Analysis of Urine and Other Bodily Fluids PDF

LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela I. CEREBROSPINAL FLUID EXAMINATION - AIDS related Cerebrospinal fluid (CSF) is a clear, colorless fluid Hemorrhage which chiefly dialysate from the blood (...

LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela I. CEREBROSPINAL FLUID EXAMINATION - AIDS related Cerebrospinal fluid (CSF) is a clear, colorless fluid Hemorrhage which chiefly dialysate from the blood (a plasma - trauma ultrafiltrate), and is relatively similar to serum but - Anticoagulant therapy differs in its concentration of the major - Leakage of aortic aneurysm constituents. Metabolic - uremia The major constituents are: - Myxedema 1. The protein - which is extremely low with no Rheumatoid dss fibrinogen MI 2. The glucose - which is approximately two thirds that of the blood sugar. SLE 3. The chloride — which is about 25% higher than the plasma chloride. Methods of collection Pericardiotomy Another difference of cerebrospinal fluid from Pericardiocentesis plasma is that many of the crystalloids in the CSF are of different concentrations. For example, sodium FUNCTIONS Of THE FLUID and chloride high levels while potassium, calcium, bicarbonate, phosphate, sulfate and glucose are of 1. For the protection of the brain from injury by lower levels. acting as a fluid buffer (cushion). 2. It acts as a medium of exchange for the transfer of dia-lyzable material between the bloodstream THE PHYSIOLOGY AND SOURCES OF CEREBROSPINAL and the spinal cord. FLUID: 3. It equalizes the pressure between the brain and The cerebrospinal fluid is formed in the highly the spinal cord. vascular choroid plexuses (tufts of capillary 4. It serves as an excretory channel in the elimination blood vessels) in the ventricles of the brain by of products of nervous metabolism. filtration (secretion and diffusion) from the blood plasma. A small portion is secreted by the ependymal cells lining the ventricle. This fluid COLLECTION OF THE FLUID then enters the subarachnoid space through the The spinal fluid is collected by a physician by foramina of Luselika and Magendie, circulating making a spinal puncture. Aside from all upward over the cerebral hemispheres sa wel sa clean and sterile equipment and materials downward over the spinal cord, and entering for use, precautions are also be taken; such venous blood (blood stream) through arachnoid as avoidance of blood in the collection, villi of the dural sinuses. adding anticoagulant if necessary, and immediate performance of the tests to be LOCATION OF THE FLUID done. 1. Internally it fills the ventricles of the brain (ventricular fluid), the cisternae, (cisternal fluid) and TYPES OF PUNCTURE: the canal of the spinal cord. 1. Lumbar puncture - It is a relatively safe and simple 2. Externally it fills the space between the pia and procedure, but it should not be done unless there arachnoid membranes surrounding the brain and are definite indications. Puncture is between the spinal cord (spinal fluid). third and the fourth vertebrae of the lumbar region. Indications: A) Diagnosis Pericardial fluid 1) To obtain spinal fluid for study Normal volume: 10-50 ml Pericardial effusion: presence of excess blood or 2) To estimate intracranial pressure tissues fluid w/in the pericardial sac. 3) To test for spinal block Produced during: 4) To introduce air or a lipoidal substance. - inflammatory process B) Therapeutic - Malignant process 1) To introduce serum, penicillin, streptomycin, or - Hemorrhagic process anesthetic substance. Common causes 2) To remove blood or irritative exudates. Infection Contraindications: - bacterial pericarditis - TB A) Subtentorial tumors - fungal 1|Page Source: Book/notes of Ma’am Tan  LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela B) Presence of generally increased intracranial 8. Acid-base balance pressure. a) PC02 47.9 mm. Hg. 2. Cisternal puncture, (cistera magna or b) НСО3 22.9 mEg/L suboccipital) 9. Total protein 15.45 mg./100 ml. This is somewhat more dangerous than a lumbar puncture and is usually done only under the a) Lumbar 20-40 mg./100 ml. following conditions: b) Cisternal. 15-25 mg./100 ml. a) Blocked spinal canal c) Ventricular 5-10 mg./100 ml. b) Deformity of the vertebrae 10. Fibrinogen - none c) Infections of the tissues of the back 11. Electrophoretic separation of lumbar fluid, mean 3. Ventricular puncture values: This is done for last resort, although frequently done a) Prealbumin 4.6 土 1.3% in infants whose fontanelles are still open, but rarely b) Albumin 49.5 ‡ 6.5% in adults except in connection with ventriculography. c) Alpha - globulin 6.7 ‡ 20% d) Alpha 2 — globulin 8.3 ‡ 2.1% e) Beta and tetraglubin 18.5 ‡ 4.8% AMOUNT TO BE COLLECTED: f) Gamma globulin 11.2 ‡ 2.7% 12. Calcium (lumbar) 2.32 mEq./L 1. At least 8 to 10 cc. of fluid is necessary for a 13. Magnesium 2.20 mEq/L complete. examination. 14. Creatinine 0.4-15 mg./100 ml: 2. It should be collected in 3 sterile, chemically clean test tubes, numbered 1, 2, and 3. 15. Glutamic oxalacetic transminase 0 - 19 units 3. The first drops are placed in tube 1 and may 16. Lactic dehydrogenose. 8.50 units contain some blood from the puncture. 17. Phospholexose isomerase 0.4.2 Bodansky units a) This fluid should not be used, unless it is necessary for bacteriological examination (smear and 18. Cells 1-5 cells/cu. mm. (lymphocytes) culture). b) Presence of blood affects all tests except for MACROSCOPIC OR PHYSICAL EXAMINATIONS chlorides. 4. Collect 7 cc. in tube 2 for serological, 1. Amount. bacteriological, and chemical tests (Hematologic- differential count and biochemical tests) a) The normal amount is roughly estimated to be 5. Collect about 2 cc. in tube 3 for cell count and about 1 cc. per pound of body weight. Also roughly qualitative protein tests (colloidal gold test). estimated to be about 100 to 150 cc. everyday production. 6. When the fluid obtained is xanthochromic (canary yellow), it is advisable to add a trace of b) There is increased amount in acute and chronic lithium oxalate to tubes 2 and 3 to prevent clotting. congestion of the meninges due to increased 7.The cell count and examination for bacteria and transudation of plasma through the capillaries and sugar must be done at once, while the remaining probably to increased permeability of the choroid tests can be delayed several hours if the specimen plexus. is kept in the refrigerator. c) There is also an increased amount in acute and 8. Venous blood should be drawn at the same time chronic infections due to the production of as the spinal fluid if chemical tests are to be done inflammatory exudate and to increase permeability especially for chloride and sugar. of the capillaries. COMPOSITION OF NORMAL SPINAL FLUID: 2. Pressure: 1. Glucose 45-100 mg./100 ml. a) Normal pressure for the horizontal position varies 2. Urea 8-28 mg./100 ml. between 70 and 200 mm. of water (0-8 mm. of mercury) with the average of 100 to 150 mm. 3. Sodium 117-137 mEg/L 4. Potassium 2.33-4.59 mEg/L 3. Color: 5. Phosphorus, inorganic 1.2-2.1 mg./100 ml. a) Normally the fluid is as clear and colorless as 6. Uric acId 0.07-2.8 mg./100 ml. distilled water. 7. Chlorides 113-127 mEq/L b) Bright red due to fresh blood from a vessel (710-790 mg./100 ml.) punctured while inserting the needle; upon centrifugation the super-natant fluid is clear. 2|Page Source: Book/notes of Ma’am Tan  LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela c) Dull red or brown (depending on the age of the 5) Complete coagulation with xanthochromia lesion) skull or in some intracranial hemorrhages and without hemorrhage occurs in Froin's syndrome chronic hemorrhagic pachmeningitis. After 48 hours (spinal subarachnoid block) hemolysis begins, so that a hemorrhage of at least Test for Fibrinogen (Screening) this age is recognized by the yellow or red color of a) Add 1/3 volume 10% NaOH to a few ce. of spinal the supernatant fluid after centrifugation. fluid and shake. d) Yellow (xanthochromic) may be due to blood b) Observe fine fibrin flocculation that ultimately pigments resulting from disintegration of RBC within condenses on the surface. the subarachnoid space or to altered permeability Sediment: Normally no sediment. Often present in of the blood plasma which under normal conditions are excluded meningitis. 6. Reaction: The normal reaction is slightly alkaline Sodium fluoride pH 7.30 - 7.45. - presukatine for CSF specimen to be mailed (long 7. Specific Gravity: The normal specific gravity is fluoride/ml CSF) 1.006-1.008. - prevents glycolysis by inhibiting enzymatic activity Glucose - in pyogenic & tuberculous meningitis Queckenstedt Test The test is to confirm the presence of any e) Greenish or grayish due to pus cells in severe subarachnoid block. inflammatory reactions and in acute meningitis. Normally, if both jugular veins are manually compressed CSF pressure rises rapidly to over 300 4. Transparency: mm. CSF rapidly returns to normal when compression ceases. In sinus thrombosis, Normal spinal fluid is clear. Fewer than 200 white subarachnoid block at the foramen magnum, or cells/cu. mm. does not give rise to macroscopic a mass lesion at the spinal canal, the rise of CSF clouding of the fluid. may be decreased or delayed. For positive Haziness is produced by 200-500 white cells/cu. results - delayed or decreased rise of CSF mm., and over 500 white cells/cu. mm., cause pressure. turbidity. a) In acute meningitis, the fluid may exhibit varying CHEMICAL EXAMINATION degrees of cloudiness, from slight turbidity to the I. PROTEINS-GLOBULIN (QUALITATIVE TESTS): capacity of pure…pus. b) In the less acute stage of epidemic meningitis, it General Considerations: is sometimes quite clear. a) The protein of chief interest is globulin. c) It is usually clear in tuberculosis and syphilitic b) The test for globulin is value less when meningitis, tubes, poliomyelitis and encephalitis. applied to fluid containing blood, owing to the presence of serum 5. Coagulation: globulins. a) Normal spinal fluid does not coagulate. c) If the fluid is cloudy, it should be centrifuged and b) Abnormal: the clear supernatant fluid used for the test. d) Globulin is increased in meningitis and latent 1) The fluid clots when there is an increase in proteins syphilis. including fibrinogen 2) Numerous small clots (coagulate) occur in 1. Nonne- Apelt Test for Globulin: paresis. 3) A "cobweh" or pine tree or "weblike" clot Principle: A 50% saturated solution of ammonium dedicated to coagulum is typical of tuberculous sulfate precipitates globulin. meningitis, it forms on the surface of the fluid and Reagent: Merck' Purified neutral ammonium sulfate extends down the middle of the tube. 85 gms. Distilled water 100 сс. Boil and filter as soon as it is cooled. a) Twelve or more hours may be required for its for- mation. Procedure: b) The absence of pellicle does not exclude a) Place 1 ml. of saturated solution of ammonium tuberculous meningitis. sulfate in a test tube. c) In purulent meningitis, large clot are seen. b) Add 1 ml. of spinal fluid and mix by inverting the tube. 4) Heavy coagulation and sediment occur in acute sup. purative meningitis. c) Allow the tube to stand for three minutes. d) Fluid that contains a normal amount of globulin will remain clear or become slightly opalescent. 3|Page Source: Book/notes of Ma’am Tan  LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela If an excess of globulin is present, a cloudy c) Heat to boiling. precipitate will form. d) Add 0.2 ml. normal sodium hydroxide. e) Allow to stand for about 10 minutes. 2. Ross-Jones Test f) Precipitation is positive reaction, however in some Principle: Same as Nonne-Apelt. case, precipitates opalescence are delayed for an Procedure: hour or more. Faint opalescence is normal a) Place 2 cc. of ammonium sulfate to the bottom of the tube Total Proteins - Quantitative Test b) Add 1 cc. of spinal fluid using medicine dropper Principle: The protein is precipitated from the spinal (overlay). fluid with acid lungetate, dissolved and c) A clear-cut, thin grayish-white rings appears at precipitated. The nitrogen is determined on an the zone of contract for positive reaction. Under aliquot portion after being put into solution with the aid of sodium hydroxide. normal condition, the ring will appear after 5 minutes. General Considerations: d) Observe for 3 minutes then mix the solution. a) Presence of blood gives false high values. e) Interpretations. b) Presence of bacteria gives unreliable results. + - ring in 3 minutes and no trace after mixing. c) Delay of analysis increases the value detained + + - ring in 3 minutes and faint opalescent after unless fluid is kept sterile and tightly corked. mixing. ++ + - ring in 3 minutes and definite cloud after Quantitative Test for Total Protein: mixing. 1. Direct Test ++++ - ring in 3 minutes and heavy cloud after a) Prepare a test tube (label and tube unknown mixing. and the other blank). b) Place 1 ml. of water to tube marked blank and 1 Albumin Test: ml. of spinal fluid to tube marked unknown. a) Shake the content of the tube used in the d) Read the value by spectrophotometer method globulin test and filter. then cal. culate as follows. b) Acidify with 1 drop of 10% acetic acid and boil. Gm. of protein X 1,000 c) A slight cloudiness is normal. 20 d) Interpretations: = mgs. protein per 100 ml. spinal fluid Negative - a slight cloudiness 2. Sicard-Cantelouble Test + - a definite cloudiness with fine precipitate. This technique uses a special graduated 21 cm. long + + - a flocculant precipitate in a slightly cloudy fluid. tube having a 7 mm. diameter. ++ + - a heavy flocculent precipitate in a clear fluid. The graduation in the bottom is 0.2 cc. and the topmost is 4 cc. Pandy's Test Principle: The reagent precipitates albumin and globulin. Procedure: Reagent: Phenol crystals 10 gms. Distilled water 100 a) Place 4 cc. of spinal fluid into a test tube. cc. b) Heat to 60° or 80°C. Procedure. c) Add 12 drops of 33% trichloracetic acid. a) Place 1 cc. of saturated aqueous solution of d) Allow to stand for 5 minutes, then invert the tube phenol in a small test tube. a few times. b) Add 1 large drop of spinal fluid. e) Let stand for 24 hours and read the quantity of c) A bluish white cloud immediately forms as soon the sediment. as the drop nixes with the reagent in increased Interpretations: globulin. 1st graduation equal to 0.22 gm. protein/L d) Normal fluids may show a faint trace but this 2nd graduation equal to 0.44 gm. protein/L should be reported as negative. 3rd graduation is equal to 0.56 gm. protein/L 4th graduation equal to 0.71 gm. protein/L 4. Noguchi's Test (Qualitative Detection of Proteins): 5th graduation equal to 0.85 gm. protein /L a) Place 0.2 ml. of spinal fluid into a test tube. The normal value of protein does not exceed 0.30 b) Add 1 ml. of a 10% solution of butyric acid in gm. protein/L. normal salt solution. 4|Page Source: Book/notes of Ma’am Tan  LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela This stabilizes the color, eliminating steps II. SUGAR: (e) and (f). e) To stabilize the color, place the tubes into the General Considerations: boiling water both again for 2 minutes. Time. a) The test for sugar must be performed within one- f) At the end of 2 minutes, return to the room half hour after withdrawal of the fluid, because temperature water bath to cool. glucose gradually decomposes on standing. b) Blood for glucose determination must be drawn g) Dilute to the 25 ml. mark with distilled water. at the same time. Stopper and mix thoroughly by inversion. c) If possible, the spinal fluid and blood for this test h) Pour into cuvette and read against the blank at should be drawn before breakfast. 420 millimicrons. d) The glucose in spinal fluid is normally 60% of that in blood. Calculations. O.D. (unknown) X Mg. standard X 500 = Mg. % Qualitative Test: glucose. 1. Benedict's Test O.D. (standard) a) Place 0.5 cc. of Benedict's qualitative reagent to a test tube. NOTE: The standards are made so that a 2.0 ml. volume will contain the following mg. contribution: b) Add 4.5 cc. of distilled water. a) (0.05 mg./ml.) 0,1 mg. equivalent to 50 mg. % c) Heat to boiling. b) (0.10 mg./ml.) 0.2 mg. equivalent to 100 mg. % d) Add 1 cc. of spinal fluid. c) (0.02 mg./ml.) 0.4 mg. equivalent to 200 mg. % e) Boil again for 1 to 2 minutes Calculate the glucose, using the standard that reads closest to the unknown. f) Allow to cool. Interpretations: III. CHLORIDES: Normal sugar in CSF - turbid greenish yellow Absence of sugar (pathological) - no change of General Considerations: color. a) Blood for Chloride determination should be Excess of protein but no sugar - deep purplish-violet drawn at the same time the spinal fluid is withdrawn. or pinkish-violet, color. b) The chlorides in spinal fluid is normally 25% higher Qualitative Test: than that in the blood. Nelson-Somogyi Test Test - Quantitative: Blank (1 tube) 1. Schales and Schales a) Pipet 2.0 ml. of distilled water into sugar tube. Procedure the same as in the Chemistry procedure. Standards (3 tubes) b) Pipet 2.0 ml. of working standard (0.05 mg/ml.) into sugar IV. ALBUMIN-GLOBULIN RATIO TEST - LANGE'S COLLOIDAL GOLD TEST tube. c) Pipet 2.0 ml. of working standard (0.20 mg./ml.) Principle: The changes in color of the colloidal gold into sugar tube. are the result of differences in Unknown; (1 tube per test) the aggregation of colloidal particles due to varying a) Pipet 2.0 ml. of spinal fluid into each tube. quantities of gamma globulin in the spinal fluid. To all tubes: General Considerations. a) Add 2.0 ml. of alkaline copper. Mix. a) spinal fluid containing blood cannot be used, b) Place tubes into boiling water both for 8 minutes. because variable reactions and false positive Time. reaction is mostly to occur. c) After 8 minutes, place tubes into room b) If the spinal fluid cell count is above normal, the temperature water bath to cool. Do not disturb the fluid should be centrifuged and the supernatant precipitate. fluid used for the test. d) Add 2.0 ml. of the phosphomolybdie acid. Mix. Procedure: (The alternate method: acid. a) Arrange a series of 12 chemically clean, dry, test Mix. tubes in a rack with an opaque glass back. Add 3.0 of alternate phosphomolobyc 5|Page Source: Book/notes of Ma’am Tan  LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela b) Place 0.9 cc. of freshly prepared 0.4% NaCL NOTE. A trace of blood in the CSF may give rise to a solution in the first test tube and 0.5 cc. in each of meningitis curve, while bacterial contamination the next 10 tubes. makes the result unreliable c) Place the 0.85 ce. of a 1% NaCL solution in the 12th tube. PREPARATION OF COLLOIDAL GOLD SOLUTION: d) To the first tube add O.I ce. of the spinal fluid, General Considerations: which must be free from any trace of blood a) Use chemically clean glasswares. 1) Mix well by sucking the fluid up into the pipet and b) Clean again with aqua regia (1 volume HNO3 to expelling it 4 times, and transfer 0.5 cc. to the 3 volume second tube. HCI) and rinse thoroughly first with single distilled 2) Mix in a similar manner and transfer 0.5 cc. to the water then with double distilled water. third tube; repeat this in each successive tube to c) All chemicals should be Merck's "Blue Label". and including the tenth tube. d) The solutions should be made in double distilled 3) Discard 0.5. cc. from the tenth tube. water and the sodium citrate solution made fresh each time. 4) The 11th and 12th tubes are controls and contain no spinal fluid. Borowskaja's Modification of Lange's Test e) To each of the 12 tubes, add 2.5 cc. of the colloidal gold solution: the 11th tube serves as a Procedure: control for the esta-bility of the colloidal gold, while a) Add 10 cc. of a 1% solution of acid yellow gold the 12th tube is a control for the sensitivity of the chloride to 950 cc. of double distilled water in a 2 colloidal gold. liter beaker. (The 12th tube should become colorless in an hour's b) Heat to 90 degrees time). centigrade and add 50 cc. of a 1% f) Let stand overnight and read the result (color changes) solution of sodium citrate. the next morning. c) Boil, stirring constantly until a dark-red color appears, then watch carefully to the daylight until a g) The reaction in each tube is reported in the order cherry red color appears without any evidence to in which the tubes stand. from 1 to 10. They may be blue. The longer the solutin boils, the more sensitive charted as follows: it becomes. normal reaction 0000000000 d) Allow the solution to cool slowly. Paretic curve or Zone 1 seen in insane and multiple myeloma patients 5555543000 e) After the solution has stood for 24 hours, it must be checked with a known positive and negative spinal Luetic or Tabetic or Zone II, seen in syphilitie fluid. patients 0003333000 Meningitis or Zone III, seen in TB meningitie patients 0000244440 Mastic Test: Curve in brain tumor and tuberculosis meningitie Principle: The principle of this test is similar to that of patient 0000243000 the LANGE’S Colloidal Test. Although the solution is much easier to prepare satisfactorily than the colloidal gold, and a little blood in the CSF or mild NOTE: bacterial contamination does not affect the result, Paretic curve shows a mark change in the first 3 no diagnostic patterns are given but merely one tubes abnormal type response. Luetic curve shows a greatest change in 3rd and 4th tubes. Meningitic curve shows greatest change in the Reagents: Mastic Solution. last three tubes. Gum Mastic 10 gms. U.S.P. Absolute alcohol 100 сс. Add 18 cc. of absolute alcohol, mix well and Reading and Interpretations: pour rapidly into 80 cc. of distilled water. The tubes are scored by the appearance of the fluid in the tube. Alkaline-Saline Solution: Unchanged deep red 0 Make a 1.25% solution of sodium chloride (C. P.) in Red to blue 1 distilled water, and to each 99 ce. of this solution, Lilac to purple 2 add 1 cc. of a 0.5% solution of potassium carbonate Deep blue 3 in distilled water. Pale blue (partial precipitate) 4 Procedure: Colorless (complete) 5 a) Take a series of six small test tubes. 6|Page Source: Book/notes of Ma’am Tan  LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela b) In the first tube, place 1.5 ec. of the alkaline-saline c) Add 2 or 3 drops of 2% solution of formalin. solution and in each of the others place 1 cc. d) Shake the tube and allow the mixture to stand for c) To the first tube, add 0.5 cc. of the spinal fluid 4 to 5 minutes. which must be completely free from blood. e) Add carefully (over lay) to form a supernatant d) Mix by sacking the fluid up into the pipet and layer 1 to 2 ce. of 0.06% solution of sodium nitrite. expelling it. f) Allow the mixture to stand for two to three minutes. e) Transfer the 1 cc. to the second tube. g) Positive reaction is indicated by the presence of f) Again mix and transfer 1 cc. to the third tube and violet rings at the zone of contact of two layers. continue down the line to the fifth tube, discarding Negative reaction if brown ring or absence of a the 1 cc. portion which is removed from this. colored ring. g) Leave the sixth tube with an alkaline-saline NOTE. A purple ring is given by bloody, purulent or solution alone to serve as a control. xanthochronic fluids. The test is positive in a very H) Finally, add 1 cc. of the mastic solution to each large percentage of tuberculous meningitis. but it is tube. i) not diagnostic. It is usually negative in syphilitic I) Mix well and set aside at room temperature for 12 meningitis, poliomyelitis and brain tumor. to 24 hours, or in the incubator for six to twelve hours. j) Tubes in which the reaction is complete will show 2. Levinson's Test a heavy precipitate with clear supernatant fluid. Reagents: a) A 1% solution of mercuric chloride. Colloidal Benzoin Test b) A 3% solution of sulfosalycylic acid. This test is similar to many respects to the mastic test. It is not specific for neurosyphilis, but does Procedure: give practically the same results as the more a) Place 1 mm. of CSE to each of two. complicated colloidal gold test. b) Add 1 ce. of 2% aqueous solution of sulfosalycylic acid to test tube 2. Reagents: c) Stopper the tubes and allow them to stand at Benzoin Solution room temperature for 24 hours. Sumatra benzoin resin 1 gm. Absolute alcohol 10 сс. d) Measure the heights of the sediments. e) Normal CSF has less than 2 mm. sediments. Filter the clear supernatant liquid after 48 hours. This Abnormal - increase protein; stock solution is prepared every time you use it. Sediment in mercuric chloride is light and featherly and forms slowly. Stock Solution Sediment in sulfosalycylic acid is compact and Add 3 cc. of stock solution drop by drop with heavy and forms rapidly. In tuberculous meningitis, constant shaking to 20 cc. of double distilled sediment in mercuric chloride is 2 mm. or more and water. Heat 36°C in water both with constant 2 or more times that in the sulfosalicylic acid tube. shaking. In suppurative meningitis, sediment in Salt Solution sulfosalicylic acid tube is greater than in the Make a 0.01% solution of sodium chloride in a mercuric chloride tube. double distilled water Accurate Colorimetric Total Protein Determination - Dennis - Ayer Method CHEMICAL TESTS FOR TUBERCULOSIS MENINGITIS: (QUANTITATIVE - TURBIDITY OF TEST) 1. Tryptophane Test Procedure: Reagents: a) A few cc. of CSF a) Concentrated hydrochloric acid b) Add a few cc. of sulfosalicylic acid. b) A 2% solution of formalin (1:20 dilution of formalin- c) Proteins are precipitated and read in water) photoelectric colori-meter, if not photoelectric colorimeter, read with visual colorimeter. c) A 0.06% solution of sodium nitrite. d) Blood, bacteria, and fungi cause high protein Procedure: reading here. a) Place 2 or 3 ce. of spinal fluid in a large test tube. b) Add 15 to 18 ce. of concentrated hydrochloric MICROSCOPIC EXAMINATION acid. 7|Page Source: Book/notes of Ma’am Tan  LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela TOTAL AND DIFFERENTIAL COUNTS IN Method for Counting Mixture of White and Red Cells: CEREBROSPINAL FLUID: To find the true white cell count when the Cells should be counted as seen as the cerebrospinal fluid is bloody, perform red and specimen is received, since they rapidly white cell counts on the patients blood as well as deteriorate. The method of counting will vary on the cerebrospinal fluid specimens multiply the with the number of cells expected. ratio of the red cell count of the fluid to the red cell count of the fluid to the red cell count of the blood by the blood leukoyte count of the spinal Method For Low White Cell Count fluid. The spinal fluid appears completely clear. Example: Procedure: RBC of blood 5 million and of spinal fluid 25,000 Transfer 1 drop well-mixed undiluted fluid to a WBC of blood 12,000 and of spinal fluid counting chamber and count all the cells in 9 large 70/mm^3. squares. 25,000 X 12,000 5,000,000 Since this represents a volume of 9/10 mms, the 70 - 60 = 10 white cells/mm^3 represents the true result is multiplied by 10/9 to obtain the number of CSF white cell count. cells/mm For practical purposes the multiplication Normal blood will add 1 leukocyte for each 700 may be obtained. red cells. Example: A total of 200 cells is counted in 9 large squares. The Significance of Total White Cell Count: corrected count is 200 X 10/9 = 222 cells/mm. The normal spinal fluid is essentially free of cells, Method for Moderate White Cell Count. containing from 0-5 cells/mm^3, chiefly small Diluting Fluid: lymphocytes. Infants at the age of a few weeks Crystal violet 0.2 gm. may have as many as 30 lymphocytes/mm^3 in the CSF. Glacial acetic acid 10.0 ml. Distilled water to 100 ml. Variations in Diseases: Procedure: The total cell count is usually normal in multiple a) Mix specimens thoroughly. If not very cloudy or sclerosis, epilepsy, brain tumor, meningismus, bloody, draw CSF to mark 1 in a white cell counting and cerebral arteriosclerosis. It is elevated in pipet and then draw diluting fluid to mark 11, syphilis (29-100), viral meningitis, and 50% of producing a dilution of 1:10. incephalitis, and reaches highest in pyogenic meningitis b) Mix, discard 1/3, and place 1 drop on each side Sometimes the cell count and the protein are of the blood counting chamber, as in the method not increased together. for leukocyte counting chamber. and add the The cells are increased without a corresponding results of all 8 squares counted. increase in protein in aseptic meningitis, and the protein is Calculation: increased without an increase of cells in Froin, or blockage, syndrome and in Guillain-Barre Divide the sum by 8 to obtain the syndrome. number/single large square. An increased cell count must always be Multiply by 10 X 10 = 100 to obtain the number confirmed by a stained smear and a differential of cells in count. a 1 mm^3. The first 10 converts the number found in 0.01 mm, to the number found in 1.0 mm^3, and the second 10 represents the Preparation of Smear for Differentio! Count: dilution factor. Various methods of cell concentration have If the total cell count is low or moderate a rough been described. estimate of the differential count can be Centrifugation is the easiest but least obtained by classifying the cells seen in the satisfactory, as cells may be damaged. counting chamber. Sedimentation techniques are probably best, but the necessary equipment is not Method for High White Cell Count: commercially available. Sedimentation of cells directly onto the slide. appears to be superior to the Millipore technic. If centrifugation is used as The white cell count on a purulent cerebrospinal the method of concentration, the sediment is fluid is doue as outlined for white cells on the smeared similar to 1 drop of blood in the peripheral blood. preparation of a blood film. Whatever concentration method is used, the cells are 8|Page Source: Book/notes of Ma’am Tan  LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela stained with Wright or Papanicolaou stain in Large Intestine - capable of absorbing differentially counted. approximately 3000 mL of H2O. When the An increase in lymphocytes was originally called amount of water reaching the large intestine pleocytosis, a term that now includes the exceeds this amount, it is excreted with the solid increase of all or any type of cell. fecal material, producing diarrhea. Constipation, on the other hand, provides time BACTERIOLOGICAL EXAMINATION: for additional water to be reabsorbed from the fecal material, producing small, hard stools. 1. Smears A. Direct Diarrhea 1) Centrifuge the fluid at a high rate of speed for 15 Defined as an increase in daily stool weight minutes. above 200 g with increased liquidity and 2) Make a smear, dry, fix with heat and stain by frequency of more than three times per day Gram's Method. (>3x). B. Smear for tubercle Bacilli Diarrhea lasting less than 4 weeks is deemed as acute, and diarrhea persisting for more than 4 Fungi - increased in number in torula meningitis. weeks is termed chronic diarrhea. Parasites - larvae of Trichinela a) Secretory Diarrhea Caused by Bacterial, viral, and II. FECALYSIS (FECAL ANALYSIS) protozoan infections PURPOSES: b) Osmotic Diarrhea For the early detection of gastrointestinal (GI) Incomplete breakdown or reabsorption of food bleeding, liver, biliary duct disorders, presents increased fecal material to the large maldigestion/malabsorption syndromes, & intestine, resulting in the retention of water and inflammation. electrolytes in the large intestine Detection and identification of pathogenic bacteria and parasites. MACROSCOPIC STOOL CHARACTERISTICS Color/ Possible Cause PHYSIOLOGY Appearance Digestive enzymes: Trypsin, Chymotrypsin, Amino Black Bismuth (antacids) Peptidase, and Lipase. Iron Therapy Charcoal Bile salts provided by the liver aid in the digestion Upper gastrointestinal of fats. bleeding A deficiency in any of these substances causes Red Lower gastrointestinal the inability to digest and, therefore, to reabsorb bleeding certain foods w/c may the increased the Beets and food coloring undigested/unabsorbed material leading to Rifampicin symptoms of maldigestion/ malabsorption. Pale yellow, white, Barium sulphate Approximately 9000 mL of ingested fluid, saliva, gray Bile-duct obstruction gastric, liver, pancreatic, and intestinal Green Biliverdin/oral antibiotics secretions enter the digestive tract each day. Green vegetables 500 to 1500 mL of this fluid reaches the large Bulky/frothy Pancreatic disorders intestine Bile-duct obstruction About 150 mL is excreted in the feces Ribbon-like Intestinal constriction Mucus/blood- Colitis streaked mucus Dysentery Malignancy Constipation MICROSCOPIC EXAMINATION OF FECES Is performed to detect the presence of leukocytes associated with microbial diarrhea and undigested muscle fibers and fats associated with steatorrhea. Fecal Leukocytes Primarily neutrophils are seen in the feces in conditions that affect the intestinal mucosa, 9|Page Source: Book/notes of Ma’am Tan  LECTURE | MIDTERM Transcribed by: Jimarfil & Ivy | Organized by: Angela such as ulcerative colitis and bacterial Nephrosclerosis – primary site of injury; vascular dysentery. system of the kidney. Microscopic screening is performed as a Pyelonephritis – inflammation of the kidney and its preliminary test to determine whether diarrhea is pelvis. being caused by invasive bacterial pathogens Renal calculi – concretions in any part of the urinary including Salmonella, Shigella, Campylobacter, tract which are usually composed of mineral salts Yersinia, and E. coli. 1. U.A. Staphylococcus aureus, Vibrio spp., viruses, and 2. CaC2O4 parasites = do not cause the appearance of fecal leukocytes. 3. Cystine crystals Specimens can be examined as wet 4. Phosphates preparations stained with methylene blue or as dried smears stained with Wright’s or Gram stain. U.A. and Urates - colored yellow to brownish red Methylene blue staining is the faster procedure and are moderately hard but may be more difficult to interpret. CaC2O4 – most common; very hard dark Lactoferrin latex agglutination test colored and have a rough surface. - For the detection of fecal leukocytes. PO4’s – usually pale and friable H2NCONH2 -Urine - Remains sensitive in refrigerated and frozen specimens. The presence of lactoferrin, a component of granulocyte secondary granules, is indicative of an invasive bacterial pathogen. Muscle Fibers − Aids in diagnosis and monitoring of patients with pancreatic insufficiency, such as in cases of cystic fibrosis. ↑ Striated fibers = seen in biliary obstruction & gastrocolic fistulas. Specimen is emulsified with 10% alcoholic eosin, w/c enhances the muscle fiber striations. Undigested fibers have vertical and horizontal striations. Partially digested fibers exhibit striations in only one direction, and digested fibers have no visible striations. 10 undigested fibers are considered increased. Patients should be instructed to include red meat in their diet prior to collecting the specimen. ADDITIONAL INFO FOR KIDNEY DISEASES Glomerular Disorder – majority are of immune origin resulting to immunologic disorders throughout the body. IgA Circulate in the blood stream and are deposited in the glomerular membrane. Components of the immune system, including: Neutrophils, Lymphocytes, Monocytes, and Cytokines are then attracted to the area producing changes and damage to the membrane. Glomerulonephritis – associated with the finding of blood, protein, and cast in urine. Nephritis (Bright’s disease) - A degenerative and inflammatory condition of the kidney Nephrosis - The tubules are the site of primary injury. 10 | P a g e Source: Book/notes of Ma’am Tan