Seminal And Vaginal Fluid PDF

Topic 7: Seminal and Vaginal Fluid 3rd Year, 1st Semester Transcriptions | MED221 Prof. Arwin R. Aparente | MLSAUBFC 3. Forensic analysis (alleged rape) COURSE OUTLINE:...

Topic 7: Seminal and Vaginal Fluid 3rd Year, 1st Semester Transcriptions | MED221 Prof. Arwin R. Aparente | MLSAUBFC 3. Forensic analysis (alleged rape) COURSE OUTLINE: SIDE NOTES Seminal and Vaginal Fluid Helpful in investigations on rape cases Physical, Chemical, and Microscopic Analysis Disease Correlations 4. Qualifications for Artificial Insemination Programs SEMINAL FLUID Artificial Insemination - process wherein the sperm will be introduced to the egg to REASONS FOR SEMINAL FLUID ANALYSIS achieve pregnancy through in vivo 1. Fertility Testing fertilization Most common cause of male infertility is Usually sa couples who have troubles in varicocele getting pregnant Varicocele - hardening of veins that drain the Kukuha ng semen, inject sa egg testes Baka mababa ang motility ng sperm kaya ○ Causes blood from the adrenal vein to hindi nakakabuo ng baby flow into the spermatic vein 5. Determining quality in semen/sperm banking Varicocele - parang varicose veins sa babae Kapag may problem talaga sa guy Hindi lang sa phlebotomy nakikita yung Almost similar with blood banking veins (artery and venous), mayroon din sa ○ Syempre may checking din if healthy reproductive system ka as a donor ○ Spermatic vein - vein in male Expensive, same with artificial reproductive organ; dito dumadaan insemination yung blood Common sa celebrities, e.g. Richard Will be incapable of producing semen if Gomez magkaroon ng obstruction sa testes ○ Nagdodonate ng sperm sa sperm because hard yung pinaka vein bank ○ Diagnosis: INFERTILITY Mayroon ding guys na before sila 2 or 3 samples be collected not < 7 days or > “mawala sa mundong ‘to” ay nagdodonate 3 weeks apart with 2 samples abnormal samples considered significant 6. Paternity 2. Post-vasectomy semen analysis Vasectomy - surgical removal of all or part of the vas deferens for the purpose of male sterilization Only concern is the presence or absence of spermatozoa Vas deferens - daluyan ng semen; thick walled muscular tube that transports sperm from the epididymis to ejaculatory duct Dapat walang makitang sperm under microscopy if nag undergo ng vasectomy - SUCCESSFUL SURGERY MED221  SEMINAL AND VAGINAL FLUID COMPOSITION OF SEMINAL FLUID Thick alkaline mucus that neutralizes acidity from the prostatic secretions and Produced in the the vagina seminiferous tubules (SPERMATOGENESIS) Cowper’s gland - bulbourethral gland Spermatogenesis is Acid-base balance the maturation series, during sexual 5% similar to RBCs intercourse since Spermatozoa vagina is neutral + Sertoli cells serve as nurse sperm is acidic cells for developing sperm 5% Cowper’s Pag nagcombine, cells Gland magkakaroon ng 90 days - sperm neutralization maturation in epididymis Pag nawalan ng (sperm becomes motile) normal flora ang vagina, nagiging Produced in the seminal alkaline vesicles Pag nagka-alkalinity Provides nutrients for tapos nag-eme ng sperm and the fluid semen, hindi Rich in Fructose - energy nagkakaroon ng anak for motility ○ Acidic vagina, 60-70% acidic sperm Seminal Fluid Para maging energized, we consume carbohydrates; same with semen and fructose Acidic fluid that contains ○ Acid phosphatase - used in rape cases (INCREASED AMOUNT) ○ Zinc ○ Citric acid ○ Other enzymes For coagulation and SPERMATOGENESIS liquefaction Sertoli cell - nurse, nagnonourish ng 20-30% sperm There are enzymes sa Prostate Divides into spermatogonium semen na nagcacause Fluid DIvides to spermatocyte (primary and ng pamumuo/coagulum secondary) Semenogelin - Becomes spermatids - no flagellate yet protein/enzyme na ○ Spermiogenesis - The process the nagpapabuo sa spermatids becomes flagellate/gets sperm flagellate For protection sa Becomes spermatozoon antisperm antibodies Out the epididymis, becomes sperm cell Pag nagliquefy, normal ang prostate MED 221  SEMINAL AND VAGINAL FLUID SEMEN COLLECTION ○ Kasi if during coitus/sex ka Important part of analysis magcocollect, hindi mo masisigurado Sexual abstinence - 2-3 days, not more yung lahat ng volume ng semen ang than 5 days makukuha ○ Sa pre-cum pa lang, mataas na Overabstinence - tumataas ang volume concentration ng sperm. Kung hindi ng sperm, decrease motility yun makukuha because of sex, mababa ang sperm count ○ Sa masturbation, beginning until end Collection of entire ejaculate macocollect Specimen should be delivered to the Condom method - lubricant is antisperm laboratory within 1 hour of collection at room Most of the sperm are contained in the first temperature portion of the ejaculate, making complete Take note of the time of specimen collection collection essential for accurate testing of both specimen receipt and liquefaction fertility and post-vasectomy specimens. Analysis should be done after liquefaction (usually 30-60 minutes) Specimen awaiting analysis should be kept MISSING THE FIRST PORTION OF EJACULATE at 37C If it does not liquefy, treat with amylase or Decreased sperm count, pH falsely increased, bromelain sample will not liquefy MISSING THE LAST PORTION OF If mahihiya si patient magcollect, allowed EJACULATE naman magcollect sa bahay pero dapat within 1 hour maibalik agad sa lab Decreased semen volume, sperm count falsely Take note of labeling!! increased, pH falsely decreased, sample will ○ Liquefaction - analysis should be not coagulate done after liquefaction (30-60 minutes) Why 37C? Optimal temperature na PHYSICAL AND MICROSCOPIC ANALYSIS - buhay sila SEMINAL FLUID ○ Nilalagay sa kili-kili yung container after collection kasi nga 37C Physical Analysis Amylase/bromelain - nagdidilute ng semen since viscous siya Pearly white, grayish-white, ○ Alternative: Dulbecco's phosphate COLOR translucent (normal) buffer saline solution OR alpha-chymotrypsin ODOR Musty, bleach ○ Indicate sa result for liquefaction kung anong ginamit Increased White turbidity - TURBIDITY infection (increased WBC) METHODS OF COLLECTION Red or Brown - RBCs or blood Masturbation Most convenient Yellow - prolonged COLORATION abstinence, urine, drugs Coitus interruptus Withdrawal technique *Flavin deposition Use of non-lubricant Condom Technique containing rubber or Normal: 2-5 mL silastic condom 1mL (abnormal Harr) VOLUME Can reach to 15 mL Increased in prolonged Masturbation is better than coitus abstinence interruptus MED 221  SEMINAL AND VAGINAL FLUID Acrosomal cap occupies ½ of the sperm’s Decreased in infertility, incomplete collection head and covers ⅔ of the sperm’s nucleus Depends on various Midpiece measures 7 um physiologic factors ○ Hydration status Motility ○ Frequency of Reading at 20 hpf, on 37C or RT, wet mount ejaculation prepared on pre-warmed slide and cover slip ○ etc. Settle for a while from freezer (preservative) Numerical Grading: 0-4 ○ 0 for Watery 95% Immediately after ejaculation ○ 4 for Gel-like VISCOSITY Normal: pours in droplets 50% - Within 1 Hour ○ “Creamy gelatinous” Normal Increased viscosity = decreased sperm motility 25-40% After 3-6 hours Normal: 7.2-8.0 0% After 12 hours Increased pH = infection pH Decreased pH = more QUALITY > 2.0 prostatic fluid is present Morphology MOTILITY GRADING (WHO Criteria) Papanicolau stain/Wright-Giemsa stain - GRADE DESCRIPTION differentiates morphology of semen ROUTINE CRITERIA of >30% Normal forms 4.0 a Rapid, straight line motility KRUGER’S STRICT CRITERIA of >14% Slower speed, some lateral Normal forms 3.0 b movement Measure the head, neck, and the tail Head - normal shape is oval Slow forward progression, 2.0 b Midpiece - contains the mitochondria noticeable lateral movement Tail - for motility 1.0 c No forward progression 0 d No movement Viability Abnormalities of sperm heads and tails: MODIFIED BLOOM’S TEST Reagent: Eosin and Nigrosin Stain ○ Living sperm: Unstained, Bluish white (75%) ○ Dead sperm: Red colored CONDITIONS OBSERVED IN VIABILITY TESTING Necrospermia Presence of dead spermatozoa Double head and pinhead - most Oligospermia Decreased number of common/seen spermatozoa Tapered head - varicocele Azoospermia Absence of spermatozoa MED 221  SEMINAL AND VAGINAL FLUID Chemical Analysis/ (+) Dark Brown Rhombic Crystals BARBIERO’S TEST NORMA DECREASED ANALYTE Very specific - tests for Spermine L VALUE VALUES INDICATE Reagents: Saturated Picric Acid + TCA Lack of seminal (+) Yellow leaf-like Crystals fluid vol. (STRASINGER) POST-VASECTOMY SEMEN ANALYSIS Ejaculatory >13 umol/ Vasectomy - the surgical procedure of cutting Fructose duct ejaculate obstruction or the vas deferens so that the ejaculate will not pathology if vas contain any sperm deferens The only concern is the presence or absence (HUBBARB) of sperm cells Done 2 months after vasectomy, continued Neutral >20 mU/ Disorders of the α-glucosidase ejaculate epididymis until 2 consecutively monthly specimens show no sperm >2.4 Zinc umol/ Lack of ejaculate prostatic fluid/prostatitis >52 umol/ (HUBBARB) Citric Acid ejaculate Acid >200 U/ Lack of phosphatase ejaculate prostatic fluid Semen Fructose - tested within 2 hours for frozen to prevent fructolysis Screening Test - Resorcinol test; (+) Orange-red color Retrograde Ejaculation - sperm is Forensic Semen Analysis expelled in the bladder instead of urethra; presence of many sperm in urine Microscopic exam Fluorescence under UV light ○ Live sperm: green MICROBIAL ANALYSIS ○ Dead sperm: orange Round cells - leukocytes and spermatids Acid phosphatase (immature sperm cells) Glycoprotein p30 - more specific method Peroxidase - positive granulocytes are the ABO Blood Grouping predominant WBCs in semen, therefore it can DNA Analysis also be further differentiated from Spermatogenic cells and Lymphocytes by Peroxidase stain DNA analysis - investigation of rape cases Tests for Chlamydia trachomatis, Mycoplasma ○ Viability of sperm within48 hours hominis, Ureaplasma urealyticum ○ After 7 days, only spermatids will be Normal Value: 1 million WBCs/ml - infection ○ >1 million Spermatids/mL - disrupted spermatogenesis FLORENCE TEST Not specific - tests for Choline Reagents: Iodine crystals + KI MED 221  SEMINAL AND VAGINAL FLUID Stages of Spermatogenesis 1. Spermatogonium (earlies) 2. Primary Spermatocyte 3. Secondary Spermatocyte 4. Spermatid 5. Spermatozoon ANTISPERM ANTIBODIES Detected in semen, cervical mucosa, or serum Cause of male antisperm antibodies: MISCELLANEOUS TESTS ○ Ruptured blood SPINBARKEIT TEST ○ Testes barrier Test for the tenacity of mucus *Sperm cells exposed to male’s immune system Presence of male antisperm antibodies can be SIMS HUHNER TEST observed during a routine semen analysis Post-coital test Sperm-agglutinating antibodies cause sperm Test for the ability of sperm cells to penetrate to stick to each other in a head-to-head, the cervical mucosa head-to-tail, tail-to-tail pattern ○ In microscopic exam, agglutination is graded as: few, moderate, or many TESTING FOR ABNORMAL SEMEN ANALYSIS RESULT CAUSE TEST Decreased Eosin-Nigrosin MIXED AGGLUTINATION REACTION motility with Viability stain normal count Detects the presence of IgG antibodies Lack of seminal Semen sample + AHG reagent + Latex Decreased vesicle support Fructose levels particles or treated RBCs coated with IgG count medium Normal: 30 organisms or cells/HPF Reaction enzymes essential for ovum penetration TESTS AMINE (WHIFF) TEST Additional: Drop of saline specimen + 1 drop of 10% Semen diluting fluid - CHILLED WATER KOH solution (+) fishy amine odor VAGINAL FLUID Due to volatilization of amines when KOH is To identify the existence of pathologic added and suggests bacterial vaginosis conditions that may affect the reproductive capability of a female POTASSIUM HYDROXIDE (KOH) TEST To identify compelling signs of sexual assault Rest for 5 minutes to dissolve epithelial and and abuse, that is vital for the alleged rape blood cells cases 10% glycerin is added to prevent specimen deterioration VAGINAL SWABBING LPF: yeast pseudohyphae; HPF: smaller Cotton: Toxic to Neisseria gonorrhoeae blastospores Wood: Toxic to Chlamydia trachomatis Calcium alginate: inactive herpes simplex GRAM STAINING virus (HSV) GOLD STANDARD in identifying the causative Room temp: Trichomonas vaginalis, Neisseria organisms for bacterial vaginosis gonorrhoeae L. acidophilus: large gram-positive rods Refrigerated: C. trachomatis, HSV G. vaginalis and Bacteroides spp.: small gram-variable rods or gram-negative rods Physical Characteristics Mobiluncus spp.: curved gram-variable rod Normal: white with flocculent discharge Bacterial vaginosis: thin, homogenous Nuget’s Gram Stain Criteria white-to-gray discharge Candida infections: white “cottage cheese” pH ○ Differentiates the causes of vaginitis ○ Performed before placing the swab into saline or KOH solutions ○ Normal values: 3.8 - 4.5 Normal flora in vag: 0-3 Microscopic Examination Intermediate: 4-6 Bacterial vaginosis: 7 or more Coverslip is placed on a specimen to exclude air bubbles Examined microscopically using LPF and HPF VAGINAL DISORDERS objective with brightfield microscope Bacterial Vaginitis Most common cause of vaginitis MED 221  SEMINAL AND VAGINAL FLUID Occurs when there is an imbalance in the ratio ○ Genital itching or burning of normal vaginal flora ○ Dyspareunia, dysuria As the vaginal pH becomes alkaline, ○ Abnormal thick, white, curd-like vaginal lactobacilli are replaced by an overgrowth of discharge other organisms ○ pH remains normal (3.8 - 4.5) Characterized with malodor and increased ○ Negative amine test abnormal vaginal discharge With microscopic examination, KOH wet prep 4 features must be present for diagnosis: and gram stain ○ Thin, white, homogeneous discharge Budding yeasts and pseudohyphae form Vaginal fluid pH > 4.5 Large numbers of WBCs, lactobacilli Positive Amine (Whiff) test Large clumps of epithelial cells Presence of clue cells on microscopic exam Treatment: Gram stain - gold standard for determining the ○ Azole antifungal agents ratio of each bacterial morphotype and offers a definitive diagnosis Treatment: ○ Metronidazole (Flagyl) DESQUAMATIVE INFLAMMATORY VAGINITIS ○ Metronidazole gel Characterized by profuse purulent vaginal ○ Clindamycin cream discharge, vaginal erythema, and dyspareunia Β-hemolytic gram-positive streptococci can TRICHOMONIASIS be cultured from most patients T. vaginalis is transmitted by sexual pH > 4.5 intercourse Negative amine test Frequently occurs with gonorrhea and With wet mount and gram stain chlamydia infections Large numbers of WBCs and RBCs Characterized by a green-to-yellow frothy Occasional parabasal and basal cells vaginal discharge, malodor, pruritis, irritation, Squamous epithelial cells dysuria, dyspareunia, and vaginal mucosa Reduced or absent lactobacilli replaced by erythema gram-positive Strawberry cervix due to punctuate Treatment: 2% clindamycin hemorrhages Diagnosed with wet mount examination and ATROPHIC VAGINITIS microscopically visualizing the motile Caused by thinning of the vaginal mucosa trichomonad because of reduced estrogen production and pH > 4.5 decreased glycogen production Positive for amine test Symptoms: Culture: Diamond’s medium ○ Vaginal dryness and soreness Treatment ○ Dyspareunia ○ Metronidazole ○ Inflamed vaginal mucosa ○ Tinidazole ○ Purulent discharge Microscopic evaluation is similar to that of DIV CANDIDIASIS Treatment: estrogen replacement Occurs when there is a change in the vaginal environment that permits overgrowth of Candida Use of broad-spectrum antibiotics, oral contraceptives or estrogen replacement therapy can cause a change in the vaginal environment Symptoms: MED 221  Topic 8: Cerebrospinal Fluid 3rd Year, 1st Semester Transcriptions | MED221 Prof. Arwin R. Aparente | MLSAUBFC CEREBROSPINAL FLUID Kung ang kidney may glomerulus, si CSF is a major fluid of the body arachnoid pili naman yung nagfifilter Provides physicians with a tool by which to The four ventricles: evaluate the central nervous system ○ First and second - lateral C-shaped Indications of performing a lumbar puncture ventricles beneath the cerebral cortex and CSF examination include suspicions and ○ Third - narrow, funnel shaped between encephalitis, meningitis, multiple sclerosis, left and right thalamus neurosyphilis, and subarachnoid hemorrhage, ○ Fourth - diamond-shaped, has four among other disorders openings where CSF drains into the subarachnoid space and central canal FORMATION AND PHYSIOLOGY of spinal cord The brain and the spinal cord is lined by the meninges, which consists of three layers Formation of CSF Produced in the choroid plexuses of the two lumbar ventricles and the third and fourth ventricles In adults, approximately 20 mL of fluid is produced every hour CSF Volume ○ 90 to 50 mL - Adults ○ 10 to 60 mL - Neonates Arachnoid granulations/villae - site of OUTER LAYER reabsorption of circulating fluid in the brain DURA MATER Lines and protects [!] The chemical composition of CSF does not “Hard mother” in Latin the skull and resemble an ultrafiltrate of plasma due to vertebral canal selective filtration under hydrostatic pressure and active transport secretion FILAMENTOUS LAYER Known ultrafiltrate of plasma With ARACHNOID Kidneys subarachnoid “spiderweb-like” Synovial fluid space Where the CSF flows BLOOD-BRAIN BARRIER Very tight-fitting junctures of the endothelial THIN INNERMOST cells in the choroid plexuses LAYER Prevent the passage of many molecules PIA MATER Adheres to the “Gentle mother” in Latin cerebrum, closer to the brain Produces plexuses Brain and spinal cord are connected, doon dumadaan ang CSF going through meninges Inflammation of meninges = possible meningitis MED221  CEREBROSPINAL FLUID procedure FUNCTIONS OF CSF Supply nutrients to the nervous tissue Microbiology Provide better Remove metabolic wastes and addtl. 4 exclusion of skin Serologic contamination Produce a mechanical barrier to cushion the tests brain and spinal cord against trauma CSF Total Volume SPECIMEN COLLECTION AND HANDLING Indications for examination of CSF include CNS malignancy, demyelinating diseases, ADULTS: NEONATES: meningeal infection, and subarachnoid 85 to 150 mL 10 to 60 mL (BRUNZEL) hemorrhage 90 to 150 mL Collection: LUMBAR PUNCTURE (STRASINGER, Contraindication to performing this procedure HENRY & GRAFF’S) is the presence of infection at the puncture 140 to 170 mL site (OLD STRASINGER & HUBBARD) CSF Appearance MAJOR APPEARANCE CAUSE SIGNIFICANCE Crystal Normal The most common site for lumbar puncture is Clear the intervertebral space between L3 and L4 WBCs Meningitis The lumbar puncture site is thoroughly cleansed and a local anesthesia is applied. Meningitis The needle is seated in the dura mater Hazy, Disorders CSF pressure is measured using a graduated turbid, affecting milky, Microorganism blood-brain manometer attached to the syringe cloudy s, Protein barrier Normal pressure: Production ○ 50 to 180 mmHg - Opening pressure of IgG within CNS ○ 10 to 30 mmHg - Closing pressure Typically, 10 to 20 mL of CSF is slowly Radiographic removed into the three or four tubes that are Oily contrast media numbered sequentially Hemorrhage Bloody RBCs Traumatic Tests are least tap affected by Chemical and blood and Disorders 1 Serologic bacteria FROZEN affecting introduced as a Protein tests blood-brain result of the tap Clotted barrier procedure Clotting Introduced by Microbiology REMAIN 2 Factors traumatic tap Laboratory AT RT Disorders Least likely to affecting contain cells REFRIG Protein 3 Cell count blood-brain introduced by ERATED Pellicle the spinal tap barrier MED 221  CEREBROSPINAL FLUID Types of CSF Clotting Tubercular Factors meningits Xanthochromic - due to degradation product PINK Slightly amount of Clear CSF oxyhemoglobin YELLOW Oxyhemoglobin ORANGE Heavy hemolysis Xanthochromia - term used to describe CSF supernatant that is pink, orange, or yellow Turbid CSF RBC Degradation products - most common ○ Depending on the amount of blood ○ Length of time it has been present ○ Color will vary from pink (very small amount of oxyhemoglobin), to orange (heavy hemolysis) to yellow (conversion of oxyhemoglobinuria Other causes of xanthochromia ○ Elevated serum bilirubin, presence of the Xanthochromic CSF pigment carotene, markedly increased protein concentrations, and melanoma pigment. ○ Xanthochromia that is caused by bilirubin ○ w/ color due to the immature liver function is also commonly seen in infants particularly premature infants. Xanthochromic Old hemorrhage Hemoglobin Lysed cells from Bloody CSF traumatic tap RBC degradation Bilirubin Elevated serum bilirubin ○ Subarachnoid hemorrhage level - all CSF are the same Carotene Increased serum levels color; even (red) Disorders affecting Protein Hemorrhage vs Traumatic Tap blood-brain barrier Bloody CSF can of indication of: Mineral Meningeal ○ Intracranial hemorrhage ○ Puncture of blood vessels during spinal tap (Traumatic tap) May matatamaan at matatamaan na blood vessel sa lumbar si doctor. MED 221  CEREBROSPINAL FLUID TRAUMATIC INTRACRANIAL CSF CELL COUNTING TAP HEMORRHAGE The leukocyte - cell count that is routinely Distribution of Uneven Even performed on CSF specimens. Blood (1>2>3) (1=2=3) RBC counts - usually determined only when a Clot Formation Positive Negative traumatic tap has occurred and a correction for leukocytes or protein is desired. Xanthochromic Clear Xanthochromic Supernatant ○ Calculated by performing a total cell count and a WBC count and subtracting the ○ Traumatic Tap WBCcount from the total count, if Uneven due to necessary. rupture of blood WBC count - should be performed vessels during immediately, because: collection ○ WBCs (particularly granulocytes) and Tube 1 = reddish RBCs begin to lyse within 1 hour. Tube 2 = lighter ○ Disintegrate within 2 hours. Tube 3 = clear DISTRIBUTION OF Specimens that cannot be analyzed BLOOD immediately should be refrigerated. ○ Intracranial Hemorrhage Even Methodology Tube 1, 2, 3 = all Normal adult CSF Value: 0-5 WBCs/uL reddish/red Newborn: 30 mononuclear cells/uL There is already Specimens with 200 WBCs or 400 RBCs/uL bleeding inside the may appear clear. brain. Neubauer Counting Chamber - is routinely used for performing CSF cell counts. ○ Traumatic Tap Electronic Cell Counters - NOT used for Positve performing CSF cell counts There is fibrinogen ○ Owing to high background counts and due to rupture of poor reproducibility of low counts. blood vessels Healing Automation CLOT FORMATION Increases precision, standardizatin, and faster ○ Intracranial turnaround time for results. Hemorrhage Various automated instruments. Negative because ○ ADVIA 210i (Siemens Healthcare the bleeding is Diagnostics Incorporated, Deerfield, IL) inside the brain. Feed lang ng feed sya nag mag ccount ○ Traumatic Tap ○ Sysmex XE-5000 (Sysmex Corporation, The only problem Mundelein, IL) with traumatic tap ○ Iris iQ200 with Body Fluids Module (Iris is the ruptured Diagnostics, Chatsworth, CA) XANTHOCHROMIC blood vessels SUPERNATANT ○ Beckman COulter LH780 ○ Intracranial ○ UniCell DxH800 (Beckman Coulter, Inc.) Hemorrhage Even distribution of blood MED 221  CEREBROSPINAL FLUID Calculating CSF Cell Counts 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 1 𝑠𝑞𝑢𝑎𝑟𝑒 = 𝑐𝑒𝑙𝑙𝑠/𝑢𝐿 Can be used fir both diluted and undiluted specimens Can be used for sperm count (0.4 uL) 4 large squares (0.1 uL) large center square Differential Count on a CSF Specimen Cellular Constituents Differential count - performed on a stained Primary lymphocytes and monocytes smear and not from the cells in the counting ○ Adults: chamber. Ratio of predominance of Specimens should be concentrated - to lymphocyte:monocyte = 70:30 ensure that the maximum number of cells is ○ Children’s ratio is reversed available for examination. Occasional neutrophils are normal Specimen concentration include: Pleocytosis - increased amount of normal ○ Sedimentation, filtration, centrifugation, cells and cytocentrifugation. ○ Considered abnormal, in the finding of ○ The specimen is centrifuged for 5-10 immature leukocytes, eosinophils, plasma minutes. cells, macrophages, increased tissue ○ Suspended sediment stained with cells, and malignant cells Wright’s stain. Neutrophils = bacterial meningitis Better: Papanicolaou stain Lymphocytes and Monocytes = viral, 100 cells = reported in percentage tubercular, fungal, parasite origin ○ (ex. 75% and 100%) If 40 years old. Methodology ○ Slightly increased Turbidity Albumin is predominant, prealbumin is second Automates instrumentation available predominant. Nephelometry Alpha globulins-haptoglobin and ceruloplasmin. Protein Fractions ○ Can be seen in CSF protein Diagnosis of neurologic disorders associated Transferrin if major beta globulin. with abnormal CSF protein often requires TAU Transferrin Fractin, carbohydrate-deficient measurement of the individual protein transferrin seen in CSF, not seen in blood. fractions. ○ Used to identify CSF (Transthyretin) Either: ○ TAU protein is unique, only in CSF MED 221  CEREBROSPINAL FLUID ○ Damage to the integrity of the blood-brain Leukemia, lymphoma, viral, HIV: bands in both barrier If mataas meaning nabasag yung BBB MNEMONICS 𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑚𝑔/𝑑𝐿) MSNEG 𝐶𝑆𝐹/𝑠𝑒𝑟𝑢𝑚 𝑎𝑏𝑢𝑚𝑖𝑛 𝑖𝑛𝑑𝑒𝑥 = 𝑆𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑔/𝑑𝐿) MS - Multiple Sclerosis ○ Increased production of IgG N - Neurosyphilis, neoplasm E - Encephalitis 𝐶𝑆𝐹 𝐼𝑔𝐺 (𝑚𝑔/𝑑𝐿)/𝑆𝑒𝑟𝑢𝑚 𝐼𝑔𝐺 (𝑔/𝑑𝐿) 𝐼𝑔𝐺 𝐼𝑛𝑑𝑒𝑥 = 𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑚𝑔/𝑑𝐿)/𝑆𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑔/𝑑𝐿) G - Guilan Barre Syndrome Detection of Oligoclonal Bands Myelin Basic Protein Primary purpose for performing CSF protein Indicates recent destruction of the myelin electrophoresis is to detect oligoclonal bands. sheath that protects the axons of the neurons ○ Represent inflammation within the CNS. (demyelination). Located in the gamma region of the protein Determines the course of multiple sclerosis. electrophoresis. Also provide a valuable measure of the Simultaneous serum electrophoresis must be effectiveness of current and future treatments. performed. Immunoassay techniques are used for measurement. Glucose NV: 60-70% of the plasma glucose Blood glucose: ○ Drawn about 2 hours prior to the spinal tap Equilibration between the blood and fluid. Clinical Significance Determine the causative agents in meningitis. Bacterial meningitis = decrease glucose + increase neutrophils Tubercular meningitis = dec. glucose + inc. lymphocytes Viral meningitis = Normal glucose + inc. lymphocyte Electrophoresis and Immunophoretic Techniques Multiple Sclerosis (MS) - no bands in serum, 2 or more oligoclonal bands in CSF ○ Compare also to IgG index. Encephalitis, Neurosyphilis, Guilain-Barre, and neoplasms may be give same pattern. ○ Consider symptoms MED 221  CEREBROSPINAL FLUID CHEMISTRY VALUES COMMENT CLIN. SIGNIFICANCE TEST Determine the causative Blood glucose: agents in meningitis. ○ Drawn about 2 Bacterial meningitis = dec. hours prior to glucose + inc. neutrophils NV: 60-70% of the Glucose the spinal tap Tubercular meningitis = plasma glucose Equilibration dec. glucose + inc. between the blood lymphocytes and fluid. Viral meningitis = Normal glucose + inc. lymphocyte Bacterial tubercular, fungal Destruction of tissue Diagnosis and meningitis = >25 within the CNS owing to management of meningitis mg/dL oxygen deprivation Lactate cases Bacterial = >35 (hypoxia) causes the Used to monitor severe mg/dL production of increased head injuries Viral meningitis = CSF lactic acid levels. 35 mg/dL) presence of excess ammonia in the CSF. Other illnesses: Reye's syndrome CSF Chemistry Test CHEMICAL REFERENCE SIGNIFICANCE OF SIGNIFICANCE OF SUBSTANCE CONCENTRATION INCREASED DECREASED VALUE, NORMAL CSF CONCENTRATION CONCENTRATION Meningitis Protein 15 to 45 mg/dL Hemorrhage CSF leakage Multiple sclerosis 60% to 70% of plasma Bacterial, tubercular, and Glucose None concentration fungal meningitis >35 mg/dL: Bacterial Lactate 10 to 24 mg/dL Meningitis None >35 mg/dL: Some Glutamine 8 to 18 mg/dL disturbance of consiousness MED 221  CEREBROSPINAL FLUID Microbiological Test TEST FOR FUNGAL CULTURE GRAM STAIN MENINGITIS PARASITIC (Confirmatory) (C. neoformans) India ink Streptococcus preparation pneumonia Gram stains = Haemophilus starburst influenza 24hrs = pattern Escherichia coli Naegleria Bacterial Latex Neisseria fowleri 6 weeks = agglutination = meningitidis Acanthamoeba Tubercular more sensitive Streptococcus method agalactiae Confirmatory = Listeria culture monocytogenes LAF Serological Test Performed to detect the presence of neurosyphilis TEST COMMENT Recommended by the CDC to diagnose Venereal Disease Research Laboratories (VDRL) neurosyphilis Most sensitive Fluorescent Treponemal Antibody Absorption Care must be taken to prevent contamination with (FTA-ABS) blood, because the FTA-ABS remains positive in the serum of treated cases of syphilis The Rapid Plasma Regain (RPR) Less sensitive than VDRL MED 221