CMNOTES PDF Clinical Microscopy - Urine Analysis

CLINICAL MICROSCOPY URINE I. URINALYSIS -Analysis of urine Physically,Chemically and Microscopically -Frederik Dekkers: discovered albuminuria in 1694 -Richard Bright: introduced the concept of urinalysis A. URINE COMPOSITION -95% water...

CLINICAL MICROSCOPY URINE I. URINALYSIS -Analysis of urine Physically,Chemically and Microscopically -Frederik Dekkers: discovered albuminuria in 1694 -Richard Bright: introduced the concept of urinalysis A. URINE COMPOSITION -95% water -5% solutes -Urea is a metabolic waste product produced in the liver from the breakdown of protein and amino acids; accounts for nearly half of the total dissolved solids in the urine; major organic solute present in urine -Chloride is the major inorganic solid dissolved in urine followed by Na and K -To determine whether a particular fluid is urine, the specimen can be tested for its UREA and CREATININE content since both of these substances are present in much higher concentrations in urine than in other body fluids B. URINE VOLUME -Normal daily urine output is 1,200 to 1,500 mL, although the range 600 to 2,000 mL is considered normal taking into account the factors that influence urine volume like fluid intake, fluid loss, variations in the secretion of ADH and need to excrete increased amounts of dissolved solids. -ANURIA-cessation of urine; it is sometimes defined as being 1% is significant; the percentage of Eosinophils in 100-500 cells is determined -LYMPHOCYTES: associated with early renal transplant rejections -MONONUCLEAR CELLS: Lymphocytes, Monocytes, Macrophages, and Histiocytes -When mononuclear cells constitute 30% or more of a deff count, Chronic Inflammation is indicated 3. Epithelial Cells a. Squamous -largest cell found in urine sediment -Least clinically significant epithelial cell -most often associated with vaginal contamination -CLUE CELLS: Squamous epithelial cell studded with Gardnerella vaginalis b. Transitional/Urothelial/Bladder cell -originate from the lining of the renal pelvis, calyces, ureters, and bladder and from the upper portion of the male urethra -present following invasive procedures such as catheterization and are of no clinical significance -Occasionally, these cells may contain 2 nuclei -has centrally located nuclei -a helpful clue on the proper identification is the characteristic Endo-Ecto Cytoplasmic Rim  c. Renal Tubular Epithelial Cell -Columnar or convoluted(PCT); Round or oval(DCT) and Cuboidal(CD) with an eccentric nucleus, possibly bilirubin stained or hemosiderin laden -most clinically significant of the epithelial cells -originate from the nephrons of the kidney -more than 2 RTE cells/hpf indicates tubular injury -OVAL FAT BODIES: lipid containing RTE cells most often found in Px with Nephrotic Syndrome -BUBBLE CELLS: RTE cells containing large, nonlipid-filled vacuoles found in Acute Tubular Necrosis 4. BACTERIA -Indicative of UTI; Correlated to Nitrite and LE rgt strip result 5. YEAST -Often dound in Px with DM -Confused with RBC (in yeast, look for budding) -Primary yeast: C. albicans -A true yeast infection should Be accompanied By the presence of increased WBCs 6. PARASITES -Most commonly encounters are T. vaginalis -S. haematobium ova is also seen (large terminal spine) -E. vermicularis when seen indicates fecal contamination 7. SPERMATOZOA -Not reported in routine urinalysis (or reported Based on laB protocol) -Correlated with a positive protein result in reagent strip test 8. MUCUS -Protein material produced By the glands and epithelial cells of the lower genitourinary tract -Made up of Tamm-Horsfall protein(Uromodulin) -Thread-like structures with low refractive index -No clinical significance 9. CASTS -The only sediment that is unique to the kidneys; formed within the PCT and CD -Should Be examined under subdued light -Located along the edges of the coverslip and reported as average per 10lpf -Visualization is enhanced by Phase Contrast Microscopy except Fatty casts because they are more visualized vividly in Polarizing Microscope -Major constituent of casts is Tamm-horsfall Protein(Uromodulin) -CYLINDROIDS are structures with tapered ends formed at the junction of the Ascending Loop of Henle and the DCT. They have the same significance as casts. In fact presence of urinary casts is termed Cylindruria -Factors involved in Cast Formation include Urinary stasis, Increased Acidity, and High solute concentration a. Hyaline Cast -Most frequently seen cast -NORMAL: 0 to 2 per lpf b. RBC CASTS -Primarily associated with Glomerulonephritis -Also observed in urine of individuals after participating in a strenuous exercise -They appear orange-red in color(Henry: Yellow under LPO) -Examination under high power magnification should concentrate on determining that a cast matrix is present, thereby differentiating the structure from a clump of RBCs. Because of the serious diagnostic implications of RBC casts, the actual presence of rbcs must also be verified to prevent the inaccurate reporting of nonexistent rbc casts. It is highly improbable that rbc casts will be present in the absence of free-standing rbcs and a positive reagent strip test for blood. -Granular dirty brown casts representing hemoglobin degradation products such as methemoglobin are associated with the Acute Tubular Necrosis often caused by the toxic effects of massive hemoglobinuria that can lead to Renal Failure -Most fragile cast c. WBC CASTS  -Primary marker for distinguishing Pyelonephritis (Upper UTI) from lower UTI -Also present in Acute Interstitial Nephritis d. BACTERIAL CASTS -Seen in Pyelonephritis -Can resemble granular cast e. EPITHELIAL CELL CASTS -Represent Renal Tubular damage -similar to RTE cells, they are associated with heavy metal and chemical or drug-induced toxicity, viral infections and allograft rejection f. FATTY CASTS -Associated with Nephrotic Syndrome, Toxic Tubular Necrosis, DM and Crush Injuries -Confirmed though the use of Polarized Microscopy -Cholesterol demonstrates characteristic Maltese Cross formation under polarized light and TAG and neutral fat stain orange with fat stains -Fats do not stain with Sternheimer-Malbin stain g. GRANULAR CASTS -Can be coarse or fine (SIDENOTE: Coarsely Granular Casts- hematuria in cases of Renal Papillary Necrosis; Fine Granular Casts- represent Calcium Phosphate precipitants in Hyperparathyroidism -The origin of the granules in nonpathologic conditions appear to be from the lysosomes excreted by RTE cells during normal metabolism -Represent disintegration of cellular casts h. WAXY CASTS -Appear fragmented with jagged ends and have notches in their sides as a result of brittle and highly refractive cast matrix; Represent extreme urine stasis -Formed from the disintegration of Granular Casts i. BROAD CASTS (Renal Failure Casts) -Represent extreme urine stasis -All types of casts may occur in the broad form; The most commonly seen broad casts are granular and waxy -Bile-stained broad, waxy casts are seen as the result of the tubular necrosis caused by Viral Hepatitis SIDENOTE:  CAST DISINTEGRATION: CELLULAR CAST GRANULAR CAST WAXY CAST 10. CRYSTALS -Formedby the precipitation of urine solutes -Precipitation is subject to changes in temperature(Low); Solute Concentration(High); and pH(Low/Acid) -Urine pH is a valuable aid(first consideration) in the identification of crystals -All abnormal crystals are found in Acidic pH -Polarized microscopy a. NORMAL CRYSTALS ACID URINE 1. URIC ACID -Found in Acidic urine, Yellow-brown in color, Alkali soluble -Can assume the widest variety of shapes: Rhombic or four-sided flat plates(Whetstones); prisms; oval forms with pointed ends(Lemon-shaped); Wedges; Rosettes; barrel-shaped; and irregular plates -Rarely they are colorless and hexagonal, resembling cystine. Unlike Cystine, they show birefringence with polarized light. Cystine is soluble in dilute HCl, UA is not.  -The average Uric acid excretion by adults is 500-600mg/day -Seen in Px with Leukemia who are receiving Chemotherapy, in Px with Lesch Nyhan Syndrome and Px with gout 2. AMORPHOUS URATES -Yellow to reddish brown or brick dust granules and are the only normal crystals found in acidic urine that appear colored -Frequently found in refrigerated specimens; Soluble in heat and alkali -Pseudocasts; resemble granular casts 3. CALCIUM OXALATE (ACID/NEUTRAL) -Enveloped/Pyramidal; oval -Normally found after ingestion of tomatoes, spinach, rhubab, garlic, orange, asparagus and vitamin C. Oxalic acid is one of the breakdown products of vitamin C and oxalic acid precipitates ionized calcium -Majority of Renal calculi are composed of Calcium oxalate -TWO FORMS: a. Monohydrate(Whewhelitelite)- Oval/Dumbbell/Hourglass; Ethylene Glycol poisoning b. Dihydrate(Whedelite)- Envelope; Most common form ALKALINE 4. AMORPHOUS PHOSPHATE -White precipitate; Granular appearance 5. CALCIUM PHOSPHATE (APATITE/BUSHITE) -Colorless irregularly shaped crystal; found in alkaline/neutral urine; soluble in dilute HAc -May appear as colorless, Flat rectangular Plates or thin Prisms often in rosette forms, “pointing fingers”. The Rosette forms may be confused with sulfonamide crystals when the urine pH is in the neutral range. Calcium phosphate crystals dissolve in dilute HAc and Sulfonamides do not 6. TRIPLE PHOSPHATE (STRUVITE/NH4 Mg PO4) -Coffin-lid crystals; soluble in dilute HAc -Colorless three to six-sided prisms with oblique ends referred to as coffin lids. As they disintegrate, the crystals may develop feathery or fern-like appearance; they may form sheets or flakes -Stones made up of Struvite are radiodense and lodge in the renal pelvis, forming an outline of the structures resembling the Antlers of a deer—“Staghorn Calculi” -Birefringent under polarized microscope 7.AMMONIUM BIURATE -Thorny apple appearance because of their appearance as spicule-covered spheres found in Alkaline urine -Yellow-brown in color; souluble in dilute HAc -Resemble other urates in that they dissolve at 60degC and convert to uric acid when HAc is added -Almost always encounterd in old specimens 8. CALCIUM CARBONATE -Distinguished by the formation of gas(Effervescence) after the addition of HAc -Dumbbell/Hourglass b.ABNORMAL CRYSTALS 1. CYSTINE -CYSTINURIA: an inherited metabolic disorder that prevents reabsorption of cystine by the renal tubules -Colorless hexagonal plates -Soluble in dilute HCl (UA is insoluble) -Positive confirmation is made using the Cyanide Nitroprusside test(Suliman Test) 2. CHOLESTEROL -Rectangular plate with a notch in one or more corners (Staircase) -Associated with Nephrotic Syndrome -Crystals do not stain with fat stain but are highly birefringent with polarized light -At times, found as a film in the surface of urine, not in the sediment -Soluble in ether, chloroform and hot alcohol  3. RADIOGRAPHIC DYE -Very similar appearance to cholesterol crystals and also are highly birefringent -Increased SG: 1.050 -Example: Meglumine Diatrizoate 4. LIVER DISORDER CRYSTALS: a. BILIRUBIN b. TYROSINE-fine colorless to yellow needles; found in IEM; Momer’s Test; Millon’s Test c. LEUCINE-concentric circles and radial striations(flowerette); when present should be accompanied by tyrosine crystals; soluble in hot alkali or alcohol; Salkowski Test 5. SULFONAMIDE -Occurs in a variety of shapes. Shapes most frequently encounterd include needles, rhombics, whetstones, sheaves of wheat and rosettes with colors ranging from colorless to yellow-brown -Tests for confirmation include Diazo Reaction(+ magenta) and Lignin Test -Asociated with Anuria 6. AMPICILLIN -Colorless needles that tend to form bundles following refrigeration SIDENOTES:  OTHER CRYSTALS: 1. HIPPURIC ACID CRYSTALS result from metabolism of benzoic acid and resemble the “coffin lid” appearance of Triple Phosphate. They are colorless needles and four-sided prisms; should be distinguished from phosphates(Struvite and Apatite) 2. HEMATIN-small, biconvex whetstone seen with hemoglobinuria 3. SODIUM URATES-“Peacock Tail” 4. CALCIUM PHOSPHATE-“Cigarette butt”  URINARY SEDIMENT ARTIFACTS 1. STARCH GRANULE-contamination from gloves; highly refractile spheres that can form maltese cross; has a dimpled center; resemble fat droplets when poloarized, producing maltese cross formation; confused with rbcs; most common contaminant in urine and other body fluids 2. LYCOPODIUM –similar in appearance to starch and is used as a dusting powder 3. OIL DROPLETS and AIR BUBBLES-highly refractile and may resemble rbcs 4. POLLEN GRAINS-appear with a cell wall and occasional concentric circles 5. FIBERS-mistaken as casts but they often polarize unlike casts  TELESCOPE SEDIMENT -Term to describe the simultaneous uccorence of elements of Glomerulonephritis and those of Nephrotic Syndrome in the same urine specimen:  Red Cells  Red Cell Casts  Cellular Casts  Broad Waxy Casts  Lipid Droplets  Oval Fat Bodies  Fatty Casts -Such sediment maybe found in Collagen Vascular Disease (notably Lupus Nephritis) and Subacute bacterial Endocarditis, Renal Failure.  Addis Count Normal Values:  RBCs- 0 to500 000  WBCs and Epithelial Cells- 0 to 1 800 000  Hyaline Casts- 0 to 5 000  Urine sediments are quantitated or graded or averaged based on subjective evaluation on either lpf or hpf Table26. Reporting of Urine Sediments URINE SEDIMENT GRADING OBJECTIVE Squamous Epithelial Cells Mucus Rare, Few, Moderate, Many LPF Transitional Cells Yeast Bacteria Rare, Few, Moderate, Many HPF Trichomonas Normal Crystals RTE Cells Oval Fat bodies Average # HPF RBCs WBCs Casts Abnormal Crystals Average # LPF Table27. Microscopic Quantitations LOW POWER FIELD HIGH POWER FIELD EPITHELIAL CELLS MUCUS THREADS NORMAL CRYSTALS BACTERIA Rare 0-5 Rare 0-1 Rare 0-2 Rare 0-10 Few 5-20 Few 1-3 Few 2-5 Few 10-50 Moderate 20-100 Moderate 3-10 Moderate 5-20 Moderate 50-200 Many >100 Many >10 Many >20 Many >200 II. RENAL DISEASES Table28. Summary of Glomerular Disorders DISORDER ETIOLOGY CLINICAL PRIMARY OTHER COURSE URINALYSIS SIGNIFICANT RESULT TESTS Acute Deposition of Rapid onset of Macroscopic ASO titer glomerulonephritis immune complexes hematuria and hematuria Anti-group A formed in edema Proteinuria Streptococcal conjunction with Permanent renal Red blood cell casts enzymes group A Damage Granular Casts Streptococcus infection on the glomerular membranes Rapidly Progressive Deposition of Rapid onset with Macroscopic BUN Glomerulonephritis immune complexes glomerular damage hematuria Creatinine from systemic and possible Proteinuria Creatinine immune disorders progression to Red blood cell casts Clearance on the glomerular ESRD membrane Goodpasture’s Attachment of Hemoptysis and Macroscopic Antiglomerular syndrome cytotoxic antibody dyspnea followed hematuria basement formed during viral by hematuria Proteinuria membrane respiratory Possible Red blood cell casts antibody infections to progression to glomerular and ESRD alveolar basement membranes Wegener’s Antineutrophilic Pulmonary Macroscopic ANCA Granulomatosis cytoplasmic symptoms hematuria autoantibody binds including Proteinuria to neutrophils in hemoptysis develop Red blood cell casts vascular walls first followed by producing damage Renal involvement to small vessels in and possible the lungs and progression to glomerulus ESRD Henoch-Schonlein Occurs primarily in Initial appearance Macroscopic Stool occult blood Purpura children following of purpura followed hematuria  viral respiratory by blood in sputum Proteinuria infections; a and stools and Red blood cell casts decrease in eventual renal platelets disrupts involvement vascular integrity Complete recovery is common but may progress to renal failure IgA Nephropathy Deposition of IgA Recurrent Early stages: Serum (Berger Disease) on the glomerular macroscopic Macroscopic or Immunoglobulin A membrane hematuria microscopic resulting from following exercise hematuria increased levels of with slow Later stages: serum IgA progression to Chronic chronic glomerulonephritis glomerulonephritis (Most common cause) Membranous Thickening of the Slow progression to Microscopic Antinuclear glomerulonephritis glomerular Nephrotic hematuria antibody membrane Syndrome or Proteinuria HBSAg following IgG Possible remission FTA-ABS test immune complex deposition associated with systemic disorders Membranoproliferative Cellular Noticeable Hematuria Serum complement glomerulonephritis proliferation progression to Proteinuria levels affecting the chronic capillary walls or glomerulonephritis the glomerular to nephrotic basement syndrome membrane, possibly immune mediated Chronic Marked decrease in Noticeable decrease Hematuria BUN glomerulonephritis renal function in renal function Proteinuria Serum creatinine resulting from progressing to renal Glucosuria Creatinine glomerular damage failure Cellular and clearance precipitated by Granular casts Electrolytes other renal Waxy and broad disorders casts Nephrotic Syndrome Disruption of the Acute onset Heavy proteinuria Serum albumin electrical charges following systemic Microscopic Cholesterol that produce the shock hematuria TAG tightly fitting Gradual Renal tubular cells podocyte barrier progression from Oval Fat bodies resulting in other glomerular Fat droplets massive loss of disorders and then Fatty and Waxy protein and lipids to renal failure casts Minimal Change Disruption of Frequent complete Heavy proteinuria Serum albumin Disease podocytes remission following Transient Cholesterol (Lipid Nephrosis) occurring primarily corticosteroid proteinuria TAG in children treatment Fat droplets following allergic reactions and immunizations Focal Segmental Disruption of May resemble Proteinuria Drug abuse Glomerulosclerosis podocytes in Nephrotic Microscopic HIV tests certain areas of syndrome or hematuria glomeruli minimal change  associated with disease heroin and analgesic abuse and AIDS Alport Syndrome Genetic disorder Slow progression to See Nephrotic showing lamellated Nephrotic Syndrome and thinning of Syndrome and Microalbuminuria glomerular ESRD basement membrane Diabetic Nephropathy Deposition of Progression to See chronic Blood glucose (Kimmelstiel-Wilson glycosylated ESRD glomerulonephritis disease) proteins resulting (Most common from poorly cause) controlled blood glucose levels Table30.Summary of Tubulointerstitial Disorders DISORDER ETIOLOGY CLINICAL PRIMARY OTHER COURSE URINALYSIS SIGNIFICANT RESULTS TESTS Acute Tubular Damage to the renal Acute renal Microscopic Hemoglobin Necrosis tubular cells caused dysfunction usually hematuria Hematocrit by ischemia or toxic resolved when the Proteinuria Cardiac Enzymes agents underlying cause is RTE cells and casts corrected Hyaline, granular, waxy and broad casts Fanconi’s Inherited in Generalized defect Glucosuria Serum and urine Syndrome association with in renal tubular Possible Cystine electrolytes cystinosis and reabsorption crystals Amino acid hartnup disease or requiring supportive chromatography acquired through therapy exposure to toxic agents Cystitis Ascending bacterial Acute onset of Leukocyturia Urine culture infection of the urinary frequency Bacteriuria bladder and burning Microscopic resolved with hematuria antibiotics Mild proteinuria Increased pH Acute Infection of the Acute onset of Leukocyturia Urine culture Pyelonephritis renal tubules and urinary frequency, Bacteriuria Blood cultures interstitium related burning, and lower White blood cell to interference of back pain resolved casts urine flow to the with antibiotics Bacterial casts bladder, reflux of Microscopic urine from the hematuria bladder and Proteinuria untreated cystitis Chronic Recurrent infection Frequently Leukocyturia Urine culture Pyelonephritis of the renal tubules diagnosed in Bacteriuria Blood cultures and interstitium children, requires White blood cell BUN caused by structural correction of the casts Creatinine abnormalities underlying Bacterial casts Creatinine clearance affecting the flow of structural defect Granular, waxy, urine Possible progression broad casts  to renal failure Hematuria Proteinuria Acute Interstitial Allergic Acute onset of Hematuria Urine Eosinophils Nephritis inflammation of the renal dysfunction Proteinuria BUN renal interstitium in often accompanied Leukocyturia Creatinine response to certain by a skin rash White blood cell Creatinine clearance medications Resolves following casts discontinuation of medication and treatment with corticosteroids SIDENOTES:  RENAL CALCULI (Kidney Stones) may form in the calyces and pelvis of the kidney, ureters and bladder. Conditions favoring the formation of Renal Calculi are similar to those favoring formation of urinary crystals including Temperature, pH, solute concentration and urinary stasis. Approximately 75% of the Renal Calculi are composed of Calcium oxalate or phosphate. Magnesium ammonium phosphate, uric acid and cystine are the other primary calculi constituents. Least common are cystine calculi and are seen in conjunction with hereditary disorders of cystine metabolism. The presence of microscopic hematuria resulting from irritation to the tissues by the moving calculus is the primary urinalysis finding.  Uric Acid and Urate Stones- Yellow to brownish red and are moderately hard  Phosphate Stones- Pale and Friable  Calcium Oxalate Stones- Very hard, often dark color and typically have a rough surface  Cystine Stones- Yellow-brown and feel somewhat greasy  IL-6 is a valuable marker of kidney stone disease Table29. Qualitatives Test for Renal Calculi/Stones STONES REAGENTS POSITIVE RESULT Uric Acid Sodium Carbonate, UA reagent Deep blue Color (Folin Reagent with PTA) Acid extract, Sodium Hydroxide, Yellow-Orange Color Neocuproine, HAc, Copper Sulfate Carbonate HCl(Carbonate) Foaming/ Formation of Tiny Oxalate And Upon addition of MnO2 bubbles (Oxalate) Phosphate Ammonium molybdate in HNO3 Distinct Yellow color after heating Acid extract, ammonium molybdate, Ferrous ammonium Blue Color sulfate, Sulfuric Acid Calcium HCl and NaOH White Cloud Acid Extract, NaOH, Clacein, Pale Yellow Color KOH Magnesium HCl, NaOH and p- Blue Color Nitrobenzeneazoresorcinol inNaOH Ammonium HCl, NaOH and Nessler’s Solution Orange-brown Color Cystine NH4OH, NaCN, and Na Beet-red/Magenta color Nitroprusside III.METABOLIC DISORDERS -Overflow disorders result from the block of a normal metabolic pathway that causes increased plasma concentrations of nonmetabolized substances -Unlike the overflow-type diseases, Renal-type aminoacidurias do not have high levels of the amino acid in the blood because the primary defect is in the Renal Tubular Reabsorption Mechanism -Blood collected by infant heel puncture is initially tested for IEM -Testingfor many substances is now performed using Tandem Mass Spectrophotometry (MS/MS). -PHENYLALANINE-TYROSINE DISORDERS: PKU, Tyrosyluria, Melanuria, Alkaptonuria -BRANCHED CHAIN AMINO ACID DISORDERS: MSUD, Organic Acidemias -TRYPTOPHAN DISORDERS: Indicanuria(Hartnup Disease), 5-HIAA  -CYSTINE DISORDERS: Cystinuria, Cystinosis, Homocystinuria -MUCOPOLYSACCHARIDE DISORDERS: Hurler, Hunter, Sanflippo A. PKU -Deficiency of enzyme Phenylalanine Hydroxylase -Mousy/Musty odor of urine due to accumulation of Phenylpyruvic Acid (Phenylacetic acid) -Microbiologic test: Guthrie Test (bacterial inhibition test); uses B. subtilis -Chemical Test: FeCl3 tube test; (+)blue green color -Phenistix Rgt Strip: Ferric NH4 Sulfate- cyclohexylsulfamic acid MgSO4; (+)gray-green color -Mental retardation, Fair skin (Albino) -Interruption of the pathway also produces children with fair complexions—even in dark- skinned families—owing to the decreased production of tyrosine and its pigmentation metabolite melanin. -Once discovered, dietary changes that eliminate phenylalanine , a major constituent of milk from the infant’s diet can prevent the excessive build up of serum phenylalanine and can thereby avoid damage to the child’s mental capabilities B. TYROSYLURIA -Deficiency of enzymes Fumarylacetoacetate hydrolase, Tyrosine trnsaminase or p- hydroxyphenylpyruvate oxidase -Increased p-hydroxyphenylpyruvic acid and p-hydroxyphenyllactic acid in urine -Nitro-naphthol test: (+)orange-red -FeCl tube test- (+)Transient green Table30. Types of Tyrosyluria TYPE ENZYME DEFICIENCY MANIFESTATION; CLINICAL COURSE Type 1 Fumarylacetoacetate Hydrolase Generalized renal Tubular disorder and progressive renal failure; Rickets; Hepatoma; Acute intermittent porphyria Type 2 Tyrosine Aminotransferase Corneal erosions and lesions of the (Richner-Hanhart Syndrome) palms, fingers, and soles caused by crystallization of tyrosine in the cells; Increased urinary phenolic acids Type 3 p-hdyroxyphenylpyruvic acid Mental retardation  Figure3. Phenylalanine and Tyrosine Metabolism C. ALKAPTONURIA -Urine of patient darkens after becoming alkaline from standing at RT -Deficiency of Homogentisic Acid Oxidase(Alkapton) -Homogentisic Acid Test: (+)black color -FeCl3: Transient deep blue color -Clinitest: Yellow precipitate/orange/red ppt D. MELANURIA -Associated with melanuria -Urinary melanin causes darkening of urine after exposure to air -Darkening is due to conversion of 5,6-dihydroxyindole->melanogen->melanin -Test: Sodium Nitroprusside test; (+)Red E. ORGANIC ACIDEMIA -Include propionic, isovaleric and methylmalonic academia F. INDICANURIA -Can be seen in Hartnup Disease (blue diaper syndrome) -Tryptophan is converted to indole -Excess indole is reabsorbed from intestines to blood and into liver -In liver, it is converted to indican, then excreted in urine -Indican in urine is colorless until exposed to air, being converted to indigo blue \-Test: Obermayer Test: (+)Violet color with choloroform G. 5-HYDROXYINDOLE ACETIC ACID -Metabolite of serotonin; Serotonin is produced from tryptophan by argentaffin (enterochromaffin) cells in the intestine and is carried through the body primarily by the platelets -elevated in urine of Px with argentaffinoma -Test is based on the development of a purple color specific for 5-Hydroxyindoles with nitrous acid and 1-nitroso-2-naphthol -Ethylene Dichloride is used to remove interfering chromogens -Patients must be given explicit dietary instructions prior to the collection of any sample to be tested for 5-HIAA because serotonin is a major constituent of foods such as bananas,  pineapples, chocolates, plums or walnuts, avocado and tomatoes or medications containing guaifenesin—these may produce false-positive elevations Figure4. Tryptophan Metabolism H. MAPLE SYRUP URINE DISEASE -Caused by failure to inherit the gene that codes for the enzyme responsible for the oxidative decarboxylation of Leucine, Isoleucine and Valine -Test: DNPH; (+)Yellow/White ppt I. CYSTINURIA -Marked elevation of cytine in urine -Caused by inability of renal tubules to reabsorb cystine -Renal-type; Calculi formation; Aside from cystine, Lysine, Ornithine and Arginine are also not reabsorbed J. CYSTINOSIS -The incomplete metabolism of cystine results in crystalline deposits of cystine in many areas of the body, including the cornea, bone marrow, lymph nodes, and internal organs -A major defect in the renal tubular reabsorption mechanism (Fanconi syndrome) also occurs -Overflow-type; There is gradual progression to Renal Failure -Positve result for reducing substances (Clinitest) K. MUCOPOLYSACCHARIDOSIS -Caused by deficient activity of enzyme necessary for the degradation GAG (Glycosaminoglyca) -Accumulation of incompletely metabolized polysaccharide portions in connective tissues and increased excretion in urine -Products most frequently found in urine are dermatan sulfate , keratan sulfate, and heparin sulfate -Includes Hurler, Hunter and Sanflippo disease -Test: CTAB and Acid albumin test; (+)White Turbidity Metachromatic Staining spot Test; (+)blue spot L. LESCH NYHAN SYNDROME -Sex-linked recessive deficiency of the enzyme Hypoxanthin guanine Phosphoribosyl Transferase -Causes orange sands in diapers  M. CARBOHYDRATE DISORDERS -Galactosuria, Lactosuria, Fructosuria N. PORPHYRINE DISORDERS -Disorders of Porphyrine metabolism -Can be inherited or acquired from eryhtrocytic and hepatic malfunctions or exposure to toxic agents -The solubility of the porphyrin compounds varies with their structure. ALA, Porphobilogen, and uroporphyrinare the most soluble and readily appear in the urine. Coproporphyrin is less soluble but is found in urine whereas protoporphyrin is not seen in urine. -Fecal analysis has been usually performed for the detection of coprporphyrin and protoporphyrin -CDC recommends analysis of whole blood for the presence of Free Erythrocyte Protoporphyrin (FEP) as a screening test for lead poisoning -An indication of the possible presence of Porphyrinuria is the observation of a red or port wine color to the urine after exposure to air -Urine in lead poisoning has normal urine color but has red fluorescence on wood’s lamp -Screening tests for Porphyrinuria: 1. Ehrlich Reaction -ALA and Porphobilinogen; Acetylacetone must be added to the specimen to convert the ALA to porphobilinogen prior performing Ehrlich Test 2. Fluorescence under UV light in the 550-600nm range -Uroporphyrin, Coproporphyrin, Protoporphyrin IV. PREGNANCY TESTING -HUMAN CHORIONIC GONADOTROPIN is a glycoprotein composed of alpha and beta subunit. -The Alpha subunit of HCG is similar with LH, FSH, and TSH -The beta subunit is used to differentiate HCG from the three hormones -Produced by the placenta -Marker for pregnancy; Serum HCG levels rise rapidly in early gestation; Peak levels are attained in the latter part of the first pregnancy -Increased during the 1st trimester but decreases during the 2nd and 3rd trimester -Marker for Testicular Carcinoma -INCREASED HCG: Down syndrome, Choriocarcinoma, Molar Pregnancy -DECREASED HCG: Ectopic pregnancy -Pregnancy tests: biological test, Immunoassay, RIA, Immunometric Assay SIDENOTES: Table31. Biological Tests for Pregnancy TEST ANIMAL USED MODE OF POSITIVE RESULT INJECTION 1. Ascheim-Zondek Immature female mice Subcutaneous Formation of hemorrhagic follicles and corpora lutea 2. Friedman Mature female virgin rabbit Marginal ear vein Hyperemic uterus and corpora hemorrhagica 3. Hogben Female toad (Xenopus laevis) Lymph sac Oogenesis South African clawed frog- carries eggs throughout the year 4. Galli-Mainini Male frog 9Rana pipiens or Subcutaneous Spermatogenesis Rana clamitans, leopard or grass frog); Male toad (Bufo bufo or B. Americanus) 5. Frank-Berman Immature female rats Subcutaneous Ovarian hyperemia 6. Kupperman Female Rat Intraperitoneal Ovarian hyperemia  IMMUNOASSAY for Pregnancy: -Complement Fixation, Hemagglutination inhibition, Latex particle agglutination inhibition, Direct agglutination, Enzyme immunoassay  HOMEBASED PREGNANCY TEST: -Principle: Enzyme Immunoassay -Label: ALP -Sensitivity: 25mIU/mL -Because the test cassette contains all necessary reagents, this is called Immunochromatography -If the resulting color is pink or fading, repeat test within 48 hours V. URINALYSIS AUTOMATION a. Chemical Testing of Urine -DIPSTICK READER: based of Reflectance Photometry; light reflection from the tests pads decreases in proportion to the intensity of color produced; the light is reflected to a photodetector and an analog.digital converter; the instruments compare the amount of light reflection with that of known concentrations b. Microscopic Testing of Urine -SYSMEX (UF-100): laser-based flow cytometry along with impedance detection, light scatter, and fluorescence -IRIS (International Remote Imaging Systems): based on Auto-particle recognition  Minimum Urine Volumes: 1. IRIS IQ200 series- 2mL 2. IRICELL- 4mL 3. IRIS Workstation- 6mL VI. CEREBROSPINAL FLUID -Fluid circulating the Central Nervous System first recognized by Cotugno in 1764 -FUNCTIONS: Supply nutrients to nervous tissue; Remove metabolic wastes; Mechanical barrier to cushion the brain and spinal cord against trauma -CSF PRODUCTION:  Choroid plexuses (70%)  Ventricular ependymal lining and subarachnoid space (30%) -Rate of production is 20mL/hr or 500mL/day -CSF VOLUME:  90-150mL in adults  10-60mL in neonates -Not an ultrafiltrate of plasma -CSF ionic components: H+, K+, Ca2+, Mg2+, HCO3- -MENINGES line the brain nd spinal cord:  Dura mater- outer layer that lines the skull and vertebral canal  Arachnoid Villi- filamentous (spider-like) inner membrane  Pia mater- thin membrane lining the surfaces of the brain and spinalcord A. SPECIMEN COLLECTION AND HANDLING -CSF is collected by lumbar puncture between the third and fourth or fifth lumbar vertebrae (Graff’s: The most common site used for lumbar puncture is the interval space between L3 and L4; Children and infants, between L4 and L5) -Typically 10-20mL of CSF is collected -NORMAL OPENING PRESSURE(Measured by Manometer)  Adults: 90-180 mm of H20  Infants and Young Children (30 min, store at 37degC Tube 3 Hematology Refrigerated SIDENOTES:  A fourth tube may be drawn for the Microbiology laboratory to provide better exclusion of skin contamination or for additional serologic tests. Supernatant that is left over after each section has performed its tests may also be used for chemical or serologic tests  If only one tube is collected or if small volume is collected, Microbiology procedures should be performed first or ask the physician for priority test to be performed.  Glass tubes should be avoided because cell adhesion to glass affects the cell count and differential process immediately  Refrigeration is contraindicated for culture specimens because fastidious bacteria will not survive  Identification of infectious meningitis particularly bacterial is the most important indication of CSF examination B. APPEARANCE -NORMAL: Crystal clear -A cloudy, turbid, or milky specimen can be the result of an increased protein or lipid concentration, but it may also be indicative of infection with the cloudiness being caused by the presence of WBCs -XANTHOCHROMIA is a term used to describe CSF supernatant that is pink, orange or yellow. It is caused by the presence of RBC degradation products, elevated serum bilirubin, presence of pigment carotene, markedly increased protein (>150mg/dL) concentration and melanoma pigment. Xanthochromia caused by bilirubin is due to immature liver function Table33. Traumatic Tap versus Intracranial Hemorrhage TRAUMATIC TAP INTRACRANIAL HEMORRHAGE  Uneven distribution of blood  Even distribution of blood  Clear Supernatant  Xanthochromic supernatant  No Erythrophages  Presence of Erythrophages  Clot Formation  No clot formation  Negative D-Dimer test  Positive D-Dimer test C. MICROSCOPIC EXAMIATION Adults usually have a predominance of Lymphocytes to -NORMAL WBC count: Monocytes (70:30), whereas the ratio is essentially reversed in  Adult- 0-5WBCs/uL children.  Newborns- 0-30 WBCs/uL -The cell count that is routinely performed on CSF specimens is the leukocyte count. RBC counts are usually determined only when a traumatic tap has occurred and a correction for leukocytes or protein is desired. No RBCs should be present in normal CSF. Any cell count should be performed immediately -DILUTION:  For total Cell count, clear specimens may be counted undiluted. Diluted specimens are made with normal saline  For Wbc count, lysis should of Rbcs should be done using 3% glacial HAc; addition of methylene blue to the diluting fluid stains the Wbcs providing better differentiation between neutrophils and mononuclear cells -Turbidity starts at:  WBC >200/uL  RBCs >4 000/uL Table34. Dilutions for Unclear specimens Slightly Hazy 1:10 Hazy 1:20 Slightly Cloudy 1:100  Cloudy/Slightly bloody 1:200 Bloody/Turbid 1:10 000 -COMPUTATION: Cell/uL = No. of cells counted x Dilution x DF OR Cells/uL = No. of cells counted x Dilution No. of squares counted No. of squares counted x Vol. of 1 Sq. -CORRECTIONS FOR CONTAMINATION: WBC (Added) = WBC (blood) x RBC (CSF) *An approximate WBC count can then be RBC (blood) obtained by subtracting the “added” WBCs from the actual count.  When peripheral Blood RBC and WBC counts are in the normal range, many laboratories choose to simply subtract 1 WBC for every 700 RBCs present in the CSF. -CSF DIFFERENTIAL COUNT  Performed on a stained smear  Specimen should be concentrated prior to the preparation of smear. Methods include: 1. Sedimentation- not routinely used 2. Filtration- not routinely used 3. Centrifugation- 5-10 minutes 4. Cytocentrifugation- recommended method for diff counts in all body fluids; addition of albumin increases the cell yiel and decreases cellular distortion  100 cells should be counted, classified and reported in percentages  For low WBC counts, report only the number of cells seen  PLEOCYTOSIS refers to increased number of WBCs in the CSF -CSF CELLULAR CONSTITUENTS Table35. Summary of CSF Cellular Constituents Neutrophils Indicate B acterial Meningitis Neutrophils with pyknotic nuclei indicate degenerating cells Nucleated RBC Result of bone marrow contamination during spinal tap Lymphocytes and Increased in Multiple Sclerosis; Viral Meningitis; Tubercular Meningitis and Fungal Monocytes Meningitis Eosinophils Parasitic infection, Fungal infection, introduction of foreign material in CNS Macrophages Appear within 2-4 hours after RBCs enter the CSF Indicative of previous hemorrhage Phagocytoses RBCs, appearance of Hemosiderin granules or hematoidin crystals NON PATHOLOGIC: These cells are from diagnostic procedures Choroidal Cells -from epithelial lining of the choroid plexuses Ependymal Cells -from linings of ventricles and neural canal Spindle-Shaped Cells -from linings of arachnoid Blasts of leukemia Malignant cells of hematologic origin Lymphoma Cells Metastatic carcinoma Malignan cells of non-hematologic origin cells D. CHEMICAL TESTS 1. PROTEIN -Most frequently performed chemical test in CSF -NORMAL: 15-45mg/dL -More than 80% of the CSF protein content is derived from blood plasma in concentration of less than 1% plasma level -Major CSF Proteins:  Albumin- makes up majority of CSF protein as in serum  Prealbumin- second most prevalent protein in CSF in contrast with serum (Ig)  Transferrin- major beta protein in CSF; a separate carbohydrate deficient tau transferrin called Tau is also present in CSF and not in serum  IgG and IgA- IgG is the major gamma globulin found in CSF; the latter only contain a small amount of IgA  -Proteins NOT found in CSF:  IgM  Fibrinogen  Beta-Lpp -Elevated CSF protein results: Meningitis, Hemorrhage, Primary CNS tumors, Multiple Sclerosis, Guillain-barre Syndrome, Neurosyphilis, Polyneuritis, Myxedema, Cushing Disease, Connective Tissue Disease, Diabetes, Uremia, Decreased endocrine function(Thyroid and Parathyroid) -Decreased CSF protein results: CSF leakage/trauma, Recent puncture, Rapid CSF production, Water intoxication, High intracranial pressure, High protein resorption by the arachnoid villi -Correction for Protein: Subtract 1mg/dL of protein for every 1 200 RBCs counted is acceptable (Henry: In the presence of a normal peripheral blood RbC count and serum protein, the correction amount to 1 WBC for every 700 RBCs and 8 mg/dL protein for every 10 000 RBCs/uL -Tests: 1. Tubidimetric: TCA and SSA 2. Dye-binding: Coomassie brilliant blue and Poceau S 3. Precipitation: Sodium Sulfate, benzethonium Chloride and Zephiran 4. Colorimetric: Lowry 5. Qualitative Tests: Ross Jones, Nonne-apelt, Pandy’s Test -CSF/SERUM ALBUMIN INDEX is used to assess the integrity of the blood brain barrier  CSF Serum Albumin index = CSF albumin (mg/dL) Serum albumin (g/dL)  Value of 0.70 represents IgG production within the CNS -Electrophoresis is primarily performed to detect Oligoclonal bands -MULTIPLE SCLEROSIS: presence of two or more oligoclonal bands in the CSF that are not present in the serum and accompanied by an increased IgG index. -Other neurologic disorders including encephalitis, neurosyphilis, Guillain-barre syndrome and neoplastic disorders also produce oligoclonal banding that may not be present in the serum 2. GLUCOSE -CSF glucose is 60-70% that of plasma glucose -Normal CSF Glucose: 50-80mg/dL -blood glucose must be drawn in comparison to CSF glucose; Results should be compared with plasma level ideally following a 4-hour fast; The blood glucose should be drawn about 2 hours prior to the spinal tap to allow time for equilibration between the blood ad fluid. -The diagnostic significance of CSF glucose is confined to the finding of values that are decreased in relation to plasma values -Decreased (Hypoglycorhacchia) in bacterial, Tubercular and fungal (normal to decreased) meningitis -Normal in Viral and Fungal (normal to decreased) meningitis -Increased CSF glucose is of no clinical significance, reflecting increased blood glucose levels within 2 hours of lumbar puncture. A traumatic tap may also cause a spurious increase in CSF glucose 3. LACTATE -NV: 11-22 mg/dL (Graff’s) -Increased in bacterial, tubercular and fungal meningitis as well as in hypoxia 4. GLUTAMINE -NV: 8-18 mg/dL (Henry: 5-20 mg/dL) -Produced from ammonia and alpha-ketoglutarate by the brain cells -Increased synthesis of glutamine is caused by the excess ammonia that is present in the CNS, therefore, it provides an indirect test for the presence of excess ammonia in the CSF -Falsely elevated results: bloody/Hemolyzed/Xanthochromic specimen -Preferred over the direct measurement of CSF ammonia because the concentration of glutamine remains more stable than the concentration of volatile ammonia in the collected specimen -CSF glutamine test is a frequently requested procedure for patients with coma of unknown origin -Approximately 75% of children with Reye Syndrome have elevated CSF glutamine levels; Also elevated in liver disorders (Hepatic Coma)  E. MICROBIOLOGY TESTS -Used to identify the cause of meningitis -CSF culture is confirmatory test -Gram stain, Acid Fast Stain, India Ink, and latex agglutination test serve for preliminary diagnosis -Agents:  Neonates- S. agalactiae  3 months and older- N. meningitidis; S. pneumoniae  Newborn to 1 month- E. coli and other gram – bacilli  3 months to 18 years- H. influenza  Neonates, elderly, alcoholics, immunosuppresed- L. monocytogenes  ***C. neoformans- starburst pattern in Gram stain F. SEROLOGICAL TESTS -For assessment of Neurosyphilis using VDRL and FTA-ABS SIDENOTE: Table36. Major Laboratory Results for the Differential Diagnosis of Meningitis BACTERIAL VIRAL TUBERCULAR FUNGAL Elevated WBC count Elevated WBC count Elevated WBC count Elevated WBC count Neutophils present Lymphocytes present Lympocytes and Lymphocytes and monocytes present monocytes present Marked protein Moderate protein Moderate to marked Moderate to marked protein elevation elvation protein elevation elevation Markedly decreased Normal glucose level Decreased glucose level Normal to decreased glucose level glucose level Lactate level >35mg/dL Normal lactate level Lactate level >25mg/dL Lactate level >25mg/dL Pellicle formation Positive india ink with Cryptococcus neoformans Positive Gram stain and Positive immunologic test Bacterial antigens tests for C. neoformans  LD Brain tissues: Source of LD1 and LD2 Lymphocytes: LD2 and LD3 (in Viral Meningitis) Neutrophils: LD4 and LD5 (in Bacterial Meningitis, concentration is reversed: LD5>LD4>LD3>LD2>LD1) VII. SEMEN -Secretion of the male reproductive system composed of fractions contributed by testes and epididymis, seminal vesicles, prostategland and bulbourethral glands -SEMINIFEROUS TUBULES are contained in the testes; Germ cells for the production of spermatozoa are located in the Seminiferous tubules -SERTOLI CELLS provide support and nutrients for the germ cells -EPIDIDYMIS is the site of sperm maturation -Testes and epididymis contribute to 5% of the total semen volume -Seminal vesicles produce majority(60-70%) of the fluid present in semen; Fluid from seminal vesicles contain Fructose -Prostate gland contributes to 20-30% of the semen volume; It produces acid phosphatase, Citric Acid, Zinc and Proteolytic enzymes responsible for normal semen liquefaction -Bulbourethral glands contribute 5% of the total fluid volume; It produces thick, alkaline mucus which help neutralize the acid secretions of prostate gland and vagina A. SPECIMEN COLLECTION -Sexual abstinence for 2-3 days but not more than 5 days -Placed in warm sterile glass container -Delivered to the laboratory within 1 hour of collection -Specimens awaiting for analysis should be kept at 37degC -Methods of collection: Masturbation or use of nonlubricant-tcontaining rubber or polyurethane condoms -Majority of the sperm concentration are contained in the first portion of the ejaculate, making complete collection essential for accurate testing  B. SEMEN ANALYSIS Table36. Normal Values for Semen Analysis Appearance Gray-white color; Translucent Odor Musty Liquefaction 30-60minutes Volume 2-5mL Viscosity Pours in droplets pH 7.2-8 (7-8) Sperm concentration >20million/ejaculate Sperm count >40million/ejaculate Motility >50% within 1 hour Quality >2.0 or a, b, c Morphology >14% normal forms (strict criteria) >30% normal forms (routine criteria) Round cells 75% Seminal Fluid Fructose 13umol per ejaculate Agglutination None Leukocytes None bacteria Epithelial Cells None to few Neutral Alpha Glucosidase >or equal to 20umol/ejaculate Zinc >or equal to 2.4unol/ ejaculate Citric acid >or equal to 52umol/ejaculate ACP >or equal to 200 units/ ejaculate Penetration on bovine mucus >30mm SIDENOTES: -APPEARANCE  Increased white turbidity indicates the presence of white blood cells and infection within the reproductive tract  Varying amounts of Red coloration are associated with Red blood cells and are abnormal  Yellow coloration may be caused by urine contamination, specimen collection following prolonged abstinence and medications. Urine is toxic to sperm thereby affecting evaluation of motility -LIQUEFACTION  If after 2 hours the specimen has not yet liquefied, proteolytic enzymes such as alpha-chymotrypsin may be added to allow the rest of the analysis to be performed  Failure of liquefaction to occur may be caused by a deficiency in prostatic enzymes and should be reported -VOLUME  Decreased volume is more frequently associated with infertility and may indicate improper functioning of the semen-producing organs primarily the seminal vesicles  Ratings of 0 (watery) to 4 (gel-like) can be assigned to the viscosity report. Viscosity can also be reported as low, normal, and high  Increased viscosity and incomplete liquefaction impede sperm motility -pH  Increased pH is indicative of infection within the reproductive tract  A decreased pH is associated with increased prostatic fluid  Nitrozine paper is the simplest way to measure the semen pH (Graff’s). -SPERM CONCENTRATION/ COUNT  The most common dilution is 1:20  Dilution immobilizes the sperm prior counting  Diluting fluids include Distilled water, Saline, Sodium Carbonate, and Formalin  Using the Neubauer hemocytometer, sperms are usually counted in the four corner and center squares of the large center square—similar to a manual RBC count. Both sides of the hemocytometer are loaded and counted and the counts should agree within 10%. If the counts do not agree,both the dilution and the counts are repeated   Only fully developed sperm should Be counted. Immature sperm and WBCs, often referred to as “round” cells must not be included  Greater than 1 million leukocytes per mL is associated with inflammation or infection of the reproductive organs that can lead to infertility  Greater than 1 million spermatids per mL indicates disruption of spermatogenesis 1. Sperm Concentration (sperm/mL) Cells/uL = No. of cells counted x Dilution x DF *Because this formula provides the no. of cells No. of squares counted per uL, the figure must then be multiplied by 1000 to convert uL to mL. 2. Sperm Count (sperm/ejaculate) Sperm/ejaculate = Sperm concentration x Specimen volume SIDENOTE:  Makler counting Chamber is used in counting sperm undiluted; Sperms are immobilized by heat -SPERM MOTILITY  The percentage of sperm showing actual forward movement can be estimated after evaluating approximately 20 hpf  A minimum motility of 50% with a rating of 2.0 after 1 hour is considered normal. Interpretation states that within 1 hour, 50% or more sperm should be motile in categories a, b, c or 25% or more should show motility in categories a and b  Computer-Assisted Semen Analysis provides objective determination of both sperm velocity and trajectory(direction of motion) Table37. Sperm Motility Grading 4 a Rapid, straight line motility 3 b Slower speed, some lateral movement 2 b Slow forward progression, noticeable lateral movement 1 c No forward progression 0 d No movement -SPERM MORPHOLOGY  NORMAL SHAPE: Oval  NORMAL SIZE: 5um x 3um x 45um (head length, head width and flagella respectively)  The acrosomal cap (critical to ovum penetration) should encompass approximately half of the head and covers two thirds of the sperm nucleus  The midpiece is the thickest part of the tail because it is surrounded by a mitochondrial sheath that produces the energy required by the tail for motility  Abnormalities in head morphology are associated with poor ovum penetration  Abnormalities in neckpiece, midpiece and tail are associated with defective motility  At least 200 sperm should be evaluated and the percentage of abnormal sperm reported from a thinly smeared stained (Wright’s or Giemsa or Papanicolaou) slide under OIO  Sperm morphology additional parameters (Kruger’s Strict Criteria): measurement of Head, neck, tail, size of acrosome, and the presence of vacuoles -SPERM VIABILITY  Decreased sperm viability may be suspected when a specimen has a normal sperm concentration with markedly decreased motility  Viability is evaluated by mixing the specimen with an Eosin-Nigrosin stain  100 sperms are evaluated  Viable cells appear bluish white/colorless/white because they do not take up eosin  Dead cells appear Red -SEMINAL FLUID FRUCTOSE  Low sperm concentration may be caused by lack of the support medium produced in the seminal vesicles. This can be indicated by a low to absent fructose level in the semen  In the absence of fructose, sperm do not display motility in the semen analysis   TEST: Resorcinol Test; (+)Orange-red  Specimens for fructose levels should be tested within 2 hours or frozen to prevent fructolysis -ANITSPERM ANTIBODIES  The presence of antibodies in a male subject can be suspected when clumps of sperm are observed during a routine semen analysis  The Mixed Agglutination Reaction (MAR) test is a screening procedure used primarily to detect the presence of IgG antibodies. Less than 10% of the motile sperm attached to the particles is considered normal  The Immunobead test is a more specific procedure in that it can be used to detect the presence of IgG, IgM and IgA antibodies and demonstrates what area of the sperm (head, neckpiece, midpiece or tail) the autoantibodies are affecting. The presence of less than 20% of the sperm is considered normal -NEUTRAL ALPHA GLUCOSIDASE  Decreased value suggests disorder of epididymis -ZINC, CITRIC ACID, ACP  Assess the function of Prostate gland  On alleged Rape: Microscopic Examination of sperm can be done; ACP determination can also be done; Dtection of Glycoprotein p30 is the most specific method  Tests for bacterial inhibitors in the prostate: 1. FLORENCE TEST -Test for choline -Reagent: KI -(+)brown, rhombic crystals 2. BARBIERO’s TEST -Test for spermine -Reagent: Picric acid, TCA -(+)Yellow leg-like crystals or needles C. POST VASECTOMY SEMEN ANALYSIS -Specimens are routinely tested at 2 months postvasectomy and continuing until two consecutive monthly specimens show no spermatozoa -A negative wet preparation is followed by centrifugation of the specimen for 10 minutes and examination of the sediment. There is no more need for staining because you are only detecting the presence of sperm, not morphology. D. SPERM FUNCTION TESTS Table38. Sperm Function Tests Hamster Egg Penetration Sperm are incubated with species-nonspecific hamster eggs and penetration is observed microscipically Cervical mucus penetration Observation of sperm penetration ability of partner’s midcycle cervical mucus Hypo-osmotic swelling Sperm exposed to low-sodium concentrations are evaluated for membrane integrity and sperm viability In vitro acrosome reaction Evaluation of the acrosome to produce enzymes essential for ovum penetration VIII. SYNOVIAL FLUID -Join Fluid; Ultrafiltrate of Plasma; collected via Arthreocentesis -FUNCTIONS: Lubricates joints; Reduces friction; Provides nutrients to the articular cartilage; Lessen shock of joint compression occurring during activities such as walking or jogging -Contains high levels of Hyaluronic Acid (responsible for its viscosity) Table39. Characteristics of Normal Synovial Fluid Volume >3.5mL Color Colorless to pale yellow Clarity Clear Viscosity Able to form a string 4-6cm long Leukocyte count