MLS Review - AUBF - CSF & Amniotic Fluid

MEDICAL LABORATORY SCIENCE REVIEW • ANALYSIS OF URINE AND OTHER BODY FLUIDS: CSF & AMNIOTIC FLUID OUTLINE • Cerebrospinal Fluid • Amniotic Fluid o Functions o Collection and Handling o Meninges o Tests for Amniotic Fluid o CSF Production o CSF Collection and Handling o CSF Gross Analysis o CSF Cell Count o CSF Chemistry o CSF Microbiology Tests • • CEREBROSPINAL FLUID • 3rd major body fluid CSF COLLECTION AND HANDLING FUNCTIONS • • • Supply nutrients to the nervous system Remove metabolic waste in the brain Produce a mechanical barrier to cushion the brain & spinal cord against trauma • MENINGES • • • Singular: meninx Lines the brain and the spinal cord 3 Layers: o Dura Mater - outer most layer ▪ Literally means “tough mother” ▪ Directly lines the skull and vertebral canal o Arachnoid Mater - inner layer ▪ Termed due to its spider-like appearance ▪ Filamentous nature of the inner membrane provides spider-like appearance ▪ Trabeculae - filaments ▪ Subarachnoid Space • Found between arachnoid and pia mater • Where CSF circulates o Pia Mater - innermost layer ▪ Literally means “tender mother” ▪ Directly lines the brain and the spinal cord Choroid Plexus o Rate of production: 20 mL/hour of CSF ▪ Approx. 500 mL/hour; 0.3-0.4 mL/minute o Composed of tight junctions of endothelial cells which make up the BBB o Blood Brain Barrier (BBB) ▪ Prevents passage of many molecules ▪ Protect brain from toxins, infections, and other substances that can harm the brain Rate of CSF production must be regulated o Continuous filtration causes accumulation leading to pressure within the subarachnoid space Arachnoid Villi/Granulations o Regulates CSF by reabsorbing circulating CSF to the peripheral circulation o Rate of reabsorption: 20 mL/hour ▪ Equal to the rate of production of choroid plexus • • Method of Collection: o Lumbar Puncture - aka spinal tap ▪ Needle is inserted between the 3rd and 4th vertebrae or between the 4th and 5th vertebra o Cisternal Puncture -aka suboccipital puncture ▪ Puncture is directly below the occipital bone (rear of the skull) ▪ Performed in rare cases ▪ More dangerous because it is near the brain stem Volume Collected: 20 mL of CSF (maximum volume) 3 Tubes Tests/section Storage First Chemistry/serology (least Frozen affected by blood or bacteria present during collection) Second Microbiology (no longer Room temp. only contaminated/at risk for bacterial contamination during collection) Third Hematology (least likely to Refrigerator contain skin cells from collection) Fourth Microbiology (to further exclude Room temp. skin contamination or bacterial contamination) Chemistry/serology Frozen o If 1 tube only is collected, deliver the specimen: ▪ Microbiology → Hematology → Chemistry/Serology o 4th tube is optional and only filled when there is excess CSF GROSS ANALYSIS CSF VOLUME • • • Adults: 90 - 150 mL Neonates: 10 - 60 mL CSF volume is maintained by reabsorption of fluid back to circulation by arachnoid villi CSF APPEARANCE CSF PRODUCTION • • CSF - ultrafiltrate of plasma Plasma is filtered by the choroid plexus to produce CSF • • Crystal Clear - Normal Hazy/Turbid/Milky/Cloudy - WBC >200/uL, RBC >400/uL, Microorganisms, Proteins, Lipids o Indication of infection (meningitis) o Microorganisms and WBCs contribute to turbidity 1 • • Xanthochromia (abnormal discoloration of CSF) o Presence of RBC degradation products ▪ Pink - slight amount of oxyhemoglobin ▪ Orange - heavy hemolysis ▪ Yellow - conversion of oxyhemoglobin to bilirubin • Bilirachia - presence of bilirubin in CSF o Other causes: ▪ Increased: • Protein (>150 mg/dL) - yellowish CSF due to BBB • Melanin - brownish CSF due to meningeal melanocarcoma ▪ Presence of: • Carotene - orange CSF due to hypervitaminosis A o Carotene - Vit. A precursor • Rifampicin - red orange CSF • Iodine - yellowish CSF due to iodine usage during spinal tap o To check for Xanthochromia: ▪ Centrifuge CSF → check supernatant against white background Bloody - Increased RBC (>6000/uL) Traumatic Tap Intracranial (accidental puncture Hemorrhage of blood vessels) Distribution of blood Clot formation Supernatant Erythrophages Uneven 1>2>3 (1st tube has more blood compared to succeeding tubes) (+) Clear (-) (macrophage w/ ingested RBCs) D-dimer (-) Even (1 = 2 = 3) (-) Xanthochromic (+); (+) hemosiderin & hematoidin crystals (+) (fibrin degradation product) o Hematoidin - RBC degradation pigment • Viscous CSF o Metastatic mucin-producing adenocarcinoma o Cryptococcal meningitis (fungal due to C. neoformans) ▪ C. neoformans - capsulated fungi ▪ Capsule contributes to viscosity o Liquid Nucleus Pulposus CSF CELL COUNT • Any cell count must be performed immediately o WBC and RBC begin to lyse w/in 1 hour after collection o 40% of WBCs disintegrate within 2 hours WBC COUNT • • • Routinely performed on CSF Normal Values: o Adults: 0-5 WBC/uL o Neonates: 0-30 WBC/uL Diluting Fluid: 3% acetic acid with methylene blue o Acetic acid - lyse RBC (hypotonic solution) o Methylene blue - stain WBC for visualization RBC COUNT • • • • Not routinely performed o RBC is nor normally observed in CSF Performed in case of traumatic tap Bloody CSF warrants RBC count RBC count correct for WBC count & total protein conc. o subtract 1 WBC for every 700 RBCs seen o subtract 8 mg/dL in total protein conc. for every 10,000 RBCs/uL CSF DIFFERENTIAL COUNT • • Performed in stained CSF smear Specimen should be concentrated first before preparing a smear o • • • Oily - radiographic contrast media Clotted - Meningitis, Froin Syndrome, blockage of CSF circulation, traumatic tap o Clot is mainly due to fibrinogen o Froin syndrome - combination of xanthochromia, inc. protein, hypercoagulability Pellicle (Web-like surface) - Tubercular meningitis o Occurs if CSF is stored overnight inside refrigerator CSF VISCOSITY • • Routine Centrifugation ▪ CSF is centrifuged for 5-10 minutes • Supernatant: removed; may be used for other tests (chemistry, serology) • Sediment: used for smear preparation o Sediment is smeared → air dry at RT → stain using Wright stain ▪ 100 cells are classified ▪ Reported in terms of percentage o Cytocentrifugation ▪ Fluid is added to a conical chamber ▪ Cells are forced into a monolayer within a 6 mm diameter circle on the slide ▪ Addition of 30% albumin: • Increases cell yield or recovery • Decreases cellular distortion o Sedimentation o Filtration Predominant Cells in CSF o Pleocytosis - increase of normal cells in CSF Water-like - Normal 2 Cells Lymphocytes/ Monocytes Clinical Significance Normal (70:30 in adults; 30:70-80 in neonates) Pleocytosis (↑↑) - Viral, Tubercular, & Fungal Meningitis; Multiple Sclerosis Occasionally normal ↑↑ = Bacterial meningitis; Early case of viral, tubercular & fungal meningitis; Cerebral hemorrhage Intracranial hemorrhage Acute Leukemia Neutrophils Macrophage Blast Forms • (lymphoblast, myeloblast, monoblasts) Lymphoma Cells Plasma Cells Ependymal, Choroidal, & Spindle-Shaped Cells Malignant Cells Disseminated Lymphoma Multiple sclerosis, Lymphocyte reactions Diagnostic procedures (neurosurgery, pneumoencephalography/removal of CSF in meninge to improve visualization of brain) Metastatic carcinoma, primary CNS carcinoma CSF CHEMISTRY CSF PROTEIN • • • • • • • • • Most frequently performed CSF chemistry test Normal value o Adults: 15-45 mg/dL o Infants: 150 mg/dL o Immature: 500 mg/dL o Non-adult CSF proteins are increased because BBB is not well intact during neonatal development and proteins are circulating with CSF Albumin - Major CSF Protein Pre-albumin - 2nd Most Prevalent Alpha Globulins - Haptoglobin. Ceruloplasmin Beta Globulins - β2-transferrin, Tau o Tau - carbohydrate-deficient transferrin; only present in CSF Gamma-Globulins - IgG, IgA Proteins not observed in CSF o IgM - filtered by choroid plexus and BBB o Fibrinogen o Lipoproteins (LDL, β-LPP) Clinical Causes of Abnormal CSF Protein Values o Elevated Results ▪ Meningitis (common cause of damage in BBB) ▪ Hemorrhage (common cause of damage in BBB) ▪ Primary CNS tumors ▪ Multiple sclerosis (↑ plasma cells producing immunoglobulins) ▪ Guillain-Barre syndrome ▪ Neurosyphilis ▪ Polyneuritis ▪ Myxedema ▪ Cushing disease ▪ Connective tissue disease ▪ Polyneuritis ▪ Uremia o Decreased results ▪ CSF leakage/trauma ▪ Recent puncture ▪ Rapid CSF production ▪ Water intoxication CSF Protein Determination o Total Protein ▪ Turbidimetric • Tricholoroacetic acid (TCA) o Uses 3% TCA o Preferred Method o Precipitates both Albumin & Globulin • Sulfosalicylic acid (SSA) o Precipitates albumin only o Uses 3% SSA o To precipitate globulin, sodium sulfate is added ▪ Dye-Binding • Coomasie Brilliant Blue o Proteins binds to dye → Dye turns from red to blue o >intensity of blue = ↑ protein o PROTEIN FRACTIONS ▪ CSF/Serum Albumin Index • Assesses integrity of BBB • Measures albumin present in CSF & serum • Index = CSF alb (mg/dL) Serum alb (g/dL) • Normal Value: < 9 • Abnormal: >9 • Interpretation o 9-14 - Slight impairment o 15-30 - Moderate impairment o >30 - Severe impairment o 100 - Complete damage to BBB ▪ IgG Index • Assess condition resulting to IgG production within CNS (multiple sclerosis = ↑ IgG) • • • Normal Value: <0.70 Abnormal: >0.70 o Indicative of IgG productive within CSF CSF Electrophoresis o Detection of oligoclonal bands in gamma region ▪ Indicates immunoglobulin production o Presence of 2 oligoclonal bands in CSF but not in serum may be due to: (MS.NENG) ▪ Multiple Sclerosis (most persistent cause) ▪ Neurosyphilis ▪ Encephalitis ▪ Neoplastic disorder ▪ Guillain-Barre syndrome o Multiple Sclerosis: ▪ Demyelinating disorder • Antibodies against myelin sheath of neurons ▪ Findings: • (+) Anti-myelin sheath autoantibody • (+) Oligoclonal band in CSF but not in serum • (+) Myelin Basic Protein (MBP) o Protein component of myelin sheath o Presence indicates myelin sheath destruction caused by MS • Inc. IgG Index 3 o • CSF GLUCOSE • • • • • Always performed in conjunction with blood glucose o Specimen for blood glucose is collected 2 hours before spinal tap to allow equilibration of CSF and plasma blood glucose Normal Values: o 60-70% of blood glucose o 65%, 2/3 of blood glucose (Strasinger’s) o 50-80 mg/dL (Henry’s) Increased: Diabetes Mellitus, in cases of increased plasma glucose Decreased: o Bacterial tubercular, and fungal meningitis ▪ Significant decrease due to bacterial use o Alterations in glucose transport across BBB o Increased used of glucose by brain cells Normal In: viral meningitis • CSF MICROBIOLOGY TESTS DIFFERENTIAL DIAGNOSIS OF MAJOR CAUSES OF MENINGITIS • CSF LACTATE • • • • Lactate - waste product of glucose metabolism o Inversely proportional to glucose Normal values: 10-24 mg/dL Increased: o Bacterial meningitis (>35 mg/dL) ▪ Production due to glucose use) o Tubercular & Fungal meningitis (>25 mg/dL) o Hypoxia (tissue destruction cause release of lactate) Normal In: viral meningitis (<25 mg/dL) CSF GLUTAMINE • • • • Glutamine - product of ammonia (NH3) and alphaketoglutarate in brain cells o Removal of ammonia in brain produces glutamine Determination can be used as indirect test for presence of excess ammonia in CSF o ↑ NH3 removal = ↑ Glutamine Normal values: 8-18 mg/dL Increased o Disturbance of consciousness (>35 mg/dL) o Reye’s syndrome ▪ 75% of cases have increased CSF glutamine CSF ENZYMES • Lactate Dehydrogenase (LDH) o Isoenzymes in CSF ▪ LD 1 & 2: predominant in brain tissues ▪ LD 2 & 3: predominant in lymphocytes ▪ LD 4 & 5: found in neutrophils o Normal distribution in serum: 2 > 1 > 3 > 4 > 5 ▪ When 1 is predominant, it is flipped pattern ▪ Flipped pattern - associated with acute myocardial infarction or hemolytic anemia LDH Distribution in CSF: 1 > 2 > 3 > 4 > 5 ▪ When 2 is predominant, it is associated with neurologic abnormalities ▪ 5 > 4 > 3 > 2 > 1 pattern indicate bacterial meningitis due to increase in neutrophils Creatine Kinase (CK) o Increased: Stroke, multiple sclerosis, degenerative disorders, brain tumors, viral & bacterial meningitis, epileptic seizure Aspartate Aminotransferase o Increased: intracerebral and subarachnoid hemorrhage, bacterial meningitis • Bacterial Meningitis o Agents: ▪ Group B streptococci - common cause among neonates ▪ E. coli & other gram-negative bacilli - common cause among newborns to 1 month ▪ Neisseria meningitidis - common cause among children with 3 months or older ▪ Streptococcus pneumoniae - common cause among children with 3 months or older ▪ Haemophilus influenzae - cause among 3 mos. to 18 years of age ▪ Listeria monocytogenes - common among newborns, adults, alcoholics, immunocompromised/immunosuppressed o ↑ WBC: ↑ neutrophils o Protein: increased o Glucose: decreased o Lactate: increased o Others: ▪ (+) Gram Stain ▪ (+) Culture ▪ (+) Limulus Lysate Test • Detects gram negative bacterial endotoxin in body fluids & surgical instruments • Reagent: blood of horseshoe crabs (Limulus polyphemus) o Blood is color blue due to presence of hemocyanin which contains copper o Blood also contains WBCs termed as amoebocyte • Principle: presence of gram negative bacterial endotoxin, the blood of horseshoe crab will release lysate causing clumping/clot formation Endotoxin → amoebocyte → release of lysate → clumping/clot • Positive: clot formation/clumping Spirochetal Meningitis o Neurosyphilis - caused by Treponema pallidum o Neuroborreliosis - caused by Borrelia burgdorferi o Diagnosis: ▪ Nontreponemal tests (VDRL, FTA-Abs) • VDRL - recommended test by CDC ▪ ELISA ▪ Western Blot 4 • • • • Tubercular Meningitis o Agents: Mycobacterium tuberculosis o ↑ WBC: ↑ lymphocyte, ↑ monocyte o Protein: increased o Glucose: decreased o Lactate: increased o Others: ▪ (+) AFB Stain ▪ (+) Pellicle/weblike clot formation after overnight or 12-24 hours standing in refrigerator Fungal Meningitis o Agents: Cryptococcus neoformans (most frequently isolated fungal pathogen in CSF) o ↑ WBC: ↑ lymphocyte, ↑ monocyte o Protein: increased o Glucose: decreased o Lactate: increased o Others: ▪ (+) Gram Stain - starburst appearance ▪ (+) India Ink - negative/indirect stain ▪ (+) Latex Agglutination Test - detect antigens of C. neoformans in CSF Amoebic Meningoencephalitis o Agents: ▪ Naegleria fowleri - causes primary amoebic meningoencephalitis ▪ Acanthamoeba spp.- causes granulomatous amoebic encephalitis ▪ Balamuthia mandrillaris - causes granulomatous amoebic encephalitis o ↑ WBC: ↑ neutrophils o Protein: increased o Glucose: decreased o Lactate: increased o Others ▪ (+) RBCs ▪ (+) Acridine Orange Stain o Agents can be identified in Wright-stained smear ▪ Macrophages can be mistaken as the agents ▪ Acridine orange is used to differentiate WBC from amoeba • Amoeba - brick red • WBC - bright green Viral Meningitis o Agents: ▪ Enteroviruses (Coxsackievirus, Echovirus, Poliovirus) ▪ Arboviruses o ↑ WBC: ↑ lymphocytes o Protein: increased o Glucose: normal o Lactate: normal o Others: ▪ RT-PCR - gold standard for viral meningitis AMNIOTIC FLUID AMNIOTIC FLUID • Colorless to pale yellow fluid found in membranous sac surrounding the fetus known as the amnion FUNCTIONS • • • • Cushion for fetus Allows fetal movement Stabilizes temperature Proper lung development VOLUME • • • • • Normal Volume: 800-1200 mL during 3rd trimester o Amniotic fluid volume increases throughout pregnancy o Gradually decrease before delivery Placenta - ultimate source of amniotic fluid water & solutes o During 1st trimester, 35 mL of amniotic fluid is derived from circulation coming from the mother o After 1st trimester, the major constituent of amniotic fluid is fetal urine o During 3rd trimester, the fetus can already release lung fluid contributing to the volume of amniotic fluid Amniotic fluid volume is regulated by balance between the production of fetal urine and lung fluid and absorption of amniotic fluid by fetal swallowing and intramembranous flow Polyhydramnios o Increase in normal volume of amniotic fluid (>1200 mL) o Causes: ▪ Decrease fetal swallowing of urine ▪ Neural tube defects Oligohydramnios o Decreased amniotic fluid (<800 mL) o Causes: ▪ Increased fetal swallowing of urine ▪ Membrane leakage (amnion damage) ▪ Urinary tract deformities (impaired urination leading to decreased urine output) AMNIOTIC FLUID COLLECTION AND HANDLING AMNIOTIC FLUID COLLECTION • • • • Method of Collection: Amniocentesis o Safe to perform at the 14th week of gestation (early 2nd trimester) Maximum volume: 30 mL o First 2-3 mL is discarded because it may be contaminated with maternal blood, tissue fluid, cells 2nd Trimester Amniocentesis (approx. 16th week): amniotic fluid can be subjected to chromosomal studies to detect chromosome defects (Trisomy 21/Down syndrome) 3rd Trimester Amniocentesis: used for assessment of fetal lung maturity, for detection of fetal hemolytic disease AMNIOTIC FLUID HANDLING • • Test For Fetal Lung Maturity (FLM) o Specimen placed on ice during delivery o Kept refrigerated when testing is delayed Test For Cytogenetics Studies/ Microbial Studies o Kept at room temp of body temp (37 degC) 5 • Test For Hemolytic Disease Of The Newborn (HDN) o For detection of bilirubin in amniotic fluid o Bilirubin - photosensitive substance o Protect from light by: ▪ Using amber-colored bottles ▪ Covering/wrapping bottle with foil ▪ Using black plastic cover AMNIOTIC FLUID vs MATERNAL URINE • • • Amniotic fluid and maternal urine must be differentiated to determine if collected sample is truly amniotic fluid • Maternal urine may be accidentally collected due to possibility of accidental puncture of urinary bladder of mother Analyte Amniotic Fluid Maternal Urine Protein (+) (-) Glucose (+) (-) Urea <30 mg/dL >30 mg/dL Creatinine <3.5 mg/dL >10 mg/dL • Protein and glucose may be unreliable o If mother has proteinuria or glucosuria, maternal urine may be positive o Urea & creatinine - more reliable ▪ Metabolic waste products which are excreted in urine Results are plotted on a Liley Graph o Zone I - nonaffected/mildly affected fetus o Zone II - moderately affected fetus ▪ Close monitoring is necessary to prevent further complications and development of HDN o Zone III - severely affected fetus ▪ Requires intervention Interference: presence of cells, meconium, debris, hemoglobin o Peak absorbance of Hgb: 410 nm FERN TEST • • • • Detects ruptured amniotic membranes Specimen: Vaginal fluid Vaginal fluid → spread on slide → air dry at RT until completely dry → observe microscopically Amniotic Fluid: fern-like crystals due to protein and sodium chloride present in amniotic fluid TEST FOR NEURAL TUBE DEFECTS • • • AMNIOTIC FLUID COLOR • • • • • Colorless to pale yellow - Normal Blood-Streaked - Traumatic tap, abdominal trauma, intraamniotic hemorrhage Yellow - increased bilirubin (HDN) Dark Green - meconium (first fetal bowel movement inside the uterus) Dark Red-Brown - fetal death/fetal demise • TESTS FOR AMNIOTIC FLUID Spina bifida (split spine) - birth defect where there is incomplete closing of the backbone and membranes around the spinal cord (exposed spinal cord) Anencephaly - absence of major portion of brain & scalp Screening Test: Alpha-Fetoprotein (AFP) o AFP - major protein produced by fetal liver during early gestation (approx. 18 weeks) ▪ During pregnancy, AFP is increased in amniotic fluid and maternal serum o AFP continuously increases if the skin fails to close the neural tissues (Spina bifida, Anencephaly) o Increased: Spina bifida, Anencephaly o Decreased: Down syndrome Confirmatory Test: Acetylcholinesterase (AChE) o Must not be performed in bloody amniotic fluid o Blood contains AChE causing false increase TEST FOR HEMOLYTIC DISEASE OF THE NEWBORN (HDN) TESTS FOR FETAL LUNG MATURITY (FLM) • • • • HDN o Aka erythroblastosis fetalis o Occurs if there are antibodies (Anti-D) that can cross the placenta and destroy fetal RBC o Results in the presence of RBC degradation product (blilrubin) aka optical density 450 (OD450) or absorbance Absorbance of Amniotic Fluid: o Normal: increased at 365 nm → decreased at 550 nm o HDN: increased at 450 nm ▪ Increase at 450 nm is due to the maximum wavelength of bilirubin absorbance • Respiratory Distress Syndrome o Most frequent complication of early delivery o 7th most common cause of morbidity and mortality among premature infants o Caused by insufficient lung surfactants ▪ Phospholipids - major component of lung surfactants assessed in tests for FLM ▪ Lung surfactant - allow alveoli to open and close during inhalation and exhalation Lecithin/Sphingomyelin (L/S) Ratio o Reference method for testing FLM o Lecithin - primary component of lung surfactant (phospholipid) ▪ Low production during 35th week and below ▪ After 35th week, lecithin production increases 6 o • • • • • Sphingomyelin - produced at a constant rate ▪ Used as control o ≤35 week = ↑ Sph = ↓ Leci o >35 week = ↓ Sph = ↑ Leci o <1.6 L/S Ratio: <35 week o ≥2.0 L/S Ratio: mature fetal lungs (pre-term delivery is safe) o False Increase: presence of blood or meconium in amniotic fluid Amniostat-FLM o Immunologic test for phosphatidylglycerol ▪ Production of phosphatidylglycerol is parallel to the production of lecithin ▪ Phosphatidylglycerol is not affected by blood or meconium ▪ Phosphatidylglycerol is decreased among diabetic mothers Foam Test o Aka shake test o Qualitative Mechanical test in assessing FLM o Only requires shaking o 1 part Amniotic fluid + 1 part 95% ethanol → shake for 15 seconds → stand for 15 minutes o Mature Fetal Lung: presence of foam/bubbles ▪ Indicates sufficient phospholipid present o Foam Stability Index ▪ Modification of Foam test ▪ Semi-quantitative test ▪ 0.5 mL amniotic fluid + increasing amount of 95% ethanol (0.42 - 0.55 mL) at 0.01 mL increments (14 tubes) → shake for 15 seconds → stand for 15 minutes ▪ Mature Fetal Lung = foam or bubbles in tubes at ≥0.47 95% ETOH Microviscosity o Measures friction when a particle is diffused o Measured by fluorescence polarization o Surfactant-to-albumin (S/A) ratio is measured ▪ ↑ phospholipid = ↓ microviscosity o Dye bound to Surfactant = longer fluorescence, low polarization o Dye bound to Albumin = decreased fluorescence, high polarization Lamellar Body Count o Lamellar body count - stored form of phospholipids o Type II pneumocytes produce lung surfactants stored in the form of lamellar bodies o Methods: Impedance, optical scatter methods ▪ Diameter of lamellar bodies is similar to platelets ▪ Uses platelet channel of hematology analyzer o Adequate Fetal Lung Maturity: >32,000/uL OD 650 nm o Increased lamellar bodies = increased O.D./absorbance o OD of ≥ 0.150 is equivalent to: ▪ L/S Ratio of ≥2.0 (mature fetal lung) ▪ Presence of phosphatidylglycerol TEST FOR FETAL AGE • Amniotic Fluid Creatinine o <36 weeks gestation: 1.5-2.0 mg/dL o 36 weeks gestation: >2.0 ▪ 9 months 7