Analysis of Urine & Other Body Fluids - Chapter 1-A: Renal Anatomy and Physiology PDF

ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 01-A: RENAL ANATOMY AND PHYSIOLOGY Afferent The afferent (thicker) and...

ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 01-A: RENAL ANATOMY AND PHYSIOLOGY Afferent The afferent (thicker) and RENAL ANATOMY arterioles efferent (thinner) arterioles Kidneys (True Regulators) Glomerulus have varying thickness to Bean-shaped organs located in the retro- Efferent maintain the hydrostatic peritoneum (posterior abdominal wall) arterioles pressure differential needed General Parts: for glomerular filtration Peritubular Covers the proximal convol- GENERAL PARTS OF THE KIDNEY capillaries uted tubules and distal con- PART DESCRIPTION voluted tubules Renal hilus An indention by where renal PCT: immediate artery enters, and renal vein reabsorption of and ureter exits essential substances Cortex Outer layer of the kidney – from the fluid the exclusive site for plasma DCT: final adjustment of filtration urinary composition Medulla Inner layer – consists of Vasa recta Located adjacent to the as- tissues shaped into pyramids cending and descending Loops of Henle – site of Nephrons: the kidneys’ basic structural major exchanges of water and functional unit and salts to maintain the o Each kidney consists of approxima- osmotic gradient in the tely 1 to 1.5 million nephrons medulla needed for renal TYPES OF NEPHRONS concentration TYPE DESCRIPTION Renal veins - Cortical Predominant nephrons nephrons (85%) found in the cortex GLOMERULAR FILTRATION Function: removal of waste Glomerular Filtration products and reabsorption of Used to monitor kidney disease prog- nutrients ression Juxtamedulla- Longer loops of Henle that extend deep into the medulla Glomerulus: ry nephrons o Located in the Bowman’s capsule Function: urine o Characterized as a coil of approxi- concentration mately 8 capillary tufts o Serves as nonselective filter of Renal Functions: plasma substances with molecu- o Renal blood flow lar weights of less than: o Glomerular filtration § 70,000 Da (Strasinger) o Tubular reabsorption § 50,000 Da (Graff’s) o Tubular secretion Three (3) Factors in Glomerular Filtra- tion: RENAL BLOOD FLOW FACTORS IN GLOMERULAR FILTRATION Renal Blood Flow FACTOR DESCRIPTION In normal individuals (ave. BSA = 1.73 m2), Glomerular Capillary Wall: consists of structure fenestrated endothelial approximately 1,200 mL of blood perfuse cells (with pores) which are the kidneys each minute (1,200 mL/min) permeable but does not accounting for 20% to 25% cardiac output allow passage of large Renal plasma flow: 600 to 700 mL/min molecules and blood cells RENAL BLOOD FLOW Basement Membrane (basal PART DESCRIPTION lamina): further restricts Renal artery Blood enters the kidneys passage of large molecules through the renal artery CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 01-A: RENAL ANATOMY AND PHYSIOLOGY Visceral (inner) Bowman’s Capsule: consists of podo- cytes or foot cells which have fingerlike processes that intertwine and form a snake- like channel on the surface of glomerular capillaries known as filtration slits – also fur- ther restrict large molecules Shield of Negativity: nega- tively-charged shield that re- pels negatively-charged plasma proteins even if they have smaller molecular weights Albumin (primary protein in renal disease). is positively-charged can bypass this shield SUMMARY OF RAAS MECHANISMS Glomerular The hydrostatic pressure Dilates afferent arterioles, and constricts the pressure from the varying thickness of efferent arterioles the afferent and efferent ar- Stimulates sodium reabsorption in the proximal terioles overcomes: convoluted tubules (PCT) Opposing pressure of Stimulates adrenal cortex to release fluids within the aldosterone for sodium retention and Bowman’s capsule potassium excretion (DCT & CD) Oncotic pressure of Stimulates hypothalamus to release plasma proteins in the antidiuretic hormone (ADH) for water glomerular capillaries resorption (CD) The hydrostatic pressure also stimulates an autoregulatory mechanism within the juxta- TUBULAR REABSORPTION glomerular apparatus nee- Tubular Reabsorption ded to maintain the glomeru- First function affected in renal disease lar blood pressure at a rela- Proximal convoluted tubules: major site tively constant rate in the reabsorption of [65%] of plasma Renin-angiotensin-aldosterone system substance (RAAS): regulates blood flow to and within the o The body cannot always lose 120 glomerulus – it also responds to blood pressure mL of water containing nutrients and sodium concentration changes within the every minute – so once the plasma juxtaglomerular apparatus ultrafiltrate (urine) passes through the PCT, essential water and nut- Juxtaglomerular apparatus: Macula densa (in the DCT) – senses low rients are reabsorbed plasma sodium which decreases water Reabsorption Mechanisms: reten-tion and subsequent decrease in REABSORPTION MECHANISMS overall blood volume and blood Active transport: movement of substances a- pressure cross membranes and into the bloodstream by Juxtaglomerular cells (in afferent an electrochemical energy (produced from the arterioles) – release renin that reacts with the angiotensinogen substrate interaction of the substance to be reabsorbed and its carrier protein). CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 01-A: RENAL ANATOMY AND PHYSIOLOGY ACTIVE TRANSPORT decrease permeability – water could not enter SUBSTANCE LOCATION producing large volume Glucose, amino PCT of dilute urine acid, salts Chloride Ascending loop of Henle Sodium PCT and DCT. TUBULAR SECRETION Passive transport: movement of molecules a- cross a membrane by diffusion because of a Tubular Secretion physical gradient Two major functions: PASSIVE TRANSPORT TUBULAR SECRETION MAJOR FUNCTIONS SUBSTANCE LOCATION Elimination of waste products not filtered by the Water PCT, descending LH, glomerulus (e.g., medications bound to plas- and CD ma proteins eliminated in the PCT) Urea PCT and ascending Regulation of acid-base balance in the body LH through secretion of hydrogen ions (100% Sodium Ascending LH reabsorption of bicarbonate ions maintain the. physiologic blood pH 7.4) Loops of Henle: where renal concentra- Renal Tubular Acidosis: inability to pro- tion begins duce acid urine (hydrogen ions are not o Countercurrent mechanism: wa- excreted in the urine) due to: ter impermeable walls of the ascen- o Defective tubular hydrogen ion ding loop prevents excessive re- secretion absorption of water o Defective tubular ammonia Collecting Ducts: final concentration of fil- production trate through reabsorption begins in the o Defective tubular bicarbonate distal convoluted tubules (DCT) and con- reabsorption in PCT tinues in the collecting ducts (CD) Hormonal involvement: HORMONES IN REABSORPTION RENAL FUNCTION TESTS HORMONE DESCRIPTION Glomerular Filtration Tests Aldosterone Producer: adrenal cortex Otherwise known as clearance tests – measures the kidney’s capacity to remove Function: Regulates or clear a filterable substance from the absorption of sodium in the blood (thereby measuring the filtering ca- DCT pacity of the glomeruli) Antidiuretic Producer: posterior pituitary Criterion for accurate GFR measure- hormone gland ment: substance to be analyzed must (Vasopressin) Function: regulates water neither be reabsorbed nor secreted reabsorption in the DCT and CLEARANCE TESTS CD TEST DESCRIPTION High level of ADH: Urea Earliest GFR test walls of DCT and CD clearance increase permeability Advantage: present in all – water is reabsorbed urine samples producing low-volume concentrated urine Disadvantage: approximate- ly 40% is filtered – will require patients to be hydrated to Low level of ADH: ensure 2 mL/min urine flow walls of DCT and CD CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 01-A: RENAL ANATOMY AND PHYSIOLOGY Inulin Gold standard and original black) clearance reference method MDRD-IDMS (stan- dardized) – recommen- Advantage: extremely stable ded by the Nat’l Kidney polymer of fructose not reab- Disease Education sorbed nor secreted by the Program (NKDEP) tubules GFR = 175 x CreaS-1.154 x age-0.203 x 0.742 (if Disadvantage: not a normal female) x 1.212 (if body constituent – need exo- black) genous infusion by IV at Cockroft & Gault – constant rate throughout the multiply by 0.85 if the test patient is female Creatinine Most commonly used clearance method Advantage: an endogenous Cystatin C Small protein that is produced substance by all nucleated cells at a constant rate Disadvantage: several Advantage: Formulae: Independent of muscle Assuming normal body mass surface area: 1.73 m2 Recommended for ped- iatric, geriatric, diabetic, and critically ill patients Measurement of plasma o C: creatinine and serum cystatin C, clearance and creatinine provides o U: urine creati- an even more accurate nine (mg/dL) information on GFR o V: urine volume β-2-microglo- Dissociated from the human (mL/min) bulin leukocyte antigen (HLA) at o P: plasma crea- a constant rate tinine (mg/dL) Assuming deviation Advantage: a rise in plasma from normal BSA β-2-microglobulin provides a more sensitive indicator of decrease in GFR than creati- nine clearance o A: the patient’s actual body sur- Disadvantage: not reliable face area for patients with immunologic eGFR Uses: disorders and malignancy Screening of patients as Radionucleo- Injection of 125I-iothalamate part of metabolic tides determines glomerular filtra- profile tion through the disappea- Monitor patients with rance of radioactivity in renal disease plasma and enables visua- lization of filtration in one Formulae: or both kidneys – valuable Modification of Diet in for measuring viability of Renal Disease (MDRD) transplanted kidney GFR = 173 x CreaS-1.154 x age-0.203 x 0.742 (if Disadvantage: labor-inten- female) x 1.212 (if sive and expensive CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 01-A: RENAL ANATOMY AND PHYSIOLOGY Tests: Tubular Reabsorption Tests TESTS FOR SECRETION AND BLOOD FLOW Otherwise known as concentration tests TEST DESCRIPTION – determine the ability of the tubules to re- Phenolsulfon- Dye excretion test – now absorb the essential salts and water that phthalein obsolete as results are hard have been non-selectively filtered by the (PSP) test to interpret glomerulus SG of ultrafiltrate in tubules: 1.010 – af- P-amminohip- Also known as the Diodrast puric acid test – most commonly assoc- ter reabsorption, it is expected that the final (PAH) test) iated test for this function urine product is more concentrated Titratable Normal person excretes ap- CONCENTRATION (OBSOLETE) TESTS Acidity and prox. 70 mEq/day of acid in TEST DESCRIPTION Urinary NH4 + the form of: Fishberg test Patients were deprived of Acid (H+) fluids for 24 hours before Hydrogen phosphate measuring specific gravity ions (H2PO4–) (SG 1.026 or higher) Ammonium ions (NH4+) Mosenthal test Comparison of specific gra- Oral ammoni- Oral administration of ammo- vity between day and night um chloride nium chloride which metaboli- urine samples to evalulate test zes to urea and HCl – failure concentrating ability to excrete acidic urine sup- Current tests now measure specific gravi- ports diagnosis of renal tu- ty for screening, and measure osmolality bular acidosis for accurate evaluation of renal concen- trating ability URINE COMPOSITION COMMONLY PERFORMED TESTS Urine Composition TEST DESCRIPTION Specific Most useful as a screening URINE COMPOSITION* gravity procedure – influenced by 95% water the number and density 5% solutes: total solids in 24 hours (60 g) (molecular weight) of the particles 60-GRAM URINE TOTAL SOLIDS Osmolality* Influenced by the number of SOLID COMPOSITION particles in a solution only Organic solids Urea: major organic so- * Quantitative measurement of renal concentrating (35 g) lute produced from protein ability is best assessed through osmometry and amino acid metabolism Creatinine: from creatine FREEZING POINT OSMOMETERS metabolism by muscles Freezing Point Osmometers: measurement of Uric acid: from nucleic acid freezing point depression was the first princi- breakdown of food/cells ple incorporated into clinical osmometers Inorganic Chloride: major inorganic Conversions is made possible the fact that solids (25 g) solute seen in combination 1 mol (1000 mOsm) of a nonionic subs- with sodium (NaCl – princi- tance dissolved in 1 kg of water is known to lower the freezing point 1.86 degrees pal salt) Celsius Sodium Potassium Tubular Secretion and Renal Blood Flow Tests Phosphate Tests to measure tubular secretion of non- Ammonia filtered substances and renal blood flow Calcium. are tied together in that they must be mea- * To determine whether a body fluid is urine, test for sured by a substance that is secreted ra- urea and creatinine ther than filtered through the glomerulus CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 01-B: MACROSCOPIC EXAMINATION Timed specimens: URINE COLLECTION TIMED URINE SPECIMENS Specimen Container URINE DESCRIPTION Specimen container capacity: 50 mL 24-hour urine Usually used for creatinine o Allow for 12 mL of the urine sam- clearance tests (begin and ple for microscopic analysis end with empty bladder) o Additional specimen for repeat an- 12-hour urine For Addis Count alysis 4-hour urine For nitrite determination o Room for specimen to be mixed by Afternoon For urobilinogen swirling. Other containers: THREE-GLASS TECHNIQUE* OTHER URINE CONTAINERS First tube: first voided urine – examined micro- CONTAINER DESCRIPTION scopically Bags with Pediatric specimen adhesive Second tube: midstream urine – control for Large 24-hour urine bladder and kidney infection containers Third tube: post-massage prostatic fluid – exa- Sterile containers: sometimes with transfer mined microscopically, straws – transfer device with a needle and eva- Prostatic infection is indicated if it has a cuated tube holder in special sterile containers WBC and bacterial count ten (10) times than that of the first specimen Uses: If the second specimen is positive, the Individually packaged for microbiologic third specimen becomes invalid due to urine studies contamination Delayed analysis more than 2 hours after * Three-glass technique is a quantitative test that collection requires specimens to be placed in sterile containers Urine Specimen DRUG TESTING SPECIMEN* Chain-of-Custody (also Chain-of-Evidence): TYPES OF URINE SPECIMENS step-by-step documentation of handling and URINE DESCRIPTION testing of legal specimen for proper sample Random urine Most commonly received – identification starting from specimen collec- used for routine screening tion to the receipt of laboratory results Midstream For routine screening and urine bacterial culture Donor: the individual from which the specimen First-morning Ideal screening specimen is collected urine (8-hr) and the most concentrated Volume of 30 to 45 mL – useful for pregnancy test Urine and orthostatic proteinuria Temperature 32.5 to 37.7 degrees Celsius Fasting urine Second voided urine useful of Urine (second for diabetes screening and Temperature of specimen must be taken morning) monitoring within minutes after collection to confirm that 2-hr post Used for monitoring insulin the specimen is not adulterated prandial therapy of diabetes patients Catheterized Collected in sterile condi- Beyond the temperature range – record tempe- urine tions for bacterial culture rature and immediately inform supervisor or Suprapubic Bladder urine used for bac- employer of the contaminated specimen and urine terial culture and cytology recollect sample for retesting * In Drug Testing, chemical reagent strip test is NOT Pediatric Catheterized or suprapubic performed specimen urine placed in a soft and clear bag with hypoallerge- nic skin adhesive CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 01-B: MACROSCOPIC EXAMINATION URINE PRESERVATION Urine Preservation Urine specimen must be promptly trans-ported to the laboratory and should be tested within two (2) hours, or if delayed, be refrigerated or preserved using che-mical preservatives Changes in unpreserved include: CHANGES IN UNPRESERVED URINE FACTOR CHANGE Color Modified or darkened Clarity Decreased Odor Increased pH Increased Glucose Decreased Ketones Decreased Bilirubin Decreased Urobilinogen Decreased Nitrite Increased RBCs/WBCs Decreased Bacteria Increased Preservation Methods and Preservatives The most accessible and routinely per-formed preservation is by refrigeration at 2 to 8 degrees Celsius URINE PRESERVATION PRESERVATIVE ADVANTAGE DISADVANTAGE Refrigeration Does not interfere with chemical Raise specific gravity hydrometer tests Can precipitate amorphous Prevents bacterial growth for 24 hrs phosphates and urates Thymol An excellent preservative for It interferes with acid precipitation sediments and glucose tests for protein Boric acid (keeps pH at Can be used for culture Can precipitate large crystals when about 6) used in great amounts Preserves protein (albumin) and 10g Boric acid can be formed elements well used for aldosterone and cortisol Does not interfere with routine tests other than pH Formalin (rinsed into the Used for Addis count as it is an Can act as a reducing agent that will specimen container) excellent sediment preservative interfere with chemical tests for glucose, blood and leukocytes, as well as in copper reduction tests Toluene Does not interfere with routine tests It will float on the surface of specimen and can cling into pipettes and testing materials Sodium fluoride Useful for glucose as it prevents It can inhibit reagent strip test for glycolysis, and excellent for drug glucose, blood and leukocyte analysis specimens esterase (Remedy: use benzoate instead of fluoride) CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 01-B: MACROSCOPIC EXAMINATION Phenol (1 drop per Does not interfere with routine tests Gives of odor ounce of specimen) Commercial Convenient if refrigeration is not It can contain other preservatives Preservative Tablets possible such as sodium fluoride (which inhibit reagent strip tests for several Controlled composition to minimize parameters) interferences Saccomano’s fixative Used for cytology as it preserves Potential chemical hazard cellular elements 6N Hydrochloric acid Used for catecholamines Potential chemical hazard and not (HCl) acceptbable for UA testing Oliguria: decreased urine output PHYSICAL URINALYSIS OLIGURIA URINE VOLUME Physical Urinalysis POPULATION VOLUME First part of urinalysis which includes the Adults 9) – recollect sample MICRALTEST Swimming Hawkinsinuria Principle Enzyme immunoassay pool Sample Random urine Odorless Acute tubular necrosis Reagents Gold-labeled antibody β-galactosidase Chloramphenicol red ga- CHEMICAL URINALYSIS lactoside Protein (1 minute) Sensitivity 0 to 10 mg/dL The most indicative of renal disease out False (–) Dilute urine of all chemical tests for urine Correlation Simultaneous urine Normal Values: crea-tinine (provide o 0.025 Hemolytic disease of newborn Alpha-fetoprotein Hematology > Chemistry sidered as the third major body fluid * Only if a fourth tube is collected FUNCTIONS OF CEREBROSPINAL FLUID Supplies nutrients to the nervous system CSF APPEARANCE Remove metabolic wastes Provide a mechanical barrier to protect the brain CSF Appearance and spinal cord from trauma CEREBROSPINAL FLUID APPEARANCE Meninges: APPEARANCE DESCRIPTION Crystal clear Normal THE MENINGES Hazy/Cloudy/ WBC count ≥200/μL PART DESCRIPTION Turbid/Milky RBC count ≥400/μL Dura mater Lines the skull and vertebral Others: lipids, protein and (outer layer) canal microorganism Arachnoid Spiderweb-like filamentous Oily Radiographic contrast pedia layer inner membrane Clotted Meningitis, protein, clotting Subarachnoid Below the arachnoid layer – factors, Froin syndrome, space where the CSF flows blockage of CSF circulation Pia mater Lines the surface of the brain Pellicle Tubercular meningitis (innermost) and spinal cord Xanthromic (pink/yellow/orange): presence of hemoglobin degradation products CSF Production and Reabsorption: XANTHOCHROMIC SUPERNATANT CSF PRODUCTION AND REABSORPTION COLOR DESCRIPTION PART DESCRIPTION Pink Slight amount of oxyhemo- Choroid Produces cerebrospinal flu- id (CSF) by selective filtra- globin plexus tion at a rate of 20 mL/hour Yellow Oxyhemoglobin converted to Arachnoid villi Also the granulation – reab- unconjugated bilirubin sorbs CSF in the same rate Orange Severe hemolysis. it is produced Bloody: presence of RBCs (≥6,000/μL) – due Blood-Brain Pseudo-structure – protects to traumatic tap (non-pathologic) or intracra- Barrier (BBB) the brain from harmful chemi- cals and substances nial hemorrhage (pathologic) CSF Volume: BLOODY CSF FEATURE T.T.* I.H.* CEREBROSPINAL FLUID VOLUME Blood 1>2>3 1=2=3 POPULATION REFERENCE RANGE distribution Adults 90 to 150 mL Clot- + – Neonates 10 to 60 mL formation CSF Collection Supernatant Clear Xanthochro- o Volume to be collected: 20 mL mic o Method: Lumbar puncture or spinal Erythropha- – + tap ges o Puncture site: Between the 3rd, 4th D-dimer – +. and 5th Lumbar processes THREE-COLLECTION TUBE TUBE SECTION STORAGE CSF CELL COUNT 01 Chemistry/Serology Freezing CSF Cell Count 02 Microbiology Room temp. Should be performed immediately CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 03: CEREBROSPINAL FLUID If testing immediately is not possible, CSF CORRECTIONS FOR CONTAMINATION is stored at refrigerator temperature SUBTRACT RBC COUNT IMMEDIATE CSF CELL COUNT 1 WBC For every 700 RBCs/μL RBCs and WBCs lyse after one (1) hour 1 mg/dL CHON For every 1200 RBCs/μL 40% WBCs disintegrate after two (2) hours 8 mg/dL CHON For every 10,000 RBCs/μL Types of CSF Cell Counts: CSF Differential Count CEREBROSPINAL FLUID CELL COUNT Performed in stained smears using con- TYPE DILUENT centrated CSF specimens by any of the Total cell ct. Normal saline following concentration techniques WBC count 3% HAc + methylene blue CSF CONCENTRATION TECHNIQUES RBC count Subtract total cell and WBC TECHNIQUE DESCRIPTION Dilution: based on CSF clarity Centrifugation Most common in laboratories CEREBROSPINAL FLUID DILUTION that do not have a cytocentri- fuge CLARITY DILUTION Filtration Produce the least distortion Clear Undiluted (DF: 1) Sedimentation of cells (not routinely done) Slightly hazy 1:10 Cytocentrifugation: Hazy 1:20 CSF is placed in a conical chamber Slightly cloudy 1:100 Cells are forced into a monolayer of a 6- Cloudy, 1:200 mm circle on the slide slightly bloody Addition of 30% Albumin: Bloody turbid 1:10,000 o Increase cell yield/recovery Cell Count Formula: o Decrease cell distortion CSF CELL COUNT FORMULA Daily Control Slide for 30% Albumin: done to Formula: prevent contamination of CSF Albumin is mixed with 0.2 mL normal sa- line smeared on a slide Smear is stained and examined micro- scopically Where; Predominant cells in CSF: lymphocytes, DF = dilution factor monocytes, and occasional minute levels of Area = area of counting chamber in mm2 neutrophils o One large WBC square: 1 mm2 o One small RBC square: 0.04 CEREBROSPINAL FLUID WBC COUNT* mm2 POPULATION REFERENCE RANGE Depth = 0.1 (constant depth of the Neuba- Adults Predominant lymphocytes over monocytes (70:30) uer chamber of the hemocytomer) Neonates Reverse ratio of (monocytes are up to 80%) CSF WBC Count * Pleocytosis: occurs when there is increased amounts Performed routinely in CSF of normal cells which may have pathologic causes CEREBROSPINAL FLUID WBC COUNT POPULATION REFERENCE RANGE Additional Notes: Adult 0 to 5/μL Neonates 0 to 30/μL CSF RBC Count Performed only in cases of traumatic tap to serve as a correction for WBC count and protein concentrations CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 03: CEREBROSPINAL FLUID Cells in CSF: CELLS IN CEREBROSPINAL FLUID CELLS CELLS Lymphocytes (resemble C. Meningitis (viral, tubercular or fungal), and multiple sclerosis neoformans) Monocytes Meningitis (viral, tubercular or fungal), and multiple sclerosis Neutrophils Bacterial meningitis (early cases of viral, tubercular or fungal meningitis), and cerebral hemorrhage Macrophages (erythrophages) RBCs in spinal fluid and presence of contrast media Blast cells Acute leukemia Lymphoma cells (resemble Disseminated lymphoma lymphocytes with cleft nuclei) Plasma cells Multiple sclerosis and lymphocyte reactions Choroidal, ependymal, spindle- Seen following diagnostic procedures shaped cells Malignant cells Metastatic carcinomas and primary CNS carcinomas CSF CHEMISTRY Proteins in CSF CSF Protein Most commonly performed Chemistry PROTEINS IN CEREBROSPINAL FLUID test in CSF CLASS PROTEINS Major protein Albumin CEREBROSPINAL FLUID PROTEIN* 2nd Prevalent Pre-albumin/Transthyretin* POPULATION REFERENCE RANGE α-globulin Haptoglobin Adult 15 to 45 mg/dL Ceruloplasmin Neonates 150 mg/dL β-globulin Transferrin (Tau protein)** Premature 500 mg/dL γ-globulin Immunoglobulin G (primary) * Normal values of CSF tend to be method-dependent Immunoglobulin A (some) Abnormal CSF Protein Values: * Prealbumin migrates faster than albumin ** Tau protein is a carbohydrate-deficient transferring found CEREBROSPINAL FLUID PROTEIN in CSF but not in serum (and can be used to identify LEVEL CLINICAL SIGNIFICANCE CSF) Increased Damaged blood-brain bar- rier (BBB): meningitis, and Tests for CSF Protein hemorrhage TURBIDIMETRIC METHOD Immunoglobulin synthesis: 9% Trichloroacetic acid (TCA): reagent multiple sclerosis (IgG) of choice – can precipitate both albumin Other causes: polyneuritis, TOTAL PROTEIN and globulin primary CNS tumors, Guil- 3% Sulfosalicylic acid (SSA): can only lain-Barre syndrome, neuro- precipitate albumin – can precipitate glo- syphilis, myxedema, Cushing disease, connective tissue bulin, if sodium sulfate is added disease, diabetes and uremia DYE-BINDING METHOD Decreased CSF leakage/trauma Coomassie brilliant blue: as protein Recent puncture binds with the dye, color changes from Rapid CSF production red to blue (intensity of blue color is pro- Water intoxication portional to protein concentration) CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 03: CEREBROSPINAL FLUID CSF/SERUM ALBUMIN INDEX Assess integrity of blood-brain barrier Multiple Sclerosis: demyelinating disorder (BBB) causing nerve im-pulses to travel slower MULTIPLE SCLEROSIS FINDINGS Formula: Positive 2 or more oligoclonal bands in CSF but not in serum Positive anti-myelin sheath autoantibody Positive myelin basic protein (MBP)* Reference Values: Increased IgG index ALBUMIN INDEX VALUES * Myelin basic protein (MBP): protein component of the lipid-protein complex that insulates nerve fibers – in- INDEX CLINICAL SIGNIFICANCE dicative of damage in myelin sheath 9 Impaired BBB CSF Glucose 9 to 14 Slight impairment Glucose enters the CSF by selective tran- 15 to 30 Moderate impairment sport spinal tap 2 hours before >30 Severe impairment. 100 Complete impairment ✓ CEREBROSPINAL FLUID GLUCOSE Determination: CSF glucose is performed in IgG INDEX conjunction with plasma glucose 2 hours be- PROTEIN FRACTION TESTS Assess conditions associated with IgG fore the spinal tap (allow time for equilibrium production within the CNS (e.g., Multi- between CSF and blood glucose) ple sclerosis) Normal Values: CSF glucose is 60% to 70% of Formula: plasma glucose Increased Usually increased due to in- crease in plasma glucose Reference Values: Normal Seen in viral meningitis Decrease Bacterial, tubercular, and IgG INDEX VALUES fungal meningitis INDEX CLINICAL SIGNIFICANCE 0.7 IgG synthesis in CNS Useful aide in the diagnosis and manage-. CSF ELECTROPHORESIS ment of meningitis Detects oligoclonal bonds in the γ CEREBROSPINAL FLUID LACTATE region in both CSF and serum (done Inversely related to glucose – provides a more conjunctively) reliable information when initial diagnosis is ELECTROPHORETIC RESULTS difficult BANDS SIGNIFICANCE CSF: ≥2 bands Multiple sclerosis Normal Values: 10 to 24 mg/dL Serum: 0 bands Increased Bacterial meningitis (>35 CSF: (–) bands Leukemia, lympho- mg/dL) Serum: (+) bands ma, viral infections Tubercular meningitis (>25 CSF: (+) bands HIV mg/dL) Serum: (+) bands Fungal meningitis (>25. mg/dL) OTHER TESTS Normal or Seen in viral meningitis Radioimmunodiffusion (RID) decreased Nephelometry CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 03: CEREBROSPINAL FLUID CSF Glutamine CK-BB Increased: bacterial meningi- Glutamine is a product of ammonia and α- tis, brain tumor, cardiac ar- ketoglutarate during the removal of toxic rest, degenerative disorders, ammonia from CSF epileptic seizures, multiple sclerosis, stroke, viral menin- CEREBROSPINAL FLUID GLUTAMINE gitis Measurement of ammonia can be replaced by AST Increased: bacterial meningi- glutamine tis, and intracerebral or suba- More stable than volatile ammonia rachnoid hemorrhage Correlates with clinical symptoms better Normal Values: 8 to 18 mg/dL f- v6 wgldv CSF MICROBIOLOGY Increased Liver disorders: increased CSF and blood ammonia Differential Diagnosis of Meningitis Reye’s syndrome: acute en- BACTERIAL MENINGITIS cephalopathy and liver infil- WBC count: increased tration in children following Predominant: neutrophils viral infection T Protein: markedly increased limulus lyrate Disruption of conscious- t Glucose: markedly decreased ness: seen in coma of un- Lactate: >35 mg/dL known origin (>35 mg/dL) Gram stain and culture: positive Limulus lysate: positive for Gram-negative CSF Enzymes organisms (endotoxins) CEREBROSPINAL FLUID ENZYMES ENZYME DESCRIPTION TUBERCULAR MENINGITIS LDH Intracellular enzyme with WBC count: increased several isoenzymes Predominant: lymphocytes and monocytes LDH ISOENZYMES Protein: moderately to markedly increased Glucose: decreased ISOFORM LOCATION not LD1 & LD2 Brain tissues Lactate: >25 mg/dL pellicle LD2 & LD3 Lymphocytes Pellicle clot: positive LD4 & LD5 Neutrophils FUNGAL MENINGITIS Serum LDH WBC count: increased Predominant: lymphocytes and monocytes SERUM LDH PATTERNS Protein: moderately to markedly increased PATTERN LOCATION Glucose: decreased 2,113,415 Normal 2>1>3>4>5 Lactate: >25 mg/dL AMI* 1>2 India ink: positive * Also seen in hemolytic anemia Gram stain: positive starburst pattern Latex agglutination: positive for cryptococcal CSF LDH antigen CSF LDH PATTERNS PATTERN LOCATION VIRAL MENINGITIS Normal 1>2>3>4>5 WBC count: increased lymphocytes Neurologic 2>1 Predominant: lymphocytes abnormali- Protein: moderately increased 2 >7 ties Glucose: normal Bacterial & 5>4>3>2>1 Lactate: normal meningitis I-4-3-2 I- Agents: Enteroviruses. CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 03: CEREBROSPINAL FLUID Summary of Bacterial Meningitis Agents AGENTS OF BACTERIAL MENINGITIS POPULATION CAUSATIVE AGENT Birth to 1 month old Streptococcus agalactiae (also E. coli) 1 month to 5 years old Haemophilus influenzae type b 5 years to 29 years old Neisseria meningitidis >29 years old Streptococcus pneumoniae Infants, elderly, immunocompromised patients Listeria monocytogenes Summary Comparison of Meningitis Agents COMPARISON OF MENINGITIS AGENTS FEATURE BACTERIAL TUBERCULAR FUNGAL VIRAL Predominant Neutrophils Lymphocytes Lymphocytes Lymphocytes WBC Monocytes Monocytes CSF Protein Marked increase Moderate to Moderate to Moderate increase marked increase marked increase CSF Glucose Marked decrease Decrease Decrease Normal CSF Lactate Inc (>35 mg/dL) Inc (>25 mg/dL) Inc (>25 mg/dL) Normal Other information Positive: Positive: Positive: Agents: Enterovi- Gram stain Pellicle clot India ink rus (most common Culture Gram stain cause of aseptic Limulus starburst meningitis) lysate pattern Poliovirus Latex aggl’n Echovirus for cryptococ- Coxsackie- cal antigen virus CSF SEROLOGY Serologic Tests CEREBROSPINAL FLUID SEROLOGY SEROLOGIC TEST SIGNIFICANCE ELISA Used for the detection of bacterial antigens Latex agglutination VDRL CDC-recommended for detection of neurosyphilis CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 04: SEMINAL FLUID PROSTATE FLUID 20%-30% 20-301 SEMINAL FLUID. Location: prostate gland – produces high con- Purpose of Seminal Fluid Analysis centrations of enzymes needed for liquefac- PURPOSE OF SEMINAL FLUID ANALYSIS tion and coagulation PURPOSE DESCRIPTION PROSTATIC FLUID CONSTITUENTS Fertility tes- Varicocele (most common Acid phosphatase ting cause of male infertility): Citric acid hardening of the veins that Proteolytic enzymes drain out the testes causing Zinc the blood from the adrenal. vein to flow into the spermatic ALKALINE MUCUS 05% 0.5% vein (tapered head of sperm) Location: bulbourethral (Cowper’s) gland – pro- Post-vasecto- Vasectomy: surgical removal duces the alkaline mucus needed to neutra- my analysis of all or parts of the vas (duc- lize the acidic prostate fluid and vagina tus) deferens for the purpose of male sterility Specimen Collection Medico-legal For suspected cases of rape Require sexual abstinence for 2 to 7 (ave. cases 3) days Methods of Collection: Physiology METHODS OF COLLECTION Semen is composed of four (4) fractions METHOD DESCRIPTION contributed by the; (a) testis and epididymis Self-produc- Best and preferred method (b) seminal vesicles (c) prostate gland and tion (Mastur- (d) bulbourethral (Cowper’s) gland bation) SEMINAL VOLUME Coitus inter- Not reliable – first portion of 51. SPERMATOZOA 5% ruptus the ejaculate may not be col- Location: testis and epididymis – site of lected spermatogenesis (maturation of sperm) Condom Usually discouraged but if (Silastic) done, we must use either (a) SPERM MATURATION STAGES non-lubricant-containing method STAGE MATURATION rubber (b) polyurethane con- Spermatogonia (stem cell) Approximately doms or (c) Silastic condoms Spermatocytes 70 days Vaginal For medico-legal cases of Spermatid* aspiration suspected rape Spermatozoa (mature) Approximately Should be transported to the laboratory 20 days within one (1) hour at room temperature TOTAL 90 days or body temperature (armpits/breast) * Once spermatogenesis is complete, immature (nonmotile) sperm is taken to the epididymis – Time of specimen collection and receipt where it fully matures and develop flagella (motile) should be noted 701 SEMINAL FLUID 60%-70% Specimen waiting for analysis should be 60 -. Location: seminal vesicles – produces the bulk kept at 37 degrees Celsius of seminal volume which has high concentra- Analysis is only made after liquefaction tions of fructose and flavine fructose 4 flavine (30 to 60 minutes after collection) o If sample fails to liquefy within SEMINAL FLUID CONSTITUENTS two (2) hours, the following should PRODUCT DESCRIPTION be added: Fructose Provide energy needed by INDUCERS OF LIQUEFACTION the flagella to propel Equal volume of physiologic Dulbecco’s through the vaginal canal phosphate-buffered saline Flavine Gives semen its gray-opa- Proteolytic enzymes: amylase, bromelain or lescent color alpha-chymotrypsin. CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 04: SEMINAL FLUID MACROSCOPIC EXAMINATION Viscosity/Consistency (related to liquefaction) Appearance SEMEN VISCOSITY/CONSISTENCY* SEMEN APPEARANCE VISCOSITY DESCRIPTION APPEARANCE DESCRIPTION Normal Poured in droplets (not Grayish-trans- Normal (with characteristic stringy) lucent musty or bleach-like odor Abnormal Stringy and forms a viscous Increased Possible infection thread longer than 2 cm white turbidity (increased WBCs) * Reported as: 0 (watery) or 4 (gel-like); may also be reported as: low, normal, or high Red/brown Presence of blood (increased RBCs) Yellow Urine (toxic to sperm) pH contamination SEMEN pH Prolonged abstinence pH DESCRIPTION Medications Normal 7.2 to 8.0 Increased pH Possible infection Volume Decreased pH Increased prostatic fluid SEMEN VOLUME Ejaculatory duct obstruction VOLUME DESCRIPTION Poorly-developed seminal Normal 2 to 5 mL vesicles Increased Prolonged abstinence volume Decreased Infertility* volume Incomplete collection * May be due to impairment of one of the semen-pro- ducing organs, most especially the seminal vesicles MICROSCOPIC EXAMINATION Sperm Concentration Usually performed under phase-contrast or brightfield microscopy, expressed in millions/mL Reference Values: SPERM CONCENTRATION REFERENCE VALUES CONCENTRATION REFERENCE RANGE Normal 20 to 250 million/mL (Strasinger 6th Edition) 20 to 160 million/mL (Strasinger 4th Edition) Borderline 10 to 20 million/mL Counting Chambers: SPERM CONCENTRATION COUNTING CHAMBERS CHAMBER DESCRIPTION Makler counting chamber Used for undiluted specimen – divided into two (2) portions: Heated portion to immobilize sperm cells Unheated portion for other parameter evaluation. Neubauer counting chamber Uses diluents (most common – 1:20) before char- ging both chambers of the hemocytometer (val- ues must agree within 10%) CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 04: SEMINAL FLUID Dilution: done to immobilize sperm cells (may also contain stains to differentiate sper- matids and leukocytes) Diluents: SPERM CONCENTRATION DILUENTS Sodium bicarbonate and formalin Chilled tap water Distilled water Saline sperm count = sperm con ✗ sperm volume (millions / ejaculate) Formula: (sperm et ) ( DF ) sperm concentration = ( Area) ( Depth) (millions 1mL) Where; DF = dilution factor Area = area of counting chamber in mm2 o One large WBC square: 1 mm2 o One small RBC square: 0.04 mm2 Depth = 0.1 (constant depth of the Neubauer chamber of the hemocytomer) Sperm Count 3 b Slower speed, some lateral Total sperm in millions per ejaculate movements Reference Values: 2 b Slow forward progression, noticeable lateral movements SPERM COUNT REFERENCE VALUES 1 c No forward progression COUNT REFERENCE RANGE 0 D No movement Normal >40 million/ejaculate Reference Values: Formula: SPERM MOTILITY REFERENCE VALUES SPERM COUNT FORMULA >50% motile sperms with a grading of at least 2.0 or b Computer-Associated Semen Analysis (CASA) Sperm Motility Parameters measured: Evaluated in terms of speed and direction Methods: CASA MEASURED PARAMATERS Velocity SPERM MOTILITY METHODS Trajectory (direction) Estimate percentage of sperm with actual rapid Concentration progression in 20 high power fields (HPFs) Morphology Examine 200 sperms per pre-warmed slide and obtain percentage of different categories Sperm Morphology of motility using a manual counter Sperm Anatomy: Criteria on Sperm Motility: SPERM CELL ANATOMY SPERM MOTILITY CRITERIA STRUCTURE DESCRIPTION ROU. WHO DESCRIPTION Acrosomal Should occupy half of the 4 a Rapid forward progression cap* head and cover 2/3 of the nucleus CLEOTABADA  ANALYSIS OF URINE & OTHER BODY FLUIDS CHAPTER 04: SEMINAL FLUID Head Oval-shaped (5 μm long and 3 μm) Sperm Viability/Vitality Midpiece** Contains mitochondria for Method: examination of 100 sperm cells energy (7 μm long) on an Eosin-Nigrosin stained smear of Flagellar tail** Approximately 45 μm long semen * Acrosomal cap produces the enzyme acrosin which is critical for ovum penetration ** Neckpiece connects SPERM VIABILITY/VITALITY the midpiece and tail SPERM DESCRIPTION Abnormal Sperm Morphology: Living sperm Unstained, remain bluish white SPERM CELL ANATOMY Dead sperm Take up stain, appear red ABNORMAL DESCRIPTION against a purple Head (and ac- Poor penetration background rosomal cap) Reference Values: Neck/midpiece/ Affects motility tail SPERM VIABILITY REFERENCE VALUES VIABILITY DESCRIPTION Criteria on Sperm Morphology: Normal >50% live sperm (6th ed.) SPERM MORPHOLGY CRITERIA >75% live sperm (5th ed.) CRITERIA DESCRIPTION Routine Normal: >30% normal forms criteria of sperm Kruger’s strict Normal: >14% normal forms criteria* of sperm * Kruger’s strict criteria requires measurement of head, neck and tails using a stage micrometer or via mor- phometry Anti-sperm Antibodies ANTI-SPERM ANTIBODIES* ANTIBODY DESCRIPTION Male Anti-sperm Antibodies Abnormality: decreased motility with clumping Tests: TESTS FOR MALE ANTI-SPERM ANTIBODIES TEST DESCRIPTION Mixed Aggluti- Reaction: nation reaction Semen + AHG + latex particles Semen + AHG + RBC coated with IgG Detection: IgG Normal Value: 40 million/ejaculate Formula: Motility >50% 2.0 or b quality ROUND CELL FORMULA Morphology >30% normal (routine) >14% normal (Kruger’s) Viability >50% live sperm MAR