Ramida Amornsitthiwat, M.D. Biological fluids Ramida Amornsitthiwat, M.D.

Biological fluids Cerebrospinal fluid Amniotic fluid Seminal fluid Saliva Human milk

Cerebrospinal fluid (CSF) brain and spinal cord total volume : 125-150 mL daily production : 400-500 mL (20 mL/hr) synthesis : 70% choroid plexus (active transport & ultrafiltration) : 30% ependymal cells subarachnoid space, ventricular system (space between arachnoid & pia mater)

CSF circulation lateral ventricles (choroid plexus) foramen of Monro third ventricle cerebral aqueduct fourth ventricle foramen of Luschka & Magendi basal cisterns & subarachnoid space reabsorption at venous sinus via arachnoid granulations

CSF functions physical support of brain protection of brain chemical stability removes waste products of cerebral metabolism

Blood brain barrier (BBB) physiologic barrier (circulating blood & CSF) - endothelial cell of brain capillaries (tight junction, no intercellular pores) - end-feet of astrocytes (astroglia) Infiltration - fat-soluble drugs (e.g. anesthetic & alcohol) - glucose, urea & creatinine - penicillin & streptomycin - proteins

Examination of CSF Lumbar puncture (LP)

Examination of CSF Lumbar puncture (LP) Indications diagnostic - CNS infection eg. meningitis, encephalitis - hemorrhage - malignancy - demyelinating disease treatment - spinal anesthesia - antibiotics - chemotherapy follow up

Examination of CSF Lumbar puncture (LP) Contraindications local infection at lumbar puncture site intracranial mass lesion (brain herniation) large brain abscess brain tumor subdural hematoma intracranial hemorrhage thrombocytopenia

Examination of CSF Lumbar puncture (LP) Complications headache : CSF leakage 2. cerebellar or tentorial herniation :increase intracranial pressure 3. paralysis 4. meningitis 5. accidental puncture of the spinal cord

CSF analysis pressure gross appearance microscopic examination chemical analysis

CSF analysis Pressure: 70 – 180 mmH2O Increase intracranial pressure 1. brain edema e.g.cerebral ischemia, meningitis, encephalitis 2. increase CSF volume e.g. hydrocephalus 3. bleeding e.g. intracerebral hemorrhage or subarachnoid hemorrhage 4. mass lesion e.g. brain tumor, brain abscess

Increased CSF volume increased CSF production normal hydrocephalus increased CSF production 2. obstruction of CSF circulation 3. decreased CSF reabsorption

CSF analysis 2. Gross appearance: clear / colourless Abnormal findings cloudy, turbid meningitis WBC > 200-300 /mm3 bloody traumatic tap subarachnoid hemorrhage intracranial hemorrhage xanthochromia subarachnoid hemorrhage (SAH)

Bloody CSF SAH traumatic tap Supernatant (หลังปั่น) xanthochromia ใส WBC < 1:1000 ~ 1:1000 (peripheral blood) three-tube test สีแดงคงเดิม สีจางลงตามลำดับ red cells lysis oxyhemoglobin (pink) bilirubin (yellow) onset : 2-4 hrs หลังจากเกิด SAH

Xanthochromia สาเหตุอื่นๆ 1. jaundice free and conjugated bilirubin in CSF total plasma bilirubin > 4-6 mg/dL 2. dietary hypercarotenemia 3. CSF protein > 150 mg/dL 4. drug e.g. rifampicin

CSF analysis 3. microscopic examination WBC 0-5 cells/mm3 Abnormal findings - leukocytes - neutrophils in bacterial meningitis - lymphocytes in viral meningitis - tumor cells - bacteria, fungi, yeast

Chemical analysis of CSF Protein - 12 - 60 mg/dL - high protein ( > 65 mg/dL) - permeability of BBB eg. meningitis - traumatic tap, SAH - obstruction of CSF - CNS IgG synthesis - low protein : no significance

Chemical analysis of CSF Glucose - plasma glucose  CSF (active transport & diffusion) - high glucose : hyperglycemia - low glucose (<40 mg/dL or 40% serum glucose) * meningitis – bacterial, TB, fungal * hypoglycemia, SAH, neurosyphilis * CNS leukemia & lymphoma causes 1. abnormal transportation of glucose 2. increased glycolytic activity 3. glucose comsumption of leukocytes and microorganisms 50-80 mg/dL (60-70% or 2/3 of serum glucose)

Chemical analysis of CSF Lactate anaerobic metabolism 10-22 mg/dL CNS hypoxia CSF lactate meningitis - cerebral infarction hypotension brain injury bacterial meningitis > viral meningitis

Chemical analysis of CSF LDH (lactate dehydrogenase) - meningitis, leukemia, cerebral ischemia ammonia & glutamine - hepatic encephalopathy - ammonia : toxic to CNS - ammonia + glutamate glutamine IgG - multiple sclerosis, neurosyphylis Tumour markers 1. carcinoembryonic antigen (CEA) 2. -fetoprotein (AFP) 3. human chorionic gonadotropin (hCG)

Meningitis causes - bacteria virus tuberculous parasite fungal e.g.Cryptococcus Brudzinski’s sign Kernig’s sign

Meningitis tuberculous (L) 50-400 clear viral bacterial Cryptococcal 50-500 +/- turbid eosinophilic cells glucose (mg/dL) protein (mg/dL) macroscopy meningitis (L>P) tuberculous

Amniotic fluid : cushioning the fetus : fetal movement : promoting muscular/skeletal development : controlling temperature : diagnostic tool for health & maturity of fetus

Amniotic fluid early pregnancy - amniotic membrane late pregnancy - fetal urine (16+ wk.) - fetal respiratory tract - desquamated fetal cell Absorbed via fetal swallowing and amniotic membrane maximal volume 1000 mL at gestational age 36 - 38 weeks (term pregnancy = 37- 42 weeks) 25

Abnormalities of amniotic fluid 1. volume 1.1 oligohydramnios - < 400 mL - renal agenesis, abnormality of urinary tract 1.2 polyhydramnios - > 2000 ml - acute : fetal heart failure, hydrops fetalis - chronic : anencephaly, esophageal atresia 2. color blood, meconium, dark red-brown 26

Gross examination of amniotic fluid color condition เหลืองใส (หรือขุ่นเล็กน้อย) normal เหลืองมาก HDN* เขียว : meconium stain (ขี้เทา) fetal hypoxia แดงคล้ำ fetal death *HDN : hemolytic disease of the newborn (erythroblastosis fetalis) 27

Amniocentesis 1. Transabdominal 2. Transcervical 3. Cul de sac (prenatal diagnosis) 1. Transabdominal 2. Transcervical 3. Cul de sac Complication of amniocentesis 1. Trauma 3. infection 2. Hemorrhage 4. amniotic fluid leakage  abortion 0.5-1%  preterm labour 28

Amniotic fluid analysis cytologic & DNA studies - chromosomal disorders - sex biochemical analysis - alpha 1-fetoprotein (AFP) - phospholipids - bilirubin 29

Amniotic fluid analysis

Indications for chromosome study มารดาอายุ > 35 ปี เมื่อถึงวันครบกำหนดคลอด 2. มารดาและ/หรือบิดามีภาวะโครโมโซมผิดปกติ 3. มีบุตรที่มีภาวะโครโมโซมผิดปกติในครรภ์ก่อน 4. มีบุตรพิการแต่กำเนิดโดยไม่ทราบสาเหตุ 5. มีบุตรเสียชีวิตในครรภ์โดยไม่ทราบสาเหตุ 6. บิดามารดาเป็นพาหะของโรคถ่ายทอดทางกรรมพันธุ์แบบลักษณะด้อย 31

Amniotic fluid analysis α 1- fetoprotein (AFP) yolk sac and fetal liver - high level : neural tube defects low level : Down syndrome can be detected in maternal serum 32

Amniotic fluid analysis Phospholipids amphipathic molecule form micelle & lipid bilayer lecithin (dipalmitoylphosphatidylcholine - DPPC) - phosphatidylglycerol (PG), sphingomyelin (S) lung surfactant

Roles of Lung surfactant surfactant decreases surface tension pulmonary compliance alveolar collapse Respiratory distress syndrome (RDS) Fetal lung maturity - L/S ratio phosphatidylglycerol foam stability or shake test L/S < 1.5 immature L/S 1.5-1.9 intermediate L/S ≥ 2 lung maturity 34

Shake test (foam stability test) Amniotic fluid + 95% ethanol shake 30 sec ดูฟอง ตั้งทิ้งไว้ 15 sec Qualitative test lung surfactant  foam stability 35

ลูกคนแรก Rh +ve เลือดมารดา Rh -ve ลูกคนที่ 2 Rh +ve Amniotic fluid analysis Isoimmunization : fetal-maternal blood incompatibility (hemolytic disease of the newborn) ลูกคนแรก Rh +ve Ag เลือดมารดา Rh -ve sensitized : ตรวจพบ Rh Ab ในเลือดมารดา ลูกคนที่ 2 Rh +ve Rh Ab ของแม่ผ่านรกมาทำปฏิกิริยากับ Rh +ve ในเลือดของลูก RBC hemolysis bilirubin (scan spectrophotometer ที่ 450 nm) 37

Semen , Seminal fluid a suspension of spermatozoa in the seminal plasma 38

Semen testis 5% spermatozoa seminal vesicle 60% สารน้ำเหนียว, pH กลาง-ด่าง, มี fructose ซึ่งเป็นแหล่งพลังงานของ sperm prostate gland 20% คล้ายน้ำนม, pH เป็นกรดอ่อน(~6.5) ผลิต acid phosphatase & proteolytic enzyme (coagulation & liquefaction) epididymis, vas deferens, bulbourethral gland 10-15% สร้าง protein ที่จำเป็นสำหรับ การเคลื่อนที่ของ sperm 39

microscopic examination Semen analysis gross examination microscopic examination Indications ประเมินภาวะมีบุตรยาก (infertility) ประเมินผลของการทำหมันชาย (vasectomy) ประเมินผลประกอบคดีทางนิติเวช (forensic medicine) 40

Gross examination of semen volume : 2-5 mL coagulation process - fibrin-liked precursor (seminal vesicle) - clotting enzyme (prostate) liquefaction process - proteolytic enzyme (prostate) 41

Microscopic examination of semen sperm count normal 60-150 million/mL oligospermia < 20 million/mL azoospermia : no spermatozoa motility motile spermatozoa 65-80 % sperm morphology normal morphology 80-90 % 42

Analysis of semen การประเมินผลของการทำหมันชาย - หลังทำหมัน 6 เดือน ไม่ควรตรวจพบ sperm การตรวจอสุจิประกอบคดีทางนิติเวช 1. sperm cytology - 7 วัน หลังร่วมเพศ 2. acid phosphatase activity - prostate gland - 3 วัน หลังร่วมเพศ - high sensitivity 43

Saliva Production Composition 500-1500 mL/day salivary gland parotid : serous sublingual : mucous > serous submandibular : serous > mucous - autonomic nervous system physiological/pathological condition Composition inorganic compounds : hypotonic fluid organic compounds : uric acid protein compounds : amylase,IgA hormones : catecholamines

Biochemical laboratory analysis of saliva viral and bacterial infections - genome and antibodies detection 2. cancer e.g. c-erb-2 soluble fragment 3. pharmaceutical and drug abuse 4. hormones - cortisol 5. DNA test - biomarker profiling or forensic identification 6. sialochemistry analysis - heavy metal

Mumps contagious disease : virus children age 2-12 years respiratory secretions, saliva fever, headache, painful and swollen parotid gland at the face, neck and jaw complications : orchitis : meningitis : encephalitis

Human milk Stage of lactation 1. colostrum (first few days) - immunologic compound (secretory immunoglobulin A) 2. transitional milk (5 days-2 weeks) - rumped up milk 3. fully mature milk (4-6 weeks)

Human milk Composition Nutrition component macronutrient protein : casien, Haptocorrin (transcobalamin II), α-lactalbumin, whey fat : palmatic acid and oleic acid lactose micronutrient - vitamin : low vitamin K

2. Bioactive component 3. Immunologic factors bioactive factors - bile-salt stimulated lipase - epidermal growth factor - growth factors anti-infective factors secretory immunoglobulin A (sIgA) white blood cells whey proteins (lysozyme and lactoferrin) oligosacccharides