerosisDiagnosis is difficult – no one specific testCSF Protein electrophoresisLooking for oligoclonal bandsMyelin Basic ProteinAbnormal protein that indicates demyelinationof neuron axonsMeasurement used to monitor course of disease and effectiveness of treatmentIgG levels (both serum and CSF)IgG index = CSF IgG mg/dl) / serum IgG (g/dl) CSF albumin (mg/dl) / serum albumin (g/dl) NV < 0.77Albumin (both serum and CSF)IgG synthesis rate.Multiple sclerosis is an autoimmune disease that affects the central nervous system. Symptoms vary, because the location and severity of each attach can be different . Episodes can last for days, weeks, or months. In between attacks the patient may have few or no symptoms.Diagnosis of multiple sclerosis is difficult and often comes about after everything else has been eliminated.Some laboratories offer a multiple sclerosis panel of tests to be used in support of the clinical findings, patient history and other data.For most of the analytes tested, you must test both the serum and CSF in parallel. In the electrophoresis, there is a direct comparison where you look for ‘oligoclonal bands’ in the CSF that are not present in the serum.Myelin Basic Protein is an abnormal protein and its presence indicates the demyelination of neuron axons.A comparison of the serum and csf levels of albumin, and immunoglobulin G can also provide useful information in making a diagnosis and monitoring the progression of multiple sclerosis.
36 Cerebrospinal Fluid (CSF) - glucose Selectively transported across blood-brain barrierNormal values: 60-70% of blood glucoseSTAT procedure, glycolysis reduces level quickly.Procedure performed as for blood specimenDecreased levels seen in bacterial & fungal meningitisHypoglycemiaBrain tumorsLeukemiasDamage to CNSThe next csf chemistry test is glucoseGlucose is Selectively transported across blood-brain barrierNormal csf glucose level 60-70% of the patient’s current blood glucose.It is very important that the csf glucose be analyzed quickly . As any organisms or cells present in the csf will quickly reduce the glucose level. When testing, the patients blood glucose should also be tested in parallel to provide a reference or expected value.The csf glucose Procedure is performed in the same manner as for blood specimenDecreased levels of CSF glucose are part of the hallmark findings seen in bacterial & fungal meningitisA patient who is hypoglycemic ( has low blood sugar) will have low CSF glucose.Other reasons for decreased CSF glucose include Brain tumors, Leukemias and as a result of Central nervous system damage.
37 Cerebrospinal Fluid (CSF) CSF LactateNormal values = mg/dLIncrease as result of hypoxiaBacterial meningitis. Head injuryCSF GlutamineNormal 8-18 mg/dLIncreased levels associated with increases in ammonia (toxin)CSF EnzymesLactate dehydrogenase (LDH or LD)5 isoenzyme types; LD1&LD2 are in brain tissueCreatine kinase (CPK or CK)Isoenzyme CK3/ CK-BB from brain tissueFollowing cardiac arrest, patients with CSF levels <17 mg/dL have favorable outcome.CSF Lactate Normal values mg/dLIncrease levels occur as result of hypoxia – or low oxygen, as may be seen in cases of Bacterial meningitis and Head injuryMeasuring CSF and blood lactate levels in parallel can be used to assist in differentiation between bacterial and viral meningitis.Repeat testing is useful in determining whether a treatment or therapy is effective as the lactate Levels return to normal quickly if therapy is effectiveHemolysis or xanthrochromica will cause an increased resultGlutamine is another chemistry test performed on CSFIncreased levels above the Normal 8-18 mg/dL is associated with increases in ammonia (toxin)Occasionally measurement of LDH and CK isoezymes are ordered as part of the analysis of the cerebral spinal fluid.For Lactate dehydrogenase - of its 5 isoenzyme types; LD1&LD2 are found in brain tissue.Creatine kinase (CPK or CK) Isoenzyme CK3/ CK-BB is primarily from brain tissueFollowing cardiac arrest, patients with CSF CK levels <17 mg/dL have favorable outcome.
38 Differential Diagnosis of Meningitis by Laboratory Results BacterialViralTubercularFungalIncreased WBC countNeutrophilsLymphsLymps & MonosLymphs & MonosMarked ↑ proteinMod. ↑ proteinMod-Marked ↑ proteinMarked ↓ glucose↔ normal glucose↓ glucoseNormal to ↓ glucoseLactate > 35 mg/dLLactate normalLactate > 25 mg/dL+ gram stainsPellicle formation+ India ink with Cryptococcus neoformans+ bacterial antigen tests+ immunological test for C. neo.I have consolatatedI strongly encourage you to spend some time evaluating this chart that compares the various lab results seen in bacterial, viral tubuarlular and fungal meningitis.
39 Cerebrospinal Fluid (CSF)- microbiology Gram stain – Extremely important for early diagnosis of bacterial meningitisEven when well performed, 10% false negatives occurUse of Cytospin to concentrate specimen increases sensitivityCultures- Aerobic & Anaerobic. Culture blood at same timeOrganismsNewbornsE. coli & group B Strep.ChildrenStreptococcus pneumoniaeHemophilus influenzaeNeisseria meningitidisAdults -Staph. aureus (if a shunt is present)ImmunocompromisedCryptococcus neoformans,Candida albicans, Coccidioides, orany opportunistic organismOne of your unit objectives addresses the usefulness of the direct csf gram stain and organisms that are common causes of meningitis in newborns, children, adults and immunocompromised patient populations. You should find this slide most useful in addressing these areas.Mixed cells and intracellular bacteria
40 Cerebrospinal Fluid (CSF) India-ink / nigrosin preparationNegative stain to view the encapsulated Cryptococcus neoformans (often AIDs /immunocompromised complication)Instead of stain, can also use dark field microscopy for same effect.These direct procedures 25-50% sensitivityPrefer latex agglutination tests, better resultsAnother classical, but somewhat outdated test performed on csf is the India-ink / nigrosin stain preparationThis procedure has been used to identify the presence of Cryptococcus neoformans.While once relatively rarely encountered, this organism is now commonly found in the csf of AIDS patients .It is also seen in other immunocompromised patient populations.The cryptococcus organism is surrounded by a capsule.When the specimen containing the organism is mixed with the stain and viewed under the microscope it can be seen or detected by how its capsule pushes away the stain particles making the organism appear to glow.The same affect can be achieved by using a dark-field microsckope instead of the negative India ink / nigrosin stain.Because These direct procedures have 25-50% sensitivity , serological latex agglutination tests are preferred.
41 Cerebrospinal Fluid (CSF) SerologyVDRL (Veneral Disease Research Laboratory)For detection of neurosyphilisOn CSF test low sensitivity, but great specificityFTA-Abs also used on CSF, more sensitive, but must prevent blood contamination.SerologyThe VDRL test is used for the preliminary detection of neurosyphilis.This test has low sensitivity, but high specificity.That means that it may not detect all cases, but a positive result is a true result.Another serological test for syphilis is the FTA-Abs which stands for fluorescent treponemal antibody absorbed which is used to detect antibodies to treponema pallidum – the organism that causes syphilis.This test is more sensitive than the VDRL, but it use in the diagnosis of neurosyphilis is still controversial as it is extremely sensitive to the effects of contamination with blood.FTA-Abs also used on CSF, more sensitive, but must prevent blood contamination.
42 Differential Diagnosis of Meningitis by Laboratory Results BacterialViralTubercularFungalIncreased WBC countNeutrophilsLymphsLymps & MonosLymphs & MonosMarked ↑ proteinMod. ↑ proteinMod-Marked ↑ proteinMarked ↓ glucose↔ normal glucose↓ glucoseNormal to ↓ glucoseLactate > 35 mg/dLLactate normalLactate > 25 mg/dL+ gram stainsPellicle formation+ India ink with Cryptococcus neoformans+ bacterial antigen tests+ immunological test for C. neo.I again, provide you a copy of the chart of information that can assist in differentiating the different types of meningitis.And finally, a few comments about the future of CSF testing.Polymerase Chain Reaction / (PCR) testing is being rapidly developed to aid in the diagnosis of meningitis.PCR brings to the table, both high sensitivity and specificity in the detection of many viral, bacterial, tubercular and fungal organisms as seen in CSF.When compared to standard procedures, PCR testing is fast, and can be done with small volumes of CSF.Although PCR testing is expensive, there is a potential for cost savings by decreasing overall diagnostic testing . And the time it can safe may mean saving patient lives because appropriate therapy can be started much faster.PCR and other molecular methodology will soon be found throughout the clinical laboratory .This concludes the session on Cerebral spinal fluid analysis.PCR has been especially useful in the diagnosis of viral meningitis. PCR of the CSF has a sensitivity of 95 to 100 percent, and a sensitivity of 100 percent for herpes simplex virus type 1, Epstein-Barr virus, and enterovirus.14 PCR is faster and more sensitive than culture for enterovirus meningitis.22 When PCR is positive for enterovirus, it allows earlier hospital discharge and less intervention.23 [Evidence level B: retrospective chart review]PCR is the most sensitive means of diagnosing CMV infections of the CNS,21 and it has been suggested that PCR should replace brain biopsy as the gold standard for herpes encephalitis.24PCR has a sensitivity of 54 to 100 percent and a specificity of 94 to 100 percent for tuberculous meningitis, and could replace acid-fast bacillus smear and culture as the test of choice.25 PCR is sensitive for acute neurosyphilis but not for more chronic forms.21 PCR also is being studied as a diagnostic tool for bacterial meningitis and other infections of the CNS.12
43 Cerebrospinal Fluid (CSF) CSF Quality ControlCommercial quality control samples availableEND of CSF
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