UNIT 4 CSF, SEMEN, SEROUS FLUID, SYNOVIAL FLUID, AMNIOTIC FLUID, FECAL ANALYSIS

what are the major functions of CSF? (learning objective)

1. supplies nutrients to nervous tissue

2. removes metabolic wastes

3. maintains intracranial pressure

4. provides a mechanical barrier that cushions the brain and spinal cord against trauma

what are the three layers in the meninges? describe them

1. dura mater → outer layer which lines the skull and vertebral canal

2. arachnoid → filamentous inner membrane

3. pia meter → thin membrane that lines the surfaces of the brain and spinal cord

describe the flow of CSF and where it is produced

• CSF produced in choroid plexus of the two lumbar ventricles, third, and fourth ventricle

  • produces ~20 mL/hour in adults

• CSF flows through the subarachnoid space (located between arachnoid and pia mater)

• circulating CSF is reabsorbed back into the blood capillaries at an equal rate to its production in the arachnoid granulations/villae

  • this is done to maintain the volume of 90-150 mL in adults and 10-60 mL in neonates

what is the choroid plexus?

capillary networks that forms CSF from plasma (CSF is not an ultrafiltrate of plasma) through mechanisms of selective filtration under hydrostatic pressure and active transport secretion

what is lined throughout the body and what do they allow regarding CSF?

the capillary walls throughout the body are lined with endothelial cells that are loosely connected which allows passage of soluble nutrients and wastes between the plasma and tissues

what is contained in the choroid plexus? what do the tight-fitting structures prevent?

• contains endothelial cells which are tight-fitting junctures that prevents passage of various molecules

  • the tight fitting structures are termed as the blood-brain barrier

• the junctures of the endothelial cells prevent passage of helpful substances like antibodies and medications

why is it essential to maintain the integrity of the blood brain barrier?

the blood-brain barrier protects the brain from chemicals and other substances from circulating in the blood and potentially harming the brain tissue

how is the blood-brain barrier disrupted?

caused by diseases like meningitis and multiple sclerosis which allows leukocytes, proteins, and other chemicals to enter the CSF

what procedure collects CSF and where? what are the precautions when collecting CSF?

• collected through lumbar puncture between the third and fourth or fourth and fifth lumbar vertebra

  • volume removed is based on the patients volume and opening pressure 

• measurement of intracranial pressure and careful technique to prevent infection or damage to the neural tissue (can hit a capillary which would indicate a traumatic tap)

how is the CSF specimen collected? describe them (learning objective)

• tube 1 → for chemical and serological tests

  • least affected by blood or bacteria that is introduced during the tap procedure and will have more cells and tissues

  • if cannot be stat tested → freeze

• tube 2 → for microbiological tests

  • more consistent of what CSF is

  • if cannot be stat tested → leave at room temp

• tube 3 → for cell count

  • goes to heme and will least likely contain cells that are introduced by the spinal tap procedure

  • if cannot be stat tested → refrigerate for up to 4 hours

• use the leftover fluid/fourth tube for additional testing (micro or additional serological tests)

CSF IS STAT & generally 2 mL is taken in both adults and neonates

what would normal CSF contain? (learning objective)

• normally crystal-clear

• adults will normally have 0-5 WBCs per mL but is higher in children (building their immune system)

  • 30 mononuclear cells per uL is normal in neonates

• lymphocytes and monocytes are normally found in CSF

  • 70:30 in adults and reversed in children (30:70)

• normally has very little protein (15-45 mg/dL and higher in infants and those over 40)

• normal conc. of CSF glutamine → 8-18 mg/dL

what is the major terminology to describe the appearance of CSF? (learning objective)

• cloudy or turbid

• milky

• xanthochromic

  • pink

  • orange

  • yellow

• hemolyzed/bloody

what is the significance of cloudy, turbid, or milky CSF? (learning objective)

• increased conc. of proteins or lipids but may be indicative of infection

• cloudiness = presence of WBCs

what are the other appearances of CSF? what are they caused by? (learning objective)

• oily = caused by radiographic contrast media

• clotted and pellicle → caused by protein or clotting factors

  • protein major significance = disorders affecting the blood-brain barrier

  • if it is just clotted → introduced by traumatic tap (fibrinogen increase)

  • if it is clotted because of pellicle → tubercular meningitis

define xanthochromia and state its significance (learning objective)

• xanthochromia means yellowish discoloration of the cerebrospinal fluid and has various colors like pink, orange and yellow

  • pink = very slight amount of oxyhemoglobin

  • orange = heavy hemolysis

  • yellow = conversion of oxyhemoglobin to unconjugated bilirubin

• xanthochromia can be caused by various things:

  • RBC degradation products (hemoglobin and bilirubin)

  • elevated bilirubin levels (yellow), jaundice (bilirubin)

    • commonly seen in infants (especially premature) due to their immature liver function

  • carotene due to increased serum levels

  • increased concentration of protein

• its significance is it can be used to differentiate if the CSF specimen was caused by intracranial hemorrhage or a traumatic tap

how would you tell if a CSF specimen was caused by an cerebral hemorrhage or a traumatic tap? be descriptive (learning objective)

1. uneven blood distribution → a cerebral hemorrhage will have even blood distribution compared to a traumatic tap where blood will be the heaviest in tube 1 and slowly decrease in tubes 2-3 or 4

   • RBC counts on all three tubes is not reliable

2. clot formation → clots may form in the CSF if it was collected by a traumatic tap due to the introduction of plasma fibrinogen

   • if it came from a cerebral/intracranial hemorrhage no clot will form because there isn’t enough fibrinogen

3. xanthochromic supernatant = pink supernatant would indicate a cerebral hemorrhage due to blood being present for longer than 2 hours

   • supernatant would be clear if it was from a traumatic tap because there is no time for RBCs to degrade (applies to a very recent cerebral hemorrhage too)

     • introduction of serum protein from the traumatic tap could cause the CSF to appear xanthochromic

what cell count is most routinely done on CSF specimens? how are RBC counts calculated? why should any cell count be performed immediately?

• leukocyte (WBC count) is performed routinely

  • RBCs are not normally seen but it is still reported and will only be counted when a traumatic tap happened and a correction for leukocytes and proteins is wanted

  • CSF specimens can appear clear if there 200 WBCs or 400 RBCs

• RBC count = total cell count and a WBC count and substracting WBC count from the total count if necessary

• should be performed immediately due to lysis of WBCs (particularly granulocytes) and RBCs begin in an hour

  • 40% of leukocytes will disintegrate after 2 hours

Neubauer counting chamber is used routinely to perform CSF cell counts

describe the Neubauer method for determining cell counts in CSF (learning objective)

• used for blood cell counts and CSF cell counts manually to determine the number of cells per microliter and specimens can be counted diluted or undiluted

  • eliminates the need to correct for the volume counted

• formula = # of cells counted x dilution factor (1 if undiluted) ÷ number of squares counted (how many did you count in both chambers) x volume/square

  • formula will vary depending which squares are counted (were they counted in RBC square or WBC square) → WBC square volume = 0.1 uL; RBC square volume = 0.004 uL

  • if counts are greater than 1000 → reported one decimal place out x 10³

• purpose of any calculation = converts the number of cells that are counted in a specific/known volume to what number of cells would be in 1 uL of solution

how would you determine if the cell counts for each chamber are within 20%? if RBCs were also counted in all 9 large squares, what would the formula be? how would you find WBC and RBC/mm³?

• |first count - second count| ÷ first count x 100

• if RBCs were counted in all 9 squares… # of cells counted x DF ÷ 18 × 0.1 = RBC/uL

• to find WBC and RBC/mm³ = # of cells counted x DF ÷ total # of squares counted x 0.1 = WBC or RBC/mm³

  • if the average number of cell was counted then you wouldn’t use total number of squares counted, only how many you counted in one chamber

how are dilutions made for total cell counts if needed? how would you perform only a WBC count on CSF specimens?

• dilutions are made with normal saline, mixed by inversion, then loaded into the Neubauer chamber (hemocytometer) with a pasteur pipette

  • cells are counted in all 9 squares on both sides

  • dilutions are only needed if there is more than 50 cells seen

• must lyse RBCs with 3% glacial acetic acid

  • can add methylene blue to stain the WBCs to differentiate between neutrophils and mononuclear cells

what is pleocytosis? why is it valuable?

• presence of increased numbers of lymphocytes and monocytes and is considered abnormal

• pleocytosis of normal cells (neutrophils, lymphocytes, or monocytes) are valuable in determining the cause of meningitis

  • high CSF WBC count if neutrophils are predominant → bacterial meningitis

  • high CSF WBC count if lymphocytes and monocytes are moderately elevated → meningitis of viral, tubercular, fungal, or parasitic origin

what is also considered abnormal in CSF?

• immature leukocytes

• eosinophils

• plasma cells

• macrophages

• increased tissue cells

• malignant cells

what are the cell forms that differ from those that are found in blood?

• macrophages

• choroid plexus

• ependymal cells

• spindle-shaped cells

• malignant cells

describe the leukocytes content of the CSF in bacterial, viral, tubercular, and fungal meningitis (learning objectives)

• in bacterial meningitis, it would have a high WBC count with a predominance in neutrophils

  • neutrophils may have phagocytized bacteria

• in viral, tubercular, and fungal meningitis, it would have a moderate WBC elevation (<50 WBC/uL) with both lymphocytes and monocytes

  • reactive lymphocytes with an increased dark blue cytoplasm and clumped chromatin is often seen with normal cells in viral infections

  • increased neutrophils can be seen in the early stages (1-2 days)

what may neutrophils have in CSF? what is lost more rapidly lost in CSF relating to neutrophils? what may resemble nucleated RBCs relating to neutrophils and when are NRBCs seen? what also may be seen after a traumatic tap?

• neutrophils may have cytoplasmic vacuoles after cytocentrifugation (cellular distortion)

• granules are lost more rapidly in CSF

• neutrophils with pyknotic (dense, round nucleus) nuclei (degenerating cells) may resemble NRBCs

  • to differentiate… NRBCs only have one nucleus and neutrophils have multiple

  • NRBCs are seen due to bone marrow contamination during lumbar puncture and is found in 1% of specimens

• capillary structures and endothelial cells could be seen after a traumatic tap

what else is clinically significant for neutrophils?

• CNS hemorrhage, repeated lumbar puncture, injection of medications or radiographic dye

  • little clinical significance

• CNS infarction

• cerebral abscess

• metastatic tumors

where else would you see increased lymphocytes? what would be indicative of multiple sclerosis and other degenerative neurological disorders? what else is clinically significant with lymphocytes?

• seen in cases of both asymptomatic HIV infection and AIDs

• moderately elevated WBC count (<50 WBCs per uL) with increased normal and reactive lymphocytes and plasma cells

• parasitic infections

what are increased eosinophils in CSF associated with?

• parasitic infections

• fungal infections → mostly Coccidioides immitis

• introduction of foreign material, including medications and shunts into the CNS

what is the purpose of macrophages in CSF? when will macrophages appear? what do macrophages tend to have? what do increased macrophages indicate?

• purpose = remove cellular debris and foreign objects like RBCs

• will appear within 2 to 4 hours after RBCs enter the CSF and is seen often in repeated taps

• macrophages tend to have more cytoplasm than monocytes in peripheral blood

• increased macrophages → indicative of previous hemorrhage

  • further degradation of the phagocytized RBCs caused by the hemorrhage result in dark blue or black iron-containing hemosiderin granules… yellow hematoidin crystals means further degradation and represent unconjugated bilirubin

where are choroidal (nonpathogically significant) cells from? how are they seen? what is the appearance of their nuclei?

• from epithelial lining of the choroid plexus

• seen singularly and in clumps

• nuclei has a uniform appearance

  • nucleoli is usually absence

where are the ependymal (nonpathogically significant) cells from? what is the appearance of their cell membrane and their nucleoli? how are they frequently seen?

• from lining of ventricles and neural canal

• less defined cell membrane and nucleoli are present

• frequently seen in clusters

where are spindle-shaped (nonpathogically significant) cells from? how are they usually seen? where may they be seen?

• from lining cells from arachnoid

• usually seen in clusters

• may be seen in systemic malignancies

what cells are frequently seen in acute leukemia (hematologic origin) regarding CSF?

• lymphoblasts

• myeloblasts

• monoblasts

what do lymphoma cells (hematologic origin) indicate when seen in CSF? what do they resemble? how do they appear?

• indicates dissemination from lymphoid tissue

• resembles large and small lymphocytes

• usually appears in clusters of large, small, or mixed cells based on the classification of the lymphoma

  • nuclei may appear cleaved and prominent nucleoli are present

where would metastatic carcinomal cells be from (nonhematologic origin)?

• lung

• breast

• renal system

• GI system

what would cells from primary CNS tumors include (nonhematologic origin)?

• astrocytomas

• retinoblastomas

• medulloblastomas

fusing of cell walls, nuclear irregularities, and hyperchromatic nucleoli are seen in the clusters of malignant cells

how would abnormal results occur regarding CSF?

• alterations in the permeability of the blood-brain barrier

• increased production or metabolism by the neural cells in response to a pathological conditions

state the reference values for CSF total protein and name some pathologic conditions that produce an elevated CSF protein (learning objective)

• total protein value is 14-45 mg/dL but is somewhat method dependent

  • higher found in infants and those over 40

• three pathologic conditions that produce an elevated CSF protein

  • meningitis and hemorrhage conditions (damage to blood brain barrier)

  • IG production within the CNS

  • decreased normal protein clearance from the fluid

  • neural tissue degeneration

  • multiple sclerosis

  • guillain-barre syndrome

what are the other pathological conditions that produce elevated CSF protein?

• primary CNS tumors

• neurosyphilis

• polyneuritis

• myxedema

• cushing disease

• connective tissue disease

• polyneuritis

• diabetes

• uremia

what decreases the results of CSF protein?

• recent puncture

• CSF leakage/trauma

• rapid CSF production

• water intoxication

what is the second most prevalent fraction in CSF? what are the globulins?

• transthyretin (previously called prealbumin) is the second most prevalent fraction in CSF

  • albumin makes up most of the CSF protein in serum

• globulins

  • alpha globulin → most has haptogloblin and ceruloplasmin

  • beta globulin → transferrin is the major beta globulin present

    • separate carb-deficient transferrin fraction is referred to as “tau” and is only seen in CSF

  • gamma globulin → mostly IgG with only a small amount of IgA

what is normally not found in CSF?

• IgM

• fibrinogen

• beta lipoprotein

what is the primary purpose of performing CSF protein electrophoresis? how would you ensure that the oligoclonal bands are due to neurological inflammation?

• to detect oligoclonal bands which represent inflammation within CNS

  • located in the gamma region which indicates IG production

• simultaneous serum electrophoresis must be performed

how would you determine what disease is what based on the oligoclonal banding pattern?

• no bands in serum but bands in CSF = multiple sclerosis

  • bands will NOT go away in MS remission (persists for life)

• bands in serum and bands in CSF = leukemia, lymphoma, viral, HIV

  • represents both systemic and neurological involvement

• bands may not be present in serum but bands in CSF = encephalitis, neurosyphilis, Guillain-Barre syndrome, neoplastic disorders

  • key distinction from MS is that their bands will eventually go away unlike MS

presence of oligoclonal banding has to be considered in conjunction with clinical symptoms

what is myelin basic protein? what does the presence of myelin basic protein in CSF indicate? how can the course of MS be monitored and where else can it be utilized?

• major component of the myelin nerve sheath surrounding the axons of nerves in the nervous system

• presence means recent destruction of myelin sheath that protects the axons of the neurons (demyelination)

• course of MS can be monitored by measuring the amount of MBP present in the CSF

  • can be valuable when measuring the effectiveness of current and future treatment

what are the causes of increased MBP?

• CNS trauma

• encephalopathies

• Guillain-Barre syndrome

• lupus

• brain tumors

state the reference values for CSF glucose and name the possible pathologic significance of decreased CSF glucose (learning objective)

• reference ranges are ~60-70% of plasma glucose

  • if plasma glucose is 100 mg/dL then reference CSF glucose would be ~65 mg/dL

• low CSF glucose could signify increase use of glucose by brain cells or there is an impaired/alterations in glucose transport across the blood-brain barrier

  • low CSF glucose can determine the causative agents in meningitis

    • markedly decreased CSF with an increased WBC count and large % of neutrophils = bacterial meningitis

    • if WBCs are leukocytes = tubercular meningitis

  • normal glucose but increased number of lymphocytes = viral meningitis

what happens to the glucose if plasma is elevated? why doesn’t this apply to low glucose? how would you accurately evaluate CSF glucose?

• elevated CSF glucose is always a result of plasma elevations

  • doesnt apply to low glucose because of CNS pathology… independent of glucose

• to accurately evaluate CSF glucose, run a blood glucose test for comparison

  • should be drawn ~2 hours before the spinal tap to allow for equilibration between blood and fluid

why would CSF lactate be evaluated? what has high conc. of lactate?

• diagnosing and managing meningitis & monitor patients who have severe head injuries

• RBCs have high conc. of lactate so results that are falsely elevated could be due to xanthochromic or hemolyzed fluid

what would be the lactate levels in bacterial, tubular, and fungal meningitis?

• CSF lactate levels greater than 25 mg/dL = bacterial, tubercular, and fungal meningitis

  • occurs more consistently than decreased glucose and provides more reliable info when initial diagnosis is hard

• CSF lactate levels of greater than 35 mg/dL is seen more in bacterial meningitis

• CSF levels of less than 25 mg/dL is seen in viral meningitis

CSF lactate levels will stay elevated during the initial treatment but will rapidly fall when treatment is successful (sensitive method for evaluating the effectiveness of antibiotic therapy)

what are the other causes for an increased level of lactic acid in CSF?

• tissue destruction within the CNS due to oxygen deprivation (hypoxia)

• anything that decreases oxygen flow to tissues

how is glutamine produced and what does it do? what is elevated CSF glutamine associated with?

• glutamine is produced from ammonia and alpha-ketoglutarate by the brain cells to remove toxic ammonia

• elevated CSF glutamine = liver disorders which results in increased blood and CSF ammonia

  • elevated in children with Reye syndrome (~75%)

  • disturbance of consciousness seen in glutamine levels >35 mg/dL (requested in patients who are in a coma for an unknown reason)

what does it mean that conc. of ammonia in CSF is indirectly proportional to alpha-ketoglutarate? what does that mean for glutamine?

• conc. of ammonia goes up then supply of alpha-ketoglutarate goes down

• glutamine can not longer be produced to remove the ammonia and coma ensures

why does the microbiology lab analyze CSF? what would be needed for a positive ID? what is culturing used for?

• analyzes CSF to ID what the causative agent is in meningitis

• positive ID = microorganism must be recovered from the fluid by growing the microorganism in the appropriate culture medium

  • tubercular meningitis ID = 6 weeks; bacterial meningitis = 24 hours

• culturing is used for confirmatory rather than diagnosis

what are the organisms that are frequently encountered on a gram stain?

• Streptococcus pneumoniae (gram + cocci)

• Haemophilus influenzae (pleomorphic gram - rods)

• E. coli (gram - rods)

• Neisseria meningitidis (gram - cocci)

• Streptococcus agalactiae (gram + cocci) and Listeria monocytogenes (gram + rods)

when would acid-fast staining or fluorescent antibody stains be done?

not usually performed on specimens and is only done when tubercular meningitis is suecpeted

• positive report from mycobacteria is extremely valuable due to the length of time to culture Mycobacteria

name and classify the microorganism associated with a positive India ink prep

Cryptococcus neoformans which is a fungi that causes fungal meningitis

• complication of AIDS

• produced a classic starburst pattern and may be seen more often than a positive india ink

what is the latex agglutination test (immunologic assay) regarding CSF? what should be done to the results because of false positives?

• more sensitive method for the detection of C. neoformans than india ink

• results should be confirmed by culture and demonstration of organisms by India ink

what microorganisms latex agglutination and ELISA are also available for?

• Streptococcus group B

• H. influenzae type B

• S. pnuemoniae

• N. meningitidis A, B, C, Y, and W135

• Mycobacterium tuberculosis

• C. imminitis

• E. coli K1 antigens

what is Naegleria fowleri?

an opportunistic parasite (amoeba) that is found in ponds, small lakes, and chlorinated swimming pools that enters the nasal passages and migrates along the olfactory nerves to invade the brain

what is a necessary diagnostic procedure for syphilis? what is the recommended way for specificity regarding neurosyphilis?

• detecting antibodies that are associated with syphilis in CSF is a necessary diagnostic procedure

• Venereal Disease Research Laboratory (VDRL) which looks for reagin (not direct)

  • should be accompanied by a positive serum Fluorescent Treponemal Antibody Absorption (FTA-ABS) for confirmatory

evaluate pertinent laboratory data to determine whether a suspected case of meningitis is of bacterial origin (learning objectives)

bacteria meningitis

• predominately neutrophils with a highly elevated WBC count

• marked protein elevation

• markedly decreased glucose level

• lactate level of >35 mg/dL

evaluate pertinent laboratory data to determine whether a suspected case of meningitis is of viral origin (learning objectives)

viral meningitis

• moderately WBC count with a predominance in lymphocytes

• moderate protein elevation

• normal glucose level

• normal lactate level

evaluate pertinent laboratory data to determine whether a suspected case of meningitis is of fungal origin (learning objectives)

fungal meningitis

• elevated WBC count with both lymphocytes and monocytes

• moderate to marked protein elevation

• normal to decreased glucose level

• lactate level of >25 mg/dL

  • positive india ink with Cryptococcus neoformans

• positive immunological test for C. neoformans

evaluate pertinent laboratory data to determine whether a suspected case of meningitis is of tubercular origin (learning objectives)

tubercular origin

• elevated WBC count with both lymphocytes and monocytes

• moderate to marked protein elevation

• decreased glucose level

• lactate level of >25 mg/dL

• has pellicle formation

semen consists of four components… what male structures contribute to seminal fluid?

1. testes and epididymis

2. seminal vessels

3. prostate gland

4. bulbourethral glands

normal semen must have all four components

explain how sperm is produced and where it matures

• the testes contain the seminiferous tubules for the secretion of sperm

  • scrotum is at an optimal (low) temp for the development of sperm

• sertoli cells give support and nutrients to the germ cells as they undergo spermatogenesis

  • germ cells are in the epithelial cells of the seminiferous tubules

• when spermatogenesis is done, the immature, nonmotile sperm enters the epididymis to mature and develop flagella

  • takes ~90 days to mature

• sperm stays in epididymis until ejaculation → propelled through ductus deferens and to the ejaculatory ducts

what is the importance of the seminal vesicles for seminal fluid? what does the seminal fluid contain that is made by the seminal vesicles?

• seminal vesicles make up 60-70% of the semen volume and is a transport medium for the sperm

  • also produces proteins for semen coagulation

• seminal fluid contains high conc. of fructose and flavin for the sperm

  • fructose = gives energy to propel through the female reproductive tract

  • flavin = gives semen their gray appearance and UV fluorescence (blue to yellow)

what is the importance of the prostate glands for seminal fluid? what does the seminal fluid contain that is made by the prostate glands?

• helps propel the sperm through the urethra by contracting during ejaculation & produces ~20 to 30% of the semen volume that is acidic

  • located below bladder and surrounds upper urethra

• the milky, acidic fluid has high conc. of acid phosphatase, citric acid, zinc, and proteolytic enzymes

  • responsible for the coagulation and liquefaction of the semen after ejaculating

what is the importance of the bulbourethral glands for seminal fluid?

contributes about 5% of the semen volume as a thick, alkaline mucus to neutralize the acidity from the prostate and vagina (sperm motility would be affected if it was not alkaline)

explain the proper collection and handling of semen specimens (learning objective)

• collect semen only through masturbation in a warm sterile glass or plastic container after a period of abstinence for 2-7 days and completely collect the semen (first portion has the most sperm)

  • first portion gone → sperm count down, pH falsely up, specimen wont liquefy

  • last portion gone → sperm count up, volume down, pH falsely down, specimen wont clot

• if collected at home, deliver to lab within an hour and keep at 37C (body temp)

• record patient name, DOB, period of sexual abstinence, completeness of specimen, if there were any difficulties, time specimen was collected and specimen receipt

state the macroscopic parameters reported in a routine semen analysis (learning objective)

appearance

• gray-white color that is opalescent and viscous (musty odor)

volume

• 2-5 mL with a lower reference limit of 1.5 mL

viscosity

• pours droplets within 60 min (0-4 rating)

pH

• 7.2-8.0 with a lower reference limit of greater than 7.2 (measure within an hour)

liquefication

• did it liquify within 30-60 mins?

state the microscopic parameters reported in a routine semen analysis (learning objective)

sperm concentration and count

• greater than 20 to 250 million per mL with a lower reference limit of 15 million per mL (sperm concentration)

• greater than 40 million per ejaculate with a lower reference limit of 39 million per ejaculate (sperm count)

motility

• greater than 50% within an hour with a lower reference limit of 40% within an hour

morphology

• greater than 30% normal forms with a lower reference limit of 4% normal forms

sperm quality

• greater than 2.0

sperm vitality (% alive)

• greater than 75% with a lower reference limit of 58%

round cells in sperm

• less than 1.0 million per mL

what is the normal appearance of semen and the other appearances semen can appear as? (learning objective)

• semen should be gray-white, translucent with musty odor

• increased white turbidity = presence of WBCs and infection within reproductive tract

  • could culture if necessary

  • differentiate WBC from spermatids

  • could use LE reagent strip test for presence of WBC

• diff amounts of red = presence of RBCs (abnormal)

• yellow coloration = urine contamination, specimen collected after prolonged abstinence, medication

what does it mean if the semen does not liquefy within 60 minutes? what should be done if it has not liquefied within 2 hours? what could be present in liquefied specimens?

• if it does not liquefy within 60 mins… deficiency in prostatic enzymes and should be recorded

• if not liquefied within 2 hours → equal volume of Dulbecco’s phosphate-buffered saline (DPBS) or proteolytic enzymes like alpha-chymotrypsin or bromelain should be added

  • may affect biochemical tests, sperm motility, and sperm morphology so record if these were used

  • dilution with bromelain has to be accounted for when calculating sperm concentration

• jelly-like granules but has no clinical significance

semen analysis cannot proceed if there is no liquefaction

when would you see increased semen volume? what is linked with decreased semen volume and what could it indicate?

• increased = prolonged periods of abstinence

• decreased = linked with infertility and could indicate improper function of one of the semen-producing organs, primarily the seminal vesicles

  • consider incomplete specimen collection

how could the viscosity of the semen specimen be measured? how is viscosity reported and rated?

• use a pipette → easily drawn in pipette and forms small discrete droplets that don’t appear clumped or stringy

  • droplets that form threads that are longer than 2 cm → highly viscous and abnormal

• reported as low, normal, or high with ratings from 0-4 (0 = watery; 4 = gel-like)

what could increased viscosity and incomplete liquefaction hinder testing for?

• sperm motility

• sperm concentration

• antisperm antibody detection

• measurement of biochemical markers

what does an increase pH mean? what is a decreased pH associated with? what are the ways you can test pH of a semen specimen?

• increase pH → infection within reproductive tract

• decreased pH → increased prostatic fluid, obstruction of ejaculatory duct, poorly developed seminal vesicles

• testing can be done with pH pad of reagent strip, pH paper (4-10), pH meter device

describe the Neubauer method for sperm counts, including the use of diluting fluid and its purpose (learning objective)

sperm is counted with the Neubauer counting chamber (hemocytometer) like how CSF is counted

• only fully developed sperm are only counted in the 5 RBC squares and is commonly diluted to 1:20 (DF = 20)

  • duplicates must be within 10% and average of the two counts are used in calculation

  • sperm is diluted with sodium bicarbonate and formalin to immobilize the sperm and preserve cells before counting but good results can be achieved with saline and DI water

calculation

• average sperm counted x 20 (dilution) ÷ 0.004 × 5 = # sperm/uL x 1000 mL = # of sperm/mL (sperm concentration)

• sperm concentration x volume in mL = # of sperm/ejaculate (total sperm count)

what kind of microscopy is used to count sperm? what are “round” cells? how can staining help when counting semen?

• counts are performed using either phase or bright field microscopy

• round cells = immature sperms (spermatids) and WBCs

  • not included in counting but their presence could be significant and may need to be ID’d and counted separately

• stains that are in the diluting fluid can help tell the difference between spermatids and leukocytes & help with visualization

  • can be counted in the same way as mature sperm

  • counts greater than 1 million leukocytes per mL = inflammation or infection of the reproductive organs and could lead to infertility

  • presence of greater than 1 million spermatids per mL = disruption of spermatogenesis

what can the disruption of spermatogenesis be caused by?

• viral infections

• exposure to toxic chemicals

• genetic disorders

what is critical for fertility regarding the motility of the sperm? how is sperm motility evaluated? what kind of sperm would need further evaluation and why?

• sperm is capable of forward, progressive movement as the sperm must propel themselves forward through cervical mucosa → uterus → fallopian tubes → ovum

• evaluated subjectively by looking at an undiluted specimen and assessing the % of motile sperm and quality of the motility within an hour of collection

  • % of sperm showing forward movement can be estimated after the examination of 20 hpf’s or 200 sperm per slide or count the percentages of the diff motility categories manually

• presence of high percentage of immobile sperm and clumps… to determine vitality of the sperm or the presence of sperm agglutinins

how would one interpret/grade the motility of the sperm?

• 4 = rapid, straight-line motility

• 3 = slower speed, some lateral movement

• 2 = slow forward progression, noticeable lateral movement

  • minimum motility of 50% with a 2.0 rating after an hour is normal

• 1 = motile without forward progression

• 0 = no movement

what is the normal morphology of a sperm?

• oval shaped head that is ~5 µm long and 3 µm wide & long, flagellar tail that is ~45 µm long

• enzyme-containing acrosomal cap located at the tip of the head for ovum penetration

  • should surround ~1/2 of head and cover ~2/3 of sperm nucleus

• neck piece attaches head to tail and midpiece

• mid piece ~7µm long and thickest part of tail

  • surrounded by a mitochondrial sheath that produces energy that is needed for the tail to be motile

what are the abnormalities of the head portion of the sperm? what about the tail? what about the neck piece?

head

• double heads

• giant and amorphous heads

• pinheads

• tapered heads

• constricted heads

tail

• doubled

• coiled

• bent

neckpiece

• could be long which causes the sperm head to bend backwards and interfere with the motility

how is the sperm morphology evaluated? what are the stains?

• thinly smeared (like how a blood smear is done), stained slide until oil immersion

  • at least 200 sperms should be evaluated and % of abnormal sperm is reported

• stained with Wright’s, Giemsa, Shorr, Papanicolaou stain

how is sperm vitality evaluated? what are the conditions that would be associated with sperm vitality?

• mixing specimen with eosin-nigrosin stain, prepping smear, counting number of dead cells (appears red against purple background) in 100 sperm using bright-field or phase contrast

  • living cells arent stained with eosin-nigrosin and remain a blueish-white

• presence of a lot of vital but immobile cells = defective flagellum

  • a lot of immotile and nonviable cells = epididymal pathology

what would a low sperm concentration be caused by and how does this relate back to fructose? how could semen specimens be screened for fructose?

• could be caused by lack of support medium produced in the seminal vesicles which indicates that the fructose level is low or absent in the semen

• use the resorcinol test which produced an orange color if fructose is present

  • normal quantitative lvl is equal or greater than 10 micromol per ejaculate

what abnormalities could cause low fructose levels?

• seminal vesicles

• bilateral congenital absence of the vas deferens

• obstruction of the ejaculatory duct

• partial retrograde ejaculation

• androgen deficiency

how do antisperm antibodies appear? what do antisperm antibodies do to the sperm in men and women?

• appears when the blood-testes barrier is disrupted through surgery, vasectomy reversal, trauma, and infection

  • antigens will appear on the sperm and produced an immune response that damages the sperm → affects fertility of both men and women (mostly men)

• sperm-agglutinating antibodies causes the sperm to stick to each in a head-to-head, head-to-tail, or tail-to-tail and suspected when clumps of sperm are observed

  • women can produce the antibodies because of an immune response and can be tested by mixing the semen with female cervical mucosa or serum

what is an overview of the mixed agglutination reaction (MAR) test and the immunobead test regarding antisperm antibodies?

mixed agglutination reaction

• screens for the presence of IgG antibodies and uses IgG antihuman globulin (AHG) and suspension of latex particles or treated RBCs coated with IgG

• AHG binds to antibody on sperm and antibody on the particles/RBCs → <10% of motile sperm with attached particles is normal

immunobead test

• used to detect presence of IgG, IgM, and IgA antibodies more specifically and detects which area of the sperm is being affected by the antibodies

• if antibodies are on the head, it blocks the penetration & if antibodies are on the tail, it blocks motility

what are the ways the lab determine whether sperm is actually present in the specimen?

1. examining the vaginal fluid and enhancing the specimen with xylene and examined under phase microscopy

   • sperm is motile up to 24 hours

   • non-motile sperm will be present for up to 3 days

   • sperm will die and only heads will remain up to 7 days

2. detecting for the presence of prostatic acid phosphatase

3. detection of seminal glycoprotein p30 (prostatic specific antigen)

what is an overview of microbial and chemical testing in semen specimens?

microbial testing

• tests for Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum and routine aerobic and anaerobic cultures if more than 1 million leukocytes are present

chemical testing

• disorder of the epididymis → decreased neutral alpha-glucosidase, glycerophosphocholine, and L-carnitine

• lack of prostatic fluid → decreased zinc, citric acid, glutamyl transpeptidase, and acid phosphotase

why is it essential to analyze sperm after a vasectomy? how is postvasectomy semen analysis done?

• detecting for the presence of sperm because it only takes on sperm for fertilization

  • DO NOT OVERLOOK A SINGLE SPERM

• done beginning at two months postvasectomy and continuing until two consecutive monthly specimens show no spermatozoa

  • uses wet prep phase microscopy for presence of motile and nonmotile sperm

  • negative wet prep → centrifuge specimen for 10 mins then examine sediment

what are the three closed cavities and what are they lined by (be specific)? what is a pneumonic to remember these?

• pleural, pericardial, and peritoneal and lined by two serous membranes

  • parietal membrane → lines cavity of wall

  • visceral membrane → covers organs within the cavity

• pneumonic → Loving Hearts Bathe in Serous fluid

  • lungs = pleural

  • heart = pericardial

  • belly/abdomen = peritoneal

what is the purpose of serous fluid? how much serous fluid should be present?

• serous fluid provides lubrication between the two serous membranes which prevents friction that happens between them

  • like when breathing, heart beating, or organs moving around

• only a small amount is present normally

  • production and reabsorption of serous fluid is constant

explain the process of how serous fluid is formed including its reabsorption under normal conditions (learning objective)

1. the production and reabsorption of serous fluid is dependent on hydrostatic pressure and colloidal pressure (oncotic pressure) and formed as ultrafiltrates of plasma

2. colloidal (oncotic) pressure is the same in both membrane capillaries meaning fluid is pulled back at equal pressures (equal oncotic pressure and equal serum proteins)

   • because of this… hydrostatic pressure in both of the membrane capillaries causes pressure to push the serous fluid out and enter between the membranes

3. filtration of the ultrafiltrate increases oncotic pressure in the capillaries due to leaving proteins behind → pulls the serous fluid back into the capillaries to be reabsorbed

   • action of increased oncotic pressure produces a continuous exchange of serous fluid and maintains the normal amount of fluid between the serous membranes

4. the slightly different amounts of positive pressure in the membrane capillaries creates a small excess of fluid which is reabsorbed by the lymphatic capillaries that are located in the membranes

explain the easier way to remember the formation and reabsorption of serous fluid

tug of war

• under normal conditions, hydrostatic and colloidal pressure are almost balanced

  • hydrostatic pushes out and colloidal pulls in

• hydrostatic pressure wins slightly which ends up creating a small excess of fluid while colloidal pressure is pulling consistently to constantly reabsorbing the serous fluid back into the capillaries

• the lymphatic system will quickly remove the excess fluid to keep the volume normal

what does effusion mean?

disruption of the mechanism of the formation and reabsorption of serous fluid which causes an increase amount of fluid between the membranes